Synthesis and in vitro and in vivo evaluation of SiFA-tagged bombesin and RGD peptides as tumor imaging probes for positron emission tomography

Article abstract of DOI:10.1021/bc400588e

Gastrin-releasing-peptide (GRP)-receptors and ?± _v?₃-integrins are widely discussed as potential target structures for oncological imaging with positron emission tomography (PET). Favored by the overexpression of receptors on the surface of tumor cells good imaging characteristics can be achieved with highly specific radiolabeled receptor ligands. PEGylated bombesin (PESIN) derivatives as specific GRP receptor ligands and RGD (one-letter codes for arginine-glycine-aspartic acid) peptides as specific ?±_v?₃ binders were synthesized and tagged with a silicon-fluorine-acceptor (SiFA) moiety. The SiFA synthon allows for a fast and highly efficient isotopic exchange reaction at room temperature giving the [¹⁸F]fluoride labeled peptides in up to 62% radiochemical yields (d.c.) and a‰￥ 99% radiochemical purity in a total synthesis time of less than 20 min. Using nanomolar quantities of precursor high specific activities of up to 60 GBq ?mol^-1 were obtained. To compensate the high lipophilicity of the SiFA moiety various hydrophilic structure modifications were introduced leading to significantly reduced logD values. Competitive displacement experiments with the PESIN derivatives showed a 32 to 6 nM affinity to the GRP receptor on PC3 cells, and with the RGD peptides a 7 to 3 ?M affinity to the ?± _v?₃ integrins on U87MG cells. All derivatives proved to be stable in human plasma over at least 120 min. Small animal PET measurements and biodistribution studies revealed an enhanced and specific accumulation of the RGD peptide ¹⁸F-SiFA-LysMe₃-?- carboxy-d-Glu-RGD (17) in the tumor tissue of U87MG tumor-bearing mice of 5.3% ID/g whereas the PESIN derivatives showed a high liver uptake and only a low accumulation in the tumor tissue of PC3 xenografts. Stability studies with compound 17 provided further information on its metabolism in vivo. These results altogether demonstrate that the reduction of the overall lipophilicity of SiFA tagged RGD peptides is a promising approach for the generation of novel potent ¹⁸F-labeled imaging agents.

Full text of DOI:10.1021/bc400588e

Article
pubs.acs.org/bc
Synthesis and in Vitro and in Vivo Evaluation of SiFA-Tagged
Bombesin and RGD Peptides as Tumor Imaging Probes for Positron
Emission Tomography
Simon Lindner,*,† Christina Michler,† Stephanie Leidner,† Christian Rensch,‡ Carmen Wangler,†,§
̈
Ralf Schirrmacher,∥ Peter Bartenstein,† and Bjorn Wangler*,⊥
̈
̈
†Department of Nuclear Medicine, University Hospital Munich, Ludwig-Maximilians-University, 81377 Munich, Germany
‡GE Global Research, 85748 Garching, Germany
§Biomedical Chemistry, Department of Clinical Radiology and Nuclear Medicine and Molecular Imaging and Radiochemistry,
⊥
Department of Clinical Radiology and Nuclear Medicine, Medical Faculty Mannheim of Heidelberg University, 68167 Mannheim,
Germany
∥McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada
S
* Supporting Information
ABSTRACT: Gastrin-releasing-peptide (GRP)-receptors and αvβ3-
integrins are widely discussed as potential target structures for
oncological imaging with positron emission tomography (PET).
Favored by the overexpression of receptors on the surface of tumor
cells good imaging characteristics can be achieved with highly
specific radiolabeled receptor ligands. PEGylated bombesin
(PESIN) derivatives as specific GRP receptor ligands and RGD
(one-letter codes for arginine-glycine-aspartic acid) peptides as
specific αvβ3 binders were synthesized and tagged with a silicon-
fluorine-acceptor (SiFA) moiety. The SiFA synthon allows for a fast
and highly efficient isotopic exchange reaction at room temperature
giving the [18F]fluoride labeled peptides in up to 62% radiochemical
yields (d.c.) and ≥99% radiochemical purity in a total synthesis time
of less than 20 min. Using nanomolar quantities of precursor high specific activities of up to 60 GBq μmol−1 were obtained. To
compensate the high lipophilicity of the SiFA moiety various hydrophilic structure modifications were introduced leading to
significantly reduced logD values. Competitive displacement experiments with the PESIN derivatives showed a 32 to 6 nM
affinity to the GRP receptor on PC3 cells, and with the RGD peptides a 7 to 3 μM affinity to the αvβ3 integrins on U87MG cells.
All derivatives proved to be stable in human plasma over at least 120 min. Small animal PET measurements and biodistribution
studies revealed an enhanced and specific accumulation of the RGD peptide 18F-SiFA-LysMe3-γ-carboxy-D-Glu-RGD (17) in the
tumor tissue of U87MG tumor-bearing mice of 5.3% ID/g whereas the PESIN derivatives showed a high liver uptake and only a
low accumulation in the tumor tissue of PC3 xenografts. Stability studies with compound 17 provided further information on its
metabolism in vivo. These results altogether demonstrate that the reduction of the overall lipophilicity of SiFA tagged RGD
peptides is a promising approach for the generation of novel potent 18F-labeled imaging agents.
diagnostic imaging purposes in the clinic.3 Some efforts have
INTRODUCTION
■
been undertaken recently to overcome this problem with
The silicon-fluorine-acceptor (SiFA) methodology is a versatile
modified silicon-containing building blocks.4,5 Another possi-
approach for the radiolabeling of a broad range of compounds
with the positron emitting radionuclide fluorine-18 (18F). It has
bility is the introduction of polar auxiliaries into the structure to
compensate the high lipophilicity in favor of an improved
pharmacokinetic profile of the radiotracer.
We intended to investigate this approach on bombesin and
RGD (one-letter codes for arginine-glycine-aspartic acid)
analogues. Bombesin peptides are ligands of the gastrin-
been applied to small molecules, even though best results were
obtained with peptides and proteins.1,2 Its great advantages are
a fast and highly efficient isotopic exchange reaction, very mild
reaction conditions, an easy purification procedure requiring no
HPLC (high performance liquid chromatography), and high
specific activities of the radiolabeled products. However, despite
all beneficial properties, SiFA containing radiotracers have often
revealed a high lipophilicity which was shown to entail poor in
vivo characteristics and therefore limits their application for
Received: January 7, 2014
Revised: March 24, 2014
Published: March 25, 2014
© 2014 American Chemical Society
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Figure 1. Structures of SiFA-PESIN (1) and SiFA-PESIN derivatives 2−9.
releasing-peptide (GRP)-receptor which is overexpressed on
several tumor tissues such as prostate, breast, ovarian, lung,
colon, and gastrinoma cancers.6,7 For that reason, GRP
receptors are a favorable target structure for tumor visualization
with imaging techniques such as PET (positron emission
tomography). Many different bombesin derivatives have been
developed so far,8−16 but most suffer from poor pharmacoki-
netic properties. The most promising peptide is PESIN
(PEGylated bombesin), as it exhibits favorable characteristics
such as high stability in vivo and good tumor uptake.17,18 The
structure of PESIN is derived from the natural GRP-receptor
ligands gastrin-releasing-peptide and bombesin sharing the
same C-terminal region of 7 amino acids. This truncated
peptide sequence is the pharmacophore and responsible for
receptor binding. A PEG4 motif is inherently present in the
molecule which serves as hydrophilic spacer separating the
pharmacophore from remaining structural motifs and the
labeling moiety.
RGD peptide 18F-AH111585, nowadays named [18F]-
fluciclatide, exhibited good preclinical results and was
successfully translated into human studies which encouraged
us to combine its peptidic scaffold with the SiFA chemistry and
to further investigate its in vitro and in vivo properties.21−28
Herein, we report on the synthesis and radiolabeling of SiFA
tagged PESIN and RGD peptides. Hydrophilic auxiliaries were
inserted in favor of improved pharmacokinetics. In vitro and in
vivo experiments were carried out to evaluate whether this
approach is feasible.
RESULTS
■
We intended to design a series of new SiFA-tagged PESIN
derivatives with a variety of hydrophilic auxiliary amino acids
embedded into the peptide backbone (Figure 1). Starting from
uncharged sugar based residues such as glucose and lactose
derived amino acids, we focused on carboxylic acid and sulfonic
acid functionalities which are negatively charged under
physiological conditions. Furthermore, positively charged
quaternary ammonium groups and combinations thereof were
considered in the design and synthesis of the peptides.
Similar experiments were envisaged with RGD peptides, an
additional class of important bioactive molecules with high
affinity to αvβ3/αvβ5 integrins for the imaging of angio-
genesis.19,20 Integrins are cell adhesion receptors which are
upregulated in the new blood vessels of growing tumors
providing the ability to monitor tumor growth and metastasis.
Synthesis and Radiochemistry. The PESIN scaffold was
constructed via solid-phase peptide synthesis using the standard
Fmoc strategy (Scheme 1). The polar auxiliary (Aux) and a
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Scheme 1. Synthesis of [18F]SiFA-Labeled PESIN Derivatives
Figure 2. Structures of SiFA-RGD (14) and SiFA-RGD derivatives 15−17.
terminal bis-Boc-aminooxyacetic acid were attached on solid
support. After cleavage of the peptide from the resin, SiFA-
CHO (12) was coupled to the aminooxyacetic acid function-
alized peptide in the liquid phase via oxime formation to yield
the labeling precursor 13. The synthesis of SiFA-CHO (12)
was described earlier.29
Amino acids which reduced the lipophilicity of PESIN
derivatives most effectively were chosen for the generation of
novel RGD peptides (Figure 2). Among them, carboxylic acid
and ammonium based residues proved to be most appropriate
for the implementation into the complex synthesis approach.
RGD peptides were also synthesized on solid support via
Fmoc based solid phase peptide synthesis (Scheme 2). The
thioether bridge was formed by condensation of an acetic acid
functionalized thiol residue with the deprotected N-terminus.
Thallium trifluoroacetic acid yielded the disulfide by depro-
tection of both remaining thiols and concomitant oxidative
coupling. The side chain of a Lysin amino acid was connected
to PEGylated linker 21 which enabled the direct attachment of
polar amino acids (Aux) and the terminal SiFA-COOH (27).
SiFA-COOH (27) was produced by simple oxidation of SiFA-
CHO (12) with potassium permanganate and served as
labeling synthon instead of SiFA-CHO (12) in this case.29
Mass spectrometric analysis of peptides 14−17 revealed
unusual solvent adducts. Whereas the expected signals
matching M + 2H+ could be detected in the case of peptides
14 and 15, very pronounced signals were observed for all RGD
peptides matching the correct structure, but incremented by a
constant value. Experiments with different deuterated solvents
strongly indicate the formation of stable solvent adducts with
MeCN and MeOH, although an unambiguous proof of adduct
identity cannot be given at this point.
The labeling reaction is identical for PESIN and RGD
peptides and occurs at the SiFA moiety via displacement of
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Scheme 2. Synthesis of [18F]SiFA-Labeled RGD Peptides
[19F]fluoride by [18F]fluoride. The preparation of reactive
[18F]fluoride was achieved by a cartridge based drying
technique, the so-called Munich method.30 In the presence of
oxalic acid, a labeling efficiency of 75−95% was obtained within
15 min at ambient temperature. A simple C18 SPE cartridge
purification yielded the desired peptides in 41−62% decay-
corrected radiochemical yields and ≥99% radiochemical purity
(Tables 1 and 2).
Table 1. LogD, IC50 Values, and Yields of PESIN Derivatives
[18F]1−[18F]9
labeling
yield
[%]
isolated
IC50
[nM]
yield d.c.
a
a
b
b
entry
compound
logD
[%]
1
2
SiFA-PESIN (1)
2.29 0.04
1.97 0.04
32 1.1
7.5 1.1
86
94
6
3
50
54
5
1
SiFA-Asn(AcNH-
β-Glc)-PESIN
(2)
Stability Studies. All compounds were tested for stability
in human plasma of healthy donors at 37 °C. No significant
degradation was observed within 120 min for PESIN derivatives
[18F]1−[18F]9 and RGD peptides [18F]14 − [18F]17.
Lipophilicity. LogD (1-octanol/phosphate buffer 0.05 M,
pH = 7.4) values were determined as a measure of the overall
lipophilicity of the compounds. Introduction of polar auxiliaries
entailed a considerable decrease of lipophilicity for PESIN
derivatives (Table 1) and RGD peptides (Table 2).
In Vitro Binding Affinity Determination Studies.
Competitive displacement experiments were performed in
vitro to evaluate the binding affinity of the modified
compounds. PESIN derivatives were tested on PC3 cells for
binding affinity toward the GRP receptor using 125I-[Tyr4]-
Bombesin as the GRP receptor specific radioligand. IC50 values
were determined to be between 7.0 1.2 nM and 32 1.1 nM
(Table 1). RGD peptides were tested on U87MG cells for their
affinity toward αvβ3 integrins. 125I-Echistatin was applied as the
αvβ3 specific radioligand. Only low affinities in the μM range
were found on U87MG cells (Table 2).
3
4
5
6
7
8
SiFA-Ser(β-Lac)-
PESIN (3)
1.44 0.01
1.41 0.03
0.48 0.06
0.34 0.02
−0.41 0.04
−0.71 0.03
32 1.1
14 1.2
19 1.1
10 1.1
7.0 1.2
15 1.1
83
92
90
95
86
93
2
2
3
1
0
2
54
59
41
56
59
61
4
3
2
0
8
0
SiFA-Cya-PESIN
(4)
SiFA-LysMe3-
PESIN (5)
SiFA-γ-carboxy-D-
Glu-PESIN (6)
SiFA-Cya2-PESIN
(7)
SiFA-LysMe3-γ-
carboxy-D-Glu-
PESIN (8)
9
SiFA-(γ-carboxy-
D-Glu)2-PESIN
(9)
−1.22 0.06
27 1.1
91
1
62
1
a
b
Mean SD (n = 3). Mean SD (n = 2).
animal PET imaging of tumor bearing mice. Male SCID mice
with subcutaneous PC3 xenografts in the right flank were
injected with approximately 10 MBq of the respective
radiolabeled PESIN derivative and scanned dynamically over
120 min. A high tracer accumulation was observed immediately
in the liver suggesting a mainly hepatobiliary clearance. In
In Vivo Biodistribution Studies. The assessment of the
pharmacokinetic profile of the compounds was done by small
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to 3.53 1.19 for [18F]15 and 3.99 2.29 for [18F]16 after
120 min. The highest level of tumor to muscle ratio was
observed with peptide [18F]17 which was determined to be
Table 2. LogD, IC50 Values, and Yields of RGD Derivatives
[18F]14−[18F]17
labeling
yield
[%]
isolated
7.75
1.66 after 120 min. Tracer accumulation in the liver
IC50
[μM]
yield d.c.
a
a
b
b
entry
compound
logD
[%]
played a minor role, whereas accumulation in the kidneys and
bladder was predominant, which suggests a mainly renal
elimination of the tracer. The accumulation of the tracer in the
tumor was highest within the first 15 min followed by a
constant but rapid wash-out. The αvβ3 specific tumor
accumulation of RGD peptide [18F]17 was proven by
coinjection of a 2000-fold excess of nonradioactive c(RGDyK).
The tumor was clearly visible under baseline conditions, but
visualization was exclusively and almost completely suppressed
under blocking conditions (Figure 4).
1
2
SiFA-RGD (14)
−1.10 0.03
−1.51 0.03
6.7 1.1
6.5 1.1
77
87
5
4
45
57
3
8
SiFA-γ-carboxy-D-
Glu-RGD (15)
3
4
SiFA-(γ-carboxy-
D-Glu)2-RGD
(16)
−1.85 0.06
−1.97 0.12
6.4 1.0
2.6 1.1
75
82
6
6
43
43
4
6
SiFA-LysMe3-γ-
carboxy-D-Glu-
RGD (17)
a
b
Mean SD (n = 3). Mean SD (n = 2).
contrast, the tumor uptake was very poor and essentially
remained on a constant low level. Since the tumor could hardly
be distinguished from the background signal, no further
experiments with PESIN analogues were performed.
RGD peptides were analyzed in the same manner using
female SCID mice with subcutaneous U87MG xenografts in the
right flank (Figure 3). The unmodified peptide [18F]14 showed
Figure 4. Coronal and transverse small animal PET images of mice
bearing a U87MG xenograft in the right flank 120 min after injection
of 10 MBq of 18F-SiFA-LysMe3-γ-carboxy-D-Glu-RGD [18F]17 under
baseline (left side) and under blocking conditions (right side).
As the small animal PET data obtained for the RGD peptides
were promising, biodistribution experiments were performed.
The results obtained for the most promising RGD analogue
[18F]17 are shown in Table 3. Tumor uptake was initially high
with 5.30 1.05% ID/g and decreased within 3 h to 0.66
0.07% ID/g. Highest activity was observed in the kidneys,
whereas liver, gallbladder, and intestine showed rather low
activity levels suggesting predominantly renal excretion.
Accumulation of activity in the bones was relatively high at
early time points, but decreased steadily over time. In vivo
metabolite studies revealed spontaneous formation of a more
hydrophilic product already after 10 min p.i. in blood and urine
samples (Figure 5). Further degradation of the tracer in blood
was not observed. Analysis of urine samples showed a minor
signal which might indicate the release of small amounts of
[18F]fluoride. The tracer is rapidly cleared from the blood
(0.03% ID/g at 180 min p.i.) and from muscle (0.07 0.01%
Figure 3. Coronal and transverse small animal PET images of mice
bearing a U87MG xenograft in the right flank 120 min after injection
of 10 MBq of 18F-SiFA-RGD [18F]14 (left side) and 18F-SiFA-LysMe3-
γ-carboxy-D-Glu-RGD [18F]17 (right side).
a similar behavior like the PESIN analogues and was
characterized by a high tracer accumulation in the liver and
only low uptake in the tumor, whereas a marked increase of
[18F]fluoride deposition was observed in the bones. This effect
was not found in the case of the other RGD derivatives, and no
simple explanation can be given for this observation as all
derivatives comprise the same SiFA building block. The tumor
to muscle ratio decreased over time from 2.54 0.27 after 60
min to 1.64 0.31 after 120 min. Peptides [18F]15 and [18F]16
showed a slightly increasing tumor to muscle ratio over time up
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a
Table 3. Biodistribution of [18F]17 in Nude Mice Bearing U87MG Xenografts at Different Times p.i.
tissue
blood
10 min
30 min
90 min
180 min
5.31 1.38
2.13 0.49
3.25 0.71
1.90 0.27
9.17 3.95
11.9 3.69
1.05 0.29
1.31 0.42
1.93 0.32
1.73 0.32
1.44 0.40
1.60 0.25
5.30 1.05
3.92 1.25
1.04 0.29
1.45 0.37
0.66 0.15
1.17 0.20
0.92 0.15
1.57 0.33
5.85 0.74
0.36 0.06
0.67 0.17
0.76 0.10
0.85 0.16
0.62 0.38
0.96 0.14
2.30 0.50
4.55 1.77
1.62 0.26
0.14 0.02
0.17 0.03
0.37 0.04
0.58 0.08
1.21 0.24
3.97 0.57
0.18 0.08
0.44 0.19
0.50 0.09
0.59 0.20
0.15 0.06
0.74 0.10
0.92 0.10
7.17 2.95
6.81 1.93
0.03 0.00
0.09 0.01
0.25 0.04
0.43 0.04
3.20 2.09
3.01 0.12
0.11 0.02
0.36 0.09
0.33 0.08
0.59 0.13
0.07 0.01
0.53 0.07
0.66 0.07
9.67 1.12
19.08 2.42
heart
lung
liver
gallbladder
kidney
pancreas
spleen
stomach
intestine
muscle
bone
tumor
tumor/muscle
tumor/blood
a
Results are expressed as % ID/g SD (n = 5).
Figure 5. In vivo metabolism of 18F-SiFA-LysMe3-γ-carboxy-D-Glu-RGD [18F]17 in murine plasma (A) and urine (B).
ID/g at 180 min p.i.) which resulted in a favorable tumor-to-
muscle ratio of 9.67 1.12% ID/g and a high tumor-to-blood
ratio of 19.08 2.42% ID/g at 180 min p.i.
and released using a lyophilized mixture of [K⊂2.2.2]OH
redissolved in DMF. It has to be considered that the stability of
the Kryptofix complex in DMF is very limited. However, if
applied directly after solubilizing, an elution efficiency of 95% of
[K⊂2.2.2]18F from the cartridge was achieved. The eluate was
used directly for the labeling reaction. A precise amount of
oxalic acid has to be added to partly neutralize the basic milieu,
which is very important as it has a strong impact on the
efficiency of the labeling reaction. Under these conditions the
isotopic exchange proceeds without formation of byproducts,
making a final HPLC separation of the radiolabeled peptides
dispensable. From a chemical perspective, this method confers
several advantages compared to methods in the literature which
often suffer from lengthy and laborious multistep synthetic
procedures. However, another powerful one-step 18F-labeling
DISCUSSION
■
In order to adjust the pharmacokinetic properties of the peptide
PESIN, several hydrophilic residues were introduced at the N-
terminus where structural diversity is rather tolerated, as the N-
terminal region has a lower impact on biological activity.
Carbohydrates gave positive results in similar works and the
influence of charge was also addressed previously.31−33 To
maintain the biological potency, no structural modifications
were made at the pharmacophoric C-terminal binding site. The
synthesis was straightforward and enabled the production of the
peptides on solid support in good yields and high purities. The
labeling synthon SiFA-CHO (12) was attached terminally
which enables the incorporation of fluorine-18 (18F) via
nucleophilic displacement of [19F]fluoride. Usually isotopic
exchange comes along with low specific activities. However,
very low precursor quantities of only 25 nmol were used, which
abrogates this effect giving specific activities of up to 60 GBq
μmol−1. Recently, one of the major advances in the preparation
of 18F-labeled compounds was achieved with the cartridge
based drying technique of [18F]fluoride, the so-called Munich
method.30 An adapted protocol which was developed for the
labeling of SiFA tagged peptides was applied.34 Aqueous
[18F]fluoride was trapped on a strong anion exchange cartridge
approach using Al18F-chelation emerged recently.35,36
A
comparison of the latest optimized procedures is given in
Table 4.37 Both approaches get along without azeotropic drying
and HPLC purification shortening the overall synthesis time to
20 min. Most strikingly, SiFA labeling occurs at ambient
temperature allowing the labeling of even sensitive biomole-
cules, whereas Al18F labeling conditions are rather harsh. It is
also noteworthy that the yields, purities, and specific activities
achieved in this report are slightly higher.
Encouraged by the good binding affinities of the RGD
peptide [18F]-AH11185 to αvβ3 and αvβ5 integrins in vitro28
and the promising clinical results, we decided to combine its
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Tumor uptake of an Al18F labeled cyclic RGD monomer, for
example, was considerably higher,39 even though concerns
about patient safety due to the toxicity of aluminum metals in
the central nervous system remain.40,41 Thus, further refine-
ments of SiFA-tagged RGD compounds will be the focus of
further studies.
Table 4. Comparison of 18F Labeling Approaches Using
Al18F Chelation37 and SiFA Technology
parameter
yield
Al18F approach
SiFA approach
42%
41−62%
>99%
purity
>95%
specific activity
synthesis time
temperature
azeotropic drying
purification
37 GBq/μmol
20 min
60 GBq/μmol
20 min
rt
For the developed SiFA-derivatized RGD peptides, binding
affinities on a μM level were rather poor and much higher than
IC50 values reported in the literature where competition
experiments were carried out on EA-Hy926 membrane
fractions.28 Nevertheless, the tumor uptake was clearly
enhanced, which shows that the overall lipophilicity is more
decisive for the in vivo behavior of the peptide derivative than
the achieved binding affinities. Correlation of logD values and
in vivo behavior indicates that logD values below −1.5 are
needed to avoid unwanted hepatobiliary clearance. Peptide
[18F]17 exhibited not only the best receptor affinity, but also
the highest tumor uptake. It can be assumed that the additional
positive charge might have a beneficial effect on these
parameters. The blood clearance is exceptionally fast, 3-fold
faster than in case of peptide [18F]16 over 180 min. All
peptides are characterized by a rapid washout from all tissues.
This can be explained by an enforced externalization of
lipophilic compounds from the cell. A possible strategy to
overcome this effect might be the multimerization of RGD
peptides which was shown to be a powerful strategy to prevent
this process as a consequence of increased avidities due to
multivalency.42
100 °C
not req.
C18 SPE
not req.
C18 SPE
favorable properties with the advantages of the SiFA labeling
technology. However, the reported synthesis28 turned out to be
inconvenient, as we intended to gain structural diversity by the
introduction of different auxiliary amino acids into the peptide
scaffold. Thus, we developed a new synthetic approach which
enabled the entire synthesis of the peptide on solid support. A
new protecting group strategy maintained full orthogonality.
The direct attachment of SiFA-COOH (27) via SPPS proved
to be superior over the coupling step with SiFA-CHO (12) in
liquid phase, as it bypassed a troublesome purification of the
aminooxyacetic acid functionalized peptide and reduced the
overall synthesis steps at the same time. Radiolabeling of RGD
and PESIN derivatives was carried out using the same
experimental conditions.
Competitive displacement experiments revealed good
affinities of the PESIN analogs toward the GRP receptor on
PC3 cells showing that the introduction of a SiFA moiety at the
N-terminus does not impede the biological function of the
peptide, still maintaining a high affinity to the receptor in the
nM range. However, small animal PET images showed an
unfavorably high accumulation of the PESIN derivatives in the
liver, although the lipophilicity of the compounds could be
significantly reduced from 2.29 to −1.22. This result is in line
with findings by Dijkgraaf et al. who found high uptake in the
abdominal cavity with an Al18F labeled bombesin derivative in
spite of an even lower logD value of −1.47.38 Nevertheless, the
IC50 value was in the subnanomolar range and the tumor could
be clearly visualized. In this report, the lipophilic character of
the peptides seems to be too pronounced still and could not be
compensated by high receptor affinities. Furthermore, PET
images suggest that the tumors were potentially poorly
vascularized or even necrotic which also impeded the effective
visualization of the tumor.
High liver uptake was also obtained with the unmodified
RGD peptide [18F]14 which is innately rather hydrophilic
showing a logD value of −1.10. Accordingly, the tumor could
be clearly visualized. Modification of the RGD sequence and
concomitant decrease of logD values resulted in a further
relevant improvement of the pharmacokinetic profile. The
rather slow decrease of activity in bone of peptide [18F]17 may
indicate that defluorination and subsequent deposition of
[18F]fluoride in the bones has taken place. However, in vivo
metabolic stability studies did not show the presence of
[18F]fluoride in murine blood, and only low levels of
[18F]fluoride in urine were observed. Instead, tracer [18F]17
was to some extent converted into another more polar
metabolite. Therefore, the relatively high bone uptake may be
a result of several factors and is not necessarily a result of
defluorination processes alone. Due to the fast clearance from
the tissues, the obtained imaging results are rather inferior
compared to other biological data reported in the literature.
CONCLUSION
■
Herein, we report the synthesis, radiolabeling, and biological
evaluation of SiFA-tagged PESIN and RGD derivatives. Plasma
stabilities, binding affinities, and logD values were determined
for each peptide analogue. In the case of RGD peptides, it was
demonstrated that the high lipophilicity of the SiFA synthon
could be successfully compensated by the introduction of polar
auxiliary amino acid residues into the peptide scaffold in favor
of a better pharmacokinetic profile. Especially 18F-SiFA-
LysMe3-γ-carboxy-D-Glu-RGD [18F]17 revealed a high tumor
uptake and a rapid blood clearance in U87MG tumor bearing
mice by means of small animal PET and biodistribution
experiments.
EXPERIMENTAL SECTION
■
General. All reagents were purchased from commercial
sources and were used without further purification. Solvents of
at least p.a. quality were used. The resin for solid-phase peptide
synthesis and amino acids was purchased from NovaBiochem
and Bachem, coupling reagents from SigmaAldrich and Fluka.
125I-[Tyr]4-Bombesin and 125I-Echistatin were obtained from
PerkinElmer. Sep-Pak Accell Plus QMA carbonate light
cartridges and Sep-Pak C18 light cartridges were obtained
from Waters.
RPMI 1640 medium, fetal bovine serum, and L-glutamine
were purchased from PAA; GlutaMax I medium was purchased
from Gibco. Dulbecco’s phosphate buffered saline (PBS) and
bovine serum albumin were obtained from SigmaAldrich.
Analytical HPLC was done on an Agilent 1200 system using
a Chromolith Performance (RP-18e, 100 × 4.6 mm, Merck,
Germany) column. For preparative HPLC, a SymmetryPrep
(C18, 19 × 300 mm, Waters, Germany) column was applied.
ESI (Electrospray Ionization) MS spectra were measured on a
744
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Bioconjugate Chemistry
Article
Finnigan MAT95Q instrument. Calculated values and corre-
sponding found mass values are reported. The γ-counter used
for radioactivity determination was a Packard Cobra Quantum.
Small animal PET images were acquired using the Siemens
Inveon P120 Dedicated PET system (Siemens Preclinical
Imaging, Knoxville, TN, USA).
(10%). ESI-MS: m/z (M + 2H+) calcd for C86H137FN16O28SSi:
1920.9251, found: 1920.9264.
Synthesis of SiFA-Cya-PESIN (4). The following amino acids
were used: Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-
OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-
Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-PEG4-COOH,
Fmoc-Cya-OH, and bis-Boc-Aoa. Coupling of Fmoc-Cya-OH
to the peptide was achieved with 8 equiv of DIPEA. SiFA-Cya-
PESIN was isolated as white solid (6%). ESI-MS: m/z (M +
2H+) calcd for C74H117FN16O20S2Si: 1660.7825, found:
1660.7840.
Synthesis of SiFA-Cya2-PESIN (5). The following amino
acids were used: Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-His-
(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-PEG4-
COOH, Fmoc-Cya-OH, Fmoc-Cya-OH, and bis-Boc-Aoa.
Coupling of Fmoc-Cya-OH to the peptide was achieved with
8 equiv of DIPEA. SiFA-Cya2-PESIN was isolated as white solid
(6%). ESI-MS: m/z (M + 2H+) calcd for C77H122FN17O24S3Si:
1811.7764, found: 1811.7776.
Synthesis of SiFA-LysMe3-PESIN (6). The following amino
acids were used: Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-His-
(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-PEG4-
COOH, Fmoc-LysMe3-OH chloride, and bis-Boc-Aoa. SiFA-
LysMe3-PESIN was isolated as white solid (11%). ESI-MS: m/z
(M + 2H+) calcd for C80H131FN17O17SSi: 1680.9383, found:
1680.9334.
Synthesis of SiFA-γ-carboxy-D-Glu-PESIN (7). The following
amino acids were used: Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-
His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH,
Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-PEG4-
COOH, Fmoc-γ-carboxy-D-Glu(OtBu)2-OH, and bis-Boc-Aoa.
SiFA-γ-carboxy-D-Glu-PESIN was isolated as white solid (25%).
ESI-MS: m/z (M2‑) calcd for C77H115FN16O21SSi: 1678.7908,
found: 1678.7914.
Synthesis of SiFA-LysMe3-γ-carboxy-D-Glu-PESIN (8). The
following amino acids were used: Fmoc-Met-OH, Fmoc-Leu-
OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH,
Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH,
Fmoc-NH-PEG4-COOH, Fmoc-γ-carboxy-D-Glu(OtBu)2-OH,
Fmoc-LysMe3-OH chloride, and bis-Boc-Aoa. SiFA-LysMe3-γ-
carboxy-D-Glu-PESIN was isolated as white solid (10%). ESI-
MS: m/z (M + H+) calcd for C86H137FN18O22SSi: 1852.9618,
found: 1852.9626.
Synthesis of SiFA-(γ-carboxy-D-Glu)2-PESIN (9). The follow-
ing amino acids were used: Fmoc-Met-OH, Fmoc-Leu-OH,
Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-
OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-
PEG4-COOH, Fmoc-γ-carboxy-D-Glu(OtBu)2-OH, Fmoc-γ-
carboxy-D-Glu(OtBu)2-OH, and bis-Boc-Aoa. SiFA-(γ-car-
boxy-D-Glu)2-PESIN was isolated as white solid (17%). ESI-
MS: m/z (M + 2H+ + Na+) calcd for C83H125FN17O26SSiNa:
1877.8342, found: 1877.8362.
The solid-phase peptide synthesis of the RGD derivatives
was carried out using the standard Fmoc strategy on a Fmoc-
functionalized Rink Amide AM resin (loading: 0.79 mmol/g).
A solution of the amino acid (4 equiv), HBTU (4 equiv), and
HOBt (1-hydroxybenzotriazol, 4 equiv) in DMF was treated
with DIPEA (4 equiv) for 2 min at ambient temperature; the
mixture was then added to the resin bound unprotected peptide
and shaken for 45 min at room temperature. Subsequently, the
resin was washed with DMF (6×). The Fmoc protecting group
Peptide Synthesis. Solid-phase peptide synthesis for
PESIN derivatives was carried out using the standard Fmoc
strategy on an Fmoc-functionalized Rink Amide resin (loading:
0.74 mmol/g). A solution of the amino acid (4 equiv) and
HBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate, 4 equiv) in DMF was treated with
DIPEA (N,N-diisopropylethylamine, 4 equiv) for 2 min at
ambient temperature, the mixture was then added to the resin
bound unprotected peptide and shaken for 45 min at room
temperature. Subsequently, the resin was washed with DMF
(6×). The Fmoc protecting group was removed by washing the
resin twice with 50% (v/v) piperidine in DMF (1 × 2 min, 1 ×
5 min) and finally with DMF (6×). All steps were repeated
until peptide elongation was complete. Finally, the resin was
washed with CH2Cl2 (6×) and diethyl ether (3×) and dried in
vacuo. The peptide was cleaved from the resin with TFA/H2O/
TIS (triisopropylsilane) 90:5:5 (1 mL) within 45 min at
ambient temperature and precipitated in ice cold diethyl ether.
The precipitate was separated by centrifugation, washed twice
with diethyl ether, dried in vacuo, and finally purified by
preparative HPLC (0−45% MeCN + 0.1% formic acid over 30
min). After lyophilization the peptide intermediate was
dissolved in phosphate buffer (0.1 M, pH = 4, 400 μL) and
mixed with SiFA-CHO (12) (1.1 equiv) in a volume of MeCN
which is sufficient for complete dissolution of the aldehyde.
After 5 min the product was purified by preparative HPLC (0−
65% MeCN + 0.1% formic acid over 22 min) and lyophilized.
All PESIN syntheses were typically performed in a 0.05−0.30
mmol scale according to this general procedure.
Synthesis of SiFA-PESIN (1). The elongation of the peptide
chain was conducted with the following amino acids: Fmoc-
Met-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH,
Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-
Gln(Trt)-OH, Fmoc-NH-PEG4-COOH, and bis-Boc-amino-
oxyacetic acid (bis-Boc-Aoa). SiFA-PESIN was isolated as white
solid (23%). ESI-MS: m/z (M + 2H+) calcd for
C71H112FN15O16SSi: 1509.7875, found: 1509.7886.
Synthesis of SiFA-Asn(AcNH-β-Glc)-PESIN (2). The follow-
ing amino acids were used: Fmoc-Met-OH, Fmoc-Leu-OH,
Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-
OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-
PEG4-COOH, Fmoc-Asn(Ac3AcNH-β-Glc)-OH, and bis-Boc-
Aoa. After cleavage from the resin the peptide was dissolved in
MeOH and treated with NaOMe (0.5 M in MeOH) until the
pH of the mixture reached 12. The solution was shaken for 30
min at ambient temperature. After acidification with neat TFA
the solvent was evaporated in a stream of nitrogen and the
residual solid was coupled with SiFA-CHO (12) as described
before. SiFA-Asn(AcNH-β-Glc)-PESIN was isolated as white
solid (14%). ESI-MS: m/z (M + 2H+) calcd for
C83H132FN18O23SSi: 1826.9109, found: 1826.9094.
Synthesis of SiFA-Ser(β-Lac)-PESIN (3). The following
amino acids were used: Fmoc-Met-OH, Fmoc-Leu-OH,
Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-
Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-
NH-PEG4-COOH, Fmoc-Ser(Ac7-β-Lac)-OH, and bis-Boc-
Aoa. SiFA-Ser(β-Lac)-PESIN was isolated as white solid
745
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Bioconjugate Chemistry
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was removed by washing the resin twice with 50% (v/v)
piperidine in DMF (1 × 2 min, 1 × 5 min) and finally with
DMF (6×). All steps were repeated until peptide elongation
was complete. The elongation of the peptide chain was done
with following amino acids: Fmoc protected linker 20, Fmoc-
Cys(tButhio)-OH, Fmoc-Phe-OH, Fmoc-Cys(Acm)-OH,
Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH,
Fmoc-Cys(Acm)-OH, and Fmoc-Lys(Mtt)-OH. The resin
was then suspended in DMF:H2O 9:1, treated with n-Bu3P
(50 equiv) under Ar-atmosphere twice for 60 min, and washed
with DMF. Subsequently, the resin was mixed with iodoacetic
acid (4 equiv) and DIPEA (1 equiv) in DMF and shaken for 45
min. Fmoc deprotection and coupling to the acetic acid
according to standard SPPS conditions yielded the thioether.
The solid supported cyclic peptide was washed with 1.75% TFA
in CH2Cl2 at least 10 times until yellow color disappeared,
washed successively with 10% DIPEA in CH2Cl2 and DMF,
and coupled with Fmoc protected linker 21 under standard
SPPS conditions. Treatment of the resin with Tl(CF3CO2)3 (4
equiv) and DIPEA (2 equiv) in DMF for 45 min afforded the
disulfide. The bicyclic peptide was deprotected with piperidine
and coupled to another auxiliary amino acid and/or SiFA-
COOH (27) using standard SPPS procedure. The peptide was
finally cleaved from the resin with TFA (950 μL), phenol (25
mg), and CH3SO3 (25 μL) for 15 min at ambient temperature
and precipitated in ice-cold diethyl ether. The precipitate was
separated by centrifugation, washed twice with diethyl ether,
and dried in vacuo. The product was purified by preparative
HPLC (25−45% MeCN + 0.1% formic acid over 20 min) and
lyophilized. All syntheses were typically done in a 0.1 mmol
scale.
127.6, 125.7, 120.7, 70.6, 70.3, 70.3, 70.3, 70.2, 70.1, 70.1, 69.6,
69.4, 68.3, 65.9, 47.3, 40.7, 38.6. ESI-MS: m/z (M + Na+) calcd
for C31H42N2O11Na: 641.2681, found: 641.2679.
Synthesis of SiFA-RGD (14). No auxiliary amino acid was
used. SiFA-RGD was obtained as white solid (18%). ESI-MS:
m/z (M + 2H+) calcd for C81H132FN17O26S3Si: 1901.8434,
found: 1901.9114.
Synthesis of SiFA-γ-carboxy-D-Glu-RGD (15). Fmoc-γ-
carboxy-D-Glu-OH was introduced as auxiliary amino acid.
SiFA-γ-carboxy-D-Glu-RGD was obtained as white solid (15%).
ESI-MS: m/z (M + 2H+) calcd for C87H139FN18O31S3Si:
2074.8758, found: 2074.9444.
Synthesis of SiFA-(γ-carboxy-D-Glu)2-RGD (16). Two Fmoc-
γ-carboxy-D-Glu-OH residues were subsequently introduced.
SiFA-(γ-carboxy-D-Glu)2-RGD was obtained as white solid
(10%). ESI-MS: m/z (M + K+ + H+ + 2MeCN + 2MeOH)
calcd for C99H159FKN21O38S3Si: 2431.9696, found: 2432.0840.
Synthesis of SiFA-LysMe3-γ-carboxy-D-Glu-RGD (17).
Fmoc-LysMe3-OH and Fmoc-γ-carboxy-D-Glu-OH residues
were subsequently introduced. SiFA-LysMe3-γ-carboxy-D-Glu-
RGD was obtained as white solid (11%). ESI-MS: m/z (M+ +
K+ + 2MeCN + 2 MeOH) calcd for C102H170FKN22O34S3Si:
2429.0791, found: 2429.1970.
Radiolabeling. Aqueous [18F]fluoride was produced via the
18O(p,n)18F nuclear reaction by irradiation of enriched
[18O]water. [18F]Fluoride was loaded onto a SAX cartridge
(Sep-Pak Accell Plus QMA Carbonate light, 46 mg). Twenty
milliliters of air passed through the cartridge followed by 5 mL
of anhydrous MeCN and a further 20 mL of air. [18F]Fluoride
was eluted from the cartridge with 0.5 mL of [K+⊂2.2.2]OH−
(100 μmol) in anhydrous DMF. [K+⊂2.2.2]OH− was
previously prepared from Kryptofix 2.2.2 (41 mg, 110 μmol)
and 100 μL aqueous KOH (1 M, 100 μmol) and lyophilization
of the Kryptofix solution to dryness. Subsequently, 37.5 μL
oxalic acid in anhydrous DMF (0.5 M, 18.75 μmol) and 5 μL
labeling precursor in anhydrous DMF (5 mM, 25 nmol) were
added to the eluate. The labeling reaction was complete after
15 min at ambient temperature without stirring and an aliquot
was taken for reaction control and analyzed by analytical
HPLC. The mixture was diluted with 10 mL HEPES buffer (0.1
M, pH = 4) and passed through a Sep-Pak C18 light cartridge.
The cartridge was rinsed with 5 mL of water to remove solvent
residues. The purified product was eluted with 0.5 mL EtOH
and diluted with isotonic saline solution (4.5 mL). The
syntheses revealed radiochemical yields ranging from 41% to
62% after a synthesis time of 20 min and specific activities of up
to 60 GBq μmol−1. The radiochemical purities were ≥99%.
Lipophilicity. Five microliters of the purified [18F]fluoride
labeled peptide in ethanol were added to 400 μL 1-octanol and
400 μL phosphate buffer (0.05 M, pH = 7.4) and the mixture
was vigorously vortexed for 5 min. After centrifugation at 14000
rpm for 1 min aliquots (300 μL) of both layers were collected
and measured for radioactivity. The logD value was calculated
as the log ratio of the radioactivity in the organic and aqueous
layer.
Synthesis of Linker 20 and 21. A solution of diglycolic
anhydride (1 equiv, 1 mmol) in 1 mL MeCN was added
dropwise to a solution of polyethylene glycol bisamine (18, 19)
(1 equiv, 1 mmol) in 10 mL MeCN. The mixture was stirred at
ambient temperature overnight. The solvent was removed in
vacuo and the residue dissolved in MeCN:H2O 1:1 (10 mL).
DIPEA was added at 0 °C until pH reached 8 to 9
(approximately 0.5 mL), and a solution of Fmoc N-
hydroxysuccinimide ester (1.3 equiv, 1.3 mmol) in 3 mL
MeCN was added dropwise. After 1 h the mixture was warmed
to room temperature and stirred overnight. After removal of the
solvent, the product was purified by preparative HPLC (0−75%
MeCN + 0.1% formic acid 0−30 min, 75−100% MeCN + 0.1%
formic acid 30−30.5 min, 100% MeCN + 0.1% formic acid
30.5−35 min) and lyophilized (20: 47%, 21: 40%).
1
Linker 20. H NMR (400 MHz, DMSO-d6) δ (ppm) 12.85
(s, broad, 1H) 7.88 (d, J = 7.6 Hz, 2H), 7.83 (t, J = 5.6 Hz,
1H), 7.69 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.32 (m,
3H), 4.29 (d, J = 7.2 Hz, 2H), 4.20 (t, J = 6.4 Hz, 1H), 4.11 (s,
2H), 3.97 (s, 2H), 3.48 (s, 8H), 3.41 (m, 4H), 3.26 (q, J = 5.6
Hz, 2H), 3.13 (q, J = 6.0 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ (ppm) 171.4, 168.8, 156.2, 143.9, 140.7, 127.6,
127.0, 125.2, 120.1, 70.0, 69.7, 69.5, 69.1, 68.8, 67.7, 65.3, 46.7,
40.1, 38.0. ESI-MS: m/z (M + Na+) calcd for C27H34N2O9Na:
553.2157, found: 553.2155.
1
Plasma Stability. 200 μL of an injectable solution of the
radiolabeled peptide were mixed with 500 μL human plasma.
The samples were incubated at 37 °C. At 10, 30, 60, and 120
min, aliquots were taken and analyzed by analytical HPLC.
Cell Culture. PC-3 prostate adenocarcinoma cells were
grown in RPMI 1640 medium, human U87MG glioblastoma
cells in DMEM, each supplemented with 10% (v/v) fetal
Linker 21. H NMR (400 MHz, DMSO-d6) δ (ppm) 12.85
(s, broad, 1H), 7.88 (d, J = 7.6 Hz, 2H), 7.83 (t, J = 5.6 Hz,
1H), 7.69 (d, J = 7.6 Hz, 2H), 7.40 (t, J = 7.6 Hz, 2H), 7.33 (m,
3H), 4.29 (d, J = 7.2 Hz, 2H), 4.20 (t, J = 6.4 Hz, 1H), 4.11 (s,
2H), 3.96 (s, 2H), 3.48 (m, 16H), 3.41 (m, 4H), 3.26 (q, J =
5.6 Hz, 2H), 3.13 (q, J = 6.0 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ (ppm) 172.0, 169.4, 156.7, 144.5, 141.3, 128.2,
746
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Bioconjugate Chemistry
Article
bovine serum and 1% (v/v) L-glutamine at 37 °C in a
humidified CO2 (5%) atmosphere.
the tail vein under isoflurane anesthesia. Animals were sacrificed
at 10, 30, 60, and 120 min after injection, and blood and urine
were collected. Blood samples were centrifuged at 2500g, and
the supernatants and urine samples were analyzed by analytical
radio-HPLC.
Cell Binding Assay. In vitro binding affinities were
measured via competitive displacement experiments using a
Millipore Multiscreen punch kit. Millipore 96-well filter plates
were incubated with 200 μL/well Dulbecco’s phosphate
buffered saline (PBS, modified, without CaCl2 and MgCl2,
liquid, sterile-filtered, supplemented with 1 g/100 mL bovine
serum albumin) for 1 h before use. PC-3 cells were harvested,
suspended in Opti-MEM I (reduced serum medium, GlutaMax
I), and seeded in 96-well plates at 105 cells per well.
Subsequently, the cells were incubated on a shaker for 1 h at
ambient temperature with 0.1 nM 125I-[Tyr4]-Bombesin (81.4
GBq/μmol) as the GRPR-specific radioligand in the presence
of increasing concentrations (0−1 μM) of competing PESIN
derivatives in a total volume of 100 μL. After incubation the cell
pellets were washed 3 times (2 × 100 μL and 1 × 200 μL) with
Dulbecco’s phosphate buffered saline (PBS, modified, without
CaCl2 and MgCl2, liquid, sterile-filtered) using the Millipore
Multiscreen vacuum manifold for filtration. The filters were
collected and measured for radioactivity in a γ-counter.
Experiments were done at least three times in triplicate. The
50% inhibitory concentration (IC50) value was calculated by
nonlinear regression analysis using the GraphPad Prism
Software program (v 5.03).
ASSOCIATED CONTENT
* Supporting Information
■
S
Data from analytical HPLC, binding curves, small animal PET
images, and data from biodistribution experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org/.
AUTHOR INFORMATION
Corresponding Authors
■
*Phone +49 (0)89 7095 69941, fax +49 (0)89 7095 7646, e-
mail Simon.Lindner@med.uni-muenchen.de.
*Phone +49 (0)621 383 5594, fax +49 (0)621 383 1910, e-mail
bjoern.waengler@medma.uni-heidelberg.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Werner Spahl for his help with ESI-MS, Dr. Lars
Allmendinger for NMR measurements, and the members of the
laboratory for preclinical imaging for their support with animal
studies. We gratefully acknowledge financial support by the
BMBF (German Federal Ministry of Education and Research)
Leading Edge Cluster “m4 − Personalized Medicine and
Targeted Therapies”.
In vitro binding affinities of RGD peptides were measured
analogously to the PESIN derivatives. U87MG cells were
suspended in binding buffer (Tris·HCl 25 mM, NaCl 150 mM,
CaCl2 1 mM, MgCl2 0.5 mM, MnCl2 1 mM, pH 7.4, BSA
0.1%) and incubated with 0.1 nM 125I-Echistatin (81.4 GBq/
μmol) as the αvβ3 specific radioligand in the presence of
increasing concentrations (0−250 μM) of competing RGD
peptides.
REFERENCES
■
(1) Wangler, C., Kostikov, A., Zhu, J., Chin, J., Wangler, B., and
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̈
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Schirrmacher, R. (2012) Silicon-[18F]fluorine radiochemistry: basics,
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PET. Nude mice (SCID, male for PESIN derivatives, female
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injected with PC-3 or U87MG cells (5 × 106 cells/mouse)
subcutaneously into the right flank. After tumor inoculation
cells were allowed to grow for 6 to 8 weeks. Approximately 10
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under isoflurane anesthesia. Dynamic small animal PET images
were acquired over 120 min. Data were divided into time
frames from 10 s to 10 min for the assessment of temporal
changes in regional tracer accumulation. The images were
reconstructed using an OSEM3D/MAP algorithm. Regions of
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animals per peptide) were injected with U87MG cells (5 × 106
cells/mouse) subcutaneously into the right flank. After tumor
inoculation cells were allowed to grow for 6 to 8 weeks.
Approximately 1−5 MBq of the RGD tracer were injected via
the tail vein under isoflurane anesthesia. The animals were
sacrificed after different time points (10, 30, 90, and 180 min, 5
animals for each time point) and the organs of interest were
dissected and weighted. Radioactivity in the tissues was
determined with a γ-counter. Data are given as % injected
dose per g tissue.
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