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99m
[
Tc]Tc-HYNIC-BN derivatives were also developed (Figure12.9). The tracer
99m
[
Tc]Tc- ED DA-HYNIC- [Lys3]BN(1-14) exhibited tumor uptake of 0.30% ID/g at two hours
p.i. in tumor-bearing mice[143]. Also, [
99m
Tc]Tc-[HYNIC-Aca-BN(7-14)]-tricine-TPPTS, where
Aca is ε-amino-caproic acid, was developed and exhibited tumor uptake of 2.24 ± 0.64%
ID/g after 30 minutes in PC-3 tumor-bearing mice. The latter was evaluated in human
patients with established prostate cancer, but the agent failed to detect the tumors by
SPECT imaging[144, 145]. Furthermore, the tracer [
99m
Tc]Tc- [HYNIC- (Ac a - BN(7-14))2]-tricine-TPPTS, where HYNIC was conjugated to 2 Aca-BN(7-14) moieties, was developed, and
exhibited comparable tumor targeting with the monomer in tumor-bearing animals[146].
12.8.2 Somatostatin Receptor Imaging Agents
The development of somatostatin receptor (SSTR) imaging agents has been shown to be
useful in the diagnosis of SSTR-overexpressing tumors, such as neuroendocrine gastroentero-pancreatic tumors. Successful eorts were made to develop a
imaging agent by using the octapeptides octreotide, D-Phe-c(Cys-Phe-D-Trp-Lys-Thr-Cys)Thr(ol), and octreotate, D-Phe-c(Cys-Phe-D-Trp-Lys-Thr-Cys)-Thr, known to have a high
binding anity for the SSTR type-2 receptor. The HYNIC ligand was conjugated to [Tyr]
octreotide (TOC), and the respective [
HYNIC-TOC tracers were developed. The tracer [
99m
Tc]Tc-HYNIC-TOC-tricine and [
99m
Tc]Tc-EDDA-HYNIC-TOC (Figure12.10)
exhibited the highest stability, its binding anity in AR42J cell membranes was found to
be K
= 2.6 nM, and its tumor uptake in AR42J tumor-bearing mice was 9.65 ± 2.16% ID/g
D
at four hour s p.i.[147, 148]. [
99m
Tc]Tc-EDDA-HYNIC-TOC underwent clinical evaluation and
was licensed in Europe[149]. More recently, a dimeric octreotide analogue was developed, where the HYNIC chelator was conjugated to two TOC moieties, (HYNIC-TOC
a high anity for SSTR2 (IC
= 0.74 ± 0.19 nM). This tracer, [
50
99m
Tc]Tc- ED DA-HYNIC-TOC2,
exhibited high tumor uptake 13.31 ± 3.14 %ID/g at one hour p.i., although high kidney
uptake was obtained (94.40 ± 6.51%ID/g at one hour p.i.)[150].
A series of TOC and [Tyr]
3
-octreotate (TATE) derivatives were conjugated to the acyclic
tetramine chelator for labeling with technetium-99m (Demotide/Demotate series). From
this series, the tetraamine-functionalized TATE derivative [
99m
Tc-Demotate-1 (Figure12.10) exhibited a high binding anity in AR42J cell membranes
(K
=0.07 nM) as well as high in vivo AR42J tumor uptake in mice at one hour p.i. (25%
D
99m
Tc]Tc- [N
ID/g) with high tumor retention (20% ID/g) at four hours p.i.[151]. In a pilot study,
Demotate-1 was investigated in humans where tumor imaging was obtained at one hour
p.i., making it a promising radiopharmaceutical[152].
Furthermore, [
99m
Tc][Tc-N
0–1
, Asp0, Tyr3]octreotate,
4
99m
Tc-Demotate-2, was developed by inserting one aspartate in the sequence, which exhibited a high binding anity
to somatostatin receptors (IC
=3.2 nM) in CA20948 sst2r-expressing tumors. The in
50
vivo behavior of this radioligand in tumor-bearing rats was similar to the gold stan-
111
dard, [
In][In-DTPA-octreotide)][153]. In the same series, [
Demotide, and [
99m
Tc][Tc-N4-X-TATE],
99m
Tc-Demotate-4 were developed, where X was
99m
Tc][Tc-N4-X-TOC],
the (p-[(carboxymethoxy)acetyl]aminobenzyl) moiety inserted between the tetramine
99m
Tc-based SSTR
99m
Tc]Tc- ED DA-
0
,Tyr3]octreotate,
4
99m
99m
Tc-
3
), with
2
Tc-
-
Chapter 12:
99m
Tc Radiopharmaceutical Chemistry 395
H
2
Demotate1
OH
L
OH
N
NH
O
H
O
HO
HO
N
O
S
H
N
HN
N
N
N
L
Tc
L
L
99m
[
Tc]TcEDDA/HYNIC-TOC, L=EDDA,
99m
Tc-Tektrotyd
Figure 12.10 Structures of
O
O
NH
NH
NH
2
O
S
O
99m
H
N
N
H
O
O
H
N
N
H
HO
Tc-labelled SSTR2 imaging agents.
HN
O
NH
O
HN
NH
Tc
N
N
O
H
H
2
2
99m
[
Tc]Tc-tetramine-TATE
O
HN
O
NH
S
S
O
HN
O
+
HO
NH
O
HN
O
NH
HO
O
HO
99m
Tc-
chelator and the N-terminus of the peptide. Their IC50 values in AR42J cells indicated
a high binding anity for SSTR2 receptors, 0.16 ± 0.05 nM and 0.10 ± 0.04 nM for
Demotide and
99m
Tc-Demotate-4, respectively. These tracers also exhibited high tumor
99m
Tc-
uptake in AR42J tumor-bearing mice of 27.87 ± 2.07% ID/g and 29.46 ± 2.63% ID/g at
one hour p.i., for
99m
Tc-Demotide and
99m
Tc-Demotate-4, respectively, although unfavor-
ably high background activity was observed, especially in the abdomen[154].
In another approach, a series of
99m
Tc-cyclized-octreotide and octreotate derivatives
were developed, where the oxometal (technetium-99m or rhenium) was coordinated
via the two reduced sulfydryl cysteine moieties and two additional donor atoms of the
peptide sequence. The position of the additional coordinating sites greatly inuenced
the binding anity as well as the in vitro stability. The best analogue in terms of binding
anity was the Re-cyclized-TATE with IC
=29.2 ± 6.2 nM; however, the
50
99m
Tc-c yclized-
TATE analogue had poor stability[155–157].
Additionally, some eort was made to develop somatostatin receptor antagonist peptides that exhibit dierent pharmacological properties compared to the agonists octreotide and octreotate, which may translate to dierent tumor uptake and retention. One
such eort described the antagonist peptide 4-NO
DTyr- N H
with [
anity in AR42J cells (IC
cessfully targeted SSTR-positive tumors via a receptor-mediated process in AR42J-tumor-
(sst2-ANT) that was conjugated to a tridentate (N,S,N) chelator and labelled
2
99m
Tc][Tc(CO)3]+. The Re complex Re(CO)3(N,S,N- sst2-ANT) exhibited good binding
=15 ± 4 nM), and the tracer [
50
-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-
2
99m
Tc]Tc(CO)3(N,S,N- sst2-ANT) suc-
bearing mice; however, low tumor uptake was obtained, probably due to unfavorable
pharmacokinetics[158].
396 Handbook of Radiopharmaceuticals
Imaging Agents
Prostate-specic membrane antigen (PSMA) is a homodimeric type II transmembrane
metalloenzyme that functions as a glutamate carboxypeptidase and a folate hydrolase
that is overexpressed in prostate cancer. The
have been developed for the diagnosis of prostate cancer belong either to the class of
99m
Tc-labelled small-molecule inhibitors of the PSMA glutamate carboxypeptidase moiety
with glutamate-urea-X sequences of varying X amino acids or to
reactive antibody constructs with improved imaging characteristics for targeting PSMA,
compared to the older radiopharmaceutical
Series of [
99m
Tc][Tc(CO)3(L-linker-(X-urea-Glu))] chelates of the glutamate-urea-type
inhibitors with glutamate, lysine, or cysteine as X were developed with various tridentate
ligands[159–162]. In these conjugates, the linker length varied from none to 11-carbon.
From the structure-activity relationship (SAR) studies that were performed, the nature
of the chelate, as well as the length and composition of the linker, signicantly inuenced the in vitro binding anity. The chelators that exhibited higher binding anities
were those that contained additional carboxylates, such as bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) and bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)1H-imidazol-2-yl)methyl)amino (TIM), indicating that perhaps these carboxylates form
additional interactions in the PSMA binding area. On the other hand, radioligands that
contained the chelators DPA, di-(1-1H-methylimidazol-2-yl) methylamine or PAMA exhibited low binding anity[160, 161]. Four of the [
conjugates developed, with SAAC-type ligands exhibiting higher binding anities, were
evaluated in vitro in PSMA and non-PSMA expressing cancer cell lines as well as in vivo in
tumor-bearing animals, of which
99m
Tc-MIP-1404 and
ited the most promising tumor uptake in LNCaP xenografts of 11.1 ± 4.1 and 8.65 ± 1.88%
ID/g at four hours p.i., respectively. Their binding anities (K
culated to be 1.07 ± 0.89 nM and 4.35 ± 0.35 nM for
respectively[163]. A rst-in-human study in prostate cancer patients was performed for
99m
Tc-MIP-1404 and
99m
Tc-MIP-1405, and both agents facilitated the detection of radiologically proven prostate cancer in bone, lymph nodes, and the prostate gland by PSMA
targeting; however,
99m
Tc-MIP-1404 exhibited lower uptake in the kidney, which makes it
more promising for the detection of early stages of the disease in the prostate gland and
the pelvis[164]. In a phase II study,
99m
Tc-MIP-1404 (
ability to detect primary prostate cancer (in patients prior to prostatectomy) with high
accuracy (94% detection rates) by SPECT/CT[165]. In another clinical study in patients
with biochemical recurrence of prostate cancer, overall, a 70% detection rate was
obtained[166]. Currently, a phase III study is underway for this agent.
Additionally, [
99m
Tc][Tc O(N2S2 or N3S)] and [
to a Glu-urea-Lys moiety were developed with a high anity for PSMA and the ability
to visualize the tumor in PC3-PIP(PSMA+)-bearing mice with SPECT/CT imaging[167]. In
the HYNIC approach, also, the inhibitor glutamate-urea-lysine-β-naphthylalanine-HYNIC
99m
Tc-PSMA-targeted imaging agents that
99m
Tc-labelled immuno-
99m
Tc-Prostascint.
99m
Tc][Tc(CO)3(SAAC-linker-(X-urea-Glu))]
99m
Tc-MIP-1405 (Figure12.11) exhib-
) on LNCaP cells were cal-
99m
Tc-MIP-1404 and
99m
99m
Tc][Tc(HYNIC)(tricine)] probes conjugated
D
99m
Tc-MIP-14 05,
Tc-trofolastat) demonstrated its
Chapter 12:
99m
Tc Radiopharmaceutical Chemistry 397
(iPSMA) was developed. The tracer [
HO
OH
R
99m
Tc]Tc-EDDA-HYNIC-iPSMA (Figure12.11) exhibited
high tumor uptake in LNCaP-tumor-bearing mice of 9.84 ± 2.63% ID/g at three hours p.i..
Preliminary images in patients demonstrated the ability of this probe to detect tumors
and metastases of prostate cancer[168]. The ligand glutamate-urea-lysine-6-aminohexanoic acid -HYNIC (HYNIC-ALUG) was developed, and the tracer [
99m
Tc]Tc- ED DA-HYNICALUG (Figure12.11) demonstrated high tumor uptake in PSMA-positive LNCaP xenografts
as early as one hour, peaking at two hours p.i. (19.45 ± 2.14% ID/g)[169].
The monoclonal antibody J591, which binds to an extracellular antigen of PSMA,
is being evaluated for the development of radiopharmaceuticals in combination with
suitable radionuclides. A diabody derived from J591 bearing a His-tag (J591Cdia) was
developed, and it was eciently labelled with [
diabody J591C exhibited a high anity for PSMA of IC
99m
Tc][Tc(CO)3]. It was shown that the
=3.4 nM in PSMA-expressing
50
cells; and furthermore, it exhibited 12.1 ± 1.7% ID/g tumor uptake in mice bearing DU145(+PSMA) tumors at eight hours p.i.[170]. This concept shows promise and has been more
recently extended to the development of a J591(scFv) bearing a His-tag for the same
purpose[171].
12.8.4
The Arginine-Glycine-Aspartate sequence selectively binds to intergins. In particular,
radiolabelled cyclic RGD peptides have been developed as angiogenesis and tumor
imaging agents via targeting of the α
designed with two disulde bridges, was conjugated to the HYNIC ligand (Cys
Cys
and was labelled with technetium-99m. The tracers [
4C)[172] and [
umbilical vein endothelial cells where [
higher cell accumulation and lower protein binding. The in vivo tumor uptake of
H
OHO
N
H
O
R=-N-imidodiacetate, [
R=OH, [
Figure 12.11 Structures of
O
N
O
N
H
COOH
OH
O
99m
Tc]Tc(CO)3(MIP-1404)
99m
Tc]Tc(CO)3(MIP-1405)
99m
Tc-cyclic-RGD αβ3 Integrin Receptor
Imaging Agents
integrin receptors. A cyclic RGD peptide was
vβ3
3
-Cys7)HYNIC-HN-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-COOH (HYNIC-RGD-4C),
99m
Tc]Tc-Tricine-(HYNIC-RGD-
99m
Tc]Tc-EDDA-(HYNIC-RGD-4C)[173] were evaluated in vitro in human
99m
Tc]Tc-EDDA-(HYNIC-RGD-4C) exhibited
O
+
N
CO
N
N
Tc
CO
N
CO
N
O
R
99m
Tc-labelled PSMA imaging agents.
N
N
N
L
Tc
L
X= b-Nal: [
X= 6-aminohexanoic acid:
99m
Tc]TcEDDA/HYNIC-ALUG, L=EDDA
[
L
L
O
H
X
N
COOH
99m
Tc]TcEDDA/HYNIC-iPSMA, L=EDDA
H
N
HO
1
-Cys9,
O
O
NHN
O
H
HO O
O
398 Handbook of Radiopharmaceuticals
99m
[
Tc]Tc-Tricine-(HYNIC-RGD-4C) in LS174T and ACHN tumor-bearing mice was 0.75 ± 0.05
and 0.59 ± 0.10% ID/g at six hours p.i., respectively[172].
Another cyclic RGD tracer, [
99m
Tc]Tc- (Cys1-Cys9, Cys3-Cys7)H2N-Cys-Asp-Cys-Arg-Gly-
Asp-Cys-Lys-Cys-COOH, was developed and used for the imaging of bronchioloalveolar
carcinoma in mice[174].
A series of [
99m
Tc]Tc-HYNIC tracers conjugated to cyclo[Arg–Gly–Asp–D-Phe–Lys]
(c[RGDfK]) were developed. The monomeric and the dimeric derivatives (cyclo[Arg–
Gly–Asp–D-Phe–(Lys-HYNIC)] (HYNIC-c(RGDfK)) and HYNIC-Glu(cyclo[Lys-Arg-Gly-Asp-
99m
99m
Tc
Tc-
D-Phe])-cyclo[Lys-Arg-Gly-Asp-D-Phe] (HYNIC-E[c(RGDfK)
tracers [
E[c(RGDfK)
dimer HYNIC ligands to human placental α
99m
Tc]Tc-Tricine–TPPTS-(HYNIC-c(RGDfK)) and [
]) were synthesized[175]. The binding anity of the monomer and the
2
integrin was found to be (IC50) 1.0 nM and
Vβ3
]) and the respective
2
99m
Tc]Tc-Tricine–TPPTS-(HYNIC-
0.1 nM, respectively, against biotinylated vitronectin. The tumor uptake of the
dimer tracer in OVAR-3-tumor-bearing mice peaked at 5.8 ± 0.7% ID/g at one hour p.i.,
while the tumor uptake of the
utes p.i.[176]. Also, the cRGD tetramer [
was developed. The binding anity of HYNIC-E{E[c(RGDfK)]
breast cancer cell line, in a competition study against
IC
=55 ± 11 nM. The tumor uptake of the tetramer tracer [
50
(HYNIC-E{E[c(RGDfK)]
2}2
99m
Tc- monomer tracer peaked at 5.2 ± 0.6 %ID/g at 30 min-
99m
Tc]Tc-Tricine–TPPTS-(HYNIC-E{E[c(RGDfK)]2}2)
in MDA-MB-435 human
2}2
125
I-echistatin, was found to be
99m
Tc]Tc-Tric ine –TPP TS-
) was 5.60 ± 0.87 and 7.30 ± 1.32% ID/g at 5 and 120 minutes p.i.,
respectively, in MDA-MB-435-tumor-bearing mice[177]. Furthermore, new cyclic RGD
peptide dimers conjugated to triglycine (G
) and polyethyleneglycol (PEG4) linkers were
3
developed in an eort to increase the distance between two RGD motifs and improve
the radiotracers excretion kinetics. The G
G
-E[G3-c(RGDfK)]2 exhibited binding anities (IC50) of 60.3 ± 4.4 and 61.1 ± 2.1 nM in
3
U87 MG glioma cells against
125
I-echistatin. The analogous
conjugates HYNIC-E[G3-c(RGDfK)]2 and HYNIC-
3
99m
Tc tracers exhibited high
tumor uptake in MDA-MB-435-tumor-bearing mice of 8.48 ± 0.59 and 9.11 ± 1.83% ID/g for
99m
[
Tc]Tc-Tricine-TPPTS-(HYNIC-E[G3-c(RGDfK)]2) and of 8.34 ± 0.39 and 7.60 ± 0.68%ID/g
99m
for [
Tc]Tc-Tricine-TPPTS-(HYNIC-G3-E[G3-c(RGDfK)]2) (Figure12.12) at 30 minutes and
120 minutes p.i., respectively. From this study, it was concluded that the G
ful for increasing integrin α
uptake and clearance kinetic of their
conjugates HYNIC-E[PEG
binding anity of cRGD dimers and improving the tumor
Vβ3
-c(RGDfK)]2 and HYNIC-PEG4-E[PEG4-c(RGDfK)]2, where PEG4
4
99m
Tc radiotracers[178]. Analogously, the pegylated
linkers are use-
3
was 15-amino-4,7,10,13-tetraoxa-pentadecanoic acid, were developed. The ligands exhibited binding anities (IC
125
I-c(RGDyK), respectively. The analogous
U87-MG-glioma-bearing mice of 11.17 ± 1.96 and 8.31 ± 2.31% ID/g for [
TPPTS-(HYNIC-E[PEG
cine-TPPTS-(HYNIC-PEG
p.i., respectively[179]. The agent [
) of 2.9 ± 0.7 and 2.4 ± 0.7 nM in U87 MG glioma cells against
50
-c(RGDfK)]2) and 8.17 ± 0.68 and 9.74 ± 3.22% ID/g for [
4
-E[PEG4-c(RGDfK)]2) (Figure12.12) at 30 minutes and 120 minutes
4
99m
99m
Tc tracers exhibited high tumor uptake in
99m
Tc]Tc-Tric i ne -
99m
Tc]Tc-Tri-
Tc]Tc-Tricine-TPPTS(HYNIC-PEG4-E[PEG4-c(RGDfK)]2)
has since undergone a series of clinical trials and was evaluated as a potentially useful
agent to diagnose rheumatoid arthritis[180], lung cancer[181], non-small-cell lung cancer and lymph node metastases[182], osteolytic bone metastases[183], and refractory
Chapter 12:
99m
Tc Radiopharmaceutical Chemistry 399
NH
HO
HN
4
4
O
N
O
H
HN
NH
O
HN
NH
O
O
N
H
O
99m
[
L=tricine/TPPTS, X=Gly3, PEG
Figure 12.12 Structures of
thyroid cancer[184]. Furthermore, the galactosylated dimer (HYNIC-Galacto-RGD2) developed via “click” conjugation exhibited a high binding anity (IC
MG glioma cells against
Galacto-RGD
nude mice bearing U87 MG gliomas, with advantages from the previous tracers in the
imaging of the chest and abdominal regions[185].
The inuence of the linker (glutamic acid vs. iminodiacetic acid) was evaluated in
the tracer [
whose tumor uptake was found to be similar to that of the tracer with a glutamic linker
99m
[
Tc]Tc-Tricine-TPPTS-(HYNIC-PEG4-E[PEG4-c(RGDfK)]2) and demonstrated advantages in
the abdominal area[186].
The inuence of the chelator was also evaluated by developing a MAG
acetyl diglycine) chelator of the formula MAG
ited a binding anity (IC
99m
[
Tc]TcO(M AG2-G3-E[G3-c(RGDfK)]2) (Figure12.12) had a high initial tumor uptake of
12.63 ± 2.88% ID/g at 30 minutes p.i., with fast tumor washout of 4.70 ± 1.15% ID/g at
120 minutes p.i. in U87 MG-glioma-bearing mice. Due to the fast renal clearance, the
tumor-to-kidney ratio increased over time to 2.49 ± 0.25 at two hours p.i.[187]. Similarly, the ligands MAG
developed, with a binding anity (IC
against
E[c(RGDfK)]
be 11.95 ± 1.90 and 15.36 ± 2.17% ID/g at one hour p.i. in U87 MG-glioma-bearing mice.
However, despite the high tumor uptake of the complexes, the target to nontarget ratios
were lower compared to the respective highly charged HYNIC tracers, perhaps owing to
the higher lipophilicity of the MAG
and its tracer [
NH
2
O
X
N
H
N
N
N
L
L
Tc
L
L
Tc]Tc-L/HYNIC-X-E[X-c(RGDfK)]
99m
Tc-labelled cRGD imaging agents.
) exhibited a high uptake of 8.37 ± 2.13% ID/g at 30 minutes p.i. in athymic
2
99m
Tc]Tc-Tricine-TPPTS-(HYNIC-I-E[PEG4-c(RGDfK)]2) (I=iminodiacetate),
125
I-(RGDyK). The tumor uptake of the respective tracers [
) and [
2
99m
H2N
N
H
O
NH
X
NH
NHX
O
2
125
I-echistatin. The radiotracer [
) of 3.6 ± 0.6 nM in U87 MG glioma cells against
50
-PEG4-E[c(RGDfK)]2 and MAG2-PEG4-E[PEG4-c(RGDfK)]2 were
2
99m
Tc]TcO(M AG2-PEG4-E[PEG4-c(RGDfK)]2) (Figure12.12) was found to
Tc]Tc-Tricine-TPPTS-(HYNIC-G3-E[PEG4-c(RGDfK)]2) were developed.
O
N
O
H
NH
O
tracers[188]. The dimer HYNIC-G3-E[PEG4-c(RGDfK)]2
2
HN
NH
HN
O
) of 8.6 ± 2.8, 3.9 ± 0.4 nM in U87 MG glioma cells
50
OH
O
O
O
99m
[
Tc]TcOMAG2-X-E[X-c(RGDfK)]2, X=Gly3, PEG
99m
-E[G3-c(RGDfK)]2. The ligand exhib-
2-G3
O
O
N N
Tc
N
S
O
X-E[X-c(RGDfK)]
) of 20 ± 2 nM in U87
50
2
Tc]Tc- tricine-TPPTS-(HYNIC-
(mercapto-
2
125
I-c(RGDyK).
99m
Tc]TcO(M AG2-PEG4-
400 Handbook of Radiopharmaceuticals
The ligand HYNIC-G3-E[PEG4-c(RGDfK)]2 exhibited a binding anity (IC50) of 3.37 ± 0.84 nM
in U87 MG glioma cells against
(HYNIC-G
-E[PEG4-c(RGDfK)]2) was 7.94 ± 1.74% ID/g at 0.5 hour p.i. in U87MG-glioma-
3
125
I-c(RGDyK). The uptake of [
99m
Tc]Tc-Tric i ne -T P P T S -
bearing mice[189]. Also, the HYNIC ligand was combined with nicotinic acid to form the
chelator HYNIC-K(NIC), which was conjugated to E[c(RGDyK)]
and G
-E[G3-c(RGDfK)]2 via lysine. The conjugates were labelled with
3
tive tracers [
PEG
-E[PEG4-c(RGDfK)]2), and [
4
99m
Tc]Tc-Tricine-(HYNIC-K(NIC)-E[c(RGDyK)]), [
99m
Tc]Tc-Tricine-(HYNIC-K(NIC)-G3-E[G3-c(RGDfK)]2), which
, PEG4-E[PEG4-c(RGDfK)]2
2
99m
Tc]Tc-Tricine-(HYNIC-K(NIC)-
99m
Tc to the respec-
exhibited similar tumor uptake of 5.73 ± 0.40, 5.24 ± 1.09, and 4.94 ± 1.71% ID/g at 60 minutes p.i. in MDA-MB-435-tumor-bearing mice. Reduced kidney and lung uptake were
observed in these tracers compared to the [
99m
Tc]Tc-Tricine-TPPTS-HYNIC analogue,
although conformational isomers were formed with the chelator HYNIC-K(NIC)[190]. Similarly, the K(HYNIC)
as mentioned previously; and while the tumor uptake of [
E[X-c(RGDfK)]
chelator was used in combination with the dimeric RGD analogues
2
) (where X=none or 3G or PEG4) was maintained, reduced kidney, lung, and
2
99m
Tc]Tc-Tricine-(K(HYNIC)2-X-
spleen uptake were obtained[191]. Modications of RGD-HYNIC by the introduction of
negative charge from six aspartate moieties[192] as well as the development of a trimer
RGD tracer[193] did not improve the imaging properties of the respective tracers in
comparison to the previously developed ones.
A similar cyclic RGD conjugated to HYNIC was developed, cyclo[Arg–Gly–Asp–d-Tyr–
(Lys-HYNIC)] (HYNIC-c(RGDyK)), as well as the respective tracers [
c(RGDyK)), [
(Nic: nicotinic acid), [
c(RGDyK))[194]. Of these tracers, [
99m
Tc]Tc-Tricine-(HYNIC-c(RGDyK)), [
99m
Tc]Tc-Tricine-TPPTS-(HYNIC-c(RGDyK)), and [
99m
Tc]Tc-EDDA-(HYNIC-c(RGDyK)) exhibited the lowest
protein binding as well as higher cell uptake. The IC
integrin receptors was measured to be 6.0 nM against
nude mice bearing either the human melanoma M21 α
99m
Tc]Tc-Tricine-Nic-(HYNIC-c(RGDyK))
of HYNIC-c(RGDyK) for the αVβ3
50
125
I-echistatin. Tumor uptake in
-expressing cell line or a human
Vβ3
99m
Tc]Tc- ED DA-(HYNIC-
99m
Tc]Tc(CO)3(HYNIC-
non-small-cell lung carcinoma cell line A549 was found to be 2.73 ± 0.26 and 1.54 ± 0.28%
ID/g at one hour p.i., respectively. The eect of the
99m
Tc-chelate was evaluated, and
c(RGDyK) was conjugated to dierent chelators: pyrazolyldiamine (pz-c(RGDyK)), isocyanide (CN-c(RGDyK)), and cysteine (Cys-c(RGDyK)). The respective tracers were prepared:
99m
[
Tc][Tc(CO)3(pz-c[RGDyK])], the “4 + 1” tracer [
and the nitrido tracer [
mentioned [
99m
Tc]Tc-EDDA-HYNIC-c[RGDyK] exhibited the best biological and phar-
99m
Tc][TcN(PNP)(Cys-c[RGDyK])]. Of these tracers, the previously
99m
Tc][Tc(N3S-COOH)(CN-c[RGDyK])],
macokinetic properties[195]. Also, the ligands [Arg-Gly-Asp-DPhe-Lys(Nε)-His] and
[Arg-Gly-Asp-DPhe-Lys(Nε)-CPA] (CPA=cysteine-propionate) were synthesized and
radiolabelled with [
tumor-bearing mice, where the tumor uptake was found to be 2.8 ± 1.5% ID/g for [
Tc-His-cRGDfK and 4.2 ± 1.5% ID/g for [
The tracer [
99m
tate dimethyl-pyrazolyl diamine ligand (PZ) carrying the RGD sequence (IC
99m
Tc][Tc(H2O)3(CO)3]+. The tracers were administered in MDA-MB 435
99m
Tc]
99m
Tc]Tc-CPA-cRGDfK at 30 minutes p.i.[196, 197].
Tc][Tc(CO)3-cyclo -[Arg-Gly-Asp-DTyr-Lys(PZ)]]+ was developed with a triden-
=3 nM), and
50
the complex was administered in M21 melanoma tumor-bearing mice, where the tumor
uptake was found to be 2.50 ± 0.29% ID/g at 1 h p.i.[198]. Also, c(RGDfK)-(Orn)
-CGG was
3
Chapter 12:
99m
Tc Radiopharmaceutical Chemistry 401
developed, containing three ornithine moieties and the cysteine diglycine (CGG) tripeptide for
99m
Tc-labeling[199]. The tracer was evaluated in mice bearing U87MG glioma
cells at dierent days after inoculation of the tumor cells, where maximum uptake was
observed at day 21 with tumor uptake of 11.6 ± 2.1% ID/g at one hour p.i.[200].
Another RGD peptide named NC100692 was developed and conjugated to the chelator
propylene aminoxime[201]. The ligand NC100692 exhibited K
integrins from human placenta. The tracer
99m
Tc-NC100692 was evaluated in breast cancer
< 1.1 nm ol l−1 in isolated αVβ3
D
patients, which detected 86% of the lesions[202]. Also, the ligand IDA-glucosaminoDc(RGDfK) was used to prepare the tracer [
which exhibited a high binding anity to α
99m
Tc][Tc(CO)3(IDA-glucosamino-Dc(RGDfK)],
integrins with IC50 of 1.5 nM against non-
Vβ3
radiolabelled cRGDyV; tumor uptake was 1.03 ± 0.21% ID/g at one hour p.i. in mice bearing
RR1022 brosarcoma[203]. This tracer was evaluated in monitoring response to paclitaxel therapy in tumor-bearing mice[204]. A cRGD tetramer RAFT-(cyclo[RGDfK])
(RAF T-
4
RGD: regioselectivity addressable functionalised template-RGD) was conjugated to
histidine and radiolabelled with [
99m
Tc][Tc(H2O)3(CO)3]+. Biodistribution of
99m
Tc-RAFT-RGD
in tumor-bearing mice showed tumor-specic uptake of 2.4 ± 0.5% ID/g in B16F0 and in
2.7 ± 0.8% ID/g TS/A-pc tumors at one hour p.i.[205].
12.8.5 Melanocortin 1 Receptor Imaging Agents
It is known that the melanocortin 1 receptor (MC1R) is overexpressed in most human and
murine melanomas, and it is considered a suitable target for the development of melanoma imaging agents. The
of the tridecapeptide, α-melanocyte stimulating hormone (α-MSH) with the following
sequence [Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH
α-MSH sequence for receptor targeting is His-Phe-Arg-Trp.
One of the earlier eorts described a peptide analogue modied with three cysteines at positions 3,4 and 10 of the sequence (Cys
after labeling with technetium-99m via glucoheptonate transchelation, formed a metalcyclized peptide [
99m
three cysteine thiols and one amide nitrogen. The tracer [
be resistant to chemical and proteolytic degradation and had a high binding anity for
MC1R[206]. The tumor uptake of [
mice and human-melanoma (TXM-13JQ)-bearing mice was 11.32 ± 3.25 and 2.39 ± 0.42%
ID/g at four hours p.i., respectively, with tumor-to-blood ratios of 39.0 and 11.5 after coadministration of lysine[207]. In the modied tracer [
where lysine-11 was replaced by arginine, a similar tumor uptake of 11.16 ± 1.77% ID/g
at four hour s p.i. was obtained in B16/F1-melanoma-bearing mice, but with an improved
renal prole, where the kidney uptake was 11 ± 1.44 at one hour p.i. (it was 19.71 ± 1.18%
ID/g for [
99m
[
99m
Tc]Tc-CCMSH)[208, 209]. Additional modications led to the tracer
Tc]Tc- RG D -Ly s- ( A rg11)CCMSH, where the RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was
conjugated to CCMSH via a lysine linker. The modied peptide RGD-Lys-(Arg
displayed binding anities to both MC1 and α
99m
Tc-based probes for MC1R targeting are peptide analogues
Tc]Tc-CCMSH, where [
99m
Tc]Tc-CCMSH in murine-melanoma (B16/F1)-bearing
]. The required
2
3,4,10
, D-Phe7)–α-MSH
99m
Tc]oxotechnetium was coordinated via the
99m
Tc]Tc-CCMSH was found to
99m
Tc]Tc- (Arg11)CCMSH (Figure12.13),
integrin receptors, with IC50 values
vβ3
(CCMSH), which,
3–13
11
)CCMSH
402 Handbook of Radiopharmaceuticals
HN
HOOC
O
O
O
N
H
S
O
O
H2N
O
99m
[
Tc][Tc(CO)3(pz-bAla-Nle-cyclo[Asp-
DPhe-Arg-Trp-Lys]-NH
N
N
H
HN
99m
[
Tc]Tc-(Arg11)CCMSH
O
N
N
OC
Tc
CO
NH
CO
N
O
N
Tc
S
O
S
H
N
O
NH
NH
2
bAla-Nle-cyclo[Asp-DPhe-Arg-Trp-Lys]-NH2]
+
]
2
Figure 12.13 Structures of
N
O
H
N
N
H
O
O
N
H
HN
99m
Tc-labelled αMSH imaging agents.
NH
O
HN
H
N
O
H
N
N
N
L
Tc
L
NH
NH
N
L
L
O
2
99m
[
L=tricine/EDDA
HN
O
–
O
N
N
Tc
N
S
99m
[
Tc][TcMAG3-GGNle-CycMSHhex]
H
N
O
Tc]TcHYNIC-AocNle-CycMSHhex
O
H
O
N
N
O
H
O
N
H
O
HN
H2N
NH
N
O
NH
NH
O
NH
O
HN
N
O
NH
NH
O
NH
O
O
HN
HN
O
HN
O
HN
O
NH
NH
O
NH
NH
O
HN
NH
N
H
O
2
H2N
NH
HN
O
HN
O
NH
N
H
O
2
of 2.0 nM and 403 nM, respectively[210]. The tracer [
exhibited high tumor uptake of 14.83 ± 2.94% ID/g at two hours p.i. and prolonged tumor
99m
Tc]Tc- RG D -Ly s- ( A rg11)CCMSH
retention of 7.59 ± 2.04% ID/g at 24 hours p.i. in B16/F1-melanoma-bearing mice[211].
Replacement of the Lys linker with Arg, Gly, or β-Ala reduced kidney uptake[212–214],
and replacement of Gly in the RGD sequence with various amino acids was found to
improve tumor uptake as well[215–217].
In another approach, a lactam-cyclized α-MSH derivative was developed, cyclized
via lysine-aspartate. This lactam-cyclic-α-MSH peptide was conjugated to the pyrazolyl
diamine tridentate chelator (pz) and labelled to form the tracer [
Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH
(Figure12.13). This tracer exhibited high
2
99m
Tc]Tc(CO)3-pz-βAla-
tumor uptake and retention of 9.26 ± 0.83 and 11.31 ± 1.83% ID/g at one and four hours
p.i., respectively. Unfortunately, high renal and liver uptake were also observed, of
32.12 ± 1.57 and 22.86 ± 1.17% ID/g at four hours p.i., respectively[218]. The lactamcyclized short hexapeptide {c[Asp-His-DPhe-Arg-Trp-Lys]-CONH
conjugated to various chelators suitable for
and HYNIC. The IC
values of the peptides MAG3-GGNle-CycMSH
50
CycMSHhex, and HYNIC-GGNle-CycMSH
99m
Tc-labelling: MAG3, Ac-Cys-Gly-Gly,
were 1.0 ± 0.05, 1.2 ± 0.19, and 0.6 ± 0.04 nM,
hex
Chapter 12:
}, CycMSH
2
, AcCG3-GGNle-
hex
99m
Tc Radiopharmaceutical Chemistry 403
hex
was
respectively, in B16/F1 melanoma cells. The respective
GGNle- CycMSH
GGNle-CycMSH
(Figure12.13), [
hex
hex
, and [
99m
Tc]Tc- ED DA-HYNIC- GGNle -CycMSH
F10 melanoma-bearing mice. The tracer [
99m
Tc]Tc- AcCG3-GGNle- CycMSH
99m
Tc]Tc- ED DA-HYNIC- GGNle -CycMSH
99m
Tc-tracers [
hex
99m
Tc]Tc- MAG3-
99m
, [
Tc]Tc(CO)3-HYNIC-
hex
were evaluated in B16/
exhib-
hex
ited the highest tumor uptake of 14.14 ± 4.90% ID/g and the fastest urinary clearance of
91.26 ± 1.96% ID at two hours p.i.[219]. In addition, the insertion of 8-aminooctanoic acid
linker (Aoc) between HYNIC and the peptide sequence improved the tumor uptake and
pharmacokinetics of [
99m
Tc]Tc- ED DA-HYNIC- AocNle-Cyc M SH
(Figure12.13), which dis-
hex
played melanoma uptake of 22.3 ± 1.72% ID/g at two hours p.i. and tumor-to-kidney ratios
of 3.29, 3.63, and 6.78 at 2, 4, and 24 hours, respectively[220]. It also exhibited improved
melanoma uptake in human-melanoma-bearing mice, and it detected melanoma lung
metastases in B16/F10 melanoma-bearing mice by SPECT/CT[221, 222].
The linear α-MSH analogue [Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys-NH
] was
2
conjugated to suitable tridentate chelators via the N-terminus or ε-Lys of the peptide[218, 223–225]. The respective [
values compared to the cyclized analogues; however, the tracer [
7
Phe
,Lys11(pz-Tc(CO)3)]α-MSH
4– 11
99m
Tc][Tc(CO)3]+ tracers exhibited lower tumor uptake
99m
Tc][Ac-Nle4,Asp5,D-
, with the chelator conjugated via the ε-Lys, exhibited the
highest values among them with tumor uptake of 5.88 ± 2.11 and 4.24 ± 0.94% ID/g at one
and four hou rs p.i., respectively[223].
12.8.6 Neurotensin Receptor Imaging Agents
Neurotensin (NT) (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) tridecapeptide
and its derivatives have been used in the development of
receptors that are overexpressed in tumors, such as pancreatic, lung, and colon carcinomas. Many eorts have focused on the development of NT derivatives with improved
metabolic stability since the original peptide and its fragments are metabolized quickly
in vivo. The following derivatives have been selected among those that were developed,
based on their improved properties and contain the NT(8-13) sequence Arg
11
Tyr
-Ile12-Leu13:
99m
Tc-tracers for imaging of NT
8
-Arg9-Pro10-
99m
NT-II: [
NT-XI: [
NT-XII: [
NT-XVIII: [
NT-XIX: [
In these [
Tc]Tc(CO)3]-(Nα-His-Ac)-Arg-Arg-Pro-Tyr-Ile-Leu[226]
99m
Tc]Tc(CO)3](Nα-His-Ac)-Lys-ψ(CH2NH)-Arg-Pro-Tyr-Tle-Leu[227] (Figure12.14)
99m
Tc]Tc(CO)3]-(Nα-His-Ac)-Arg-NMeArg-Pro-Tyr-Tle-Leu[228]
99m
Tc]Tc(CO)3]-(Nα-His-Ac)-Lys(shikimic)-Arg-NMeArg-Pro-Tyr-Tle-Leu[229]
99m
Tc]Tc(CO)3]-(Nα-His-Ac)-Arg-NMeArg-Pro-Dmt-Tle-Leu[229]
99m
Tc][Tc(CO)3]+-tracers, the peptide was conjugated to the Nα-histidinyl
acetate chelator (Nα-His-Ac). Modications in the original sequence include (i)
replacement of Arg
8
with Lys and a reduction of the peptide bond to CH2NH between
the positions 8 and 9 (NT-XI) and (ii) replacement of isoleucine (Ile) by tert-leucine (Tle)
at position 12 (NT-XI, NT-XII, NT-XVIII, NT-XIX). NT-XII was stabilized further by methylation of the bond between Arg at positions 8 and 9. In NT-XVIII, the shikimic acid
404 Handbook of Radiopharmaceuticals