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118 Nuclear Medicine Physics

Macrocyclic ligands are, by deﬁnition, cyclic polydentate ligands where the

donor atoms belong to the macrocyclic ring and/or to their pendant arms. The

main characteristics that differentiate macrocyclics from their linear analogs

are the high stability of the complexes formed, their speciﬁcity and selectivity

for the cations that they coordinate, and the slower formation and dissociation

kinetics of their complexes. In fact, polyfunctional macrocyclic ligands show a

very selective coordination proﬁle, which is strongly pH dependent, and form

very stable complexes with several metal ions. The chelate stability depends

on the relationship between the cavity size, the metal ionic radius, the rigidity

of the ligands, and the nature of the coordinating groups. In particular, the

ligand 1,4,7-triazacyclononane-N,N





-triacetate (NOTA) forms chelates of

high stability with Ga

[61]. In fact, the Ga(NOTA) complex has proved to

stay intact ina1Mnitric acid solution for a period of at least 6 months [62].

The high stability of the complex of Ga

is a consequence of the excellent

ﬁtting of the small metal ion (ionic radius 0.76 Å) into the cavity of the NOTA

ligand. This class of ligands binds the metal ion efﬁciently [63], protecting it

from competitor ligands, in particular transferrin (the blood serum protein

that transports iron), which is the main competitor for Ga

in the blood

stream.

As has already been stated, gallium and iron share a very similar chemistry:

charge, ionic radius (62 pm for Ga

and 65 pm for Fe

), and the preference

for the coordination number 6. Transferrin, a plasma protein, has two sites of

binding to iron that also have high afﬁnities for Ga

, and this protein is

presentin high concentrations in plasma, 2.5 ×10

−3

M. The binding constants

for gallium and indium to transferrin are log K(Ga-tf) = 20.3 and log K(In-tf)

= 18.74 [63], respectively. Thus, when injecting

in the form of gallium

citrate (or as another low stability complex), more than 90% of this metal is

complexed by the transferrin.

111

In radiopharmaceuticals, due to its larger ionic radius, 81 pm, and

also its higher coordination number (normally it forms complexes with

coordination number 7), it is preferable to use the octadentate macrocyclic

DOTA (1,4,7,10-tetraaza-cyclododecane, N,N







-tetraacetate), which

forms more stable complexes with this metal [45] (Figure 4.14).

4.8.2.2 Gallium and Indium Radiopharmaceuticals

Ga, in the chemical form of galliumcitrate, was initially used in the detection

of inﬂammation, but this technique became less important when methods

of radiolabeling leucocytes (with

99m

Tc-HMPAO or with

111

In-oxine) were

developed. However, [

Ga]Ga-citrate is still in current use in the diagnosis of

some chronic inﬂammations, in the diagnosis of some inﬂammatory processes

such as sarcoidosis, and in patients with low level of leucocytes, such as AIDS

patients.

In oncology, the citrate of gallium-67 still has a role in the diagnosis of some

tumors of soft tissues, especially in some types of lymphomas. Although it

Radiopharmaceuticals 119

OHOH

FIGURE 4.14

Structure of the macrocyclics NOTA (1,4,7-triazacyclononane-N,N





-triacetate) and DOTA

(1,4,7,10-tetraazacyclododecane-N,N







-tetracetate).

is known that 90% of gallium injected in the blood stream in the form of

gallium citrate is complexed by transferrin, the mechanism of incorporation

of gallium into tumor cells is not yet known. One hypothesis states that this

can be due to the uptake of the complex

Ga-transferrin by the transferrin

receptors of tumor cells, but various other mechanisms can be present or may

coexist with that one.

With the increased number of PET centers and with the establishment of

the importance that

Ga could assume in molecular imaging, the interest in

new ligands for gallium has reemerged and even increased, particularly those

involving bifunctional chelators [64,65].

Until recently,

111

In was almost exclusively used for labeling of leukocytes

and platelets through the complex between

111

and 8-hydroxyquinoline

(oxine) [66]. One of the few exceptions listed in the literature was the use of

111

In-DTPA to evaluate alterations of the blood–brain barrier [67]. Today, this

radioisotope has acquired additional importance in the labeling of antibodies

and peptides (see Section 4.9) [30,45].

111

forms a liposoluble complex with oxine (8-hydroxyquinoline)

and

penetrates in this form into the cell membrane. After diffusion, the complex

111

In-oxine(Figure 4.15) dissociates and the

111

bindsto cytoplasmatic and

nuclear proteins [66]. Although the complex

111

In-leukocyte suffers liver and

spleen uptake, the absence of uptake in the kidneys, bladder, and gallbladder

makes this method of great applicability in middle-low abdomen and chest

scintigraphy.

Reference has also been made, although more rarely, to the use of

immunoglobulin G (IgG) labeled with

111

In in the diagnosis of certain partic-

ular types of inﬂammation (e.g., inﬂammation of the joints) [68]. The complex

111

In-HIG presents the great advantage that it is not taken up in the bone mar-

row. The complex

111

In-oxine is still used to label platelets with a view of the

detection of thrombi [69]. The method developed to label platelets with

111

may also be used in their labeling with

Ga [70].

120 Nuclear Medicine Physics

111

In(Ox)

111

In(Ox)

111

+ 3Ox Ox

Leukocyte or platelet

FIGURE 4.15

Structure of the In-oxine complex and the cell labeling mechanism.

4.8.3 Copper Radioisotopes and Copper Coordination Chemistry

There are several copper radioisotopes with suitable characteristics both for

diagnostics and therapy:

Cu,

Cu, and

Cu. These radio-

isotopes have very different nuclear properties, with half-lives ranging from

9.6 min (

Cu) to 62 h (

Cu), β

and/or β

decay.

Cu is a β

−

emit-

ter with 40% γ emission and hence a suitable therapeutic radionuclide.

However, for diagnostic purposes the most interesting copper radioisotope

Cu. Due to its relatively long half-life (12.8 h),

Cu is mainly used

in PET imaging of biomolecules with very long biological half-lives, in

particular monoclonal antibodies and peptides [71]. Its decay scheme com-

bines β

(19%), β

−

(40%), and electron capture (41%), allowing pretherapeutic

dosimetry estimation. The

Cu radionuclide can be produced in a nuclear

reactor by a

Zn(n,p)

Cu reaction or by the

Ni(p,n)

Cu mechanism in a

cyclotron.

The copper ion exists in only two relevant oxidation states, Cu(I) and

(II). The Cu(II), the most used in the production of radiopharmaceuticals,

prefers to coordinate to compounds containing atoms of nitrogen (amines,

Schiff bases, and pyridines) or sulfur. The chelating agents more commonly

used to complex copper, particularly in the form of bifunctional com-

plexes, include the 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic

acid (CPTA), the 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid

(TETA), and the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

(DOTA) (Figure 4.16) [30].

Radiopharmaceuticals 121

Cyclam

TETA

Cyclen DO2A

DOTA

OH OH

FIGURE 4.16

Macrocyclics used to coordinate copper radioisotopes in radiopharmaceuticals.

4.9 Monoclonal Antibodies and Peptides in

Molecular Imaging

4.9.1 Monoclonal Antibodies

Monoclonal antibodies (mAbs) are proteins with molecular weights averag-

ing 160 kDa. In the early 1970s, these antibodies were initially labeled with

iodine radioisotopes [72]; but today it is more common to use a bifunctional

chelator conjugated to the antibody to complex the radioisotope of choice. The

majority of the mAbs used for tumor targeted belong to the IgG (immuno-

globulin G) family and are formed by two large heavy chains and two small

light chains bounded by disulﬁde bonds.

The usefulness of mAbs in tumor targeting depends on several factors: (1)

the antibodies cannot induce cross reactivity with the normal tissues; (2) their

antigens should be present in a good distribution at the cell membrane; (3) the

antibodies should present a high afﬁnity for the antigen; (4) no free antigens

should be present in the plasma; (5) the tumor should be well vascularized; (6)

the antibody should have rapid clearance from the nonspeciﬁc locals factors;

122 Nuclear Medicine Physics

and (7) the chelate and the conjugate chelate-mAb should present high in vivo

stability.

The high molecular weight of the antibodies implies a slow blood hepato-

biliary clearance. To reduce these disadvantages, mAbs fragments have been

produced with molecular weights 10–100 kDa, which maintain, at the same

time, the biological properties of their intact precursors; these fragments

receivedthe designation of Fab [73]. However, although Fabs reduce the back-

ground due to a more rapid blood clearance and excretion, at the same time,

their tumor uptake is also reduced when compared with the intact mAbs.

For use in radiolabeled mAb, a ligand should fulﬁll the following criteria:

(1) the ligand, covalently coupled to the protein, should rapidly complex the

radioisotope at physiological conditions and (2) the complex formed should

be kinetically and thermodynamically stable under in vivo conditions, in par-

ticular relation to demetalation promoted by other cations that are present in

the blood (e.g., Ca

,Zn

, and Mg

Macrocyclic ligands accomplish these requirements with regard to the

radioisotopes mainly used for mAbs radiolabeling,

111

In,

67/68

Ga, and

[59,73]. There are several examples in the literature of bifunctional derivatives

of NOTA and DOTA coupled to antibodies and radiolabeled with

111

In,

Ga,

Cu, and

Cu [62,74].

Although much enthusiasm and developments have been devoted to this

area, until now the FDA has only approved three monoclonal antibod-

ies for human use:

111

In-DTPA-B72.3 (ONCOScint),

111

In-DTPA-7E11.C53

(Proctascint), and IMMU-4 labeled with

99m

Tc (CEA-Scan) [75].

4.9.2 Peptides

Although mAbs show high tissue afﬁnity and speciﬁcity, they have sev-

eral disadvantages: slow blood clearance, slow delivery to the target, and

a low target to nontarget ratio. Their relatively high molecular weight limits

their localization and diffusion, increasing the length of time between their

administration and imaging acquisition. For these reasons, the introduction

of radiolabeled bioactive peptides in molecular imaging sounds promising,

particularly for oncology [76,77]. Peptides, due to their small size, allow rapid

blood clearance and a high tumor-to-background ratio.

Many peptides are known to have a regulatory, inhibitory, or stimulatory

role in a variety of cellular functions, in both normal and tumor tissues.

Peptides act as hormones, neurotransmitters, neuromodulators, and growth

factors, in addition to performing other functions. They act at very low con-

centrations (from nanomolar to femtomolar), and their action is mediated by

binding to cellular membrane receptors. Most of these receptors belong to the

super-family of G protein coupled receptors, membrane single chain proteins

with seven transmembrane domains, The extracellular parts of the receptor

contain two highly conserved cysteine residues, which form disulﬁde bonds

to stabilize the receptor structure. The extracellular parts are responsible for

the ligand binding, whereas the intracellular domain couples to the G protein.

Radiopharmaceuticals 123

Many tumors express receptors for different peptides, often a large num-

ber, and many of these receptors are known to mediate regulatory growth

effects in vitro. Some types of tumors also respond to an inhibitory effect or

to a growth-promoting effect induced in vivo by peptides [45]. This effect

constitutes an important approach to the clinical treatment of tumors in

humans. An important example is the use of analogs of somatostatin, whose

receptors are overexpressed in neoplastic tissues. Since 1979, radiolabeled

somatostatin analogs have been successfully used both in diagnosis and in

internal radiotherapy [78].

The radiolabeling techniques of small peptides are very similar to those

used for MAb labeling. A natural peptide or an analog is covalently bound

through a spacer to a chelator, which allows the radiometal complexation. In

an alternative approach, the peptide itself contains a prosthetic group able to

be labeled with an iodine radioisotope or with

F for PET imaging [79]. In

most of the natural peptides, the molecular recognition site is limited to very

speciﬁc locations, which provides a clear advantage by allowing modiﬁca-

tion of other peptide regions to make the radiolabeling possible. However,

due to the small size of most of the peptides, the introduction of a prosthetic

group or a chelator may cause relevant changes in the peptide biodistribu-

tion, and this can also dramatically decrease its receptor afﬁnity. Strategies

to diminish the interaction between the chelate and the biological binding

site include the introduction of a spacer between the chelate and the biolog-

ical active portion of the peptide to prevent steric or electronic hindrance in

the speciﬁc binding to the receptor. Considering the possible pharmacologi-

cal effects of a peptide, even if injected in very small quantities, it is crucial

that the radiopeptide is produced with very high speciﬁc activity to guaran-

tee that the radiopharmaceutical will not have any biological activity. This

is the reason that it is of fundamental importance to improve the labeling

efﬁciency, which depends on factors such as ligand concentration, chelation

kinetics, and the presence of trace concentrations of other metals inside the

labeling solution. Another drawback of the in vivo utilization is the rapid

proteolysis of peptides in plasma by endogenous peptidases and proteases.

To circumvent this problem, peptides must be modiﬁed in order to decrease

their enzymatic recognition, in particular by introducing

D-aminoacids or

nonusual aminoacids [80].

The majority of peptides developed for molecular imaging are analogs of

somatostatin. The naturally occurring somatostatin consists of two peptides,

one with 14 amino acids, SS14, and another with 28 amino acids, SS28. There

are cellular receptors of somatostatin in several organs and tissues, as this

neuropeptide presents multiple physiological functions, which are mainly

inhibitory: secretioninhibition of the growthhormone, glucagon, insulin, gas-

trin, and other peptides. It also has an inhibitory function of the immune sys-

tem together with a neuromodulator role in the central nervous system [81].

Since the endogenous somatostatins cannot be used in imaging due to their

low plasma and tissue stability, there has been a need to synthesize somato-

statin analogs that are more resistant to biological degradation. One such

124 Nuclear Medicine Physics

Ala—Gly—Cys—Lys—Asn—Phe

(a) (b)

Cys—Ser—r—Phe

Phe

Trp

Lys

r

r(ol)—Cys

D Phe—Cys

r

Lys

DTrp

Phe

FIGURE 4.17

Structures of (a) somatostatin-14 and (b) the somatostatin analog octreotide. The amino acids

within the oval constitute the recognition site of the receptor.

analog is octreotide (

111

In-DTPA-octreotide, Octreo-Scan) [82]. Figure 4.17

represents the amino acid sequence of somatostatin and its analog octreotide.

Other somatostatin analogs, such as DOTATOC [45] (Figure 4.18), have been

successfully used in the visualization (labeled with

111

In,

67/68

Ga,

99m

Tc, and

Cu) [45,83] and therapy (labeled with β

−

emitters such as

Yor

174

Lu) of

neuroendrocrine tumors and their metastasis [84].

Other regulatory peptides for which it is known that there are receptors in

several types of tumors have also been the subject of considerable inves-

tigation with the objective of obtaining new radiopeptides. Among these

peptides, we should include the α melanocyte-stimulating hormone (αtMSH),

the vasoactive intestinal peptide (VIP), the substance P (SP), the cholecys-

tokinin B (CCK-B), gastrin, neurotensin (NT), and bombesin (BN) [85]. All

these peptides mentioned have multiple regulatory functions, either at the

central nervous system or at the peripheral nervous system. They also have in

NH2

H N

FIGURE 4.18

Chemical structure of DOTATOC.

Radiopharmaceuticals 125

common the fact that their action is mediated by speciﬁc receptors belonging

to the super-family of receptors associated with the G protein.

4.10 Radiopharmaceutical Quality Control: Radiochemical

Purity, Specific Activity, and Radiolysis

The chemical purity of a radiopharmaceutical can be deﬁned as the percent-

age of the total sample radioactivity that is present in the desired chemical

form of the radiopharmaceutical. For instance, if the radiopharmaceutical

is the

99m

Tc-HMPAO, the chemical purity measures the quantity of

99m

coordinated to HMPAO and compares this value with other forms of

99m

present in the sample (e.g., pertechnetate). The radiochemical purity of a

radiopharmaceutical should be close to 95%, as the presence of impurities

with a different biodistribution invalidates a diagnostic test based on scinti-

graphic images. Radiochemical purity in the majority of cases is determined

by separation methods, such as chromatographic techniques, radio-HPLC

and TLC (thin-layer chromatography), or electrophoresis.

The chemical purity of a radiopharmaceutical can also be compromised

by a radiolytic breakdown process releasing unbound radionuclides. For this

reason, it is necessary to establish a compromise between the maximum activ-

ity desirable for biologically active radiopharmaceuticals and the possibility

that this amount of radiation promotes the radiopharmaceutical dissociation

by radiolysis.

Further, the ionizing radiation can break the water molecules down to pro-

duce hydrogen peroxide and free radicals. The presence of these oxidizing

species can compromise the radiolabeling with

99m

Tc, because, as described in

Section4.8.1, the

99m

Tc obtained from thegenerator needs to be reduced before

radiolabeling. This is one of the reasons that the reductor (usually tin chloride)

is present in large excess in the labeling kits. It is necessary to point out within

this context that speciﬁc activity is deﬁned by the ratio between the radionu-

clide activity and the quantity of ligand present in the sample (MBq/μmol).

Since most of the radiopharmaceuticals are administrated by injection (IV),

the product should be prepared under sterile and apyrogenic conditions and

regularly tested for these two parameters.

4.11 Advances in Radiopharmaceutical Development and

New Trends in Radiochemistry

Molecular imaging (using both PET and SPET radiotracers) will encompass

in the future imaging of gene expression,protein expression, protein function,

126 Nuclear Medicine Physics

and physiological function [86]. There is increasing enthusiasm and intense

research interest in the determination of patterns of gene expression that

encode normal processes and in understanding how these patterns change

with disease. In addition to spatiotemporal information, a radiolabeled PET

tracer can provide information as to whether a particular gene product, such

as a receptor or enzyme, is active. The latter information, combined with the

capability for the concurrent analysis of normal and disease tissues and the

assessment of regional variations, makes PET and NM techniques exciting

imaging tools for diagnostics and evaluation of patient response to therapy.

In oncology, the noninvasive monitoring of the dynamics of cell proliferation

by nucleoside tracers will someday become a prerequisite for the develop-

ment of molecular-targeted drugs and their postmarketing evaluation [87].

In conclusion, in the near future we will use more radiopharmaceuticals that

will allow us to image the basic processes underlying diseases rather than

detecting the effects of an installed pathology.

The PET and other molecular imaging methods are being increasingly used

in the pharmaceutical industry. The appropriate use of molecular imaging in

drug discovery and development could signiﬁcantly speed up the develop-

ment process, thus saving money for patients and health care systems by

helping the industry in the extrapolation of in vitro data to in vivo applica-

tion [88]. In addition, radiopharmaceutical development will beneﬁt from

methods already established in the pharmaceutical industry, such as com-

binatorial techniques, rational design, and high-throughput testing. These

approachesprovidea modernalternative tothe rationalconception ofligands,

and very large libraries of candidate molecules can be randomly synthesized

and simultaneously screened to identify those with the desired property.

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