Improving the stability of peptidic radiotracers by the introduction of artificial scaffolds: Which structure element is most useful?

Article abstract of DOI:10.1002/jlcr.3315

Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for ⁶⁸Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually. In this contribution, the influence of different structure elements on the stability of ⁶⁸Ga-labeled BBN_(7-14) analogs was comparatively assessed by an in vitro serum stability assay with the aim to identify those structure elements being most useful for peptide radiotracer stabilization. It was found that the introduction of artificial scaffolds and the overall molecular design substantially affect the stabilities of the resulting peptidic radiotracers.

Full text of DOI:10.1002/jlcr.3315

Research article
Received 26 May 2015,
Revised 11 June 2015,
Accepted 26 June 2015
Published online 27 July 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3315
Improving the stability of peptidic radiotracers
by the introduction of artificial scaffolds: which
structure element is most useful?
Lisa Bacher,a,b† Gabriel Fischer,b,c† Shanna Litau,b,c Ralf Schirrmacher,d
Björn Wängler,c Marko Baller,a and Carmen Wänglerb
*
Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single
Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such
as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option
to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In
order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and
to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric
bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for 68Ga-labeling.
The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of
the introduced molecular scaffolds on the peptides’ serum stabilities. The results of the evaluations showed that the
introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the
resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results
point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually.
Keywords: 68Ga; peptide; radiotracer; serum stability; molecular design
overexpressed on various cancers5,6 are promising radioligands
Introduction
for the visualization of these cancer types but were often
observed to be degraded quite quickly in vivo.7–10 Nevertheless,
Radiolabeled peptides are a very potent substance class
especially for the specific in vivo imaging of malignant diseases
it could be shown for bombesin as well as other susceptible
with Single Photon Emission Computed Tomography and
radiolabeled peptides such as cRGD and gastrin derivatives that
Positron Emission Tomography as well as therapy as they are
able to address receptors or other surface proteins over-
these can profit in terms of stability and in vivo pharma-
cokinetics from the introduction of certain artificial linker
expressed on cancer cells.1,2 By their target-specific accumu-
lation, they enable favorable target-to-background-ratios and
thus images of high quality combined with sensitive target
visualization. A prerequisite in order to obtain a suitable peptidic
radiotracer is—apart from a specific and preferably strong target
interaction and stable radionuclide introduction—a high
in vivo stability of the radiotracer over the course of imaging.
However, as frequently being derived from endogenous
structures.11–14 However, the comparative assessment of the
influence of different commonly applied linkers and beyond
these other molecular scaffolds on the achievable stabilities of
a specific peptidic radiotracer has not been performed so far.
aUniversity of Applied Sciences, Campus Zweibrücken, Zweibrücken, Germany
regulatory compounds, radiolabeled peptides can be prone bBiomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine,
to a considerable degradation after in vivo administration.3,4
Medical Faculty Mannheim of Heidelberg University, Mannheim, Germany
This can result in an insufficient accumulation of the peptidic
radiotracer or its fragments in target tissues combined with
cMolecular Imaging and Radiochemistry, Department of Clinical Radiology and
Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University,
high background radioactivity levels being problematic in Mannheim, Germany
case of peptidic ligands radiolabeled with diagnostic but
dDepartment of Oncology, Division Oncological Imaging, University of Alberta,
Edmonton, Canada
especially therapeutic radionuclides. Thus, a high stability of
the radiolabeled peptide is a prerequisite for a suitable
imaging agent and efficient in vivo target visualization and
*Correspondence to: Carmen Wängler, Biomedical Chemistry, Department of
Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of
Heidelberg University, Mannheim, Germany.
furthermore
substances.
a critical factor for therapeutically applied
E-mail: carmen.waengler@medma.uni-heidelberg.de
Radiolabeled analogs of bombesin (BBN) which bind with high
affinity to the gastrin-releasing peptide receptor (GRPR) †Both authors contributed equally to this work.
J. Label Compd. Radiopharm 2015, 58 395–402
Copyright © 2015 John Wiley & Sons, Ltd.

L. Bacher et al.
products were isolated as white solids after lyophilization. Gradients used
for HPLC purification and synthesis yields for each compound are given
below. 2 (BBN(7–14)-Cys): gradient: 20–50% MeCN + 0.1% TFA in 8 min,
Rt = 1.9 min, yield: 49% (38.6 mg), MALDI-MS (m/z) for [M + H]+
(calculated): 1043.31 (1043.27); [M + Na]+ (calculated): 1065.27 (1065.25);
[M + K]+ (calculated): 1081.25 (1081.36). 3 (BBN(7–14)-PEG3-Cys): gradient:
20–50% MeCN + 0.1% TFA in 8 min, Rt = 4.1 min, yield: 53% (28.8 mg),
MALDI-MS (m/z) for [M + H]+ (calculated): 1290.92 (1291.55); [M + Na]+
(calculated): 1312.86 (1313.54); [M + K]+ (calculated): 1328.88 (1329.64).
4 (BBN(7–14)-Ahx-Cys): gradient: 20–50% MeCN + 0.1% TFA in 8 min,
For such a comparative investigation, bombesin represents a
suitable model peptide as it exhibits only a limited in vitro and
in vivo stability meaning that the stabilizing or destabilizing
effect exerted by a certain molecular design can be monitored
well.
The aim of this study was therefore to systematically assess
the influence of commonly used linkers, bioconjugation scaffolds
and peptide multiplicities on the in vitro serum stabilities of
respectively modified radiolabeled BBN(7–14) derivatives. This
approach was intended to comparatively assess the influence Rt = 3.2 min, yield: 22% (28.2 mg), MALDI-MS (m/z) for [M + H]+
(calculated): 1156.42 (1157.42); [M + Na]+ (calculated): 1178.42 (1179.41);
of these molecular scaffolds on the resulting peptide radiotracer
stabilities and to identify those structure elements being most
useful for peptide radiotracer stabilization.
[M + K]+ (calculated): 1194.41 (1195.51).
5 (BBN(7–14)-ACMP-Cys):
gradient: 20–30% MeCN + 0.1% TFA in 8 min, Rt = 4.5 min, yield: 37%
(16.4 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 1184.03 (1184.45);
[M + Na]+ (calculated): 1205.95 (1206.43); [M + K]+ (calculated): 1221.91
(1222.54). 6 (BBN(7–14)-ACMP2-Cys): gradient: 20–30% MeCN + 0.1%
TFA in 8 min, Rt = 4.0 min, yield: 45% (22.3 mg), MALDI-MS (m/z) for [M
+ H]+ (calculated): 1323.83 (1324.63); [M + Na]+ (calculated): 1345.53
(1346.61). 7 (BBN(7–14)-MAA): gradient: 35–60% MeCN + 0.1% TFA in
8 min, Rt = 2.2 min, yield: 24% (16.5 mg), MALDI-MS (m/z) for [M + H]+
(calculated): 1014.49 (1015.22); [M + Na]+ (calculated): 1036.40 (1037.21);
[M + K]+ (calculated): 1052.40 (1053.31).
Experimental
General
All commercially available chemicals were of analytical grade and were
used without further purification. Resins for peptide synthesis, coupling
reagents, Fmoc-protected amino acids and Fmoc-NH-PEG3-COOH
(PEG = polyethyleneglycol) were obtained from Merck. Fmoc-
aminohexanoic acid, TCEP and maleimide-NODAGA (NODAGA = (1,4,7-
triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) were purchased
from SigmaAldrich and CheMatech, respectively. S-trityl-mercaptoacetic
acid,15 the S-tBu-thio-protected maleimide dimer 1316 and DOTA-PESIN
Synthesis of the NODAGA-modified BBN(7–14) monomers 8–12
A solution of the thiol-modified BBN(7–14) derivatives 2–5 and 7 (2.8–
7.8 μmol) in H2O:MeCN 1:1 + 0.1% formic acid (FA)(250–400 μL) was
added to solid NODAGA-maleimide (2.5 eq.), and the pH of the solution
was adjusted to 6.5–7.0 using PB (phosphate buffer, 0.2 M, pH 7.2, 110–
470 μL). After 5-min reaction time, the crude products were purified by
semipreparative HPLC and isolated as white solids after lyophilization.
Gradients used for HPLC purification and synthesis yields for each
compound are given below. 8 (BBN(7–14)-Cys-NODAGA): gradient: 20–
70% MeCN + 0.1% TFA in 8 min, Rt = 3.2 min, yield: 49% (5.1 mg),
MALDI-MS (m/z) for [M + H]+ (calculated): 1541.42 (1541.77); [M + Na]+
(calculated): 1563.42 (1563.75); [M + K]+ (calculated): 1579.42 (1579.86).
9 (BBN(7–14)-PEG3-Cys-NODAGA): gradient: 20–30% MeCN + 0.1% TFA
in 8 min, Rt = 6.2 min, yield: 71% (3.5 mg), MALDI-MS (m/z) for [M + H]+
(calculated): 1787.65 (1789.05); [M + Na]+ (calculated): 1809.63 (1810.03);
[M + K]+ (calculated): 1825.75 (1826.14). 10 (BBN(7–14)-Ahx-Cys-
NODAGA): gradient: 20–30% MeCN + 0.1% TFA in 8 min, Rt = 5.4 min,
yield: 73% (4.5 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 1653.16
(1654.92); [M + Na]+ (calculated): 1675.09 (1675.90); [M + K]+ (calculated):
1691.05 (1692.01). 11 (BBN(7–14)-ACMP-Cys-NODAGA): gradient: 20–
30% MeCN + 0.1% TFA in 8 min, Rt = 4.3 min, yield: 75% (4.0 mg),
MALDI-MS (m/z) for [M + H]+ (calculated): 1680.89 (1681.95); [M + Na]+
(calculated): 1702.98 (1702.93); [M + K]+ (calculated): 1718.04 (1719.04).
12 (BBN(7–14)-MAA-NODAGA): gradient: 20–50% MeCN + 0.1% TFA in
8 min, Rt = 4.1 min, yield: 25% (3.1 mg), MALDI-MS (m/z) for [M + H]+
(calculated): 1512.24 (1512.72); [M + Na]+ (calculated): 1534.21 (1533.71);
[M + K]+ (calculated): 1550.18 (1549.81).
17
were synthesized according to published procedures. Peptides were
synthesized following a standard Fmoc-based solid phase peptide
synthesis protocol.18 For analytical and semipreparative HPLC
chromatography, a Dionex UltiMate 3000 system (Idstein, Germany)
equipped with a Gabi-Star (Raytest) radioactivity detector was used
together with a Chromolith Performance (RP-18e, 100–4.6 mm, Merck)
and a Chromolith SemiPrep (RP-18e, 100–10 mm) column, operated with
a flow of 4 mL/min. MALDI (Matrix-Assisted Laser Desorption/Ionization)
spectra were obtained using a Bruker Daltonics Microflex system.
Synthesis of 2-(4-(Fmoc-amino)piperidin-1-yl)acetic acid
(Fmoc-ACMP-OH) 1
To a suspension of 4-(Fmoc-amino)-piperidine hydrochloride (2.0 g,
5.6 mmol) and K2CO3 (2.3 g, 16.8 mmol) in acetone (20 mL) was slowly
added a solution of bromoacetic acid (1.0 g, 7.24 mmol) in acetone
(5 mL). The mixture was allowed to react over four days before the
solvent was evaporated. The residue was taken up in HCl (1 M, 10 mL)
and the suspension filtered and the remaining solid washed with
acetone (5 mL). The solid product obtained as slightly yellow solid after
drying (791.4 mg, 37%). 1H-NMR (DMSO-d6) (δ, ppm; J, Hz): 7.89 (d, 2H,
J3 = 7.6); 7.70 (d, 2H, J3 = 7.3); 7.50 (d, 1H, J3 = 6.7); 7.42 (dd, 2H, J3 = 7.6
and 7.5); 7.34 (dd, 2H, J3 = 7.5 and 7.3); 4.34 (d, 2H, J3 = 6.4); 4.22 (t, 1H,
J3 = 6.4); 3.94 (s, 2H); 3.57–3.49 (m, 1H); 3.45–3.05 (m, 4H); 2.00–1.70 (m,
4H). 13C-NMR (DMSO-d6) (δ, ppm; J, Hz): 167.25; 155.37; 143.75; 140.65;
127.51; 126.95; 125.01; 120.03; 65.16; 55.35; 50.81; 46.64; 39.93; 28.20.
MALDI-MS (m/z) for [M+H]+ (calculated): 380.63 (381.18).
Synthesis of the thiol-bearing BBN(7–14) dimer intermediates
14–19
Synthesis of the thiol-modified BBN(7–14) derivatives 2–7
A solution of the thiol-modified BBN(7–14) derivatives 2–7 (6.5–27.0 μmol,
The peptide derivatives were synthesized on solid support by standard 2.5 eq.) in H2O:MeCN 1:1 + 0.1% FA (250–400 μL) was added to the S-tBu-
Fmoc-based solid phase peptide synthesis using standard thio-protected maleimide dimer 13 (2.6–10.8 μmol), and the pH of the
a
commercially available Rink amide resin, Nα-Fmoc-amino acids and solution was adjusted to 6.5–7.0 using PB (0.2 M, pH 7.2, 110–470 μL).
Fmoc-linker-amino acids (1, Fmoc-PEG3-OH or Fmoc-Ahx-OH) where After 5-min reaction time, the crude products were analyzed by HPLC
appropriate. The so obtained fully protected peptide derivatives were
further reacted under the same conditions with Fmoc-Cys(Trt)-OH or S-
in order to confirm the reaction to be complete. To the solution was then
added an excess of solid TCEP (2–2.5 eq.), and the reaction progress was
trityl-mercaptoacetic acid on resin to obtain the thiol-modified protected monitored by analytical HPLC. The reactions were commonly finished
peptides. The resulting linker- and cysteine- or MAA-modified peptides
were cleaved from the solid support using mixture of TFA
(trifluoroacetic acid):TIS (triisopropylsilane):H2O (95:2.5:2.5) for 60 min,
within 5–6 h, and the crude products were purified by semipreparative
HPLC and isolated as white solids after lyophilization. Gradients used
for HPLC purification and overall synthesis yields for each compound are
a
suspended in diethyl ether and purified by semipreparative HPLC. The given below. 14 ((BBN(7–14)-Cys)2-SH): gradient: 20–40% MeCN + 0.1%
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 395–402

L. Bacher et al.
TFA in 8 min, Rt = 5.3 min, yield: 64% (10.3 mg), MALDI-MS (m/z) for
[M + H]+ (calculated): 2709.42 (2709.24). 15 ((BBN(7–14)-PEG3-Cys)2-SH):
gradient: 25–60% MeCN + 0.1% TFA in 8 min, Rt = 3.3 min, yield: 40%
(3.6 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 3204.54 (3203.81);
[M + Na]+ (calculated): 3225.98 (3225.80); [M + K]+ (calculated): 3242.28
(3241.91). 16 ((BBN(7–14)-Ahx-Cys)2-SH): gradient: 25–60% MeCN + 0.1%
TFA in 8 min, Rt = 3.2 min, yield: 37% (7.9 mg), MALDI-MS (m/z) for
[M + H]+ (calculated): 2934.90 (2935.55); [M + Na]+ (calculated): 2957.14
(2957.54); [M + K]+ (calculated): 2973.73 (2973.65). 17 ((BBN(7–14)-ACMP-
Cys)2-SH): gradient: 22.5–27.5% MeCN + 0.1% TFA in 8 min, Rt = 6.9 min,
yield: 49% (8.3 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 2989.47
(2989.63); [M + Na]+ (calculated): 3010.90 (3011.59); [M + K]+ (calculated):
3027.96 (3027.70). 18 ((BBN(7–14)-ACMP2-Cys)2-SH): gradient: 22.5–30%
MeCN + 0.1% TFA in 8 min, Rt = 5.4 min, yield: 41% (9.0 mg), MALDI-MS
(m/z) for [M + H]+ (calculated): 3270.11 (3270.00); [M + Na]+ (calculated):
Determination of radiotracer stabilities in human serum
A solution of the respective 68Ga-labeled product 68Ga-8–68Ga-12, 68Ga-
20–68Ga-25 or 68Ga-DOTA-PESIN (25–36 MBq, 125 μL), obtained as
described before, was added to 500 μL of human serum and incubated
at 37 °C. At 10, 20, 30, 40, 50, 60, 70, 80 and 90 min, aliquots of 50 μL of
the mixture were added to 50 μL of ethanol, and the precipitation of
serum proteins was enhanced by ice-cooling for 2 min. After 2-min
centrifugation at 2000 ×g, supernatant and precipitate were measured
for radioactivity and the supernatant was analyzed by analytical radio-
HPLC. All experiments were performed thrice separately.
Results and discussion
3292.51 (3291.96); [M + K]+ (calculated): 3308.94 (3308.07). 19 ((BBN(7–14)
-
The aim of this study was to systematically assess the influence
of different commonly applied linkers, bioconjugation scaffolds
and peptide multiplicities on the in vitro serum stabilities of
respectively modified radiolabeled BBN(7–14) derivatives. As it
was shown before in several studies, each investigating the
influence of a special linker type on radiopeptide properties,
the following structure elements were shown to be able to
positively influence binding affinities or in vivo pharmacokinetics
of peptidic radiotracers and were thus assessed in the present
study in direct comparison: polyethylene glycol (PEG) linkers
(Figure 1A),19,20 4-amino-1-carboxymethyl-piperidin (ACMP)21–23
(Figure 1B) and 6-aminohexanoic acid (Ahx) (Figure 1C).24,25
Furthermore, also the influence of the peptide multiplicity was
investigated here (Figure 1D) as a higher peptide multiplicity
might—besides improving receptor avidities17—influence the
radiotracer stability by decelerating the degradation because of
the contained artificial structure elements or their bulky
structure hampering an enzymatic attack. To successfully
synthesize such peptide multimers, a highly efficient click
chemistry reaction is warranted for peptide conjugation, and it
was shown that the Michael addition between thiols and
maleimides is especially useful for this purpose.17,26 For this type
of reaction, different thiol-bearing conjugation scaffolds can be
used. Thus, we also assessed the influence of these conjugation
scaffolds on the resistance towards enzymatic degradation and
investigated if the use of an artificial thiol conjugation scaffold
can contribute to a higher radiopeptide stability compared to a
natural one (Figure 1E). Figure 1 summarizes the structure
elements whose influence on peptide radiotracer serum
stabilities was thus comparatively assessed.
MAA)2-SH): gradient: 25–50% MeCN + 0.1% TFA in 8 min, Rt = 4.4 min,
yield: 22% (2.9 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 2650.82
(2651.15); [M + Na]+ (calculated): 2672.96 (2673.14); [M + K]+ (calculated):
2688.59 (2689.25).
Synthesis of the NODAGA-modified BBN(7–14) dimers 20–25
The respective S-tBu-thio-protected BBN(7–14) dimer intermediate 14–19
(5.6–6.7 μmol) was dissolved in H2O:MeCN 1:1 + 0.1% FA (250–400 μL)
and the pH of the solution was adjusted to 6.5–7.0 using PB (0.2 M,
pH 7.2, 110–340 μL). This solution was then added to solid NODAGA-
maleimide (2.5 eq.). After 5-min reaction time, the crude products were
purified by semipreparative HPLC and isolated as white solids after
lyophilization. Gradients used for HPLC purification and synthesis yields
for each compound are given below. 20 ((BBN(7–14)-Cys)2-NODAGA):
gradient: 20–40% MeCN + 0.1% TFA in 8 min, Rt = 5.0 min, yield: 58%
(7.1 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 3206.21 (3206.74);
[M + K]+ (calculated): 3245.61 (3243.83). 21 ((BBN(7–14)-PEG3-Cys)2-
NODAGA): gradient: 25–50% MeCN + 0.1% TFA in 8 min, Rt = 3.6 min,
yield: 13% (4.6 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 3700.83
(3699.30); [M + Na]+ (calculated): 3723.42 (3720.28); [M + K]+ (calculated):
3739.86 (3736.39). 22 ((BBN(7–14)-Ahx-Cys)2-NODAGA): gradient: 25–
50% MeCN + 0.1% TFA in 8 min, Rt = 3.5 min, yield: 23% (8.4 mg),
MALDI-MS (m/z) for [M + H]+ (calculated): 3432.90 (3433.05); [M + Na]+
(calculated): 3454.97 (3454.03); [M + K]+ (calculated): 3470.84 (3470.14).
23 ((BBN(7–14)-ACMP-Cys)2-NODAGA): gradient: 20–50% MeCN + 0.1%
TFA in 8 min, Rt = 4.1 min, yield: 73% (7.0 mg), MALDI-MS (m/z) for
[M + H]+ (calculated): 3486.79 (3487.10). 24 ((BBN(7–14)-ACMP2-Cys)2-
NODAGA): gradient: 22.5–30% MeCN + 0.1% TFA in 8 min, Rt = 5.2 min,
yield: 76% (7.9 mg), MALDI-MS (m/z) for [M + H]+ (calculated): 3766.53
(3767.47); [M + Na]+ (calculated): 3788.56 (3788.45); [M + K]+ (calculated):
3804.81 (3804.56). 25 ((BBN(7–14)-MAA)2-NODAGA): gradient: 20–50%
MeCN + 0.1% TFA in 8 min, Rt = 5.2 min, yield: 11% (2.0 mg), MALDI-MS
(m/z) for [M + H]+ (calculated): 3148.29 (3148.65); [M + Na]+ (calculated):
3170.24 (3169.64); [M + K]+ (calculated): 3186.24 (3185.74).
Synthesis of thiol-bearing BBN(7–14) derivatives
The first step towards the differently modified bombesin analogs
to be studied in vitro was the synthesis of BBN(7–14) derivatives
comprising PEG3, Ahx, one or two ACMP scaffolds as well as
two different thiol-bearing units (cysteine or 2-mercaptoacetic
68Ga-radiolabeling of the NODAGA-modified BBN(7–14)
monomers 8–12, dimers 20–25 and DOTA-PESIN for in vitro
stability tests
-
A solution of the respective NODAGA-modified BBN(7–14) analog 8–12 and acid (MAA) which both are often applied as thiol-carriers for
the modification of bioactive compounds27) used as reactive
20–25 or DOTA-PESIN in tracepure-H2O containing chelex resin (1 nmol/μL,
15 μL, 15 nmol) was added to 121À171 MBq of 68Ga3+ in a solution
functionalities during click chemistry conjugations. The synthesis
of the peptides as well as their modification with linkers and
obtained by fractioned elution of a 68Ge/68Ga generator (IGG100, Eckert &
Ziegler, Berlin, Germany or from ITG, Garching, Germany) with HCl (0.1 M
thiol-carriers was performed on solid support using a standard
or 0.05 M, 1.2 mL) and subsequent titration to pH 3.5À4.0 by addition of
Fmoc-based solid phase peptide synthesis approach together
sodium acetate solution (1.25 M, 45–105 μL). After reaction for 10 min at
with standard conjugation protocols. Figure 2 shows the
30 °C (or 99 °C in case of DOTA-PESIN), the reaction mixtures were analyzed
structures of the thiol-modified peptide derivatives which were
by analytical radio-HPLC. The radiolabeled products were purified by
obtained in overall chemical yields of 22–53% and used for the
following syntheses of the monomeric and dimeric peptidic
radiolabeling precursors.
cartridge (Waters SepPak C18 light) and obtained in radiochemical yields
of 69–84%, radiochemical purities of ≥95% and specific activities of
8.1–11.4 GBq/μmol (non-optimized).
J. Label Compd. Radiopharm 2015, 58 395–402
Copyright © 2015 John Wiley & Sons, Ltd.
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L. Bacher et al.
Figure 1. Schematic depiction of the structure elements whose influence on peptide radiotracer stabilities was comparatively assessed by in vitro serum stability tests.
Figure 2. Structures of the synthesized thiol-modified BBN(7–14) analogs 2–7 used for the syntheses of monomeric and dimeric peptidic radiolabeling precursors.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 395–402

L. Bacher et al.
Scheme 1. Schematic depiction of the synthesis pathways and structures of the monomeric and dimeric peptidic labeling precursors 8–12 (A), 20–25 (B) as well as DOTA-
PESIN (C). Reagents and conditions: A: (a) NODAGA-maleimide, H2O and MeCN (pH 6.5–7.0), 25 °C, 5 min, yields: 25–75%; B: (a) 2–7, H2O and MeCN (pH 6.5–7.0), 25 °C,
5 min; (b) TCEP, 25 °C, 5–6 h; (c) NODAGA-maleimide, H2O and MeCN (pH 6.5–7.0), 25 °C, 5 min, overall yields (steps a–c): 2.4–37%.
J. Label Compd. Radiopharm 2015, 58 395–402
Copyright © 2015 John Wiley & Sons, Ltd.
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L. Bacher et al.
Synthesis of the monomeric and dimeric labeling precursors completed, and the radiolabeled products were purified by
cartridge extraction by which the in-solution-stability of the
tracers was considerably increased over time (data not shown).
This can most probably be attributed to the ethanol which is
used for product elution and serves as protectant against ra-
The obtained thiol-modified BBN(7–14) analogs 2–5 and 7 were in
the following reacted with NODAGA-maleimide giving the
respective monomeric labeling precursors 8–12 for 68Ga-labeling
diolysis.31,32 Starting from activities of 121–171 MBq of 68Ga3+
,
(bearing different linker and conjugation scaffolds (Scheme 1A))
the labeled products 68Ga-8–68Ga-12, 68Ga-20–68Ga-25 and
68Ga-DOTA-PESIN were obtained in radiochemical yields of 69–
84%, radiochemical purities of ≥95% and non-optimized specific
activities of 8.1–11.4 GBq/μmol being sufficient for the following
in vitro serum stability studies.
in yields of 25–75%. NODAGA ((1,4,7-triazacyclononane-4,7-diyl)
diacetic acid-1-glutaric acid) was chosen as chelating agent as
it enables—in contrast to the more frequently applied DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid)
—the labeling with 68Ga3+ at ambient temperature within
minutes while yielding gallium-complexes of high in vivo
stability as well.28,29
In order to obtain the corresponding chelator-modified BBN(7–14)
dimers, the thiol-bearing monomers 2–7 were reacted with a
dendritic maleimide-dimer scaffold structure (13)16 comprising
a protected thiol functionality for further derivatization with the
chelator. The resulting peptide dimers were in the following first
reacted with TCEP (tris(2-carboxyethyl)phosphine hydrochloride)
giving the thiol-bearing intermediate products 14–19 in yields of
22–64% and subsequently with NODAGA-maleimide, yielding
the BBN(7–14)-dimer labeling precursors 20–25 (Scheme 1B) in
yields of 11–73%.
In addition to the differently modified monomeric and dimeric
BBN(7–14) analogs 8–12 and 20–25, DOTA-PESIN (BBN(7–14)-PEG3-
DOTA) (Scheme 1C) was synthesized and intended to serve as a
reference compound in the following in vitro serum stability
studies as it was shown before to exhibit a relatively high
in vivo stability compared to other GRPR-binding BBN(7–14)
analogs.13
The serum stability of the 68Ga-labeled radiotracers was in the
following determined in human serum. For this purpose, an
aliquot containing 25–36 MBq of the respective cartridge-
purified radiotracer 68Ga-8–68Ga-12, 68Ga-20–68Ga-25 or 68Ga-
DOTA-PESIN was added to 500 μL of human serum and
incubated at 37 °C for 90 min. Every 10 min, an aliquot of 50 μL
was taken, serum proteins were precipitated with the same
volume of ethanol at 0 °C, precipitate and supernatant were
measured for radioactivity and the supernatant was analyzed
by radio-HPLC. From the measurements of precipitate activities
after 90 min of incubation with serum, the expected trend of a
significantly lower serum protein binding of peptide monomers
compared to dimers could be found (68Ga-8–68Ga-12: 2.8
2.5%, 68Ga-20–68Ga-25: 9.1 4.0%). Figure 3 gives an overview
of the results of the serum stability tests for all studied tracers
which will be discussed in detail below. As can be observed from
these data, the serum stabilities of the radiotracers strongly vary
and depend on the molecular design of the respective
radiolabeled peptide analog.
In detail, it can be observed that in general, dimeric
structures (Figure 4B) tend to exhibit a higher stability towards
enzymatic degradation than the corresponding monomers
(Figure 4A) as the tested dimeric radiotracers comprising
68Ga-radiolabeling and determination of radiotracer
stabilities in human serum
The 68Ga-labeling of the monomeric and dimeric labeling cysteine as conjugation scaffold (68Ga-20–68Ga-24) exhibited
precursors 8–12 and 20–25 as well as DOTA-PESIN was stabilities between 21 4% and 97 2% after 90-min
performed using the fractioned elution method of 68Ge/68Ga incubation with serum, and the corresponding monomers
generators30 and a simplified radiolabeling protocol requiring 68Ga-8–68Ga-11 only showed intact tracer percentages of 26
no heating step28 in case of the NODAGA-comprising peptide
1% up to 55 2% within the same time interval. This higher
analogs 8–12 and 20–25. In contrast, DOTA-PESIN was labeled serum stability observed for the peptide dimers can most
at 99 °C as the chelator DOTA allows for the complete probably be attributed to the artificial structure elements
incorporation of the radiometal at elevated temperatures only. contained in the dendron core and/or the steric hindrance
After 10-min reaction time, the complexation of 68Ga was exerted by the bulky structure of the dendritic substances
Figure 3. Graphic representation of the stabilities of the 68Ga-labeled BBN(7–14) monomers 68Ga-8–68Ga-12, dimers 68Ga-20–68Ga-25 and 68Ga-DOTA-PESIN in human
serum.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 395–402

L. Bacher et al.
Figure 4. Results of the comparative assessment of the influence of the peptide multiplicity and linker structures on the resulting radiotracer serum stabilities.
A: stabilities of the BBN(7–14) monomers 68Ga-8–68Ga-11 and B: stabilities of the corresponding BBN(7–14) dimers 68Ga-20–68Ga-24.
which might impede the enzymatic attack. This effect of
Interestingly, the Ahx linker had an invariant negative effect
increased stability observed for multimers of susceptible on the serum stabilities of the respectively modified peptide
peptides is supported by a very recent study showing a monomers and dimers 68Ga-10 and 68Ga-22 as compared to
considerably higher in vivo stability of a DUPA-Pep trimer the corresponding tracers 68Ga-8 and 68Ga-20 comprising
compared to its respective monomer.33 The authors of this no linker: 68Ga-10 showed a 7% stability decrease compared to
study thus conclude that metabolically unstable peptides 68Ga-8 and 68Ga-22 showed a 24% stability decrease compared
may profit from multimerization in terms of in vivo stability.
Furthermore, the introduction of artificial linker structures has
to 68Ga-20.
A structure element which displayed invariant stabilizing
the potential to result in general in products of increased effects was the artificial conjugation scaffold MAA which
stability compared to those tracers not containing any linker. produced peptide analogs of considerably higher stability than
So exhibited e.g. 68Ga-BBN(7–14)-PEG3-Cys-NODAGA (68Ga-9) a
by 9% increased stability compared to 68Ga-BBN(7–14)-Cys-
NODAGA (68Ga-8) and 68Ga-(BBN(7–14)-ACMP-Cys)2-NODAGA
(68Ga-23) a by 52% increased stability compared to 68Ga-
(BBN(7–14)-Cys)2-NODAGA (68Ga-20).
Apart from these findings, the tested linkers showed
inconsistent effects on the resulting serum stabilities, as e.g.
ACMP and PEG3 linkers were found to have contrary effects on
stabilities in peptide monomers and dimers: so exhibited the
PEG3-containing monomer 68Ga-9 a by 9% increased stability
compared to monomer 68Ga-8 without linker whereas the
PEG3-containing dimer 68Ga-21 exhibited a by 10% decreased
stability compared to the corresponding dimer 68Ga-20 without
linker. As well, the ACMP-containing monomer 68Ga-11 showed
a by 20% decreased stability compared to monomer 68Ga-8
without linker; the ACMP-containing dimer 68Ga-23 however
showed
a by 52% increased stability compared to the
corresponding dimer 68Ga-20 without linker. Furthermore, in
case of peptide dimers, the introduction of one ACMP linker
(68Ga-23) resulted in a highly stable product (97 2% intact after
90 min), whereas the according introduction of two ACMP linker
units (68Ga-24) resulted in a lower stability of the tracer of only
74 4% intact product after 90-min incubation with serum.
These contradictory findings may be a result of conformational
changes caused by the introduced linkers, potentially
enveloping or exposing a susceptible part of the peptide
molecules to the degrading enzymes.
Figure 5. Graphic representation of the influence of the applied conjugation
scaffold on the resulting peptidic radiotracer stabilities.
J. Label Compd. Radiopharm 2015, 58 395–402
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

L. Bacher et al.
[6] J. C. Reubi, S. Wenger, J. Schmuckli-Maurer, J. C. Schaer, M. Gugger,
Clin. Cancer. Res. 2002, 8, 1139–1146.
the corresponding cysteine-modified ones (Figure 5). The use of
MAA instead of cysteine could in both peptide multiplicities
increase the serum stability from 46 3% to 95 0.9% in case
of the monomers 68Ga-8 and 68Ga-12 as well as from 45 5%
to 95 0.4% in case of the dimers 68Ga-20 and 68Ga-25 after
90-min incubation. For comparison, 68Ga-DOTA-PESIN which
was tested as a relatively stable reference exhibited a stability
of 85 2% under the same conditions.
[7] S. Richter, M. Wuest, S. S. Krieger, B. E. Rogers, M. Friebe, R.
Bergmann, F. Wuest, Nucl. Med. Biol. 2013, 40, 1025–1034.
[8] Y. Ma, M. Yang, H. K. Gao, G. Niu, Y. J. Yan, L. X. Lang, D. O.
Kiesewetter, X. Y. Chen, Amino Acids 2012, 43, 1625–1632.
[9] P. J. Marsouvanidis, T. Maina, W. Sallegger, E. P. Krenning, M. de Jong,
B. A. Nock, J. Nucl. Med. 2013, 54, 1797–1803.
[10] X. Z. Zhang, W. B. Cai, F. Cao, E. Schreibmann, Y. Wu, J. C. Wu, L. Xing,
X. Y. Chen, J. Nucl. Med. 2006, 47, 492–501.
[11] J. Y. Shi, Y. Zhou, S. Chakraborty, Y. S. Kim, B. Jia, F. Wang, S. Liu,
Theranostics 2011, 1, 322–340.
[12] P. Kolenc-Peitl, R. Mansi, M. Tamma, T. Gmeiner-Stopar, M. Sollner-
Dolenc, B. Waser, R. P. Baum, J. C. Reubi, H. R. Maecke, J. Med. Chem.
2011, 54, 2602–2609.
Conclusions
A general answer as to which linker structure should be
introduced into a susceptible peptide in order to obtain a
peptidic radiotracer of optimized stability cannot be given, and
besides the applied linker type, the particular molecular design
was found to have a considerable influence on the degradation
of the peptidic tracers in human serum.
However, some trends could be identified how certain
scaffolds influence peptidic radiotracer stabilities in case of
bombesin analogs: (i) the introduction of an artificial linker unit
such as ACMP or PEG3 can have a considerably stabilizing effect,
(ii) the use of 6-aminohexanoic acid as linker seems to negatively
influence the resulting radiotracer stability, whereas (iii) dimeric
peptides appear to be of higher stability than monomeric ones
and (iv) for the synthesis of thiol-modified peptides applicable in
Michael addition click chemistry reactions, mercaptoacetic acid-
derivatized peptidic tracers in general exhibit a considerably
higher stability compared to cysteine-modified ones.
[13] H. W. Zhang, J. Schuhmacher, B. Waser, D. Wild, M. Eisenhut, J. C. Reubi,
H. R. Maecke, Eur. J. Nucl. Med. Mol. Imaging 2007, 34, 1198–1208.
[14] M. Jamous, M. L. Tamma, E. Gourni, B. Waser, J. C. Reubi, H. R.
Maecke, R. Mansi, Nucl. Med. Biol. 2014, 41, 464–470.
[15] C. Z. Tang, L. J. Li, Z. Pang, Z. B. Yuan, Chinese J. Chem. 2009, 27, 195–201.
[16] G. Fischer, B. Wängler, C. Wängler, Molecules 2014, 19, 6952–6974.
[17] S. Lindner, C. Michler, B. Wängler, P. Bartenstein, G. Fischer, R.
Schirrmacher, C. Wängler, Bioconjug. Chem. 2014, 25, 489–500.
[18] C. Wängler, B. Waser, A. Alke, L. Iovkova, H. G. Buchholz, S. Niedermoser,
K. Jurkschat, C. Fottner, P. Bartenstein, R. Schirrmacher, J. C. Reubi, H. J.
Wester, B. Wängler, Bioconjug. Chem. 2010, 21, 2289–2296.
[19] S. Liu, Bioconjug. Chem. 2009, 20, 2199–2213.
[20] Z. Varasteh, U. Rosenstrom, I. Velikyan, B. Mitran, M. Altai, H.
Honarvar, M. Rosestedt, G. Lindeberg, J. Sorensen, M. Larhed, V.
Tolmachev, A. Orlova, Molecules 2014, 19, 10455–10472.
[21] R. Mansi, K. Abiraj, X. J. Wang, M. L. Tamma, E. Gourni, R. Cescato, S.
Bemdt, J. C. Reubi, H. R. Maecke, J. Med. Chem. 2015, 58, 682–691.
[22] M. de Visser, H. F. Bernard, J. L. Erion, M. A. Schmidt, A. Srinivasan, B.
Waser, J. C. Reubi, E. P. Krenning, M. de Jong, Eur. J. Nucl. Med. Mol.
Imaging 2007, 34, 1228–38.
Apart from these general trends found, the obtained results
point to the necessity to empirically assess the influence of
artificial structure elements on the stability for each individual
susceptible peptidic radiotracer.
[23] R. Mansi, X. J. Wang, F. Forrer, B. Waser, R. Cescato, K. Graham, S.
Borkowski, J. C. Reubi, H. R. Maecke, Eur. J. Nucl. Med. Mol. Imaging
2011, 38, 97–107.
[24] J. J. Parry, R. Andrews, B. E. Rogers, Breast Cancer Res. Tr. 2007, 101,
175–183.
[25] P. K. Nanda, U. Pandey, B. N. Bottenus, T. L. Rold, G. L. Sieckman,
A. F. Szczodroski, T. J. Hoffman, C. J. Smith, Nucl. Med. Biol. 2012,
39, 461–471.
Acknowledgements
[26] C. Wängler, S. Maschauer, O. Prante, M. Schafer, R. Schirrmacher, P.
Bartenstein, M. Eisenhut, B. Wängler, ChemBioChem 2010, 11,
2168–2181.
[27] G. T. Hermanson. Bioconjugate Techniques. Academic Press: San
Diego, 1996, 60–62.
[28] C. Wängler, B. Wängler, S. Lehner, A. Elsner, A. Todica, P. Bartenstein,
M. Hacker, R. Schirrmacher, J. Nucl. Med. 2011, 52, 586–591.
[29] E. W. Price, C. Orvig, Chem. Soc. Rev. 2014, 43, 260–290.
[30] W. A. P. Breeman, M. de Jong, E. de Blois, B. F. Bernard, M.
Konijnenberg, E. P. Krenning, Eur. J. Nucl. Med. Mol. Imaging 2005,
32, 478–485.
[31] T. Fukumura, R. Nakao, M. Yamaguchi, K. Suzuki, Appl. Radiat.
Isotopes 2004, 61, 1279–1287.
[32] P. J. H. Scott, B. G. Hockley, H. F. Kung, R. Manchanda, W. Zhang, M. R.
Kilbourn, Appl. Radiat. Isotopes 2009, 67, 88–94.
[33] Z. Baranyai, D. Reich, A. Vagner, M. Weineisen, I. Toth, H. J. Wester, J.
Notni, Dalton Trans. 2015, 44, 11137–11146.
The authors acknowledge financial support by the German
Research Foundation (WA-3555), the Ministry of Science,
Research and the Arts of the State of Baden-Württemberg and
the German Federal Ministry of Education and Research (BMBF)
within the Framework ‘Forschungscampus: public–private
partnership for Innovations’—Research Campus M2OLIE.
References
[1] M. Schottelius, H. J. Wester, Methods 2009, 48, 161–177.
[2] M. Fani, H. R. Maecke, S. M. Okarvi, Theranostics 2012, 2, 481–501.
[3] D. J. Kwekkeboom, W. H. Bakker, P. P. M. Kooij, J. Erion, A. Srinivasan,
M. de Jong, J. C. Reubi, E. P. Krenning, Eur. J Nucl Med. 2000, 27,
1312–1317.
[4] H. C. Cai, P. S. Conti, J. Labelled Compd. Rad. 2013, 56, 264–279.
[5] V. Ambrosini, M. Fani, S. Fanti, F. Forrer, H. R. Maecke, J. Nucl. Med.
2011, 52, 42s–55s.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 395–402