Abstract. Radiolabeled, receptor-specific peptides are becomingincreasingly popular as targeting vectors for the design anddevelopment of new diagnostic and therapeutic radio-pharmaceuticals. The over-expression of functioning receptors ona variety of human cancers makes this method of drugdevelopment a viable tool for tumor targeting in vivo. This reviewdescribes some of the more recent efforts that are currentlyunderway towards development of new receptor-specificradiopharmaceuticals. Diagnostic/therapeutic radionuclides,specific metal co-ordinating ligands/chelating systems, spacertechnology, radiolabeling protocols, and specific peptides/peptideconjugates will be discussed in detail.

Clinical applications of radiolabeled monoclonal antibodies in

recent years (1-4) have demonstrated the potential of site-

directed, biologically-active compounds for development of new

and successful diagnostic and therapeutic radiopharmaceuticals

for human cancers. However, the pitfalls of slow clearance from

blood serum/non-target tissue, and the less-than-desirable

tumor uptake have limited the clinical applications of these

conjugates (1-5). Therefore, radiolabeled, biologically-active,

receptor-specific peptides continue to hold some promise for

diagnosis or treatment of certain human cancers (6-14). This is,

in part, due to the recent successes of Octreoscan® ([111In-

DTPA-octreotide]), a peptide-based radiopharmaceutical that

is currently being used to image neuroendocrine tumors (15).

For example, the clinical utility of Octreoscan® has catalyzed

research efforts in radiolabeling other biologically-active

peptides with various therapeutic or diagnostic nuclides,

including 90Y, 188Re, 64Cu, 111In, 105Rh and 99mTc (15-20).

Lower molecular weight, receptor-specific peptides are

more suitable for delivery of diagnostic or therapeutic

radionuclides to specific binding sites when compared to

monoclonal antibodies. For example, peptides exhibit

distinct advantages over their higher molecular weight

‘cousins’ including rapid clearance from blood serum, ease

of penetration into tumor vascular endothelium, and

increased diffusion rates into human tissue (21-25). As a

result of their small size, receptor-avid peptides possess

relatively low immunogenicity (21-25). Furthermore, high

affinity receptors, expressed on a variety of neoplastic cells,

have been identified for a number of small, receptor-avid

peptides (21-26). Therefore, certain radiolabeled, receptor-

specific peptides are ideal candidates as new diagnostic

and/or therapeutic radiopharmaceuticals.

Diagnostic Radionuclides

Metallic radionuclides can be utilized in gamma scintigraphy

and positron emission tomography (PET) to diagnose

neurological disorders, heart perfusion, renal disorders and

specific cancers. The requirements for SPECT (Single Photon

Emission Computed Tomography) imaging radioisotopes are,

ideally, ready availability, a short physical half-life, and

emission of a single gamma (Á) photon in the 100-250keV

range (27). The physical half-life of diagnostic radiometals is

of utmost importance in radiopharmaceutical development. If

the radiometal is not readily available (i.e., available from a

radionuclide generator), the half-life of the radionuclide must

be sufficiently long to accommodate travel/delivery,

radiopharmaceutical preparation and possible purification, and

dose delivery to the patient. However, while the half-life should

be long enough to overcome these particular concerns, it

should also be as short as possible in order to deliver a minimal

dose to non-target tissue within the patient (27). PET imaging

9

Correspondence to: Dr. Charles J. Smith, Radiopharmaceutical

Sciences Institute and the Department of Radiology. University of

Missouri-Columbia School of Medicine, 143 Major Hall,

Columbia, MO 65211, U.S.A. e-mail: smithcj@health.missouri.edu

Key Words: Peptides, therapy, diagnosis, radionuclide, somatostatin,

bombesin, review.

in vivo 19: 9-30 (2005)

Review

Radiometallation of Receptor-specific Peptides for Diagnosis and Treatment of Human Cancer

MICHAEL F. GIBLIN1,2, BHADRASETTY VEERENDRA2 and CHARLES J. SMITH1,2,3

1The Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO 65201; 2Department of Radiology, University of Missouri-Columbia School of Medicine, Columbia, MO 65211;

3University of Missouri-Columbia Research Reactor Center, Columbia, MO 65211, U.S.A.

0258-851X/2005 $2.00+.40

radiometals are positron-emitting (‚+) radionuclides. Positron

emission results in the production of two 511 keV gamma

photons emitted at an angle of 180Æ. Therefore, PET requires

coincidence counting over many angles around the body axis

of the patient. PET radionuclides often have relatively short

half-lives and are produced by either an accelerator or

cyclotron, limiting their ready availability (27). Table I lists a

variety of selected, metallic radionuclides that have suitable

physical characteristics for SPECT or PET imaging.

SPECT imaging radionuclides. 99mTc continues to be the most

versatile radioisotope in nuclear medicine applications today.

This is due primarily to ready availability, ideal nuclear

decay/imaging characteristics (t1/2 = 6.04h, EÁ = 143keV

(89%)), and well-established radiolabeling chemistries (28-29).

In fact, 99mTc accounts for more than 85% of all diagnostic

applications performed in medical facilities each year. 99mTc is

available at a relatively low cost as Na99mTcO4- radionuclide

and is produced via the 99Mo/99mTc generator system. 99Mo

has a half-life of 2.78 days and decays by ‚- emission to99mTc/99Tc. Na99mTcO4

- is eluted from the generator using

isotonic saline solution. While the 99mTc solution is not

entirely carrier-free, the specific activity of the eluted

precursor is considered to still be very high and is ideal for

radiometallation of biologically-active molecules (30).

Indium-111 is a cyclotron-produced radionuclide with a

sufficiently long half-life (67.9h) to be considered readily

available for site-directed radiopharmaceutical preparation.

In-111 is produced by irradiation of Cd-111 via the111Cd(p,n)111In reaction. In-111 emits two imagable

photons (171keV, 89% and 245keV, 95%) and decays by

electron capture (27). In3+ is considered to be a hard metal

center, preferentially complexing to hard donor atoms such

as oxygen and nitrogen (27). An example of a FDA-

approved, peptide-based, site-directed radiopharmaceutical

used routinely for clinical application is Octreoscan®, an111In-containing diagnostic agent for identification of

neuroendocrine tumors (15).

Gallium radioisotopes continue to receive considerable

interest as SPECT or PET imaging radionuclides. 67Ga is a

cyclotron-produced radionuclide having suitable physical

characteristics for diagnostic imaging. For example, 67Ga

has a half-life of 3.261 days, and emits a host of gamma

photons ranging in energy from 91 to 388keV (27,31). Like

In3+, Ga3+ is also considered to be a hard metal center.

However, in terms of the hard/soft acid base theory,

gallium is considered to be a harder center than indium,

forming complexes with harder oxygen/nitrogen atom

donors (i.e., EDTA or DTPA). Indium, on the other hand,

also forms stable complexes with sulfur atom-donating

ligands (27,32,33).

PET imaging radionuclides. 64Cu is a readily available,

cyclotron-produced radionuclide ((64Zn(n,p)64Cu) or

(64Ni(p,n)64Cu)) with attractive physical characteristics for

PET imaging and radionuclide therapy. For example, 64Cu

has a half-life of 12.7h, emits a 0.651MeV ‚+, and decays by

‚- (0.578MeV) emission (19). There are other ‚+-emitting

isotopes of copper that are also suitable for PET imaging.

However, they suffer from very short half-lives, requiring the

presence of an in-house cyclotron for availability.

As previously mentioned, gallium isotopes continue to

hold some promise as radiolabels to produce site-directed,

diagnostic radiopharmaceuticals. 68Ga is a readily available,

positron-emitting (1.899MeV) radionuclide that is produced

via the 68Ge/68Ga generator system. 68Ga has a very short

half-life (1.13h), which could limit its usefulness for

production of site-directed bioconjugates. However, a shelf-

life of ~2 years for the 68Ge/68Ga generator system (t1/2 of68Ge=270.8d) provides for an ample supply of radionuclide

for PET imaging without the need for an in-house cyclotron

facility (27). 66Ga is another gallium radionuclide with ideal

physical characteristics for PET imaging (34,35). However, a

9.5h half-life and the need for an on-site cyclotron facility

limits its usefulness in production of site-directed

radiopharmaceuticals.

Yttrium-86 continues to receive considerable attention as

a useful radionuclide for PET imaging. 86Y has ideal

physical characteristics (see Table I) including a 14.7h half-

in vivo 19: 9-30 (2005)

10

Table I. Selected, metal-based radionuclides (Á and ‚+) which aresuitable for diagnostic imaging.

Radionuclide Production/ Decay EÁ/E‚+(keV) Reference

Availability

Tc-99m 99Mo/99mTc IT Á, 142.7 28-31

Generator

In-111 Cyclotron EC Á, 171.3 and 15,27,31

245.4

Ga-67 Cyclotron EC Á, 93.3, 184.6, 27,31

and 300.2

Cu-64 Cyclotron EC, ‚-, Á, 1345.8 19,27,31

and ‚+ ‚-, 578,

‚+, 651

Ga-68 Cyclotron EC Á, 1077.3 27,31

and ‚+ ‚+, 1899

Ga-66 Cyclotron EC Á, 1039.3 and 27,34,35

and ‚+ 2752.2

‚+, 4153

Y-86 Cyclotron EC Á, 1076.7, 627.8, 27,36

and ‚+ and 1153.1

‚+, 1248

life, making it a readily available cyclotron-produced

(86Sr(p,n)86Y) radionuclide. Another unique feature of 86Y

is that it is a true "matched pair" of 90Y, a pure beta-

emitting radionuclide that has been investigated extensively

for use in radiotherapeutic applications (36,37).

Therapeutic Radionuclides

Therapeutic radiopharmaceuticals utilize metallic

radionuclides and specific "transport vehicles" (i.e., peptides,

Mabs, specific ligating frameworks) to deliver ionizing

radiation to diseased tissue. Particle-emitting radionuclides

tend to be very effective at delivering cytotoxic ionizing

radiation to a localized site (38-40). However, as with those

diagnostic radionuclides previously mentioned (vide supra),

there are inherent requirements that must be considered

when selecting a therapeutic radiometal for treatment of

specific disease. For example, physical half-life, ready

availability, emission characteristics, nuclear decay properties,

specific activity, and linear energy transfer (LET) of the

radionuclide must be considered (38-40).

Ideally, the physical half-life of the radionuclide should be

well matched to the biological half-life of the delivery vehicle. In

other words, the physical half-life should be very similar to the

in vivo localization and clearance properties of the biomolecular

targeting vector (40-42). Furthermore, the physical half-life of

the radionuclide must once again be sufficiently long to

accommodate travel or delivery to the radiopharmacy,

radiopharmaceutical preparation and possible purification, and

dose delivery/administration to the patient. For shorter half-

lived radionuclides, ready availability and cost effectiveness is

very critical for widespread clinical efficacy (40-42).

Decay properties and emission characteristics are also to

be considered during the design and development of site-

directed, therapeutic radiopharmaceuticals. Beta (‚-)

particle emitters, Alpha (·) particle emitters, and Auger

electron emitters (not to be discussed in this brief review)

offer a diverse spectrum of effective range in tissue and

LET (38). The choice of radioisotope can depend entirely

on the application for which it is to be used. For example,

tumor size and homogeneity might influence whether or not

a low-energy, low-penetrating or high-energy, high-

penetrating radionuclide is used. Luckily, a diverse array of

metallic radionuclides is available for therapeutic

applications (vide infra) (38,43).

Lastly, the specific activity of the radionuclide should be

considered during the design and development of

radiometallated, site-directed radiopharmaceuticals for patient

use. Ideally, the isotope should be available as a carrier-free

radionuclide, free of any non-radioactive target material. The

presence of a non-radioactive metal precursor can and will

compete for complexation to ligand binding sites on the

biologically-active molecule. The non-radioactive, metallated

conjugate will be inseparable from the radiometallated

conjugate using traditional chemical methods of separation

(i.e., RP or size exclusion HPLC) and will compete for low

capacity binding sites on cancerous tissue (39,43,44).

Giblin et al: Radiolabeling of Receptor-specific Peptides

11

Table II. Selected, metal-based radionuclides (· and ‚-) which arepotentially useful for radiotherapy.

Radionuclide Production/ Decay E‚-/E·(MeV) Reference

Availability

Re-188 188W/188Re ‚- and Á ‚-, 2.118, 31,38,46

Generator 1.962

Á, 0.155

Re-186 Reactor ‚- and Á ‚-, 1.071, 31,38

0.933

Á, 0.1372

Rh-105 Reactor ‚- and Á ‚-, 0.566, 47-49

0.248

Á, 0.3192

Cu-67 Accelerator ‚- and Á ‚-, 0.577, 27

0.484, 0.395

Á, 0.185

Y-90 90Sr/90Y ‚- ‚-, 2.27 27,31,38

Generator

Lu-177 Reactor ‚- and Á ‚-, 0.497 31,38,43

Á, 0.2084,

0.1129

Pm-149 Reactor ‚- and Á ‚-, 1.072 31,38,43

Á, 0.286

Sm-153 Reactor ‚- and Á ‚-, 0.69, 31,41,43

0.64

Á, 0.1032,

0.0697

Ho-166 Reactor ‚- and Á ‚-, 1.855, 31,41,43

1.773

Á, 0.0806,

1.3794

At-211 Accelerator EC, ·, Á, 0.687, 38,53

and Á 0.6696

·, 5.868

Bi-212 224Ra/212Bi ‚-, Á, ‚-, 2.251 39.53

Generator and · Á, 0.7273

·, 6.051

Bi-213 225Ac/213Bi ‚-, Á, ‚-, 1.42, 1.02 38,55

Generator and · Á, 0.4405,

0.3237

·, 5.87, 5.55

Beta-emitting radionuclides. An exciting approach for the

development of new diagnostic and therapeutic

radiopharmaceuticals is that of the "matched pair" concept.

"Matched pairs" (i.e., 99mTc/188Re) provide the opportunity

to use information derived from routine patient diagnostic

Single Photon Emission Computed Tomography (SPECT)

studies (99mTc) to determine the receptor availability on

primary and metastatic tissues prior to administration of the

corresponding therapeutic analog (188Re). In this way,

treatment would only be administered to patients previously

demonstrating expression of the target receptor.

Furthermore, the diagnostic radiopharmaceutical would be

invaluable in pre-screening receptor-positive patients for

therapy with respect to drug pharmacokinetics, receptor

density and patient dosimetry, potentially reducing or

eliminating unsuccessful radiotherapeutic regimens (45).

Rhenium-188 continues to hold potential as an isotope for

therapeutic nuclear medicinal applications because of its

widespread availability (188W/188Re Generator) and attractive

physical characteristics (t1/2=16.94h, ‚-max=2.12MeV, and

EÁ=155keV). 186Re is another rhenium isotope that is

available for radiotherapy (38,46). 186Re (Table II) is

prepared by direct neutron irradiation of enriched 185Re in a

nuclear reactor facility. 186Re has a 3.7 day half-life, decays

by ‚- emission (E‚max=1.07MeV), and emits an imagable

gamma photon of 137keV. Although its physical

characteristics make it an attractive isotope for use in

therapeutic application, its low specific activity limits its

usefulness (31,38). 188/186Re-labeled radiopharmaceuticals often parallel the

chemistries used to develop technetium-based

radiopharmaceuticals. However, 188/186Re is used to a lesser

extent than other conventional therapeutic isotopes (i.e.,rare-earth elements) due to the complex radiolabeling

chemistries and purification protocols required for

formation of an in vivo stable radiopharmaceutical. The

formulation of rhenium-based radiopharmaceuticals, like

their technetium surrogates, most often involves complexes

in the +5 oxidation state. However, the reduction potential

of rhenium is ~200mV higher than that of technetium and,

therefore, the reaction conditions necessary for formation

of a *Re-conjugate often necessitate excess reducing agent

(i.e., Sn2+) and/or more harsh reaction conditions (i.e.,heating, pH, etc.). Furthermore, oxidative instability in vivocan be an issue for rhenium complexes/conjugates of this

type. As the radiometal *Re is more difficult to reduce, it is

also more easily oxidized, as compared to technetium (46).

Rhodium-105 has, in recent years, received considerable

interest as a therapeutic radionuclide that would be suitable

for the formulation of a site-directed radiopharmaceutical

primarily due to the formation of low-spin, d6 Rh(III)

complexes that would be stable to in vivo transligation

reactions to proteins such as transferrin (47-49). 105Rh can

be produced in high specific activity in a nuclear reactor

facility by the neutron irradiation of an enriched 104Ru

target. Chemical separation of parent 105Ru from daughter105Rh has been reported (47-49). 105Rh has a half-life of

36h, and emits two ‚- particles of medium (0.56MeV) and

low (0.25MeV) energy. Emission of two imagable gamma

photons (306 and 319keV) would allow for ex vivoevaluation of the injected therapeutic dose (47-49).

Copper-67 (t1/2=62h) is an accelerator-produced

radionuclide (67Zn(n,p)67Cu) that emits a host of ‚-

particles that are suitable for development of site-directed

therapeutic conjugates. For example, Cu-67 emits three ‚-

particles (0.577, 0.484 and 0.395 Mev) and an imagable

gamma photon of 0.185 Mev (27).

Yttrium-90 exists as a 3+ cation and exhibits labeling

chemistries very similar to 111In3+. In fact, the two species

are often mistaken for "matched pair" radionuclides. 90Y is

a pure beta-emitting radionuclide (E‚max=2.27MeV) and is

available in carrier-free form from a 90Sr/90Y generator

system. 90Y has a half-life of 2.7days, making radionuclide

shipment and subsequent preparation/purification of

radiolabeled conjugates quite feasible. Like 111In3+, 90Y is

considered to be a hard metal center, preferentially

complexing to donor ligands containing the elements

nitrogen or oxygen (27,38).

The rare-earth radionuclides, much like In3+, Ga3+ and

Y3+, exist primarily in the 3+ oxidation state and are

considered to be hard metal centers, requiring multidentate,

hard donor ligands for in vivo kinetic inertness (27,43).

Furthermore, the radiolanthanides possess very similar

labeling chemistries while offering a diverse spectrum of

nuclear decay properties (See Table II). Therefore, it is

possible to match a desired set of nuclear properties, (i.e.,t1/2, E‚max, etc.) to a particular clinical application (43). The

radiolanthanides are produced, primarily, via direct or

indirect neutron capture of an irradiated target in a nuclear

reactor facility (43,50). For example, most lanthanide

elements have sufficiently large cross sections to capture a

neutron and are, therefore, considered to be suitable targets

for production of radiolanthanide elements. The

radiolanthanides are considered to be readily available.

However, for those rare-earth radionuclides produced by

direct neutron capture, only moderate specific activities can

be obtained. However, production of no-carrier-added

radiolanthide elements (i.e., 177Lu and 149Pm) has recently

been reported, and these radionuclides do hold significant

promise for future therapeutic applications (43).

Alpha-emitting radionuclides. While ‚- particles tend to

deposit their decay energy over a range of 5-150 cell

diameters (depending upon the isotope), · particles (high-

energy helium nuclei) tend to have ranges across only a few

cells (38,51,52). Therefore, they are ideally suited for

in vivo 19: 9-30 (2005)

12

smaller diameter tumors that are more homogeneous in

nature (38). Alpha-emitting radionuclides are considered to

be high LET radiation, producing a very high density of

ionization along their linear tracks (52-54). Alpha-emitters

are therefore considered to be ideal candidates for site-

directed radiotherapeutic applications as collateral damage

to surrounding normal tissue is often minimal (52-54).

Although there are many ·-emitting radionuclides, only

a handful have been considered for therapeutic

radiopharmaceutical application (38,51). Astatine-211 is an

accelerator-produced (209Bi(·,2n)211At) radionuclide

having ideal physical characteristics for radionuclide therapy

(53). For example, 211At has a half-life of 7.21h and decays

by ·-emission (E·=5.8 and 5.2MeV). Bismuth-212 is

readily available from a 224Ra/212Bi generator system. 212Bi

has a half-life of 1.009h and decays by emission of a 6.1MeV

·-particle (53). Bismuth-213(E·=5.8 and 5.5MeV) has also

been considered to be potentially useful for

radiotherapeutic applications in nuclear medicine (55). With

a half-life of 45.6 min and ready availability from an on-site225Ac/213Bi generator system, this nuclide does hold some

promise for site-directed radiopharmaceutical development

in the near future (55).

Radiolabeling Strategies

Methods of radiolabeling proteins or peptides generally follow

one of two approaches: 1) the direct labeling method, and 2)

the indirect (bifunctional chelate) labeling approach (56). To

be successful, both radiolabeling strategies require that high

specific activity products are obtained (7). A high specific

activity radiolabeled bioconjugate is one in which the maximum

number of molecules possible possess a radionuclide.

The direct radiolabeling strategy. This method of radiolabeling

peptides is predominantly used for 99mTc/186/188Re

radiopharmaceutical production. The direct labeling strategy

offers a relatively short, easy radiosynthetic approach for

producing radiolabeled peptides or proteins. In this method of

peptide/protein labeling, naturally occurring functional groups

(i.e., -SH, -NH2, -OH, etc.) within the peptide/protein sequence

are used to complex to the radioisotope (2,57-60). While this

method offers a simplistic approach to peptide/protein labeling,

there are distinct disadvantages that must also be considered.

For example, this radiolabeling strategy often suffers from lack

of specificity. For example, radiometals can complex within or

near the binding region of the biologically-active compound,

Giblin et al: Radiolabeling of Receptor-specific Peptides

13

Figure 1. Specific ligand frameworks capable of stabilizing the Tc(V) metal center.

thereby inhibiting receptor-binding affinity. Secondly, the

reduction of disulfide bonds via external reducing agents

promotes protein degradation and, ultimately, lack of receptor

binding. Lastly, this method provides for effective radiolabeling

of large proteins/monoclonal antibodies, as smaller peptides

and peptide receptor fragments often lack reducible disulfide

bonds necessary for metal chelation. If reducible disulfide

bonds do exist in these smaller systems, they are often a

necessary component for effective biological activity (58,61).

The indirect labeling strategy. The indirect labeling method

overcomes the problems of the direct labeling strategy by

introduction of a bifunctional chelating moiety onto the

biologically-active compound at some distance away from the

region necessary for receptor binding. The bifunctional

chelating ligand serves to form a stable, high-yield complex

with the metallic nuclide, while at the same time covalently

linking the radioligand to the biologically-active region of the

protein or peptide (62). The indirect labeling strategy can be

categorized into two groups: 1) the preformed chelate

approach, and 2) the post-conjugate method of radiolabeling

(58,63). The preformed chelate approach is most often used

when the isotope-BFCA complex can only be generated under

harsh reaction conditions, such as extreme heating or pH, that

could otherwise destroy the biospecificity of the targeting

vector. The post-conjugate method of radiolabeling

biologically-active molecules is most often the method of

choice, as it offers a one-step synthetic approach to effective

labeling. This method requires the covalent attachment of the

BFCA at some point in the sequence away from the binding

site of the peptide/protein, typically on the N-terminus or on

an Â-amine of a lysine residue (62,63).

Metal-specific Bifunctional Chelating/Co-ordinating Ligand Frameworks

Entire symposia and several reviews have been devoted to

the discussions of design and development of specific

ligating frameworks necessary to stabilize a radioactive

element for in vivo applications (27-30, 38). We intend to

only discuss the general frameworks of specific ligand

entities for this review, but will refer the reader elsewhere

for more detailed information.

Technetium/Rhenium-based. Technetium/Rhenium(V)

conjugates have been extensively investigated for use as site-

directed tumor-targeting vectors for diagnosis and therapy

of human cancers. For conjugates of this type, the metal

center most often exists as the [MO]3+ core. For example,

square pyramidal complexes of the [MO]3+ core with

tetradentate ligand frameworks such as PNAO (propylene

amine oxime), N2S2 constructs (N2S2 = diaminedithiols,

diamidedithiols, or monoamidemonoaminedithiols), and

N3S triamidethiols have been reported (Figure 1) (64-69).

Radiolabeling conditions are relatively straight-forward for

conjugates of this type. For example, radiolabeling is often

performed at either ambient temperature or with minor

heating and at normal or basic pH in the presence of a

reducing agent, typically Sn2+. For the most part, 99mTc-

conjugates of this type tend to be relatively stable in

aqueous environments (64-69).

PNAO conjugates of specific biomolecules have been

reported (64). PNAO complexes of technetium are square

pyramidal, with deprotonation of the secondary amine

atoms upon co-ordination to the metal center. The terminal

in vivo 19: 9-30 (2005)

14

Figure 2. Specific ligand frameworks capable of stabilizing the Tc(I)(CO)3 metal fragment.

hydroxides of the ligand share a single proton upon metal

co-ordination, giving the complex an overall neutral charge

(70,71). Conjugates of this type are ideal from the

standpoint of preparation. For example, the conjugate can

be prepared at ambient temperature in the presence of

reducing agent, typically Sn2+. However, while the

advantages of conjugates of this type seem to be ideal from

a clinical standpoint, PNAO derivatives often suffer from

instability in aqueous solution and high lipophilicity (30).

N2S2 ligand frameworks have become the cornerstone of

production for anionic, neutral, and cationic 99mTc-

bioconjugates (30,65-67). Diamidedithols, for example,

deprotonate on the thiolate sulfurs and the amide nitrogens

to produce anionic technetium conjugates (Figure 1). On

the other hand, diaminedithiols can deprotonate on the

thiolate sulfurs and on one or both nitrogen atoms to form

neutral or cationic species. Monoamidemonoaminedithiols

contain an amide nitrogen, an amine nitrogen, and two

thiolate sulfurs. Co-ordination of the donor atoms is

tribasic, forming an overall neutral 99mTc-complex. N2S2

conjugates tend to be very stable in vivo (30,65-67).

However, 99mTc/188Re-N2S2 conjugates often suffer from

significant in vivo hydrophobicity, hence slower clearance

from blood serum and non-target tissue. Therefore,

conventional labeling of biologically-active compounds with99mTc(V) or 188Re(V), via N3S donor ligands (Figure 1) is

an attractive radiolabeling alternative (72,73). Triamidethiol

complexes of technetium have an overall zero charge. While

conjugates of this type tend to show more favorable in vivopharmacokinetics of the radiopharmaceutical when

radiolabeled with 99mTc, development of a therapeutic

surrogate (186Re/188Re) often proves futile due to

insufficient stability of the radiopharmaceutical in vivo.

Hence, there is a significant trade-off between ligands of

either N2S2 or N3S origin.

An alternative to traditional 99mTcO3+-radiolabeling of

peptides/proteins is the use of 2-hydrazinonicotinamide,

HYNIC (Figure 1), as a bifunctional complexing ligand. The

use of the 99mTc-HYNIC core was first reported by Abrams

(74), for the labeling of polyclonal IgG. Since then, HYNIC

has been conjugated to various biomolecules including

antibodies (75), chemotactic peptides (76), somatostatin

analogs (77), antisense-oligonucleotides (78), interleukin-

879 and many others (80,81). Technetium-99m binds to the

hydrazino-moiety forming a 99mTc-nitrogen bond (81,82).

As HYNIC alone cannot satisfy the coordination

requirements of Tc(V) (HYNIC can only occupy one or two

coordination sites on the radionuclide), coligands are

Giblin et al: Radiolabeling of Receptor-specific Peptides

15

Figure 3. Poly(aminocarboxylate) bifunctional chelating ligand frameworks for lanthanide and lanthanide-like elements.

necessary to complete the coordination sphere of the

technetium(V) core (76). Coligands that have been used to

improve the 99mTc-radiolabeling of HYNIC-biomolecules

include glucoheptonate (83), tricine and ethylenediamine

diacetic acid (EDDA) (75,77,81). Liu and co-workers have

described the use of ternary ligand systems to complete the

coordination environment of the Tc(V)-HYNIC metal

fragment. For example, they have used a water soluble

phosphine or an imine-N-containing heterocycle as an

additional coligand to form a ternary ligand framework

(84,85). Other ternary ligand systems that have been

reported include tricine/pyridine (77,84), tricine/nicotinic

acid (77,79,81) and tricine/trisodium triphenylphosphine-

3,3’,3"-trisulfonate (TPPTS) (80).

Current techniques for radiolabeling peptides containing

disulfide bonds with Tc-99m continue to be a problem. For

example, production of a high specific activity, well-defined

product via direct or indirect radiolabeling methods is

sometimes difficult, as disulfide bond reduction can occur. This

is by virtue of excess reducing agent (Sn2+) in the labeling

cocktail (86). Clearly, this is a problem that plagues formation

of 99mTc(V)- and 188Re(V)-bioconjugates appended with

N2S2/N3S/HYNIC chelators. More recently, an

"organometallic" radiolabeling strategy has been identified.

Recent investigations by Alberto and co-workers have led to

the development of some remarkable Tc(I) and Re(I)

chemistry (87-91). With the development of the new

organometallic triaqua ion [99mTc(H2O)3(CO)3]+, a new

avenue for the successful radiolabeling of bioactive molecules

with low-valent 99mTc/188Re has been achieved (90,91).

[*M(H2O)3(CO)3]+ (*M=99mTc or 188Re) eliminates the

problem of labeling S-S-containing biomolecules and is well

suited for preparing 99mTc-labeled bioconjugates containing

disulfide bonds since excess reducing agent (sodium

borohydride or borane-ammonia complex) is destroyed prior

to the radiolabeling procedure (87-89). Furthermore, the new

[99mTc(H2O)3(CO)3]+ aqua ion is stable over a wide range of

pH values, presumably due to the low-spin, d6 electronic

configuration of Tc/Re(I). Lastly, the lability of the three water

molecules coordinated to the fac-M(CO)3 moiety account for

the excellent labeling efficiencies with a number of donor

ligands including amines, thioethers, phosphines, and thiols

(Figure 2) (87-94). [99mTc(H2O)3(CO)3]+ is now available as

Isolink® from Mallinckrodt Inc., St. Louis, MO, USA.

Lanthanide and lanthanide-like based. Bifunctional chelating

ligands necessary to stabilize the lanthanide (i.e., 153Sm,149Pm, 177Lu) and lanthanide-like elements (i.e., 90Y and111In) for in vivo therapeutic applications are centrally

focused around the poly(aminocarboxylates) (15,16,27,43).

Central to the development of kinetically inert *M3+-

conjugates for the diagnosis or therapy of human cancers

are the polydentate-ligating amino(carboxylates) including

DTPA (diethylenetriaminepentaacetic acid), DOTA

(1,4,7,10-tetraazacyclododecane-N,N’,N",N’"-tetraacetic

acid), and TETA (1,4,8,11-tetraazacyclotetradecane-

N,N’,N",N"’-tetraacetic acid) (Figure 3).

Acyclic poly(aminocarboxylic acids) such as DTPA have

been extensively investigated as stabilizing ligand frameworks

for the radiolanthanide/lanthanide-like elements due to the

successes of Octreoscan® (111In-DTPA-octreotide) for

diagnosis of somatostatin, receptor-positive tumors (15). The

in vivo stability of ligands of this type is presumably due to

the chelating capacity of the multidentate ligand network

in vivo 19: 9-30 (2005)

16

Figure 4. Tetrathiamacrocycles capable of forming stable complexes with Rh(III).

about the metal center. Expanded co-ordination spheres for

these elements necessitate multidentate ligands for in vivokinetic inertness in order to reduce radiation toxicity to

normal tissues such as the bone marrow (43). The chelating

capacity of DTPA can be diminished if one of the pendant

arms necessary for complexation to the metal center is used

for covalent linkage to the biomolecular targeting vector. Invivo decomplexation of the metal center from the ligating

framework can result in isotopic uptake by serum proteins

and other non-target tissues such as the bone marrow. The

consequences of demetallation are undesirable and can be

lethal to the patient (43). In order to overcome the inherent

difficulties of such, many researchers have developed

functionalized derivatives of DTPA so that octadenticity

toward the metal center can be maintained (95,96).

DOTA continues to be the most widely-used ligand

framework for complexation of 3+ metallic radionuclides

(i.e., the rare-earth radionuclides) to biologically-active

molecules. Macrocyclic, polyaza amino(carboxylates) such

as DOTA and TETA tend to form complexes with

lanthanide and lanthanide-like radionuclides that exhibit a

higher degree of in vivo kinetic inertness than acyclic ligands

such as DTPA, presumably due to the inherent macrocyclic

effect of such ligand-metal complexes (15,16,27,43). Ligands

of this type, unlike DTPA and other acyclic ligand

frameworks, provide rigidity to the metallic complexes and

result in in vivo, kinetically-inert, site-directed

bioconjugates. Complexes of DOTA and TETA with

numerous cationic radionuclides have been extensively

investigated (15,16,27,43). Kinetically inert complexes/

conjugates under the most stringent of conditions have been

reported (15,16,27,43).

Radiolabeling strategies employed for complexing M3+

radionuclides to targeting vectors containing

poly(aminocarboxylate) ligand frameworks tend to follow a

universal approach (50,96-101). The radionuclide is

generally shipped in a 0.05M HCl solution as *MCl3.

Complexation of the radionuclide solution to the targeting

Giblin et al: Radiolabeling of Receptor-specific Peptides

17

Figure 5. Cyclam-based ligand framworks capable of forming stable complexes with Cu(II).

vector is often performed in ammonium acetate or

tetraethylammonium acetate solution, pH = 5.5 to 6.0.

Reaction times of 30min to 1h (80ÆC) often provide for

radiolabeling yields of ≥90% (50,100,101).

Specific ligand frameworks for copper and rhodiumradionuclides. 64Cu2+ and 105Rh3+ are two aforementioned

radionuclides that have also been investigated for

production of site-directed, diagnostic/therapeutic

radiopharmaceuticals. Venkatesh, Goswami and Li have

found that tetrathiamacrocycles (Figure 4) are able to

stabilize the Rh3+ metal center to form kinetically inert,

in vivo stable conjugates (47-49). They have suggested that

a combination of the macrocyclic effect and -acidity of

the sulfur donor atoms is responsible for stability of these

Rh3+ complexes (47-49, 102-104).

in vivo 19: 9-30 (2005)

18

Figure 6. Structures of somatostatin and somatostatin-like receptor-specific peptides.

Anderson and co-workers have investigated the relationship

between kinetic inertness and in vivo stability for peptide-

based conjugates of 64Cu2+ (19,27). They and others have

found that derivatives of 1,4,8,11-tetraazacyclotetradecane

(CYCLAM, Figure 5) produce conjugates with the desirable

pharmacokinetics, tumor uptake and in vivo kinetic inertness

to be used as site-directed tumor-targeting agents (19). For

example, *Cu2+-conjugates of CPTA (4-[(1,4,8,11-

tetraazacyclotetradec-1-yl)-methyl]benzoic acid), BAT ([6-(p-

bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-

N,N’,N",N"’-tetraacetic acid), and TETA (1,4,8,11-

tetraazacyclotetradecane-N,N’,N",N"’-tetraacetic acid) have

shown considerable promise as Mab/peptide-based tumor-

targeting vectors (Figures 3 and 5) (27).

Spacer Technology

Spacing moieties (i.e., amino acids or aliphatic spacers) are

often a critical element in site-directed radiopharmaceutical

preparation. These linkers are placed at a point between the

bifunctional chelating ligand and the receptor-binding

region of the molecule in order to preserve biological

integrity or receptor specificity. Furthermore, specific amino

acid or aliphatic spacers can also be used to tune the degree

of hydrophilicity/hydrophobicity of radiometallated

conjugates. An increase in the hydrophilic nature of a

radiolabeled peptide will serve to increase renal clearance

and decrease residence time in blood of the new

radiopharmaceuticals, imparting ideal pharmacokinetic

criteria on the radiolabeled conjugates. Liu and Edwards

have reported that an increase in the hydrophilic nature of

the conjugate can be accomplished by introduction of

"innocent" peptide sequences such as polylysine, -glycine, or

-aspartic acid residues into the peptide sequence (30).

Decristoforo has recently reported that more hydrophobic99mTc-labeled peptides are accumulated in liver tissue and

excreted predominantly by the hepatobiliary pathway (105).

Therefore, hydrophilic site-directed radioconjugates can

provide diagnostically useful abdominal images if needed.

Receptor-specific Peptides as Targeting Vectors

There is a tremendous body of research relating to the

topics of peptide-receptor scintigraphy and peptide-receptor

radiotherapy. This review necessarily touches on only a

small subset of recent results. The five classes of peptides

reviewed here have all received considerable attention in

radiopharmaceutical development efforts. However,

numerous other peptides have also been studied, including

neurotensin, cholecystokinin, E. coli heat-stable enterotoxin,

substance P, and endothelin, among others. For discussion

of these peptide-targeting vectors, as well as more detailed

information on the peptides reviewed here, readers are

referred to several excellent reviews (102, 106-109).

Somatostatin. Analogs of somatostatin (SST) are the most

successful examples of peptide-receptor imaging agents to

date (26). SST is a peptide hormone that is expressed in

both the central and peripheral nervous systems. It exists

naturally in two forms. SST-14 is a 14 amino acid peptide,

Giblin et al: Radiolabeling of Receptor-specific Peptides

19

Figure 7. Structures of VIP, BBN and ·-MSH peptides.

while SST-28 contains those same 14 amino acids, plus a 14

amino acid N-terminal extension. Each peptide possesses a

cyclic domain by virtue of a single disulfide bond (Figure 6).

There are 5 known SST receptor subtypes, SSTR1-5, which

mediate the diverse biological functions of SST. These

receptors belong to the family of G protein-coupled

receptors, are highly expressed on tumors of

neuroendocrine origin, and bind both SST-14 and SST-28

with nanomolar affinity.

Native SST has a very limited half-life in vivo (1-3 min).

As a result, much work has been devoted to the development

of SST analogs with increased stability to proteolytic

degradation, which could be used pharmaceutically for the

treatment of diseases including cancer (110). Three

octapeptide analogs, octreotide, vapreotide and lanreotide,

are currently available in the clinic (111). Each of these is a

truncated analog of SST-14, with a disulfide bond directly

adjacent to the sequence analogous to the four most critical

binding residues in SST-14, Phe-Trp-Lys-Thr. Each contains

a D-Trp substitution for the L isomer, which increases

peptide resistance to proteolytic degradation and also

increases binding affinity. The analogs differ in N- and C-

terminal residues that lie outside of the constrained disulfide

loop, and in substitutions at positions 3 and 6 of the

truncated molecule (Figure 6).

In addition to their clinical utility as antiproliferative

agents that modulate hormonal secretion, SST analogs such

as octreotide have had extensive application as radiolabeled

agents for both imaging and treatment of cancer. Since the

first 123I-labeled octreotide analog was developed (112), a

host of different labeled SST analogs have been reported.

OctreoScan® is a commercially available SST analog (111In-

DTPA-D-Phe1-Octreotide, 111In-pentetreotide) that is

routinely used for imaging of neuroendocrine tumors (15).99mTc depreotide (NeoSpect®, NeoTect®, 99mTc-p829) and99mTc-tricine-HYNIC-Tyr3-octreotide are two 99mTc-labeled

SST analogs that have been tested in the clinic (113).

Use of the DOTA chelator in SST analogs has widened

the range of radionuclides available for imaging and

treatment with radiolabeled SST analogs (114). Both 90Y-

DOTA-lanreotide and 90Y-DOTA-DPhe1-Tyr3-octreotide

(90Y-DOTATOC) have been extensively studied as internal

radiotherapeutic agents (115, 116). In order to predict the

internal dosimetry of such analogs, surrogate molecules have

been synthesized that employ radionuclides such as 86Y117.

A series of PET agents has also been synthesized using

DOTA- and TETA-SST analogs, including tracers based on68Ga (118), 86Y (119) and 64Cu (19,120). Such tracers will

allow more accurate determination of individual internal

dosimetry, thereby perhaps reducing the occurrence of side-

effects such as nephrotoxicity, which can currently be dose-

limiting for peptide receptor radiotherapy (121). Further

refinement of SST analogs is currently being studied,

resulting, for example, in molecules modified in positions 3

and 8 of octreotide to yield tracers with high affinities for

SSTR subtypes 2, 3 and 5 (122). These modified analogs may

prove to be superior vectors for the imaging and treatment

of tumors expressing these SSTR subtypes.

Bombesin. Bombesin (BBN) is a tetradecapeptide

originally isolated from the skin of the frog Bombinabombina (123). In its native form, it is C-terminally

amidated and contains an N-terminal pyroglutamate

residue (Figure 7). Gastrin-releasing peptide (GRP) is a

mammalian analog of the amphibian BBN peptide, as is

neuromedin B. Bombesin shares its 8-14 C-terminal

sequence with GRP, and this sequence (-Trp-Ala-Val-Gly-

His-Leu-Met-NH2) has been shown to be sufficient for

binding to the bombesin receptor (124).

Four GRP receptor subtypes are known – BB1

(neuromedin B receptor subtype) (125), BB2 (GRP

receptor subtype) (126), BB3 ("orphan receptor" subtype)

(127) and BB4 (128). GRP receptors are G protein-coupled,

7 transmembrane receptors which are efficiently

endocytosed upon agonist binding (129). The ability of GRP

agonists to be internalized is one factor that has led to their

use as vehicles for radionuclide imaging and therapy,

despite their proliferative effect on cells expressing GRP

receptors (130). GRP receptors are overexpressed on

various tumor types, including prostate (131, 132), breast

(133, 134), and small cell lung cancer (135). This

overexpression in various neoplasias relative to normal

tissues is another factor that has driven BBN-based

radiopharmaceutical development.

The development of GRP receptor-specific radio-

pharmaceuticals has recently been reviewed (45). Several99mTc- and 188Re-bombesin conjugates have been

synthesized using a wide range of chelators, including

DADT (136), N3S (137), P2S2 (138) and carbonyl (46)

moieties. The clinical utility of RP-527 ([99mTc(V)-N3S-5-

Ava-BBN[7-14]NH2]) as a cancer specific imaging agent

was recently demonstrated in human patients with either

prostate or breast cancer. These studies showed that

[99mTc-N3S-5-Ava-BBN[7-14]NH2] localizes in tumors with

high specificity, producing good tumor-to-normal tissue

uptake ratios and high quality SPECT images (72).

Extensive work has been done with DOTA-labeled BBN

anaolgs, both for imaging with 111In (100) and for

treatment with various radiolanthanides (101). Most

recently, PET imaging agents have been developed using

radionuclides such as 64Cu (139,140).

The majority of research into BBN-based

radiopharmaceuticals has been carried out using GRP

agonists (129, 140). Again, one reason for this has been that

GRP agonists are internalized upon receptor binding,

thereby residualizing the attached radiometal within the

in vivo 19: 9-30 (2005)

20

targeted cell. One recent study utilizing a 99mTc-labeled

GRP antagonist adds complexity to this picture (141). Since

GRP antagonists are presumably not internalized to the

degree that GRP agonists are, agonists have been expected

to more effectively residualize label within tumor cells.

However, in a study using 99mTc-labeled Demobesin-1,

tumor localization was 3-8 times higher than for various

GRP agonists at 1 h pi (45). Furthermore, at 24 h pi, 99mTc-

Demobesin-1 was well retained at the tumor site (5.24 +

0.67 %ID/g), at 3- to 5-fold higher levels than previously

reported for GRP agonists (141). This study suggests that

the concept of GRP agonists being preferable for

radiopharmaceutical development, while intuitively

compelling, may bear reexamination.

RGD-containing peptides. Peptides containing the amino acid

sequence Arg-Gly-Asp (RGD) have been used extensively to

target integrin receptors up-regulated on tumor cells and

neovasculature. The RGD consensus sequence appears in

several proteins of the extracellular matrix, including

vitronectin, fibronectin, fibrinogen, von Willebrand factor,

thrombospondin and osteopontin (142). Integrin recognition

of the canonical RGD sequence plays a prominent role in

many cell-cell and cell-ECM interactions. Integrins are cell

surface transmembrane glycoproteins that exist as ·‚

heterodimers. At least 24 different combinations of ·‚

heterodimers are known (143). The integrins of most interest

in cancer imaging and therapy contain the ·v subunit,

particularly the ·v‚3 and ·v‚5 subtypes. The structure of the

extracellular domain of the ·v‚3 integrin was determined

crystallographically to 3.1 Å resolution in 2001 (144).

The ·v‚3 integrin is known to be overexpressed in many

tumor types, and expressed at lower levels in normal tissues

(145). Both ·v‚3 and ·v‚5 subtypes are expressed in

neovasculature during angiogenesis (145). These expression

patterns form the basis of attempts to image angiogenesis

and tumor formation in vivo using RGD-based peptide-

targeting vectors. RGD peptides used to target integrin

receptors generally fall into one of three categories: linear,

disulfide-cyclized, and head-to-tail cyclized.

At least two examples of integrin targeting using linear

RGD-containing peptides have been described. In one

instance, two RGDS peptide sequences from human

fibronectin were linked in series and labeled with 99mTc

(146). A single cysteine residue intervening between the two

RGDS sequences presumably served to localize 99mTc

binding at that position within the molecule. This conjugate

was studied in fourteen melanoma patients. In this group,

eleven metastatic lesions were visualized, with high

background activity in the lung and abdomen (146). A

second study described the N-terminal labeling of an RGD-

containing peptide (KPQVTRGDVFTEG-NH2) with an

[18F]fluorobenzoyl moiety by solid phase synthesis (147).

RGD-dependent tumor uptake of this agent was not

observed in a C26/BalbC in vivo model, probably due to

both the relatively low affinity of the linear molecule for the

integrin receptor (IC50 = 20 ÌM) and to the rapid rate of

proteolytic breakdown of the construct.

Cyclization of RGD-containing peptides has been shown

to increase both the specificity and affinity of peptides for

defined integrin subtypes (148,149). In one instance,

screening of a phage display library yielded an RGD peptide

cyclized via two disulfide bonds, termed RGD-4C (150). An

abbreviated version of this cyclic RGD peptide has been

used as substrate for 99mTc labeling (151). The affinity of the99mTc-labeled peptide for the ·v‚3 integrin was estimated to

be approximately 70-fold lower than that of RGD-4C,

suggesting that some aspect of the tracer synthesis could be

responsible for a lack of specific tumor uptake in vivo.

A large number of studies have been carried out utilizing

head-to-tail cyclized RGD peptides, and this represents the

most promising class of RGD-based imaging agents (152).

Early investigations into the effect of head-to-tail cyclization

on RGD affinity for the ·v‚3 integrin led to the

identification of cyclo(RGDfV), an ·v‚3 antagonist with a

low nanomolar IC50. Further characterization led to the

observation that a bulky hydrophobic residue was required

in position 4 for maximum affinity, while position 5 was

tolerant of a range of substitutions (153).

One result of these observations was that D- or L-tyrosine

was inserted into either position 4 or 5 to yield RGD peptide

substrates for iodination reactions. The resulting 125I-labeled

peptides have been tested in vitro and in vivo to examine the

feasibility of using cyclic RGD peptides as in vivo imaging

agents (153). These peptides displayed rapid blood clearance

through hepatobiliary excretion, and tumor uptake at 1 h pi

of 1.30±0.13 %ID/g in M21 melanoma human tumor

xenografts. In an attempt to improve the pharmacokinetics

of these compounds, a glycosylated analog was synthesized

and tested (154). Substitution of a lysine residue permitted

the attachment of a carbohydrate moiety to the  amino

group of lys5. The resulting molecule showed decreased

uptake in liver and intestine relative to an analog lacking

carbohydrate, with 1.22±0.32 %ID/g vs. 11.23±1.95 %ID/g

in the liver at 1 h pi. Additionally, the glycosylated species

showed increased tumor uptake and residualization, with

2.05±0.55 %ID/g in M21 melanoma human tumor

xenografts. Similar biodistribution results were obtained

using an 18F-labeled galacto-RGD analog (155). ·v‚3-

targeted cyclic pentapeptides commonly include a lysine

residue in position 5 for the purpose of appending a diverse

array of labeling moieties. PET tracers have been

synthesized by appending a PEG- [18F]fluorobenzoate

domain (156). Metal chelators such as DTPA and DOTA

have also been used to coordinate a range of radionuclides

useful for imaging and therapy (157, 158).

Giblin et al: Radiolabeling of Receptor-specific Peptides

21

Increasing attention is currently focused on the development

of dimeric or multimeric cyclic RGD peptide constructs.

Multimerization is expected to increase the apparent affinity

of targeting vectors for their cognate receptors due to avidity

effects. In one instance, a dimeric cyclo(RGDfK) construct was

synthesized by bridging two lysine  amino groups with a

DOTA-glutamic acid moiety (159). This peptide, when labeled

with 111In, showed high receptor-mediated tumor uptake of

7.5%ID/g at 2 h pi in an OVCAR-3/nude mouse in vivo model.

Receptor-mediated uptake in nontarget tissues such as spleen,

liver, and lung was also surprisingly high in this study. In a

second study, cyclo(RGDfE) multimers were formed using

variable-length PEG spacers linked to a diaminopropionic

acid/lysine backbone (160). Monomeric, dimeric, and

tetrameric forms were synthesized and labeled with 18F.

Biodistribution of the dimer compared favorably with that of

an 18F-labeled galacto-RGD analog.

Vasoactive intestinal peptide. Vasoactive intestinal peptide

(VIP) is a 28 amino acid peptide neurotransmitter with a

wide range of biological activities in mammals (161). VIP is a

member of a larger family of peptide hormones that includes

secretin, glucagon, peptide histidine methionine amide,

growth hormone-releasing factor (GRF), and pituitary

adenylate cyclase-activating peptide (PACAP) (162). VIP

receptors are highly expressed in various tumor types (163),

including intestinal adenocarcinomas (164) and breast

cancers (165). Recognition of this fact has led to the use of

labeled VIP analogs for peptide receptor scintigraphy (166).

VIP action is mediated through two known receptor

subtypes, VPAC1 and VPAC2. VPAC1 and VPAC2 are

members of the G protein-coupled receptor (GPCR)

superfamily. These receptors can be distinguished

pharmacologically through the use of subtype-specific VIP

analogs. Such experiments have demonstrated the

predominance of VPAC1 expression in the majority of

human tumors (163).

The use of radiolabeled VIP analogs for in vivo imaging

of cancer has been documented in numerous studies. VIP

analogs have been labeled with 123I (164, 167), 99mTc (168),64Cu (169), and 18F (170). 123I-labeling occurs at tyr10

and/or tyr22 of the native molecule (Figure 7), while

incorporation of 99mTc and 64Cu occurred via addition of C-

terminal chelating moieties (169). 123I-VIP was studied in

over 200 patients, demonstrating the ability to specifically

localize intestinal adenocarcinomas and carcinoid tumors invivo. The 123I-VIP preparations used were purified by RP-

HPLC prior to injection, and even so caused a small,

transient blood pressure drop due to the vasodilatory effect

of VIP (164). High receptor-mediated uptake was also

observed in the lungs in these studies due to high expression

of VIP receptors in lung acini, which reduces the ability to

observe pulmonary lesions.

Much current effort is being expended on the

development of VIP analogs with specificity for VPAC1 and

VPAC2 subtypes (162, 171), and with increased stability

toward in vivo proteolytic degradation (172). For example, a

modified VIP analog was synthesized with 9 alanine

substitutions at positions 2, 8, 9, 11, 19, 24, 25, 27 and 28

(171). This analog was equipotent to the wild-type peptide

at VPAC1 receptors (IC50=1.6±0.1 nM), yet showed

significantly increased stability to proteolytic degradation.

Incorporation of an arginine residue in the nonessential

position 8 of VIP resulted in a fluorescent dye-labeled

analog with increased tumor-targeting capacity over dye-

labeled wild-type VIP (172). Continuing structure/function

studies will provide new lead compounds for development

of VIP-based imaging agents.

·-MSH. ·-Melanocyte stimulating hormone (·-MSH) is an

N-terminally acetylated, C-terminally amidated

tridecapeptide [Ac-S1YSMEHFRWGKPV13-NH2] that

regulates skin pigmentation in most vertebrates (173). The

biological activity of this peptide in vivo is mediated by

interactions with the melanocortin 1 receptor (MC1R), one

of five known subtypes of G-protein-coupled melanocortin

receptors. ·-MSH receptors are expressed on melanoma

cell lines and on human melanoma tissue samples (174,

175). This, coupled with the low nanomolar affinity of ·-

MSH for its receptor and the ability of the peptide-receptor

complex to rapidly internalize, has catalyzed investigation

into the potential of ·-MSH-based imaging and therapeutic

radiopharmaceuticals. The ·-MSH-based peptide

radiopharmaceuticals studied to date can be broadly

classified as either linear or cyclic structures.

The class of linear ·-MSH peptide radiopharmaceuticals

is based upon a superpotent analog (NDP-MSH) developed

over 20 years ago (176). This is a highly potent, protease-

resistant analog identical to wild-type ·-MSH, but for the

substitution of D-Phe at position 7 and norleucine at

position 4. The first radiopharmaceutical in this class

consisted of two NDP-MSH molecules bridged by a single111In-DPTA moiety (177). Although this construct did

demonstrate specific tumor uptake, high nonspecific kidney

and liver uptake limited its clinical utility (178).

Dehalogenation of 125I-NDP-MSH in vivo (179) has been

overcome through the use of N-Succinimidyl 3-125I-

Iodobenzoate (180). Use of this reagent results in addition

of 125I-IBA to the  amino group of Lys11, and lends the

resulting labeled peptide increased inertness to

dehalogenation. The NDP-MSH molecule has also been

conjugated to the DOTA moiety to provide a targeting

vector capable of coordinating a wide range of

radioisotopes. The DOTA group in one study was attached

to the N-terminus of both NDP-MSH and an altered

octapeptide fragment of NDP-MSH and used for

in vivo 19: 9-30 (2005)

22

coordination of 111In (181). The DOTA moiety has also

been linked to the  amino group of Lys11 in a similar

octapeptide fragment of NDP-MSH and used to coordinate67Ga/68Ga (182). In a B16F1/B6D2F1 in vivo murine

melanoma model, tumor uptake of these peptides at 24h pi

was between 1.17±0.13 and 3.10±0.36%ID/g, while kidney

uptake at that time point ranged between 2.04±0.17 and

6.56±0.77%ID/g.

The cyclic ·-MSH radiopharmaceuticals under current

study are based upon a superpotent ·-MSH analog, Cys4,10,

D-Phe7-·-MSH4-13, first described in 1985 (183). Using a

rational design approach, the structure of this peptide was

modified such that incorporation of technetium or rhenium

led to ring closure and cyclization of the peptide via a

Tc(V)O3+ or Re(V)O3+ core (17). Subsequent studies have

extended this approach, for example, by altering the Lys11

residue in order to reduce nonspecific kidney uptake (184,

185). Further work has resulted in peptides that retain a

nonradioactive Re(V)O3+ core to preserve peptide stability

and affinity, while also including an N-terminal DOTA

moiety for the coordination of a more diverse array of

radionuclides (186).

Conclusion

This review is but a brief introduction into the design and

development of site-directed, diagnostic/therapeutic,

peptide-based radiopharmaceuticals. In brief, we have

described many of the important aspects necessary to

develop conjugates of this type, keeping in mind the

capacity of these conjugates to target specific human tissues.

In order to continue the development of, or improve,

existing radiopharmaceuticals of this type, a collaborative

research effort among scientists and physicians in inorganic

chemistry, organic/medicinal chemistry, analytical chemistry,

biochemistry, molecular biology, radiology and nuclear

medicine is paramount. Great strides will continue to be

made in the field of radiopharmaceutical chemistry, but only

through interdisciplinary research efforts.

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Received November 2, 2004Accepted December 17, 2004

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