Synthesis of [18F]anginex with high specific activity [ 18F]fluorobenzaldehyde for targeting angiogenic activity in solid tumors

Article abstract of DOI:10.1002/jlcr.1912

Anginex is a 33-residue peptide that has been previously demonstrated to possess antiangiogenic properties. To provide a tool to evaluate the regional biodistribution and pharmacokinetics of anginex, and possibly to provide a useful angiogenesis-targeted

Full text of DOI:10.1002/jlcr.1912

Research Article
Received 26 January 2011,
Revised 3 June 2011,
Accepted 6 June 2011
Published online 18 August 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1912
1
8
Synthesis of [ F]anginex with high specific
1
8
activity [ F]fluorobenzaldehyde for targeting
angiogenic activity in solid tumors
a
b
b
b
Scott M. Apana, Robert J. Griffin, Nathan A. Koonce, Jessica S. Webber,
c
c
a,d
Ruud P. M. Dings, Kevin H. Mayo, and Marc S. Berridge *
Anginex is a 33-residue peptide that has been previously demonstrated to possess antiangiogenic properties. To provide a
tool to evaluate the regional biodistribution and pharmacokinetics of anginex, and possibly to provide a useful angiogenesis-
1
8
targeted radiotracer, we have radiolabeled anginex with fluorine-18. High specific activity [ F]fluorobenzaldehyde (1.5–4.8 TBq
40–130Ci)/mmol) was used to label anginex via reductive amination in 76% yield. The effective specific activity of the
(
product was lower because unlabeled anginex was not separated. However, the high specific activity labeling reagent
increased the labeling yield and reduced the amount of anginex required for labeling. Regional pharmacokinetics were
measured by PET scanning in mice, demonstrating tumor uptake and low background, with up to 30% of total injected dose
localized in some tumors.
Keywords: fluorine-18; anginex; angiogenesis; tumor imaging; PET
1
6
Introduction
inducing apoptosis and thus inhibiting angiogenesis. Although
it is, therefore, an antiangiogenic peptide, it has additional
mechanisms of action. The study of inhibition of gal-1 overex-
pression could lead to the development and use of more effec-
Angiogenesis, the growth of new blood vessels from preexist1-
ing vessels, is crucial for organ growth as well as organ repair.
Angiogenesis plays a key role in normal organ development, but
it is also involved in pathological conditions including cancer,
arthritis, diabetic retinopathy, and restenosis.
genesis inhibitors have been approved for use by the US Food
and Drug Administration, and several F-18- and Cu-64- labeled
radiotracers are used to image angiogenesis.
endogenous angiogenic inhibitors include angiostatin, platelet
19
tive therapeutic agents. In view of the substantial amount of
in vitro and in vivo evidence that anginex targets the angiogenic
microvasculature in tumors and can be a potent adjuvant ther-
apy, we were interested in better understanding the tumor
uptake of this peptide in vivo.
2
–4
Several angio-
5
3
,6–10
Common
18
Anginex radiolabeled with [ F]fluorobenzaldehyde may
permit PET scanning for assessment of regional anginex biodis-
tribution and kinetics. Labeled anginex may thus serve as a trac-
er to assess angiogenic activity in tumors, which may allow
evaluation of optimal radiation therapy scheduling and tumor
targeting for therapeutic use of this or other antiangiogenic
agents. In preliminary investigations, we obtained substantial
tumor localization of labeled anginex in bone grafts of multiple
factor-4, thrombospondin, interferon-g inducible protein-10,
3
and endostatin.
Anginex was designed based on common amino acid
sequences of known endogenous inhibitors and has been shown
to inhibit endothelial cell growth more effectively than several of
1
1
these inhibitors. Anginex is a synthetic 33-mer peptide with a
molecular weight of 3900. The amino acid sequence, using stan-
dard single-letter amino acid designations, is H N-A-N-I-K-L-S-V-
2
Q-M-K-L-F-K-R-H-L-K-W-K-I-I-V-K-L-N-D-G-R-E-L-S-L-D-C(O)-NH .
2
4
,11
a
Note that the compound is a carboxyl-terminal amide.
Anginex has been shown to sensitize tumor endothelial cells to
3D Imaging, LLC, Cyclotron Suite Rm PS010, UAMS Radiology #556, 4301 W.
Markham Street, Little Rock, AR 72205-7199, USA
1
2
radiation, inhibit tumor growth up to 80% in several murine
b
Department of Radiation Oncology, University of Arkansas for Medical Sciences,
1
3–15
and human tumor models,
and may improve tumor Little Rock, AR, USA
16
response to radiation by improving tumor oxygenation. A last-
ing influence of anginex therapy on tumor growth inhibition in
combination with weekly radiation in a model of focal human
c
Department of Biochemistry, Molecular Biology, and Biophysics, University of
Minnesota, Minneapolis, MN, USA
17
d
multiple myeloma was recently observed.
Department of Radiology, University of Arkansas for Medical Sciences, Little
Rock, AR, USA
Anginex inhibits tumor growth specifically by binding to
galectin-1 (gal-1), which has been found to be overexpressed
in endothelial cells of various tumors. By binding to gal-1,
*
Correspodence to: Marc S. Berridge, 3D Imaging, LLC Cyclotron Suite Rm PS010,
UAMS Radiology #556, 4301 W. Markham Street, Little Rock, AR 72205-7199, USA.
anginex disrupts endothelial cell adhesion and migration, E-mail: MBerridge@3DImagingLLC.com
18
J. Label Compd. Radiopharm 2011, 54 708–713
Copyright © 2011 John Wiley & Sons, Ltd.

S. M. Apana et al.
1
7
myeloma as well as in a murine breast tumor model, suggest-
The N-terminal sequencing of anginex proceeded normally
18
ing that the refinement and expansion of [ F]anginex may be through the entire peptide sequence. In contrast, the N-terminal
beneficial for various aspects of oncology. The synthesis of this sequencing of any of the reaction products gave no reaction,
tracer and the imaging results reported here provide motivation indicating that the N-terminus is blocked in all products
and means to continue to investigate labeled anginex as an (> 99%), and therefore the N-terminal is the most reactive,
antiangiogenic radiotracer for use in tumor research, tumor preferentially labeled, site in the anginex molecule. This suggests
evaluation, and treatment planning.
that in this stoichiometric reaction, the N-terminus was labeled
first, followed by the reaction of additional sites.
The mass spectral fragmentation patterns of the major
63min) product suggested that this product was singly substituted.
F]Anginex was obtained via reductive amination of high This conclusion was also supported by the lack of observation of the
specific activity [ F]fluorobenzaldehyde with anginex in several products that would be expected if multiple reaction sites
Results and discussion
(
18
[
1
8
7
[
6.8Æ3.5% (n=4) chemical yield (Figure 1). Purification of had similar reactivity, by the N-terminal sequence results, and by
1
8
F]anginex for production was accomplished by C18 cartridge the product stoichiometry. In a no-carrier added radiolabeling,
purification rather than HPLC as a matter of preference. anginex is present in excess of the radiolabeled fluorobenzaldehyde.
1
8
Although [ F]fluorobenzaldehyde can also be retained by C18 Therefore, the chance of double labeling under labeling conditions is
cartridge purification, it was conveniently removed during remote. The major product of this reaction is thus the only one of
prior solvent evaporations. Neither purification method sepa- relevance to the radiolabeling conditions, and radiolabeling occurs
1
8
rated [ F]anginex from the unlabeled anginex precursor. The almost exclusively at the N-terminus.
effective specific activity of the product therefore depended
upon the amount of anginex used in the reaction and was
Concentration dependence
typically about 4GBq (100mCi)/mg, or 14.4GBq (390mCi)/mmol.
To determine the labeling position of fluorobenzaldehyde, the
amounts of both anginex and fluorobenzaldehyde were
increased from standard labeling procedure levels and were
reacted at 1:1 stoichiometric ratio due to the need for greater
detectable mass of the reacted product. Most of the anginex
peptide reacted with the labeling reagent, as evidenced by the
HPLC results shown in Figure 2. The trace at the bottom is for
18
Production batches of [ F]anginex used the entire 1-mL portion
18
of purified [ F]fluorobenzaldehyde. This resulted in a 4-fold
decrease in the concentration of unlabeled anginex precursor
(0.9vs 4mg/mL) when ‘high’ and ‘normal’ specific activity compar-
ison methods were used. As a result of this difference in concentra-
tion, the production batches also saw a corresponding decrease in
18
the yield of [ F]anginex to 20–30%. Because the overall yield was
19
labeled anginex (using nonradioactive F-fluorobenzaldehyde
in this case), whereas the partial trace at the top is labeled
anginex spiked with nearly an equimolar amount of standard
anginex peptide as indicated. Notice in the bottom HPLC trace
of the labeled peptide that apparently less than 1% of free angi-
nex peptide remains from this stoichiometric ratio reaction.
These HPLC data also indicate that the conjugation reaction
produced three species of labeled anginex in significant
amounts—a major one eluting from the HPLC at 63min
sufficient, no effort was made to increase the yield of the produc-
tion batches by either a reduction of the reaction volume or by
an increase in the amount of reagents used to a level equivalent
with the high specific activity method. The effective specific
activity could therefore also be increased, although there was
no need to do so for the purpose of this work.
Carrier dependence
(retention time), and two minor ones eluting at 75 and 117
min. Because we used reverse-phase HPLC columns, the hydro- Recent work has shown Teflon to be a major source of fluoride
20
phobicity of these species increased with increasing retention carrier in the synthesis of fluorine-18-labeled compounds.
time. The labeling reagent could react with any free amine group Prior to the identification of the source of carrier, the average
20
on the peptide (including 5 lysine e-amine groups and an mass of carrier fluoride per synthesis was 400–800nmol,
N-terminal amine), and the polarity of the peptide product would and the corresponding specific activity of many fluorine-18
be reduced as more groups were conjugated. Therefore, the minor, compounds at multiple centers worldwide was generally
21–30
later-eluting products were likely to be multiply substituted.
18–111GBq (0.5–3Ci)/mmol.
By removing all sources of Teflon
to reduce the amount of carrier to approximately 25nmol, with
20
corresponding specific activities of 30–50Ci/mmol, an expected
result would be an improvement in yield or a reduction in the
amount of precursor needed to maintain a high yield, or both.
To determine whether high specific activity fluorobenzaldehyde
18
improved the overall yield of [ F]anginex, a comparison was
performed by adding carrier fluorobenzaldehyde to adjust the
total amount of carrier to an amount that was representative
of formerly typical specific activities. This was done prior to
addition and reaction of anginex. Whereas the average mass of
carrier in fluorine-18 syntheses in prior work was 400–800
20
nmol, the amount of unlabeled fluorobenzaldehyde added as
carrier in these experiments was only 250nmol. This reduction
in the added carrier amount was due to the relatively high
molecular weight and resulting low molar amount of anginex
used for labeling (260nmol) and a desire to not enter the range
Figure 1. Synthesis of radiolabeled fluorobenzaldehyde and anginex. Ax, anginex
3
3-mer peptide (see Introduction).
J. Label Compd. Radiopharm 2011, 54 708–713
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

S. M. Apana et al.
Figure 2. HPLC analysis of the reaction product of fluorobenzaldehyde and anginex (1.1:1). Inset shows the same sample with added unreacted anginex. A singly ‘labeled’
product eluted at 63min, and a doubly ‘labeled’ product eluted at 75min.
1
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of carrier in which [ F]fluorobenzaldehyde would be in excess
over the anginex.
1
8
The yield of [ F]anginex in these carrier added syntheses was
5
8.3Æ3.6% (n=4), in comparison with a yield of 76Æ3.5%
without added carrier, indicating a significant increase (p<0.05)
in yield with high specific activity fluorobenzaldehyde under
otherwise identical conditions.
MicroPET results
An image of a mouse with human BN-myeloma xenograft is
shown in Figure 3. The highest organ uptake was observed in
liver, with some urinary excretion and residual abdominal cavity
activity remaining from the intraperitoneal (i.p.) injection also
observed. No other major organs were noted on the image.
There was a striking uptake into the dorsal tumor graft, which
grew well beyond the normal boundaries of the animal’s body
(drawn on the figure). Tumor uptake accounted for at most
3
0% of the total body distribution of labeled anginex and was
five times that of muscle background. Maximum tumor uptake
was 16% higher than maximal uptake in the liver. We have previ-
ously observed that repeated anginex treatment by i.p. injection
of 10–20mg/kg can slightly inhibit SCK (murine mammary carci-
noma) and BN myeloma tumor growth and can significantly
18
Figure 3. Saggital plane of a 20-min microPET scan of [ F]anginex 2h after i.p.
injection in a SCID-rab BN myeloma tumor-bearing mouse. Arrow indicates the
tumor/bone graft, which extends outside the normal body contour (approximate
contour indicated by the sketch).
increase radiation-induced delays in tumor growth in these facility of the University of Minnesota as a lyophilized powder
1
2,17
models.
Figure 4 shows tumor uptake in FSaII tumor-bearing and reconstituted in methanol.
HPLC analysis was performed on a Hewlett-Packard 1090
mice. This tumor, implanted on the hindlimb, accumulated
noticeably less anginex than did the BN-myeloma. However, it Series II instrument with autoinjector, diode array UV, and a Grace
should also be noted that Figure 4 includes earlier time point Econosphere 4.6Â250mm reverse-phase column eluted with
image data than Figure 3. Unabsorbed dose from the i.p. injec- 40% aqueous acetonitrile/0.2% trifluoroacetic acid at a flow rate
18
tion is more apparent in Figure 4 as an intense abdominal activ- of 1.6mL/min. Retention times (in minutes): [ F]anginex, 3.1;
18
ity than in the image shown in Figure 3, which was acquired at a [ F]fluorobenzaldehyde, 6.5. Radioactivity was measured with
longer time after injection. Decay-corrected time–activity curves a Beckman 170 flow-through detector. Radiolabeled anginex
are shown (Figure 5) for tumor and reference muscle regions of co-eluted on this system with unlabeled anginex (precursor).
interest from the FSaII tumor mouse shown in Figure 4. Uptake
1
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in tumor from the i.p. injection is slow until a modest maximum [ F]-p-Fluorobenzaldehyde
after 10min of about three times the level seen in muscle.
1
8
Synthesis of [ F]fluorobenzaldehyde was done using previously
20
18
reported methods. Briefly, high specific activity [ F]-fluoride
from 11-MeV proton bombardment of [ O]H
Experimental
18
(
2
O) was passed
O was recovered.
18
Solvents and reagents were purchased from Fisher Scientific and through anion exchange resin, and the [ O]H
Sigma-Aldrich and were used without further purification unless [ F]Fluoride was eluted with aqueous potassium carbonate
otherwise noted. Anginex was obtained from the biochemical (24mmol, 600mL), Kryptofix 2.2.2 (10mg, 26mmol) was added,
2
18
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 708–713

S. M. Apana et al.
of sodium cyanoborohydride (1.6mmol) in 100mL of methanol
ꢀ
were added, and the solution was heated at 100 C for 30min.
[
18
F]Anginex was purified by reverse-phase HPLC (Figure 6).
1
8
18
[
F]Anginex reactions with ‘normal’ specific activity [ F]
fluorobenzaldehyde
One milligram of anginex (260nmol) was dissolved in
1
[
~
00mL of methanol and added to a portion of the purified
F]fluorobenzaldehyde solution (50mL, 0.04–0.8GBq (1–20mCi),
1.25nmol), to which unlabeled fluorobenzaldehyde (250nmol)
1
8
had been previously added. The carrier quantity of 250nmol
had previously been determined to be representative of the
amount of carrier present in a typical radiolabeling experiment
Figure 4. MicroPET image planes of an FSaII (mouse fibrosarcoma) tumor on the
right hind limb following i.p. injection (left, coronal image slice; right, transaxial
image slice). The horizontal line in each image slice shows the approximate posi-
tion of the plane of the other image slice.
20
(400–800nmol) before improvements in specific activity were
introduced. To this solution, 4mL of glacial acetic acid and
1
00mg of sodium cyanoborohydride (1.6mmol) in 100mL of
ꢀ
methanol were added, and the solution was heated at 100 C
for 30min. [ F]Anginex was purified by reverse-phase HPLC
18
(Figure 6).
1
8
[
F]Anginex for imaging use
18
[
F]fluorobenzaldehyde (16–24GBq (400–650mCi), ~25nmol)
in 1mL of methanol was added to 1mg of anginex (260nmol).
Glacial acetic acid (4mL) and 100mg of sodium cyanoborohydride
(
1.6mmol) in 100mL of methanol were added, and the solution
ꢀ
was heated at 100 C for 30min. The solvent was evaporated,
and the residue dissolved in 10mL of water. The entire solution
was passed over a C18 cartridge (Waters Sep-Pak). The cartridge
was rinsed with an additional 5mL of water and dried briefly
with argon. The retained [ F]anginex was eluted with 1mL of
acetonitrile into a conical glass vial at 100 C, and the solvent
18
ꢀ
was evaporated under vacuum with argon at a flow rate of
5
5mL/min. Ethanol (2Â0.5mL) was added and evaporated to
18
remove acetonitrile. [ F]Anginex was taken up in sterile water,
and the pH was adjusted to 6–7 with 1–10mL of 1N sodium
bicarbonate.
Figure 5. Decay-corrected time–activity curves from the FSaII (mouse fibrosarcoma)
tumor and corresponding muscle reference region shown in Figure 4.
1
8
and [ F]fluoride was dried by acetonitrile evaporation. The pre-
31
cursor p-trimethylammoniumbenzaldehyde triflate (6–8mg,
ꢀ
1
9–25mmol in 1mL of DMSO) was reacted for 10min at 100 C.
After C18 cartridge (Waters Sep-Pak) purification and elution with
mL of methanol, the labeled fluorobenzaldehyde was used with-
out further purification. Each experiment could be performed with
1
18
up to 130GBq (3500mCi) of [ F]fluoride, depending on the pro-
ton beam current and duration used for the experiment. Regard-
less of the quantity of fluorine-18 produced, however, the mass
of fluorobenzaldehyde that was produced in each experiment
20
was in the range of 8–50nmol. Individual experiments were
thus performed with fluorobenzaldehyde of measured specific
activity in the range of 1.5–4.8TBq (40–130Ci)/mmol.
1
8
18
[
F]Anginex reactions with high specific activity [ F]
fluorobenzaldehyde
One milligram of anginex (260nmol) was dissolved in
Figure 6. Radiochromatogram of crude anginex following the reaction.
1
[
00mL of methanol and added to a portion of the purified
F]fluorobenzaldehyde solution (50mL, 0.04–0.8GBq (1–20mCi),
Anginex mass in the quantity injected was not detectable. The chromatogram of
1
8
18
[
F]anginex (purified product) consisted of a single mass and the corresponding
~
1.25nmol). To this solution, 4mL of glacial acetic acid and 100mg radioactivity peak and is therefore not shown here.
J. Label Compd. Radiopharm 2011, 54 708–713
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

S. M. Apana et al.
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Fluorobenzyl anginex ( F) and determination of labeling
position
Cells were harvested in log phase with 0.125% trypsin (Media-
tech Inc.), counted with a Z2 Coulter Counter (Beckman Coulter,
Brea, CA), spun, and resuspended in serum-free medium at a
Anginex (10mg, 2.6mmol) was dissolved in 1mL of methanol,
and unlabeled fluorobenzaldehyde (2.6mmol) was added. To
this solution, 40mL of glacial acetic acid and 1mg of sodium
cyanoborohydride (16mmol) in 100mL of methanol were added,
and the solution was heated at 100 C for 60min. Purification
followed the ‘[ F]anginex for imaging use’ method described
earlier.
5
concentration of 2Â10 cells/50mL. Female C3H mice were pur-
chased from Charles Rivers (Wilmington, MA), and tumor cells
were injected s.c. in the right hind limb. Mice were imaged when
tumors grew to 8–10mm in size.
ꢀ
1
8
MicroPET—BN myeloma
Analysis of the position of labeling of the anginex was
performed using an HPLC system capable of separation of
analytical quantities of the derivatized anginex products from
anginex and from each other. A Beckman Coulter System Gold
Static microPET imaging was performed on three BN myeloma
tumor graft-bearing mice at 2h after i.p. injection with 1.1MBq
18
(30mCi) of [ F]anginex tracer in a solution of 10mg/mL
unlabeled anginex. Static scans through the tumor region were
captured for 20min, and a two-dimensional OSEM image recon-
struction was used. Decay-corrected images were used to
draw regions of interest around the BN tumor and whole body
(125 Solvent Module; 166 Detector) analytical/semi-preparative
HPLC with UV detection at 220nm was used, with reverse-phase
C18 Kromasil columns and an elution gradient of 0% to 60%
acetonitrile with 0.1% trifluoroacetic acid in water. Fractions from
the HPLC were collected for analysis of m/z on a MALDI-TOF
mass spectrometer (4800 MALDI TOF/TOF, Applied Biosystems).
Fractions were also analyzed by N-terminal sequencing via
Edman degradation in a Beckman 1600N-terminal sequencer.
(Figure 3). Activities within the tumor were normalized to the
total body activity.
MicroPET - FSaII
Authentic, underivatized anginex was also added to the sample Dynamic microPET scans were performed for 45min immedi-
18
for positive chromatographic identification of the unreacted ately after i.p. injection of 36MBq (0.98mCi) of [ F]anginex in
and reacted materials.
three mice. A two-dimensional OSEM image reconstruction was
used. Images of data acquired from 6 to 45min are shown in
Figure 4. Time–activity curves (Figure 5) were generated from
regions of interest manually created over the tumor and refer-
ence tissue (muscle).
Tumors and animals
All experimental methods were approved by the Institutional
Animal Care and Use Committee at the University of Arkansas
for Medical Sciences (UAMS). Animals were housed in a
Conclusion
ꢀ
temperature-controlled environment (72Æ2 F) with a 12-h light
18
cycle and were fed with a commercially prepared diet (Harlan, [ F]Anginex was labeled in 76.8Æ3.5% chemical yield from
18
Indianapolis, IN) and provided water ad libitum. Mice were [ F]fluorobenzaldehyde. Preliminary microPET data show that
subjected to imaging analysis using UAMS microPET Focus 220 uptake in two tumor types is substantial and selective, with a
(
Concorde Microsystems, Knoxville, TN) when the tumors grew possibility of high tumor-to-background ratios. Further work to
18
to 8–12mm in size.
define the specificity of [ F]anginex for its receptor gal-1
in vivo using PET imaging and biological correlate assays is
ongoing in our laboratories and in feline head and neck
clinical studies. Radiolabeled anginex is useful as a tracer for
the development of therapeutic anginex and possibly as a
general antiangiogenic diagnostic radiotracer.
Human BN myeloma
In vitro expansion of the BN cells was performed as previously
3
2
described. Briefly, mononucleated marrow cells from human
fetal long bones (Advanced Bioscience Resources, Alameda, CA)
were used as feeder cells and cultured in flasks in low-glucose
DMEM supplemented with 10% fetal bovine serum (Hyclone,
Logan, UT) and antibiotics. SCID-rab mice were prepared as previ-
ously described. Briefly, rudiments of long bones of newborn
New Zealand rabbits (Myrtle Rabbitry, Thompson Station, TN)
were implanted s.c. in 6-week-old CB.17/Icr-SCID mice (Harlan
Sprague Dawley, Indianapolis, IN). Six to eight weeks after
implantation of the bone grafts, BN cells (0.5Â10 –2Â10 cells
in 50–100mL of PBS) were injected directly into the marrow
cavity of the implanted bone grafts through the diaphyseal
opening. Mice were imaged after tumor growth extended
outside the graft. For imaging, mice were anesthetized in an
induction chamber with 4–5% isoflurane in oxygen and main-
tained at 1.5–2.5% isoflurane in oxygen during the scan.
The current work is a proof of principle that the biodistribution
18
of [ F]anginex and its uptake in tumors can be measured. This
will allow detailed in vivo characterization of the targeting
properties of anginex and exploration of potential applications
for this nontoxic and stable peptide. Phase I/II trials of anginex
are under way in felines with spontaneous head and neck
tumors. It appears that the method established here for labeling
anginex and imaging its tumor uptake may be a valuable
addition to these comparative oncology studies to understand
tumor targeting efficiency and the basic pharmacokinetic
properties of this agent.
3
3
6
6
Acknowledgments
Supported by grant CA107160 and CA096090 from the NCI and
by the Central Arkansas Radiation Therapy Institute.
Mouse fibrosarcoma—FSaII
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