Radiosynthesis and in vivo evaluation of a F-18-labeled pancreatic islet amyloid inhibitor

Article abstract of DOI:10.1002/jlcr.1748

Formation of islet amyloid deposits contributes to the progressive loss of beta-cells in Type 2 diabetes. Islet amyloid is composed of islet amyloid polypeptide (IAPP). [(N-Me)G²⁴, (N-Me)I²⁶]hIAPP(22-27) peptide was found to bind hum

Full text of DOI:10.1002/jlcr.1748

Research Article
Received 18 December 2009,
Revised 20 January 2010,
Accepted 25 January 2010
Published online 30 March 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1748
Radiosynthesis and in vivo evaluation
of a F-18-labeled pancreatic islet
amyloid inhibitor
Gopal Pathuri, Hrushikesh B. Agashe, Vibhudutta Awasthi,
and Hariprasad Gali
Ã
Formation of islet amyloid deposits contributes to the progressive loss of beta-cells in Type 2 diabetes. Islet amyloid is
composed of islet amyloid polypeptide (IAPP). [(N-Me)G24, (N-Me)I26]hIAPP(22–27) peptide was found to bind human IAPP
with high-affinity and inhibit fibrillogenesis. We labeled [(N-Me)G24, (N-Me)I26]hIAPP(22–27) with fluorine-18 using
N-succinimidyl-4-[18F]fluorobenzoate. Results from biodistribution studies in healthy CF-1 mice at 1 h p.i. indicated that
18F-peptide was cleared efficiently (0.0470.02%ID/g remained in blood). The primary route of clearance from the body
appears to be hepatobiliary. Radioactivity accumulation in liver, intestine, and kidney was 6.772.9, 60.3718.5, and
0.270.0%ID, respectively. Other organs accumulated negligible radioactivity. As normal mice do not develop pancreatic
amyloid deposits, only background radioactivity was seen in pancreas. The radio-HPLC analysis of mouse urine at 2 h p.i.
showed that ꢀ29.3% of injected 18F-peptide excreted intact along with two additional metabolite peaks. Dynamic
microPET/CT imaging of a CF-1 mouse injected with 18F-peptide indicated that radiotracer was rapidly taken up by liver and
most of it moved into intestine within 10 min. The results provide a useful insight into the biological disposition of
[(N-Me)G24, (N-Me)I26]hIAPP(22–27) that is being developed as a pancreatic amyloid inhibitor.
Keywords: pancreatic islet amyloid; hIAPP(22–27); inhibitor; [18F]SFB; [18F]FBA; PET imaging; F-18-labeled peptide; biodistribution;
pharmacokinetics; metabolic stability
mechanism is that these fibrils form pores in the membrane that
allows extracellular calcium to enter the cell resulting in
Introduction
apoptotic cell death.13,14 However, recently it has been
Several studies suggest that the formation of pancreatic islet
amyloid deposits may be a major contributor to the progressive
loss of pancreatic beta-cells.1–4 These deposits are known to be
a characteristic pathologic feature of the pancreas in 490% of
patients with Type 2 diabetes.5–6 Pancreatic islet amyloid
deposits in humans consist mainly of b sheet fibrillar aggregates
of the 37-amino acid islet amyloid polypeptide (IAPP or
amylin).4,5,7 IAPP is a neuroendocrine hormone of the calcitonin
family of polypeptide hormones, and is produced and co-
secreted with insulin in response to beta-cell stimulation by
both glucose and non-glucose secretagogues.8 IAPP is over-
produced in states of insulin resistance, a typical feature of Type
2 diabetes. Excess IAPP promotes self-aggregation and amyloid
fibril formation that are thought to be toxic to beta-cells.4 That
islet amyloidosis is an early factor responsible for beta-cell failure
is strongly supported by the in vitro cytotoxic effect of human
islet amyloid polypeptide (hIAPP), as well as by the results from
in vivo hIAPP transgenic mouse studies.9,10 The precise
mechanisms by which hIAPP-derived fibrils induce beta-cell
death and dysfunction are still not clear. It has been suggested
that the presence of hIAPP fibrils between islet cells and
capillary endothelial cells may impair movement of nutrients
(such as glucose) from the bloodstream into beta-cells. At the
same time, insulin exocytosed by beta-cells also faces this
barrier before reaching the circulation.11,12 Another proposed
suggested that prefibrillar aggregates (or oligomers) formed
early during aggregation, and not mature amyloid fibrils, are the
cytotoxic species in Type 2 diabetes.15,16
Although the sequence of IAPP is strongly conserved over a
number of animal species, IAPP-derived amyloid is only formed
by humans, cats, and few non-human primates.17 Rodents do
not form pancreatic amyloid, although rat IAPP sequence differs
from its human analog by only six amino acid residues.
Considering that five of these six amino acid residues are
located between residues 20 and 29, amyloidogenicity of hIAPP
has been related to these residues. It has been shown that the
synthetic decapeptide hIAPP(20–29) is able to form fibrils with
morphology that is similar to the fibrils formed by the complete
hIAPP sequence.18 The ‘amyloid core’ has been further narrowed
down to the residues 22–27 (NFGAIL) that has been shown to
Small Animal Imaging Facility, Department of Pharmaceutical Sciences, The
University of Oklahoma College of Pharmacy, Oklahoma City, OK 73117, USA
*Correspondence to: Hariprasad Gali, The University of Oklahoma College of
Pharmacy, 1110 N. Stonewall Avenue, Room 301, Oklahoma City, OK 73117,
USA.
E-mail: hgali@ouhsc.edu
J. Label Compd. Radiopharm 2010, 53 186–191
Copyright r 2010 John Wiley & Sons, Ltd.

G. Pathuri et al.
ˆ
participate in hIAPP self-association into a-sheets and amyloid an important tool for both monitoring therapeutic inter-
fibril formation.19
ventions and better understanding of the natural history of the
Compounds that block cytotoxic protein/polypeptide self- disease.
assembly and amyloidogenesis or fibril formation are therefore
important targets for use in therapeutic intervention.20,21 There are
Results and discussion
two classes of hIAPP amyloidogenicity and/or cytotoxicity inhibi-
tors: (1) aromatic organic compounds such as Congo red,
rifampicin, and others that bind to amyloid fibrils and/or suppress
amyloid-fibril formation,4,22 and (2) short synthetic peptides
derived from hIAPP sequences that contain hIAPP self-recognition
domains.23 The synthetic peptides suffer from a disadvantage of
being able to undergo self-amyloidosis, which limits their
application in therapeutics development. A few modifications in
the NFGAIL sequence have been able to overcome self-amyloidosis
without altering their inhibitory effect on hIAPP fibrillogenesis. For
instance, NFGAIL has been converted into a non-amyloidogenic
and non-cytotoxic sequence by N-methylation at Gly24 and Ile.24,25
The strategy of N-methylation of peptide amide bonds has been a
well-known protein-design approach to suppress H-bonding ability
of an 4NH group and to restrict the conformation of the back-
bone. The N-methylated analog, [(N-Me)G24, (N-Me)I26]hIAPP(22–27),
conjugated with 5(6)-carboxyfluorescein on the N-terminus has
been found to selectively bind hIAPP in vitro as well as in vivo-
formed human pancreatic islet amyloid deposits in human
pancreatic tissue obtained postmortem.24,25
We have selected [(N-Me)G24, (N-Me)I26]hIAPP(22–27) peptide
based on its documented ability to selectively bind to hIAPP
in vitro (Kd = 129759 nM), as well as to the in vivo-formed
human pancreatic islet amyloid deposits in human pancreatic
tissue obtained postmortem.24,25 The goal of this study was to
evaluate its biodistribution and clearance properties by radi-
olabeling with 18F and following it in vivo by PET imaging.
We labeled [(N-Me)G24, (N-Me)I26]hIAPP(22–27) with fluorine-
18 using a [18F]SFB prosthetic group. [18F]SFB was synthesized as
shown in Figure 1 by slight modifications in the published
procedure.26,27 The non-radioactive analog, FB-[(N-Me)G24,
(N-Me)I26]hIAPP(22–27), was synthesized by reacting the peptide
with SFB in presence of triethylamine. It was purified by HPLC
and characterized by electrospray mass spectrometry. It was
used as a non-radioactive reference for characterizing [18F]FB-
[(N-Me)G24, (N-Me)I26]hIAPP(22-27)(18F-peptide) using HPLC. The
conjugation reaction of [18F]SFB and peptide was carried out in
presence of triethylamine at 901C for 35 min.
As shown in Figure 2, the HPLC retention times of [18F]SFB
and 18F-peptide were very close (11.3 min versus 10.9 min,
respectively under identical HPLC conditions). To overcome the
separation problem and obtain pure 18F-peptide for animal
studies, we added 1 M NaOH to the reaction mixture after
conjugation to completely hydrolyze the unreacted [18F]SFB.
The [18F]FBA, hydrolysis product of [18F]SFB is relatively more
hydrophilic than the precursor [18F]SFB (9.8 min versus 11.3 min,
Here we report radiosynthesis and preliminary in vivo
evaluation of F-18-labeled [(N-Me)G24, (N-Me)I26]hIAPP(22–27)
peptide in normal mice. The overall goal of this study was to
explore the possibility of using the pancreatic amyloid inhibitor
as a targeting vector for in vivo imaging of pancreatic islet
amyloid deposits using positron emission tomography (PET).
Non-invasive assessment of islet amyloid deposits would provide
[K/K222]18F, AcCN
90 oC, 10 min
O
O
+
18F
Me3N
-SO3CF3
OEt
OEt
Pr4NOH, AcCN
90 oC, 5 min
HSTU, AcCN
90 oC, 10 min
O
O
O
18F
18F
+NPr4
O N
O-
O
[
18F]SFB
a) [(N-Me)G24, (N-Me)I26]hIAPP(22-27)
TEA, DMF, 90 oC, 35 min
b) 1M NaOH, 90 oC, 5 min
18F
O
O
O
NH
N
N
NH
CONH2
18F]FB-[(N-Me)G24, (N-Me)I26]hIAPP(22-27)
NH
NH COOH
O
O
O
[
Figure 1. Radiosynthesis of [18F]FB-[(N-Me)G24, (N-Me)I26]hIAPP(22–27).
J. Label Compd. Radiopharm 2010, 53 186–191
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

G. Pathuri et al.
benzoic acids.31 The conjugation reaction is a multi-step process
that takes place in the mitochondrial matrix and catalyzed by two
enzymes, benzoyl-coenzyme A synthetase and glycine N-acyl-
transferase.32 Thus, we speculate that [18F]FBA is also metabolized
in the same fashion as other benzoic acid derivatives in liver.
Although not proven, we hypothesize that [18F]FBA is formed
in vivo after metabolic cleavage of the C-terminal amide bond of
18F-peptide. The other more hydrophilic metabolite (1st peak)
may be the product obtained from metabolic cleavage of the
amide bond between asparagine and phenylalanine. The in vivo
metabolic stability of this peptide was found to be higher than
other small linear peptide radiopharmaceuticals, probably due to
N-methylation of glycine and isoleucine.33 This high metabolic
stability is a critical aspect for its therapeutic use to inhibit
pancreatic amyloid formation.
[18F]SFB
11.3
[18F]FBA
9.8
As the retention time of 18F-peptide was very close to that of
[18F]SFB, we also performed biodistribution studies of [18F]SFB in
healthy CF-1 mice to confirm that the results obtained with
18F-peptide are not due to the presence of [18F]SFB in the
injected dose. We also performed biodistribution studies of the
expected 18F-peptide metabolite, [18F]FBA, in healthy CF-1 mice
to see its influence on the overall biodistribution profile
attributed to 18F-peptide.
18F-peptide
As seen in Table 1, the biodistribution profile of [18F]SFB and
[18F]FBA were entirely different from that observed for the
18F-peptide. Both [18F]SFB and [18F]FBA cleared from blood
primarily through renal/urinary pathway. Only 1.1970.16%ID
and 0.4470.25%ID remained in intestine for [18F]SFB and
[18F]FBA respectively at 1 h p.i. The blood clearance of [18F]FBA
was significantly faster than that of [18F]SFB. At 1 h p.i.,
5.0570.46%ID of [18F]SFB remained in blood whereas only
0.0770.03% [18F]FBA was seen after 1 h. Accumulation of
radioactivity in other tissues was significantly lower with
[18F]FBA than that of [18F]SFB. The HPLC analysis (Figure 3) of
the mouse urine at 30 min p.i. of [18F]FBA showed that it was
completely metabolized into [18F]PFH and other more hydro-
philic (unknown) products. No formation of the [18F]PFH meta-
bolite was observed with [18F]SFB. We believe that [18F]SFB may
be able to bind to the plasma protein before being taken up by
liver. As [18F]SFB is an active ester, it may react with the free
primary amine groups present on the plasma protein. The
physiological pH is suitable for this reaction. The high radio-
activity seen in blood with [18F]SFB may be due to the protein
binding. More studies are needed to confirm this conjecture.
As the biodistribution profile and clearance pathways of
18F-peptide, [18F]SFB, and [18F]FBA were found to be signifi-
cantly different, it is clear that 18F-peptide dose injected into
mice was radiochemically pure and does not contain any
unreacted [18F]SFB. Hydrolysis of unreacted [18F]SFB prior to
HPLC purification of the F-18-labeled peptide appears to be a
good choice if HPLC retention times of both [18F]SFB and F-18-
labeled peptide are very close.
10.9
0
10
20
30
min
Figure 2. Radio-HPLC chromatograms.
respectively, under identical HPLC conditions) and can be easily
separated from 18F-peptide on HPLC.
Results from in vivo biodistribution studies of 18F-peptide
performed on healthy CF-1 mice at 1 h p.i. are summarized in
Table 1. The 18F-peptide was found to be cleared efficiently from
blood. Only ꢀ0.04%ID/g remained in blood at 1 h p.i. The
primary route of radioactivity clearance from the body appears
to be hepatobiliary. About 60.3718.5%ID accumulated in
intestine, whereas liver-associated radioactivity was found to
be 6.772.9%ID at 1 h p.i. The retention of radioactivity in
kidneys (0.2270.04%ID/g at 1 h p.i.) was found to be lower than
that shown for most other small peptide radiopharmaceuti-
cals.28 Other organs accumulated negligible radioactivity.
The radio-HPLC analysis of the mouse urine collected at 2 h p.i.
of 18F-peptide showed that ꢀ29.3% (3rd peak) of the radio-
activity was excreted intact; however, two additional metabolite
peaks were observed (Figure 3). One of the metabolite (ꢀ42.3%,
2nd peak) was identified as para-[18F]fluorohippurate ([18F]PFH).
Non-radioactive PFH was synthesized and used as an HPLC
reference for characterizing [18F]PFH. Literature search showed
that [18F]PFH is an expected product from glycine conjugation
reaction of [18F]FBA in liver.29,30 Conjugation with glycine is an
important mechanism for elimination and detoxification of
several xenobiotic and endogenous carboxylic acids such as
The small animal PET/CT imaging study of 18F-peptide
performed on a healthy CF-1 mouse (Figure 4) indicated that
the peptide is rapidly taken up by liver. As shown in Figure 4,
liver was found to accumulate large amounts of radioactivity
within the first 1 min p.i.; most of this radioactivity moved into
intestines by 10 min p.i. At 60 min p.i., the radioactivity signal
only originated from liver and intestine, which confirms the
result of the biodistribution study at 1 h p.i. High uptake of
radioactivity by liver and intestine will hinder the visibility of the
pancreatic uptake (in presence of amyloid fibrils) in a PET image
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J. Label Compd. Radiopharm 2010, 53 186–191

G. Pathuri et al.
Table 1. Biodistribution (%ID/g tissue) of 18F-peptide, [18F]SFB, and [18F]FBA in healthy CF-1 mice at 1 h p.i
Organs
18F-peptide
[18F]SFB
[18F]FBA
%ID/g tissue
%ID/organ
%ID/g tissue
%ID/organ
%ID/g tissue
%ID/organ
Blood
0.04
(0.02)
0.04
(0.02)
0.07
(0.02)
3.7
(1.7)
0.10
(0.08)
0.16
(0.16)
0.22
(0.04)
0.11
(0.03)
0.32
0.10
(0.05)
0.01
(0.00)
0.01
(0.00)
6.7
(2.9)
0.01
(0.01)
0.04
(0.04)
0.15
(0.03)
1.94
(0.58)
0.38
3.7
(0.3)
2.9
(0.6)
3.5
(0.6)
0.43
(0.05)
1.1
(0.2)
0.52
(0.20)
3.5
(0.0)
0.66
(0.50)
0.17
(0.02)
0.37
(0.07)
—
5.7
(0.7)
0.34
(0.08)
0.57
(0.06)
0.65
(0.03)
0.14
(0.03)
0.06
(0.01)
1.5
(0.2)
6.4
(4.8)
0.11
(0.03)
1.2
0.05
(0.03)
0.05
(0.04)
0.05
(0.02)
0.17
(0.14)
0.11
(0.09)
0.25
(0.25)
0.89
(0.06)
0.37
(0.32)
0.12
0.07
(0.03)
0.01
(0.00)
0.01
(0.00)
0.24
(0.19)
0.01
(0.01)
0.02
(0.03)
0.35
(0.03)
3.8
(3.4)
0.07
Heart
Lung
Liver
Spleen
Pancreas
Kidney
Muscle
Stomach
Intestine
Bone
(0.19)
17.7
(4.0)
0.10
(0.27)
60.3
(18.5)
0.43
(0.11)
0.14
(0.09)
—
(0.06)
0.44
(0.25)
—
(0.2)
—
(0.14)
26.3
(14.1)
(0.63)
—
Urine
445.4
(369.2)
—
267.2
(40.9)
—
Data are presented as mean and SD in brackets (n = 3).
as the pancreas is surrounded by liver and intestine. The mice 166 absorption detector, and a Bioscan Model B-FC-300 radio-
used in this study do not develop pancreatic amyloid deposits activity detector. HPLC solvents consisted of water containing
as rodent IAPP is non-amyloidogenic. Therefore, only back- 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing
ground accumulation of radioactivity was seen in pancreas in 0.1% trifluoroacetic acid (solvent B). A Sonoma C18 column
˚
both biodistribution and imaging studies.
(ES Industries, West Berlin, NJ) 10 mm, 100 A, 4.6 ꢂ 250 mm) was
used with a flow rate of 1.5 mL/min. The HPLC gradient system
began with an initial solvent composition of 80% A and 20% B
for 2 min followed by a linear gradient to 0% A and 100% B
General
All chemicals were obtained commercially and used without in 15 min, after which the column was re-equilibrated. The
further purification. Ethyl 4-(trimethylammonium triflate)benzoate UV-absorption detector was set at 254 nm.
and N-succinimidyl-4-fluorobenzoate (SFB) were synthesized as
previously reported.34,35 [(N-Me)G24, (N-Me)I26]hIAPP(22–27) pep-
tide was synthesized by the University of Oklahoma Health
Synthesis of FB-[(N-Me)G24, (N-Me)I26]hIAPP(22–27)
Sciences Center (OUHSC) Molecular Biology-Proteomics Facility
(Oklahoma City, OK) utilizing traditional Fmoc synthesis strategy.
No-carrier-added [18F]Fꢁ was obtained from Midwest Medical
Isotopes, Inc. (Oklahoma City, OK). Electrospray mass spectral
analysis was performed at the OUHSC Molecular Biology-
A solution of SFB (1.6 mg) dissolved in dimethylsulfoxide (DMSO)
(100 mL) and a solution of [(N-Me)G24, (N-Me)I26]hIAPP(22–27)
(1.1 mg) dissolved in DMSO (100 mL) and phosphate buffered
saline (1 mL) were reacted in presence of triethylamine (10 mL)
for 24 h at room temperature. The resultant FB-[(N-Me)G24,
(N-Me)I26]hIAPP(22–27) was purified by HPLC and lyophilized to
obtain pure product as a white powder. Electrospray MS
calculated m/z for C39H54FN7O9: 783.9; found: 782.6 ([M-H]ꢁ).
1
Proteomics Facility. H NMR spectral analysis was performed on
a Varian Mercury VX-300 NMR Spectrometer at the University of
Oklahoma NMR Facility (Norman, OK). CF-1 (19–21g) male mice
were purchased from Charles River Laboratories International, Inc.
(Wilmington, MA). The microPET/CT imaging was conducted in
the OUHSC College of Pharmacy Small Animal Imaging Facility
Synthesis of para-fluorohippurate (PFH)
(Oklahoma City) using a Flex X-O/X-PET/CT scanner (Gamma A solution of SFB (12.5 mg, 52.7 mmol) dissolved in dimethylforma-
Medica-Ideas, Northridge, CA). All animal studies were conducted mide (DMF) (0.5 mL) and a solution of glycine (2 mg, 26.7 mmol)
in accordance with the protocols approved by the OUHSC dissolved in DMF (0.5 mL) were reacted in presence of diisopro-
institutional animal care and use committee.
pylethylamine (50 mL) for 18 h at room temperature. The resultant
Reversed-phase HPLC analyses were performed on a Beckman PFH was purified by HPLC and lyophilized to obtain pure product
System Gold HPLC equipped with a Beckman Model 126 pump, as a white powder (yield 76%, 4 mg). 1H NMR (D2O, 300 MHz)
J. Label Compd. Radiopharm 2010, 53 186–191
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

G. Pathuri et al.
acetonitrile) and 50 mL of K2CO3 (20 mg/mL in water). The
solvents were evaporated under a stream of nitrogen at 901C.
Azeotropic drying was repeated with 1.5 mL of acetonitrile to
generate anhydrous [K/K222]18F complex.
18
Mouse urine at 2 hr p.i. of F-peptide
10.9
6.2
Ethyl 4-(trimethylammonium triflate)benzoate (5.0 mg) dis-
solved in anhydrous acetonitrile (1 mL) was added to the dried
[K/K222]18F complex and the reaction mixture was heated at
901C in a sealed vial for 10 min. The resultant ethyl 4-
[18F]fluorobenzoate was hydrolyzed to tetrapropylammonium
4-[18F]fluorobenzoate by adding 1M tetrapropylammonium
hydroxide (50 mL) and heating the reaction mixture at 901C in
a sealed vial for 5 min. Finally, N,N,N0,N0-tetramethyl-O-(N-
succinimidyl) uronium hexafluorophosphate (12 mg) dissolved
in acetonitrile (1 mL) was added and the reaction mixture was
heated at 901C in a sealed vial for 10 min. The reaction mixture
was passed through an Oro-Sep SCX cartridge (600 mg,
ChromTech), a Sep-Paks Light Accell Plus QMA cartridge
(130 mg, Waters), and a Sep-Paks Light Alumina N cartridge
(130 mg, Waters) connected in series to remove the cationic and
anionic organic impurities and unreacted [18F]Fꢁ. The cartridges
were eluted with an additional 2.5 mL acetonitrile. The solvent
was removed under a stream of nitrogen at 901C. The dried
[18F]SFB was redissolved in acetonitrile (100 mL) and injected on
to an HPLC column. Pure [18F]SFB fraction was collected and the
solvents were removed under a stream of nitrogen at 901C. The
decay-corrected radiochemical yield of the HPLC-purified
[18F]SFB was 61.8% EOS. The radiochemical purity of [18F]SFB
was 499% as determined by HPLC.
4.0
18
Mouse urine at 30 min p.i. of [ F]SFB
5.2
6.5
8.3
2.1
18
Mouse urine at 30 min p.i. of [ F]FBA
0
0
0
0
0
0
0
0
0
0
0
0
6.0
2.1
Radiosynthesis of [18F]FB-[(N-Me)G24, (N-Me)I26]hIAPP(22–27)
(18F-peptide)
A solution of [(N-Me)G24, (N-Me)I26]hIAPP(22–27) (5 mg) in DMF
(25 mL) and triethylamine (10 mL) was added to the dry HPLC-
purified [18F]SFB vial and reacted at 901C in a sealed vial for 35 min.
About 50 mL 1M NaOH was added to the reaction mixture to
hydrolyze the unreacted [18F]SFB at 901C for 5 min. The reaction
mixture was injected on to an HPLC column and pure 18F-peptide
fraction was collected. The solvents were removed under a stream
of nitrogen at 901C. The dried HPLC-purified 18F-peptide was
reconstituted in normal saline and passed through a 0.2 mm syringe
filter. The decay-corrected radiochemical yield for the HPLC-purified
18F-peptide was 47.6% starting from [18F]SFB. The radiochemical
purity of the final product was 499% as determined by HPLC.
Total duration of the radiosynthesis starting from [18F]Fꢁ to the
injectable 18F-peptide was 253 min.
0
10
20
30
min
Figure 3. Radio-HPLC chromatograms.
Radiosynthesis of [18F]fluorobenzoic acid ([18F]FBA)
1 min p.i.
10 min p.i.
60 min p.i.
To a solution of HPLC-purified [18F]SFB in ethanol (100 mL) was
added 1M NaOH (50 mL) and allowed to react at 901C for 5 min.
The reaction mixture was neutralized with 1M HCl (50 mL) and
diluted with 1.8 mL of normal saline. The solution was passed
through a 0.2 mm syringe filter and used for animal studies. The
radiochemical purity of the final product was 499% as
determined by HPLC.
Figure 4. Small animal PET/CT images of a healthy mouse injected with [18F]FB-[(N-
Me)G24, (N-Me)I26]hIAPP(22–27).
d (ppm) 3.99 (s, 2H), 7.09 (m, 2H), 7.70 (m, 2H). Electrospray MS
calculated m/z for C9H8FNO3: 197.2; found: 195.9 ([M-H]ꢁ).
Radiosynthesis of N-succinimidyl-4-[18F]fluorobenzoate
([18F]SFB)
Biodistribution studies
[18F]SFB was synthesized manually by slight modifications to a Three healthy CF-1 mice were anesthetized with 2% vaporized
previously reported one-pot procedure.27 [18F]Fꢁ (13.5 mCi) isoflurane and injected with a dose of ꢀ30 mCi of 18F-peptide,
dissolved in water (1 mL) was added to a 5 mL V-shaped bottom [18F]SFB, or [18FFBA in 100 mL normal saline via the tail vein. The
vial containing 250 mL of Kryptofix 222 (K222, 20 mg/mL in mice were euthanized and tissues/organs were excised at 1 h after
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G. Pathuri et al.
injection. The tissues/organs were washed with normal saline, dried
by blotting on a tissue paper and transferred to pre-weighed
tubes. They were weighed and radioactivity associated with each
tissue was recorded on a Packard Cobra II automated gamma
counter (PerkinElmer Life And Analytical Sciences, Inc, Waltham,
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Acknowledgement
This work was funded by the University of Oklahoma College of
Pharmacy Startup Grant and Presbyterian Health Foundation
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