Imaging Somatostatin Positive Tumors with Tyr3-Octreotate/Octreotide Conjugated to Desferrioxamine B Squaramide Radiolabeled with either Zirconium-89 or Gallium-68

Article abstract of DOI:10.1021/acs.bioconjchem.1c00109

Radiolabeled derivatives of Tyr3-octreotide and Tyr3-octreotate, synthetic analogues of the peptide hormone somatostatin, can be used for positron emission tomography (PET) imaging of somatostatin receptor expression in neuroendocrine tumors. In this work, a squaramide ester derivative of desferrioxamine B (H3DFOSq) was used attach either Tyr3-octreotide or Tyr3-octreotate to the metal binding ligand to give H3DFOSq-TIDE and H3DFOSq-TATE. These new peptide-H3DFOSq conjugates form stable complexes with either of the positron-emitting radionuclides gallium-68 (t1/2 = 68 min) or zirconium-89 (t1/2 = 3.3 days). The new complexes were evaluated in an AR42J xenograft model that has endogenous expression of SSTR2. All four agents displayed good tumor uptake and produced high-quality PET images. For both radionuclides, the complexes formed with H3DFOSq-TATE performed better, with higher tumor uptake and retention than the complexes formed with H3DFOSq-TIDE. The versatile ligands presented here can be radiolabeled with either gallium-68 or zirconium-89 at room temperature. The long radioactive half-life of zirconium-89 makes distribution of pre-synthesized tracers produced to certified standards feasible and could increase the number of clinical centers that can perform diagnostic PET imaging of neuroendocrine tumors.

Full text of DOI:10.1021/acs.bioconjchem.1c00109

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Article
Imaging Somatostatin Positive Tumors with Tyr3‑Octreotate/
Octreotide Conjugated to Desferrioxamine B Squaramide
Radiolabeled with either Zirconium-89 or Gallium-68
Asif Noor, Jessica K. Van Zuylekom, Stacey E. Rudd, Peter D. Roselt, Mohammad B. Haskali, Eddie Yan,
Michael Wheatcroft, Rodney J. Hicks, Carleen Cullinane, and Paul S. Donnelly*
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ABSTRACT: Radiolabeled derivatives of Tyr3-octreotide and Tyr3-
octreotate, synthetic analogues of the peptide hormone somatostatin,
can be used for positron emission tomography (PET) imaging of
somatostatin receptor expression in neuroendocrine tumors. In this
work, a squaramide ester derivative of desferrioxamine B (H3DFOSq)
was used attach either Tyr3-octreotide or Tyr3-octreotate to the metal
binding ligand to give H3DFOSq-TIDE and H3DFOSq-TATE. These
new peptide-H3DFOSq conjugates form stable complexes with either
of the positron-emitting radionuclides gallium-68 (t1/2 = 68 min) or
zirconium-89 (t1/2 = 3.3 days). The new complexes were evaluated in
an AR42J xenograft model that has endogenous expression of SSTR2.
All four agents displayed good tumor uptake and produced high-
quality PET images. For both radionuclides, the complexes formed
with H3DFOSq-TATE performed better, with higher tumor uptake and retention than the complexes formed with H3DFOSq-TIDE.
The versatile ligands presented here can be radiolabeled with either gallium-68 or zirconium-89 at room temperature. The long
radioactive half-life of zirconium-89 makes distribution of pre-synthesized tracers produced to certified standards feasible and could
increase the number of clinical centers that can perform diagnostic PET imaging of neuroendocrine tumors.
chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tet-
raacetic acid).9 A current approach for the treatment of
somatostatin positive tumors is to use diagnostic PET imaging
with the gallium-68 complex of DOTA-Tyr3-octreotide
(DOTATOC) or DOTA-Tyr3-octreotate (DOTATATE) to
guide peptide receptor radionuclide therapy (PRRT) using
therapeutic radionuclides such as the β-emitting lutetium-177
or yttrium-90.6,10−12
The rapid growth in diagnostic PET imaging with gallium-68
radiopharmaceuticals has been made possible by relatively easy
access to the radionuclide from germanium-68/gallium-68
generators.13,14 The production of gallium radiopharmaceuti-
cals still requires a dedicated on-site radiopharmacy/radio-
chemistry team, as the relatively short radioactive half-life of
gallium-68 (t1/2 = 68 min) limits the potential for centralized
production and distribution. The rapid growth in gallium
INTRODUCTION
■
Neuroendocrine cells are regulated by hormones that include
somatostatin, a 14-amino-acid peptide, which has a relatively
short biological half-life. Neuroendocrine tumors are often
characterized by high levels of expression of G-coupled
somatostatin receptors (SSTR), especially the somatostatin
subtype 2 receptor.1,2 Synthetic peptide analogues of
somatostatin which retain affinity for the receptor but are
more metabolically stable are useful agents for both diagnostic
imaging and therapy of neuroendocrine tumors. An example is
an 8-amino-acid peptide called octreotide (DPhe-c[Cys-Phe-
DTrp-Lys-Thr-Cys]-Thr-ol) (Figure 1) radiolabeled with γ-
emitting indium-111, [111In]In(DTPA-octreotide) (DTPA =
diethylenetriaminepentaacetic acid), which is used for
diagnostic single photon emission computed tomographic
(SPECT) imaging of neuroendocrine tumors.3 Further refine-
ments in SSTR targeting peptides involved substitution of the
third phenylalanine for tyrosine to give [Tyr3]-]octreotide and
by replacing the C-terminal threoninol with threonine to give
[Tyr3]-octreotate (DPhe-c[Cys-Tyr-DTrp-Lys-Thr-Cys]-Thr)
(Figure 1).4−8 Positron emission tomography (PET) imaging
offers superior resolution to SPECT imaging, and this has led
to developments that take advantage of the positron-emitting
radionuclide gallium-68 and conjugates of the macrocyclic
Special Issue: State-of-the-Art of Radiometal-based
Bioconjugates for Molecular Imaging and Radiotherapy
Received: March 2, 2021
Revised: March 15, 2021
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Figure 1. Structures of octreotide, Tyr3-octreotide, Tyr3-octreotate, and DFOSq-TIDE/TATE.
radiopharmaceuticals has led to shortages of germanium-68/
gallium-68 generators in some regions. Consequently, SSTR
imaging agents that use other positron-emitting radionuclides,
including fluorine-18,15,16 copper-64,17−20 scandium-44,21,22
and terbium-15223 are of interest (for a review of next
generation SSTR PET imaging agents, see ref 24). The
relatively long radioactive half-life of positron-emitting
zirconium-89 (t1/2 = 3.3 days) is well suited for manufacturing
radiopharmaceuticals to certified standards and then regional
distributions from centralized manufacturing sites. The
relatively low positron emission translational energy of
zirconium-89 (395.5 keV) could offer better resolution than
images acquired using gallium-68 that has a higher positron
emission translational energy (830 keV).14,25,26 However, a
significant disadvantage of zirconium-89 is that the positron
emission leads to the formation of yttrium-89m, which then
decays to yittrium-89 through a relatively high-energy γ-
emission (920 keV) adding to radiation exposure. Safe
transport of zirconium-89 radiopharmaceuticals would require
more shielding than what is routinely used for shipping
fluorine-18 radiopharmaceuticals.25
A single versatile SSTR targeting ligand that could be
radiolabeled with either gallium-68 or zirconium-89 could be
useful in that the selection of radionuclide could be chosen
depending on local availability. The ability to centrally
manufacture the zirconium-89 analogue for distribution
could improve the feasibility of PET imaging at clinical sites
without on-site radiochemistry expertise. In recent work, aimed
at preparing receptor targeting ligands that can be radiolabeled
with either zirconium-89 or gallium-68, we used a version of
the bacterial siderophore, desferrioxamine B (H3DFO) that
has been modified by the addition of a squaramide ethyl ester
functional group to give H3DFOSquaramide (H3DFOSq) to
attach glutamate-ureido-lysine small molecules that target
prostate-specific membrane antigen.27 The potential of
derivatives of H3DFO to coordinate gallium-67 and gallium-
68 radionuclides has been demonstrated previously.27−32 For
example, a [67Ga][Ga(DFO)] complex was demonstrated as
being sufficiently stable in human serum for 2 days.29,33
Zirconium(IV) complexes of the squaramide ester derivative,
DFOSqOEt, are more resistant to ligand exchange than
zirconium(IV) complexes with DFO derivatives prepared using
the isothiocyanate derivative, H3DFO-C(S)-NH-PhNCS, and
have better solubility in aqueous mixtures.34,35 The coupling of
squaramide ethyl esters to the amino groups of peptides is
straightforward and does not involve the use of additional
coupling agents. In this work, we extend the approach to attach
the cyclic peptides Tyr3-octreotate and Tyr3-octreotide to
DFOSq. The gallium-68 and zirconium-89 complexes of the
new conjugates were evaluated in an AR42J xenograft model.
RESULTS
■
Synthesis of H3DFOSq-TIDE/TATE. The resin-bound
linear Tyr3-octreotide/tate peptides were prepared using
standard microwave-assisted automated solid-phase peptide
synthesis. Each amino acid coupling involved the use of the
respective Fmoc protected amino acids, 1-[bis-
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxide hexafluorophosphate (HATU) and diiso-
propylethylamine (DIPEA) as the base. Deprotection of Fmoc
groups was achieved with 20% piperidine in dimethylforma-
mide (DMF) after each cycle, but no final N-terminus Fmoc
deprotection was performed. Cyclization of the peptides
through the formation of an intramolecular disulfide bridge
between the second and seventh cysteine residues was
achieved by in situ deprotection of acetamido methyl (Acm)
group on cysteine residues followed by oxidation with iodine in
DMF. Conjugation of H3DFOSquarate to amino acids
B
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a
Scheme 1. Synthesis of H3DFOSq-TATE/TIDE Peptides
a
(i) Iodine, DMF; (ii) TFA cocktail; (iii) t-Boc-anhydride, DIPEA, DMF; (iv) 20% piperidine in DMF; (v) H3DFOSqOEt, 0.1 M borate buffer,
pH 9.0, (vi) 20% TFA in DCM.
proceeds well in aqueous borate buffer (0.1 M, pH ∼ 9) which
is not compatible with the resin used for solid-phase peptide
synthesis. Consequently, following cyclization, the peptide was
cleaved from the resin and global deprotection of the
remaining protecting groups was achieved using a standard
trifluoracetic acid mixture (Scheme 1) prior to conjugation to
H3DFOSquarate in solution. To attach a single H3DFOSq
specifically on N-terminus D-Phe1 unit, the side chain amine
group of Lys5 was protected using t-Boc anhydride followed by
removal of the Fmoc group from the N-terminus using 20%
piperidine in DMF. The resulting Lys5(t-Boc)-TATE/TIDE
peptides were conjugated to H3DFOSq by incubating the
mixture in 0.1 M borate buffer (pH ∼ 9.0, 10% DMSO) over 7
days. The deprotection of the t-Boc group on Lys5 allowed
isolation of H3DFOSq-TATE/TIDE peptides in ∼25−28%
yield (Scheme 1).
Radiolabeling of H3DFOSq-TATE/TIDE with Either
Gallium-68 or Zirconium-89. Radiolabeling of both
H3DFOSq-TATE and H3DFOSq-TIDE with [68Ga]GaIII was
achieved at room temperature in 10 min. The modified
peptides were added to aqueous mixtures of [68Ga]GaIII,
obtained from elution of a germanium-68/gallium-68 gen-
erator with 0.05 M HCl, following partial neutralization to pH
4−5 with sodium acetate buffer (1 M, pH 4.5). Even with the
use of a relatively low peptide mass (approximately 150 ng/
MBq of [68Ga]GaIII), it was possible to obtain [68Ga]-
GaDFOSq-TATE and [68Ga]GaDFOSq-TIDE in high radio-
chemical yield and purity (≥98%, 150 ng, 0.08 nmol/MBq)
requiring no purification for in vivo experiments (Figure 2).
The conditions for radiolabeling with zirconium-89 were
extrapolated from our previous work with H3DFOSq
conjugated to lysine-ureido-glutamate.27 Both peptides were
labeled with zirconium-89 at a concentration of 2 μg/MBq
(1.1 nmol/MBq). Analysis by radio-HPLC shows radio-
chemical yield of >95% in 30 min at room temperature. The
crude tracers were then purified by passage through
Phenomenex Strata-X C18 cartridges using ethanol as eluent
to give the tracers in >98% radiochemical purity (Figure 2).
The resistance of the new complexes to radiolysis was
investigated by incubating the purified radiolabeled peptides in
phosphate buffered saline at room temperature for 4 h for 68Ga
labeled peptides (18 MBq, 100 μL) or 96 h for zirconium-89
labeled peptides (2.5 MBq, 220 μL). For each peptide, analysis
of the samples by radio-HPLC suggests <5% decomposition to
more hydrophilic uncharacterized compounds with shorter
retention times, presumably due to radiolysis (Figure S11).
[89Zr]ZrDFOSq-TATE was selected for preliminary inves-
tigations with higher amounts of radioactivity (100 MBq)
which was labeled with H3DFOSq-TATE (2.5 μg) in 15 min at
75 °C to give >98% radiochemical yield. The stability of this
preparation (∼66 GBq/μmol) in a mixture of PBS, ethanol
(10%), and sodium gentisate (0.5%) was monitored by analysis
by HPLC. After 5 days, the purity of [89Zr]ZrDFOSq-TATE
was ∼75% with the major decomposition product leading to a
peak in the HPLC with a shorter retention time (Figure S13,
SI).
PET-CT Imaging and Biodistribution in SSTR2
Positive AR42J Xenograft Mice Models. [68Ga]-
GaDFOSq-TIDE and [68Ga]GaDFOSq-TATE tracers were
injected (2−3 MBq, 1 μg, 0.5 nmol) intravenously via tail vein
to AR42J (rat pancreatic cancer cell line) tumor-bearing Balb/
c nude mice. PET images were acquired at 1 and 2 h post-
injection (Figure 3a and b). The tumor uptake in the PET
images is clear for both tracers, but [68Ga]GaDFOSq-TATE
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post-injection, the tumor uptake of [68Ga]GaDFOSq-TATE
was 9.80
2.33%IA/g and administration of [68Ga]-
GaDFOSq-TIDE gave a tumor uptake of 8.81 1.03%IA/g.
The initial tumor uptake of [68Ga]GaDFOSq-TIDE reduced
from 8.81 1.03%IA/g at 1 h post-injection to 4.4 1.1%IA/
g at 2 h post-injection (Figure 4). In contrast, the tumor
uptake of [68Ga]GaDFOSq-TATE at 1 h post-injection (9.80
2.33%IA/g) is retained at 2 h post-injection (9.22 0.92%
IA/g) consistent with the PET images (Figure 3). [68Ga]-
GaDFOSq-TIDE displays a higher degree of uptake in the
kidneys (1 h, 37.56
3.50%IA/g) than [68Ga]GaDFOSq-
TATE (1 h, 15.98 3.27%IA/g).
[89Zr]ZrDFOSq-TATE and [89Zr]ZrDFOSq-TIDE were
evaluated in the same mouse model. The tracers (2−3 MBq,
6 μg, 3.3 nmol) were administered via intravenous tail vein
injection, and PET images were acquired at 1, 2, 4, and 18 h
post-injection. Both tracers display rapid clearance from blood
with low uptake in bone and muscle. At each time point, the
tumor uptake of [89Zr]ZrDFOSq-TATE (e.g., 1 h post-
injection, SUVmax 3.1
0.24) is higher than for [89Zr]-
ZrDFOSq-TIDE (e.g., 1 h post-injection SUVmax 1.1 0.07).
The coinjection of ∼20-fold excess of the respective peptides
(approximately 120 μg, 66 nmol per mouse) results in a
significant reduction in tumor uptake, suggesting the uptake of
both tracers in the tumor is receptor mediated (Figure 5).
Ex vivo biodistribution studies (Figure 6) confirmed the
observations made by inspection of the PET images. The
tumor uptake of [89Zr]ZrDFOSq-TATE (1 h, 10.4 0.5%IA/
g; 18 h 4.9
TIDE (1 h, 3.4
0.8%IA/g) was higher than [89Zr]ZrDFOSq-
0.4%IA/g; 18 h 1.7
0.1%IA/g).
[89Zr]ZrDFOSq-TIDE has higher accumulation in the liver
(1 h, 10.8 0.4%IA/g) than [89Zr]ZrDFOSq-TATE (1 h, 4.3
0.4% IA/g). Both [89Zr]ZrDFOSq-TATE (1 h, 49.9 4.6%
IA/g; and [89Zr]ZrDFOSq-TIDE (1 h, 39.79
1.8) exhibit
Figure 2. Radio-HPLC chromatograms of [89Zr]ZrDFOSq-TATE/
TIDE and [68Ga]GaDFOSq-TATE/TIDE peptides (red, radiation
detection) compared against nonradioactive analogues, [natZr]-
ZrDFOSq-TATE/TIDE and [natGa]GaDFOSq-TATE/TIDE (blue,
λabs = 254 nm, offset for clarity, gradient method: 0−100% solvent B
to A over 25 min, solvent A = 0.05% TFA in Milli-Q water and
solvent B = 0.05% TFA in CH3CN).
significant uptake in the kidneys.
DISCUSSION
■
The iron(III) siderophore desferrioxamine B (H3DFO) forms
stable complexes with both gallium(III), logβGaDFO 28.2,36 and
zirconium(IV), logβZrHDFO = 47.6.37 Hexadentate DFO forms
coordinatively saturated six-coordinate complexes with
gallium(III), and both [68Ga]GaDFOsq-TIDE and [68Ga]-
GaDFOsq-TATE are stable in the presence of human serum
albumin. In contrast to the gallium(III) complex, the presence
of a squaramide functional group in H3DFOSq-conjugates may
lead to either 7- or 8-coordinate to zirconium(IV) complexes
where the dione backbone of the squaramide functional group
also coordinates the metal ion. Analysis of a zirconium(IV)
complex of a DFOSq-conjugate by 13C NMR spectroscopy
suggested that the dione backbone does interact with the metal
ion, whereas there are no such interactions in the analogous
gallium(III) complex.27,34 Predictive computational modeling
of [Zr(DFOSqOCH2CH3)]+ with density functional theory
suggested that one of the oxygen atoms of the dicarbonyl
functional group bound to the zirconium(IV) ion.38 Both
[89Zr]ZrDFOsq-TIDE and [89Zr]ZrDFOsq-TATE are stable
in the presence of human serum albumin.
has higher tumor uptake. Quantification of the uptake in the
images using standard uptake value (SUVmax = radioactivity in
a tissue (Bq/mL) × body weight (g)/injected activity (Bq))
confirms the higher uptake for[68Ga]GaDFOSq-TATE (1 h;
SUVmax = 1.8 0.2, compared to 1.21 0.20, Figure 3c). The
higher tumor uptake of [68Ga]GaDFOSq-TATE was also
associated with a higher degree of tumor retention at 2 h post-
injection with only a 5% reduction in SUVmax, whereas the
tumor SUVmax for [68Ga]GaDFOSq-TIDE reduced by 30% at
2 h post-injection. Addition of an excess of the respective
nonradioactive peptides (20 μg, 11.1 nmol per mouse) results
in a significant reduction in tumor uptake, suggesting the
uptake of both tracers in the tumor is receptor mediated
(Figure 3).
The high tumor uptake was confirmed by an ex vivo
biodistribution study in the same mouse model where mice
were injected with either [68Ga]GaDFOSq-TIDE or [68Ga]-
GaDFOSq-TATE (2−3 MBq, 1 μg, 0.5 nmol) and then
euthanized at either 1 or 2 h after administration. The amount
of injected activity per gram of tissue (%IA/g) in the tumor
and major organs was quantified (Figure 4 and SI). At 1 h
A limitation of this study is that the relative binding affinities
of each tracer to SSTR were not measured, but blocking
studies in the mouse models demonstrated that it is highly
likely that the tumor uptake is receptor-mediated. Further-
more, multiple studies have highlighted that the SSTR binding
affinities of Tyr3-octreotide and Tyr3-octreotate are tolerant of
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Figure 3. MicroPET maximum intensity projection (MIP) and CT images of mice bearing AR42J xenograft tumors injected with (a)
[68Ga]GaDFOSq-TIDE and (b) [68Ga]GaDFOSq-TATE tracers at 1 and 2 h p.i.; (c) Tumor SUVmax analyzed from PET images and results are
shown as mean SEM, n = 3; blocking was achieved by injecting excess amount of uncomplexed peptides. Organ labels: tumor (T), kidney (K),
bladder (B).
modification at the N-terminus of the peptide with hydrophilic
functional groups and metal complexes, although it was
acknowledged that different metal complexes can lead to
small changes to relative binding affinities for SSTR.1,3,16 It is
also significant that the higher measured affinity of [68Ga]-
GaDOTATATE for SSTR2 (0.2 0.04 nM) when compared
to [68Ga]GaDOTATOC (2.5 0.5 nM)1 did not prove to be
clinically relevant in a comparative study in human
subjects.39,40 The Tyr3-octreotide and Tyr3-octreotate peptides
were synthesized with the Lys5 residue protected with a t-Boc
group using standard solid-phase peptide synthesis method-
ologies. The peptides were attached to the chelator by reaction
of H3DFOSqOEt with peptide in solution at room temper-
ature. Deprotection of the t-Boc protecting from the Lys5
residue allowed isolation of H3DFOsq-TATE/TIDE in ∼25−
28% yield.
The new DFOSq conjugates could be radiolabeled with
gallium-68 in ≥98% radiochemical yield at room temperature
in 10 min using relatively low amounts of peptide (∼150 ng/
MBq, 0.08 nmol/MBq of [68Ga]GaIII). It was also possible to
radiolabel H3DFOsq-TATE/TIDE with zirconium-89 at room
temperature with radiochemical yields of >95% with 2 μg of
ligand/MBq (1.1 nmol/MBq). Purification with a C18
cartridge allowed isolation of [89Zr]ZrDFOsq-TIDE and
[89Zr]ZrDFOsq-TATE in >98% radiochemical purity. The
radiochemical procedures reported here are similar to our
previous work on H3DFOSq-glutamate-ureido-lysine deriva-
tives.27 The initial radiolabeling with both gallium-68 and
zirconium-89 was conducted at ambient temperature, but it is
likely that heating the reaction mixtures to ∼75 °C would
further decrease the amount of peptide required to obtain high
radiochemical yields leading to higher specific activities, and
this will be investigated in further work. All four complexes
have similar lipophilicities, as estimated by their distribution
All four tracers were evaluated in a murine xenograft model
using a neuroendocrine tumor cell line with endogenous
expression of SSTR2 (AR42J). The PET images acquired
following injection of either [68Ga]GaDFOsq-TIDE or [68Ga]-
GaDFOsq-TATE (Figure 3) show that the tumor uptake of
the latter is marginally higher than the former, but both tracers
produce images with high tumor uptake. As expected, both
tracers have significant uptake in the kidneys and bladder,
consistent with the renal excretion expected for peptides. The
PET images following administration of [68Ga]GaDFOSq-
TATE display evidence of some activity remaining in the blood
resulting in a blood pool background signal from the left
ventricle of the heart (Figure 3). The higher activity in blood
following administration of [68Ga]GaDFOSq-TATE when
compared to [68Ga]GaDFOSq-TIDE was confirmed by the
ex vivo biodistribution study, but the reason for this difference
is known (Figure 4). The specificity of the receptor-mediated
uptake in the tumor was supported by a blocking study where
addition of an excess of the respective nonradioactive peptide-
DFOSq conjugates leads to a significant reduction in tumor
uptake (Figure 3a). The higher tumor uptake of [68Ga]-
GaDFOsq-TATE at 1 h post-injection (9.80 2.33%IA/g) is
retained at 2 h post-injection (9.22 0.92%IA/g). In contrast,
the tumor uptake of [68Ga]GaDFOSq-TIDE reduced from
8.81 1.03%IA/g at 1 h post-injection to 4.4 1.1%IA/g at 2
h post-injection (Figure 4). In an early demonstration of the
potential of DFO to act as a ligand for gallium-67 and gallium-
68, the ligand was coupled to octreotide through a succinyl
linker. The gallium-67/68 complexes were stable in vivo and
displayed rapid tumor uptake in Islet Cell tumor-baring rats
with 0.56%IA/g accumulating in the tumor at 1 h post-
injection.29
Direct comparisons with the tumor uptake of [68Ga]-
GaDFOSq-TATE with that of [68Ga]GaDOTATATE in
AR42J tumor xenograft models need to be approached with
caution, because reported values vary widely, presumably
because the size of the respective tumors, the amount of
peptide injected, and the mouse strain used can affect the
coefficients (Log D7.4), which range from −0.88
0.06 for
[89Zr]ZrDFOSq-TATE to −1.38 0.07 for [68Ga]GaDFOSq-
TATE (Table 1).
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peptides than for the gallium-68 analogues. This difference in
tumor uptake is confirmed by the ex vivo biodistribution
studies. Two other positron-emitting radionuclides with
radioactive half-lives that are compatible with centralized
manufacture are copper-64 (t1/2 ∼ 12.7 h) and fluorine-18 (t1/2
∼ 109 min). Direct comparisons of tumor uptake from
independent studies have to be approached with caution due
to likely differences in tumor size, the amount of agent used,
and the different time points used. The tumor uptake of
[89Zr]ZrDFOSq-TATE (10.4
0.5%IA/g at 1 h post-
injection) is less than that of a fluorine agent, Al-[18F]F-
NOTA-octreotide (NOTA = 1,4,7-triazacyclononane-1,4,7-
triacetic acid) in the same AR42J tumor model (28.3 5.7%
ID/g at 2 h after injection)15 and [64Cu]CuSarTATE, a copper
complex conjugated to octreoate, which has a tumor uptake of
61.8 2.4%IA/g 4 h after injection in AR42J tumors.20
The images show that administration of [89Zr]ZrDFOSq-
TIDE leads to higher liver uptake when compared to
administration of [89Zr]ZrDFOSq-TATE, suggesting more
hepatobiliary clearance for the former. The images acquired
following administration of [89Zr]ZrDFOSq-TATE do show
uptake in the gallbladder (particularly at 2 h post-injection),
and some activity in liver and bowel (with likely radioactive
faeces at the 4 h time point) indicative of some hepatobiliary
excretion. There is little evidence of radioactivity in bone in the
PET images, and this is confirmed in the ex vivo biodistribution
suggesting the zirconium-89 complexes are stable in vivo. The
differing biodistribution and clearance mechanisms of the
gallium-68 and zirconium-89 labeled versions of each tracer
and especially the higher kidney uptake of the zirconium
complexes can at least partially be attributed to the different
ionic charges of the metal ion (GaIII versus ZrIV).
CONCLUSIONS
■
The peptides Tyr3-octreotide and Tyr3-octreotate that bind to
SSTR were conjugated to DFOSq. The new ligands can be
radiolabeled with either gallium-68 or zirconium-89 at room
temperature to give the radioactive complexes in high
radiochemical yield. All four agents displayed good tumor
uptake and produced good quality PET images in an AR42J
xenograft model that has endogenous expression of SSTR2.
For both radionuclides, the complexes formed with DFOSq-
TATE performed better, with higher tumor uptake and
retention, than the complexes formed with DFOSq-TIDE.
The quality of the PET images acquired following admin-
istration of [89Zr]ZrDFOSq-TATE suggests further studies on
the potential of this agent are warranted. These studies should
include prospective dosimetry to assess the potential radiation
dose to nontarget organs such as the kidneys as well as more
thorough investigations of the stability of the complexes when
prepared with high levels of radioactivity. The versatile ligands
presented here can be radiolabeled with either gallium-68 or
zirconium-89 at room temperature, and in principle, this
chemistry could be readily translated to kit-based formulations.
The long radioactive half-life of zirconium-89 makes
distribution of presynthesized tracers to sites that lack the
capability for on-site production of gallium-68 tracers feasible.
Figure 4. Ex vivo biodistribution analysis, mice were euthanized at
either 1 or 2 h after injection of either [68Ga]GaDFOSq-TIDE (a) or
[68Ga]GaDFOSq-TATE (b). Tumor uptake of [68Ga]GaDFOSq-
TIDE and [68Ga]GaDFOSq-TATE and reduction in tumor uptake
following “blocking” by injecting excess peptide conjugate (c). Tracer
uptake is expressed as percent injected activity/gram of tumor, data
represents mean SEM of n = 3 mice/group.
degree of uptake. In previous work, in the same AR42J tumor
xenograft model from our laboratory the tumor uptake of
[68Ga]GaDOTATATE was 14.4
0.8%IA/g.41 In another
example, the tumor uptake of [68Ga]GaDOTATATE in AR42J
tumor xenograft models was 3.14 2.07%IA/g at 1 h post-
injection,42 but higher tumor uptakes of 24.1 4.9 and 21.6
4%IA/g have also been reported.43,44
The PET images in the AR42J xenograft model following
injection of both [89Zr]ZrDFOSq-TIDE and [89Zr]ZrDFOSq-
TATE show that both tracers provide images with high uptake
in tumor and very low background. The tumor uptake of
[89Zr]ZrDFOSq-TATE is higher than for [89Zr]ZrDFOSq-
TIDE, and there is a larger difference between the different
MATERIAL AND METHODS
■
General Experimental and Materials. Reagents were
purchased from commercial sources. H3DFOSq was synthe-
sized by a reported procedure.34 Gallium-68 was eluted with
F
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Figure 5. MicroPET (MIP) and CT images of mice bearing AR42J xenograft tumors after 1, 2, 4, and 18 h post-injection of (a) [89Zr]ZrDFOSq-
TIDE and (b) [89Zr]ZrDFOSq-TATE tracers; (c) Time activity curves for tumor SUVmax analyzed from PET images, and results are shown as
mean
SEM, n = 3 (n = 4 for blocking experiment; blocking was achieved by injecting an ∼20-fold excess of either H3DFOSq-TIDE or
H3DFOSq-TATE. Organ labels: tumor (T), liver (L), kidneys (K), bladder (B), and gallbladder (GB).
hydrochloric acid (0.05 M, 4 mL) from a 1850 MBq Ge-68/
Ga-68 Generator from ITG Isotope Technologies Garching
GmbH. Zirconium-89 was obtained from either PerkinElmer
Inc. (part #NEZ308000MC) or the Department of Molecular
Imaging and Therapy, Austin Health). ESI-MS spectra were
recorded on Thermo Fisher OrbiTRAP infusion mass
spectrometer. For details of HPLC conditions, see Supporting
Information.
distilled water (2.5%), 3,6-dioxa-1,8-octanedithiol (2.5%),
and thioanisole (2.5%) in TFA (5 mL) with gentle shaking
for 2 h at RT. The resin was filtered, and the filtrate was
sparged with N2 to reduce the volume to 1 mL, and then
diethyl ether (40 mL) was added to precipitate the peptide
which was collected after centrifugation. The crude cyclic
peptides Fmoc-TATE/TIDE were purified by HPLC (Phe-
nomenex Luna 5 μm C18(2) 100 Å, LC column 250 × 21 mm
using 0.1% TFA in Milli-Q water and acetonitrile), and the
identity of the peptides were confirmed byESI-MS. The
purified cyclic peptides Fmoc-TATE/TIDE were dissolved in
DMF (1 mL) and treated with di-tert-butyldicarbonate (5
equiv) in the presence of DIPEA (1 equiv) for 4 h at RT, and
then cold diethyl ether was added to the reaction mixture to
precipitate the product which was collected by centrifugation.
The residue was treated with 20% piperidine in DMF (1 mL)
for 30 min at RT and then precipitated again with cold diethyl
ether, and the residue was collected by centrifugation and
purified by HPLC (Column C, SI) to provide the peptides
Lys(t-Boc)-TATE/TIDE. Analytical HPLC was performed
(Column B, SI) and HPLC purity found to be >95% for both
peptides. ESI-MS: Lys(t-Boc)-TIDE (+ve ion) [M + H+] m/z
Synthesis of Lysine Side Chain Protected Tyr3-
Octreotide/Octreotate Peptides. Tyr3-octreotide or Tyr3-
octreotate linear peptide with sequence [DPhe-Cys(Acm)-Tyr-
(tBu)-D(Trp)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr-(tBu)-OL
or OH] were prepared using a CEM Liberty Blue automated
microwave peptide synthesizer. For the octreotate peptide,
Fmoc-Thr(tBu)-OH preloaded on Wang resin was used, while
for octreotide, preloaded Fmoc-Threninol(tBu) on 2-chloro-
trityl resin was used. Each coupling cycle involved HATU (1
equiv) and DIPEA (5 equiv) followed by deprotection of
Fmoc group using 20% piperidine in DMF, but no final
deprotection of N-terminal Fmoc group was performed. The
resin-bound linear peptides were cyclized using iodine (1 mg/
mg of resin) in DMF (20 mL). The resin cleavage was
performed using a solution of triisopropylsilane (2.5%),
=
1135. 4952 (experimental), calculated for
G
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Table 1. Log D7.4 for [89Zr]ZrDFOSq-TATE/TIDE and
[68Ga]GaDFOSq-TATE/TIDE
Complex
Log D7.4
[89Zr]ZrDFOSq-TIDE
[89Zr]ZrDFOSq-TATE
[68Ga]GaDFOSq-TIDE
[68Ga]GaDFOSq-TATE
−1.03 0.13
−0.88 0.06
−0.98 0.07
−1.38 0.07
temperature for 7 days, and then neutralized with 10% TFA
and purified by HPLC (Column C, SI) and the identity of
peptides were confirmed byESI-MS. The purified fractions
were treated with 20% TFA in DCM for 30 min at RT, and
then TFA was removed under N2 flow followed by HPLC
purification again. Analytical HPLC was performed (Columns
A and B, SI) and HPLC purity found to be >95% for both
peptides. ESI-MS: H3DFOSq-TIDE (+ve ion) [M + H+] m/z
=
1673. 7716 (experimental), calculated for
[C78H113N16O21S2]+: m/z = 1673.7708; H3DFOSq-TATE
(+ve ion) [M + H+] m/z = 1687.7500 (experimental),
calculated for [C78H111N16O22S2]+: m/z = 1687.7500.
Synthesis of [68Ga]GaDFOSq-TIDE. 68GaIII in HCl (630
μL, 44 MBq) was buffered with sodium acetate (1 M, 70 μL,
pH 4.5), then H3DFOSq-TIDE(6 μg in 6 μL DMSO, 3.3
nmol), and the reaction mixture was left to stand at room
temperature for 15 min; then, an aliquot was analyzed by
radio-HPLC (Column B, SI). The traces showed >98%
radiochemical yield with a radiochemical purity of >98%.
The reaction mixture was diluted with Milli-Q water (470 μL)
and then buffered with 10 × PBS buffer (130 μL, pH 7.4) to a
final volume of 1.3 mL. Six syringes (BD Ultra-Fine, 0.3 mL)
containing approximately 3.5 MBq in 200 μL (approximately
peptide mass 1 μg, 0.5 nmol) were prepared. An excess of
H3DFOSq-TIDE (∼20 equiv, approximately 20 μg, 11 nmol
per mouse) was added to each injection for the “blocking”
experiment.
Synthesis of [68Ga]GaDFOSq-TATE. 68GaIII in HCl (900
μL, 40 MBq) was buffered with sodium acetate (1 M, 100 μL,
pH 4.5), then H3DFOSq-TATE (6 μg in 6 μL DMSO, 3.3
nmol), and the reaction mixture was left to stand at room
temperature for 15 min; then, an aliquot was analyzed by
radio-HPLC (Column B, SI). The traces showed >95%
radiochemical yield with a radiochemical purity of >95%.
The reaction mixture was diluted with Milli-Q water (100 μL)
and then buffered with 10 × PBS buffer (110 μL, pH 7.4) to a
final volume of 1.2 mL. Six syringes (BD Ultra-Fine, 0.3 mL)
containing approximately 2.3 MBq in 200 μL (approximately
peptide mass 1.0 μg, 0.5 nmol) were prepared. An excess of
H3DFOSq-TATE (∼20 equiv, approximately 20 μg, 11.1 nmol
per mouse) was added to each injection for the “blocking”
experiment.
Figure 6. Ex vivo biodistribution analysis at 1 and 18 h after injection
with either [89Zr]ZrDFOSq-TIDE (a) or [89Zr]ZrDFOSq-TATE (b).
Tracer uptake is expressed as percent injected activity/gram tissue.
Data represents mean SEM of n = 3 mice/group. (c) Tumor uptake
following injection of either [89Zr]ZrDFOSq-TIDE or [89Zr]-
ZrDFOSq-TATE and “blocking” experiment, with ∼20-fold excess
of respective peptide conjuagte, data represents mean SEM of n = 4
mice/group.
Synthesis of [89Zr]ZrDFOSq-TIDE. 89ZrIV in 1 M oxalic
acid (60 μL, 60 MBq) was diluted with Milli-Q water (60 μL)
and then was neutralized (pH 6−7) with a series of small
volume additions of aqueous Na2CO3 (1 M, 4 × 10 μL).
HEPES buffer (54 μL,1 M, pH 7.0) was then added and the
solution allowed to stand for 5 min before pH was tested again.
After confirming the mixture had a pH 6−7, a solution of
H3DFOSq-TIDE (120 μg in 60 μL of 0.25 M HEPES buffer,
10% DMSO, 1.18 nmol/MBq) was added, and the reaction
mixture was left to stand at room temperature for 30 min;
then, an aliquot was analyzed by radio-HPLC (Column B, SI).
The crude tracer was purified by Phenomenex StrataX
[C54H75N10O13S2]+: m/z = 1135.4956; Lys(t-Boc)TATE (+ve
ion) [M + H+] m/z = 1149.4739 (experimental), calculated for
[C54H73N10O14S2]+: m/z = 1149.4749.
Synthesis of H3DFOSq-TATE/TIDE. Lys(t-Boc)-TATE/
TIDE (1 equiv) and DFOSq (5 equiv) were dissolved in
DMSO (100 μL), and then borate buffer (0.1 M, pH 9.0, 900
μL) was added. The solution was slowly agitated at room
H
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cartridges (C18, 60 mg) using ethanol as eluent, and fractions
containing most labeled product (30 MBq) were combined
and diluted to 8% ethanol in PBS to final volume of 1.0 mL.
The purified tracer was analyzed by radio-HPLC (Column B,
SI) and radiochemical purity of the tracer found to be >95%.
Six syringes (BD Ultra-Fine, 0.3 mL) containing approximately
2−3 MBq (approximately peptide mass 6 μg, 3.3 nmol) in 165
μL were prepared. An excess of H3DFOSq-TIDE (∼20 equiv,
approximately 120 μg, 66 nmol per mouse) was added to each
injection for the “blocking” experiment.
2H+] m/z = 880.8249 (experimental), calculated for
[C78H111N16O21S2Zr]2+: m/z = 880.8293.
Measurement of Log(D7.4). Radioactive reaction mixture
(10 μL aliquot) was added to 1-octanol (500 μL, saturated
with PBS) and PBS (490 μL, saturated with 1-octanol). The
mixture was mixed and then allowed to separate (30 min). A
portion (400 μL) from 1-octanol layer was repartitioned
against PBS (400 μL). A portion (2 μL) of each layer was
collected, and the radioactivity in each layer was quantified.
Log D7.4 = log10 of the ratio of the counts between the 1-
octanol layer and the PBS layer (each partition was repeated in
three times (mean SEM).
Stability of Radiolabeled Peptides in PBS and in the
Presence of Human Serum Albumin. The radiolabeled
peptides were incubated at room temperature in phosphate
buffered saline for 96 h for 89Zr labeled peptides (2.5 MBq in
220 μL) and for 4 h for 68Ga labeled peptides (18 MBq in 100
μL) and analyzed by radio-HPLC. To assess the stability of the
[68Ga]GaDFOSq-TIDE/TATE and [89Zr]ZrDFOSq-TIDE/
TATE in human serum albumin the complexes (10 MBq in
∼50 μL) were incubated in 2% human serum in PBS for 3 h at
37 °C, and then an equal volume of acetonitrile was added to
precipitate proteins. The precipitated proteins were separated
by centrifugation and the supernatant was analyzed by radio-
HPLC. The chromatograms (Figures S11−12, SI) reveal <5%
decomposition to more hydrophilic uncharacterized com-
pounds with shorter retention times, presumably due to
radiolysis.
Synthesis of [89Zr]ZrDFOSq-TATE. 89ZrIV in 1 M oxalic
acid (70 μL, 56 MBq) was diluted with Milli-Q water (70 μL)
and then was neutralized (pH 6−7) with a series of small
volume additions of aqueous Na2CO3 (1 M, 4 × 10 μL).
HEPES buffer (62 μL,1 M, pH 7.0) was then added and the
solution allowed to stand for 5 min before pH was tested again.
After confirming the mixture had a pH 6−7, a solution of
H3DFOSq-TATE (112 μg in 56 μL of 0.25 M HEPES buffer,
10% DMSO, 1.19 nmol/MBq) was added into the 89ZrIV
solution, and the reaction mixture was left to stand at room
temperature for 30 min; then, an aliquot was analyzed by
radio-HPLC (Column B, SI). The crude tracer was purified by
Phenomenex StrataX cartridges (C18, 60 mg) using ethanol as
eluent, and fractions containing most labeled product (28.8
MBq) were combined and diluted to 8% ethanol in PBS to
final volume of 1.0 mL. The purified tracer was analyzed by
radio-HPLC, and radiochemical purity of the tracer was found
to be >95%. Six syringes (BD Ultra-Fine, 0.3 mL) containing
approximately 2−3 MBq (approximately peptide mass 6 μg,
3.3 nmol) in 150 μL were prepared. An excess of H3DFOSq-
TATE (∼20 equiv, approximately 120 μg, 66 nmol per mouse)
was added to each injection for the “blocking” experiment.
Synthesis of [natGa]GaDFOSq-TATE. A solution of
H3DFOSq-TATE (1 mg, 0.592 μmol) and Ga(NO2)3.H2O
(0.2 mg, 0.9 μmol) in acetonitrile:water (1:1, 500 μL) was
stirred for 2 h at room temperature and then purified by
HPLC. The purified fractions were analyzed by analytical
HPLC and HRMS. ESI-MS: [natGa]GaDFOSq-TATE (+ve
ion) [M + 2H+] m/z = 878.3280 (experimental), calculated for
[C78H109Ga1N16O22S2]2+: m/z = 878.3300.
Stability of [89Zr]ZrDFOSq-TATE with Respect to
Radiolysis at Higher Activities (100 MBq). 89ZrIV (162
μL, 100 MBq, in 0.05 M oxalic acid, produced by Department
of Molecular Imaging and Therapy, Austin Health) was
neutralized to pH 4.5 with sodium acetate (1 M, 187 μL), and
then H3DFOSq-TATE(2.5 μg, 1.5 nmol, in 1:1 Milli-
Q:ethanol (25 μL)) was added and the reaction mixture was
agitated at 75 °C for 15 min. The reaction mixture was
analyzed by radio-HPLC (Column B, SI). The traces showed
>98% radiochemical yield with a radiochemical purity of >98%.
Ethanol (48 μL), sodium gentisate (60 μL, 5%), PBS (60 μL),
and Milli-Q water (58 μL) were added to give a final volume of
∼600 μL (25 ng/MBq, 0.015 nmol/MBq in PBS with 10%
ethanol and 0.5% Na-Gentisate). The stability of [89Zr]-
ZrDFOSq-TATE was monitored by radio-HPLC showing that
after 5 days >75% [89Zr]ZrDFOSq-TATE remained with
evidence of one major unidentified decomposition product
with a shorter retention time presumably due to a more
hydrophilic species (Figure S13, SI).
Synthesis of [natGa]GaDFOSq-TIDE. A solution of
H3DFOSq-TIDE (1 mg, 0.597 μmol) and Ga(NO2)3.H2O
(0.2 mg, 0.9 μmol) in acetonitrile:water (1:1, 500 μL) was
stirred for 2 h at room temperature and then purified by
HPLC. The purified fractions were analyzed by analytical
HPLC and HRMS. ESI-MS: [natGa]GaDFOSq-TIDE (+ve
ion) [M + 2H+] m/z = 871.3393 (experimental), calculated for
[C78H111Ga1N16O21S2]2+: m/z = 871.3331.
Synthesis of [natZr]ZrDFOSq-TATE. A solution of
H3DFOSq-TATE (1 mg, 0.592 μmol) and ZrCl4.2THF (0.8
mg, 2.3 μmol) in acetonitrile:water (1:1, 500 μL) was stirred
for 2 h at room temperature and then purified by HPLC. The
purified fractions were analyzed by analytical HPLC and
HRMS. ESI-MS: [natZr]ZrDFOSq-TATE (+ve ion) [M +
2H+] m/z = 887.8199 (experimental), calculated for
[C78H109N16O22S2Zr]2+: m/z = 887.8190.
PET-CT Imaging and Biodistribution. These tests were
performed as reported previously27 and are also included in the
Supporting Information.
ASSOCIATED CONTENT
■
sı
* Supporting Information
Synthesis of [natZr]ZrDFOSq-TIDE. A solution of
H3DFOSq-TIDE (1 mg, 0.597 μmol) and ZrCl4.2THF (0.9
mg, 2.3 μmol) in acetonitrile:water (1:1, 500 μL) was stirred
for 2 h at room temperature and then purified by HPLC. The
purified fractions were analyzed by analytical HPLC and
HRMS. ESI-MS: [natZr]ZrDFOSq-TATE (+ve ion) [M +
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00109.
HPLC information, information on animal experiments,
additional figures for MS spectra; selected HPLC traces
and microPET images for in vivo experiments (PDF)
I
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AUTHOR INFORMATION
ABBREVIATIONS USED
■
■
PET, positron emission tomography; SPECT, single photon
emission computed tomographic; SSTR, somatostatin recep-
tor; PRRT, peptide receptor radionuclide therapy; %IA/g, %
injected activity per gram of tissue; %AR/g, % added activity
per gram of tissue; H3DFO, desferrioxamine B; DOTA,
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; keV,
kilo electronvolt; HATU, 1-[bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophos-
phate; DIPEA, diisopropylamine; TFA, trifluoracetic acid;
MBq, mega becquerel; MIP, maximum intensity projection;
SEM, standard error of the mean; SUVmax, standardized uptake
value; Log D7.4, distribution coefficient at pH 7.4
Corresponding Author
Paul S. Donnelly − School of Chemistry, University of
Melbourne, Parkville, Victoria 3010, Australia; orcid.org/
0000-0001-5373-0080; Email: pauld@unimelb.edu.au
Authors
Asif Noor − School of Chemistry, University of Melbourne,
Parkville, Victoria 3010, Australia
Jessica K. Van Zuylekom − Research Division, Peter
MacCallum Cancer Centre, Melbourne, Victoria 3000,
Australia
Stacey E. Rudd − School of Chemistry, University of
Melbourne, Parkville, Victoria 3010, Australia
Peter D. Roselt − Centre for Cancer Imaging, Peter
MacCallum Cancer Centre, Melbourne, Victoria 3000,
Australia
Mohammad B. Haskali − Centre for Cancer Imaging, Peter
MacCallum Cancer Centre, Melbourne, Victoria 3000,
Australia; orcid.org/0000-0003-3084-2084
Eddie Yan − Telix Pharmaceuticals Limited, North
Melbourne, Victoria 3051, Australia
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Rodney J. Hicks − Research Division, Peter MacCallum
Cancer Centre, Melbourne, Victoria 3000, Australia; Sir
Peter MacCallum Department of Oncology, University of
Melbourne, Parkville, Victoria 3010, Australia
Carleen Cullinane − Research Division, Peter MacCallum
Cancer Centre, Melbourne, Victoria 3000, Australia; Sir
Peter MacCallum Department of Oncology, University of
Melbourne, Parkville, Victoria 3010, Australia
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.bioconjchem.1c00109
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare the following competing financial
interest(s): Asif Noor, Stacey E. Rudd and Paul S. Donnelly
are listed as inventors on intellectual property relating to this
research that has been licensed from the University of
Melbourne to Telix Pharmaceuticals. Rodney Hicks has shares
in Telix Radiopharmaceuticals that are held on behalf of the
Peter MacCallum Cancer Centre. Michael Wheatcroft and
Eddie Yan are employed by Telix Pharmaceuticals, the licensee
of the intellectual property related to this research.
ACKNOWLEDGMENTS
■
Telix Pharmaceuticals and the National Health and Medical
Research Council, Australia (funding to Paul S. Donnelly,
Carleen Cullinane, and Peter D. Roselt) for financial support.
Australian Cancer Research Foundation supported the
purchase of the PET/CT scanner used in this work. Susan
Jackson (Peter MacCallum Cancer Centre) for expert technical
assistance and Dr. Benjamin Blyth (Peter MacCallum Cancer
Centre) for assistance with the blocking study experiments.
Mass Spectrometry and Proteomics Facility (MSPF) (Bio21
Institute, University of Melbourne).
J
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Bioconjugate Chemistry
pubs.acs.org/bc
Article
labeled aspartic acid peptides introducing octreotate derivatives. Ann.
Nucl. Med. 33, 244−251.
NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published on the Web on March 31, 2021. The
Table of Contents/Abstract graphic was replaced, and the
corrected version was reposted on April 8, 2021.
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https://doi.org/10.1021/acs.bioconjchem.1c00109
Bioconjugate Chem. XXXX, XXX, XXX−XXX