Fluorine-18 labeled galactosyl-neoglycoalbumin for imaging the hepatic asialoglycoprotein receptor

Article abstract of DOI:10.1016/j.bmc.2009.09.017

Asialoglycoprotein receptors (ASGP-R) are well known to exist on the mammalian liver, situate on the surface of hepatocyte membrane. Quantitative imaging of asialoglycoprotein receptors could estimate the function of the liver. ^99mTc labeled ga

Full text of DOI:10.1016/j.bmc.2009.09.017

Bioorganic & Medicinal Chemistry 17 (2009) 7510–7516
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorine-18 labeled galactosyl-neoglycoalbumin for imaging the hepatic
asialoglycoprotein receptor
Wenjiang Yang a, Tiantian Mou a, Cheng Peng b, Zhanhong Wu c, Xianzhong Zhang a,
,
*
Fang Li c, Yunchuan Ma b
a Key Laboratory of Radiopharmaceuticals (Beijing Normal University), Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, PR China
b PET Center of Xuanwu Hospital, Capital Medical University, Beijing 100053, PR China
c Department of Nuclear Medicine, PUMC Hospital, CAMS and PUMC, Beijing 100730, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 July 2009
Revised 9 September 2009
Accepted 10 September 2009
Available online 15 September 2009
Asialoglycoprotein receptors (ASGP-R) are well known to exist on the mammalian liver, situate on the
surface of hepatocyte membrane. Quantitative imaging of asialoglycoprotein receptors could estimate
the function of the liver. 99mTc labeled galactosyl-neoglycoalbumin (NGA) and diethylenetriaminepentaa-
cetic acid galactosyl human serum albumin (GSA) have been developed for SPECT imaging and clinical
used in Japan. In this study, we labeled the NGA with 18F to get a novel PET tracer [18F]FNGA and eval-
uated its hepatic-targeting efficacy and pharmacokinetics. Methods: NGA was labeled with 18F by conju-
gation with N-succinimidyl-4-18F-fluorobenzoate ([18F]SFB) under a slightly basic condition. The in vivo
metabolic stability of [18F]FNGA was determined. Ex vivo biodistribution of [18F]FNGA and blocking
experiment was investigated in normal mice. MicroPET images were acquired in rat with and without
block at 5 min and 15 min after injection of the radiotracer (3.7 MBq/rat), respectively. Results: Starting
with 18Fꢀ Kryptofix 2.2.2./K2CO3 solution, the total reaction time for [18F]FNGA is about 150 min. Typical
decay-corrected radiochemical yield is about 8–10%. After rapid purified with HiTrap desalting column,
the radiochemical purity of [18F]FNGA was more than 99% determined by radio-HPLC. [18F]FNGA was
metabolized to produce [18F]FB-Lys in urine at 30 min. Ex vivo biodistribution in mice showed that the
liver accumulated 79.18 7.17% and 13.85 3.10% of the injected dose per gram at 5 and 30 min after
injection, respectively. In addition, the hepatic uptake of [18F]FNGA was blocked by pre-injecting free
NGA as blocking agent (18.55 2.63%ID/g at 5 min pi), indicating the specific binding to ASGP receptor.
MicroPET study obtained quality images of rat at 5 and 15 min post-injection. Conclusion: The novel ASGP
receptor tracer [18F]FNGA was synthesized with high radiochemical yield. The promising biological prop-
erties of [18F]FNGA afford potential applications for assessment of hepatocyte function in the future. It
may provide quantitative information and better resolution which particularly help to the liver surgery.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
18F
NGA
[
18F]FNGA
PET imaging
Asialoglycoprotein receptor
1. Introduction
been quite scarce. A more clinically acceptable radiopharmaceutical
neogalactosylalbumin (NGA) had been developed by attaching
Asialoglycoprotein receptors (ASGP-R) are well known to exist on
the mammalian liver,1 situated on the surface of hepatocyte mem-
brane. Asialoglycoprotein receptors participate in the hepatic
metabolism of serum proteins. They could recognize a glycoprotein
having galactose residues on the terminal position of the saccharide
chain, such as asialoglycoprotein. Quantitative imaging of asialogly-
coprotein receptors could estimate the function of liver. This is a
unique way to diagnose disease by a non-invasive method. ASGP
receptor imaging agent can assess the anatomy and function of the
liver, help the early diagnosis of hepatic diseases and accurate eval-
uation of functionalstatus.1,2 However, native ASGP receptor ligands
require enzymatic preparation, and in vivo clinical experience has
galactosyl units to human serum albumin (HSA) in 1980, and then
labeled with 99mTc for imaging the liver.3 Later, to make the labeling
procedure simple, diethylenetriaminepentaacetic acid galactosyl
human serum albumin (GSA) was obtained by added three to four
DTPA molecules covalently attached to the albumin backbone, and
developed as an instant kit.4 This instant GSA kit is commercially
available for routine clinical use in Japan now.2,5
The derivates of glycoprotein radiolabeled with technetium-
99m, iodine-125/131,6 and indium-1117 have been reported for
single photon emission computed tomography (SPECT) imaging
applications. Positron emission tomography (PET) imaging is the
gold standard for quantitative imaging in nuclear medicine. Vera
reported deferoxamine modified NGA to labeled with positron emis-
sion nuclides gallium-67/68.8 Biodistribution of 67Ga-DF-NGA in
rabbits was similar to 99mTc-NGA. 68Ga-DF-NGA could be used for
* Corresponding author. Tel./fax: +86 10 62208126.
E-mail addresses: zhangxzh@gmail.com, zhangxzh@bnu.edu.cn (X. Zhang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.017

W. Yang et al. / Bioorg. Med. Chem. 17 (2009) 7510–7516
7511
hepatic ASGP receptor PET imaging. However 68Ga has a high beta
emission energy which results in diminished spatial resolution and
less favorable dosimetry. Fluorine-18 is the most important posi-
tron-emitting isotope for PET imaging. It has low positron energy
and optimal physical half-life of 110 min allows for multistep radio-
synthesis, longerinvivoinvestigation. 18F-Labeledprostheticgroups
such as N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) have been
developed, which can be attached to either N-terminal or lysine
was heated at 120 °C in oil bath and the solvent evaporated with a
stream of nitrogen. Then 500 L dry acetonitrile was added to dis-
l
solve the residue and concentrated again under a stream of nitro-
gen. The azeotropic drying process was repeated three times. To
the residue was added a solution of 4 mg ethyl 4-(trimethylammo-
nium triflate) benzoate dissolved in 1 mL dry dimethyl sulfoxide,
the reaction vial was sealed and heated at 120 °C for 20 min with
magnetic stirring. For hydrolysis 0.5 mL 0.2 mol/L NaOH solution
was added and heated for additional 10 min at 120 °C. After cool-
ing for 2 min and then acidified with 0.5 mL 1 mol/L HCl, the solu-
tion diluted with water to a final volume of 10 mL and loaded onto
a Sep-Pak C18 cartridge. The cartridge was washed with 10 mL
water and blown dry with a stream of nitrogen. The radioactivity
was eluted with 3 mL acetonitrile (with 0.1% TFA) into a second
reaction vial. The sample was evaluated by radio-HPLC.
e
-amino groups with little or no loss of bioactivity of the protein or
peptide ligand,9 such as bombesin analogs10 and RGD peptide.11
Here we report the radiolabeling NGA with 18F for hepatic asialogly-
coprotein receptor imaging with PET.
2. Materials and methods
2.1. Materials
2.4. Synthesis of N-succinimidyl-4-[18F]fluorobenzoate,
[
18F]SFB
All chemicals obtained commercially were used without further
purification. No-carrier-added [18F]Fꢀ was trapped on Sep-Pak
QMA cartridge (Waters) and then eluted with 0.5 mL K2CO3
(12 mg/mL in H2O) combined with 1 mL Kryptofix 2.2.2. (Acros
Organics) (11 mg/mL in acetonitrile). Ethyl 4-(dimethylamino)ben-
zoate, methyl trifluoromethanesulfonate and tetrabutylammo-
nium hydroxide (40 wt % solution in water) were purchased from
Acros Organics. N,N,N0,N0-Tetramethyl-O-(N-succinimidyl)uronium
Ten microliters of 40% solution of tetrabutylammonium
hydroxide were added into the second vial and then evaporated
the solvent in oil bath with a stream of nitrogen. The process
was repeated three times by addition and evaporation of
0.5 mL dry acetonitrile. Finally, 15 mg TSTU in 1 mL dry acetoni-
trile was added to the residue. The vial was sealed and heated
for 5 min at 80–90 °C, and cooled for 2 min. The solution was
tetrafluoroborate (TSTU) and
L-lysine were purchased from
added 500 lL 50% acetic acid and diluted with water to a final
Sigma–Aldrich. Ethyl 4-(trimethylammonium triflate)benzoate
volume of 10 mL and loaded onto a Sep-Pak C18 cartridge. The
cartridge was washed with 10 mL water and blown dry with a
stream of nitrogen. The [18F]SFB was eluted with 2.5 mL methy-
lene chloride into a third reaction vial. The sample was evalu-
ated by radio-HPLC.
was prepared by the procedure of Guhlke et al.9
Instant thin-layer chromatography-silica gel (ITLC-sg) chro-
matographic strips were purchased from Pall Life Sciences. LabGEN
7 homogenizer was purchased from Cole-Parmer Ins, USA. Radio-
high pressure liquid chromatography (Radio-HPLC) experiments
were performed on a SHIMADZU system with SCL-10Avp HPLC
pump system (SHIMADZU Corporation, Japan) and liquid scintilla-
tion analyzer (Packard BioScience Co., USA). The Reversed-phase
2.5. Conjugation of [18F]SFB to NGA
The methylene chloride solution of [18F]SFB was evaporated to
dryness with a stream of nitrogen. The residue was taken with
Kromasil C-4 column (4.6 ꢁ 250 mm, 5
lm particle size, 300 Å,
Eka Chemicals, Sweden) was eluted at a flow rate of 1 mL/min
according to the procedure described in the experimental part.
The absorbance was monitored at 220 nm. Reversed-phase extrac-
tion Sep-Pak C18 Plus cartridges (Waters) were activated with
methanol and water before use. HiTrap Desalting column (filled
with Sephadex G25) was purchased from GE Healthcare, eluted
with 0.05 mol/L phosphate buffer (pH 7.5). Animal experiments
were carried out in Kunming mice (average weight about 20 g)
or SD rats (average weight about 300 g), obtained from the Animal
Center of Peking University. All biodistribution studies were
carried out in compliance with the national laws related to the
conduct of animal experimentation.
50
(3 mg, 0.04
for 30 min at room temperature. After reaction the crude product
was passed through a 0.22 m millipore filter and loaded onto a
l
L dimethyl sulfoxide and added 200
lL of a solution of NGA
lmol) in 0.1 mol/L borate buffer, pH 8.6 and incubated
l
HiTrap desalting column, eluted with 0.05 mol/L phosphate buffer,
pH 7.5. After purification the radiochemical purity was evaluated
by ITLC chromatography and radio-HPLC.
2.6. Synthesis of [18F]FB-Lys
The dried [18F]SFB was then redissolved in dimethyl sulfoxide
(50
lL) and added to the L-lysine (6 mg, 0.04 mmol) dissolved in
borate buffer (400
l
L, pH 8.6) and incubated for 30 min at room
temperature. The [18F]FB-Lys was evaluated by radio-HPLC with-
out further purification.
2.2. Synthesis of NGA
2.7. Synthesis of [19F]FNGA
Galactosyl-neoglycoalbumin (NGA) was synthesized according
to the procedure of Vera et al.3 Briefly, 2-imino-2-methoxyethyl-
thio-galactose was prepared, and reacted with HSA in borate buffer
(pH 8.6, 0.2 mol/L). The conjugated number of galactose per mole-
cule of human serum albumin was calculated by measuring galact-
ose concentration from the absorbance at 490 nm using the
phenol/sulfuric acid method.12 Average 38 galactose units were
attached to each HSA molecule.
N-Succinimidyl-4-[19F]fluorobenzoate was synthesized from
4-[19F]fluorobenzoic acid. To 1 mL of a 20 mg/mL solution of NGA
in 0.2 mol/L borate buffer pH 8.6 was added dropwise 40 lL of a
solution containing 3.5 mg of N-succinimidyl-4-[19F]fluorobenzo-
ate in DMSO. The solution was stirred gently for 1 h at room
temperature. After reaction, the solution was passed through a
0.22 lm millipore filter and loaded onto a HiTrap desalting
2.3. Synthesis of 4-[18F]fluorobenzoic acid, [18F]FB
column, eluted with 0.05 mol/L phosphate buffer, pH 7.5. The
collected [19F]FNGA solution was dialyzed against distilled water
for three days and lyophilized. 19FNMR(D2O) d: ꢀ108.2 (m). The
The aqueous solution of [18F]Fꢀ was added to a 10 mL reaction
vial with 6 mg K2CO3 and 11 mg Kryptofix 2.2.2.. The reaction vial
[
19F]FNGA was evaluated by HPLC with UV detection at 220 nm.

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W. Yang et al. / Bioorg. Med. Chem. 17 (2009) 7510–7516
2.8. Radiochemical analysis
2.11. MicroPET imaging
The chromatography analysis were performed on ITLC-sg strips
with ACD (0.068 mol/L citrate, 0.074 mol/L glucose, pH 5.0) as mo-
bile phase. [18F]FNGA was remaining at the point of spotting
(Rf = 0–0.1), while other radioactive impurities moved with the sol-
vent front (Rf = 0.8–1.0). Radio-HPLC was performed on a SHIMA-
DZU system with Reversed-phase Kromasil C-4 column
PET imaging was performed using a Siemens Inveon dedicated
microPET. The scanner has a computer-controlled bed and 10-cm
transaxial active and 12.7-cm axial fields of view (FOVs). SD rat
was placed near the center of the FOVs of the microPET scanner,
where the highest image resolution and sensitivity are available.
SD rat was deeply anesthetized by intraperitoneal injection of chlo-
ral hydrate. The microPET studies were performed by tail vein
(4.6 ꢁ 250 mm, 5
lm). The mobile phase was changed from 70%
solvent A (0.1% trifluoroacetic acid [TFA] in water) and 30% solvent
B (0.1% TFA in acetonitrile [ACN]) to 30% solvent A and 70% solvent
B at 30 min. [19F]FNGA was analyzed in same gradient and moni-
tored at 220 nm.
injection of 3.7 MBq (100
l
Ci) of [18F]FNGA. For comparison one
Ci) [18F]FNGA with free NGA
rat was coinjected 3.7 MBq (100
l
(10 mg/kg rats body weight) as blocking agent. MicroPET data
acquisition was started at 5 min after radiotracer injection. Dy-
namic images at 5 and 15 min time points were acquired.
After purified, [18F]FNGA was incubated at room temperature
for 4 h. The radiochemical purity (RCP) was evaluated by ITLC chro-
matography at every single hour. Plasma stability was determined
by diluted [18F]FNGA 20-fold with freshly prepared murine plasma,
and the solutions were incubated at 37 °C for 4 h. The radiochem-
ical purity (RCP) was evaluated by ITLC chromatography at every
single hour. At the end of 4 h, the plasma was passed through a
Sep-Pak C18 cartridge, and washed with 0.5 mL of water and eluted
with 0.5 mL of acetonitrile containing 0.1% TFA. The combined
3. Results
3.1. Radiosynthesis
18F-fluorination of NGA was performed using [18F]SFB (Scheme 1).
Our decay corrected radiochemical yield of [18F]SFB was 31.7 8.9%
(n = 8) in 100–110 min. [18F]SFB was purified with simple cartridge
desalting before coupling with NGA. The radiochemical purity of
aqueous and organic solutions passed through a 0.22
filter and evaluated by radio-HPLC.
lm millipore
[
18F]FB and [18F]SFB was investigated with radio-HPLC. The retention
time of [18F]FB and [18F]SFB in our gradient system was 7.2 min and
10.2 min, respectively. NGA was labeled with 18F by coupling with
2.9. Metabolic stability
[
18F]SFB under a slightly basic condition. Yields for the conjugation
Mice were intravenously injected with 7.4 MBq of [18F]FNGA. The
animals were sacrificed at 5 and 30 min after tracer injection. Blood,
liver and urine were collected. Blood samples were immediately
centrifuged for 5 min at 13,200 rpm. After removal of the superna-
correlate with initial proteinconcentration. Weobtainedlower radio-
chemical yields when the concentration of NGA is less than 5 mg/mL.
Starting with [18F]Fꢀ in Kryptofix 2.2.2./K2CO3 solution, the total reac-
tion time was about 150 20 min. The overall radiochemical yield
with decay correction was about 8–10% typical, including final purifi-
cation. Starting with 370–1295 MBq[18F]fluoride, thespecificactivity
of [18F]FNGA ranged from 1.92 TBq/mmol to 7.10 TBq/mmol. After
rapidpurifiedwithHiTrapdesaltingcolumn, theradiochemicalpurity
of [18F]FNGA was above 99% as determined by ITLC and radio-HPLC.
The retention time of [18F]FNGA was 12.3 min. [19F]FNGA was ana-
lyzed in same gradient. The retention time of [19F]FNGA was
12.1 min which is similar with [18F]FNGA (Fig. 1). Lysine was coupled
with [18F]SFB to produce radiochemical yield of more than 98% after
30 min incubation at room temperature when determined by HPLC
analyses. The retention time of [18F]FB-Lys was 4.3 min in our
gradient.
tants, the pellets were washed with 250 lL of PBS (pH 7.5). Superna-
tants of both centrifugation steps were combined and passed
througha Sep-PakC18 cartridge. Theliversamples werewashedwith
saline. After adding 4 mL PBS buffer (pH 7.5), the hepatic samples
were homogenized by a polytronhomogenizer (LabGEN 7, Cole-
Parmer Ins, USA) at full speed for 5 min. The resulting homogenate
was centrifuged for 60 min at 14,000 rpm. Supernatants were
passed through a Sep-Pak C18 cartridge. The urine samples were
directly diluted with 0.5 mL of PBS and then passed through a Sep-
Pak C18 cartridge. All the cartridges were washed with 0.5 mL of
water and eluted with 0.5 mL of acetonitrile containing 0.1% TFA.
The combined aqueous and organic solutions passed through a
0.22 lm millipore filter and then injected onto radio-HPLC using a
In vitro stability, the result measured by ITLC showed that
[
18F]FNGA can be stable over 4 h in saline at room temperature,
flow rate of 1 mL/min and a gradient as described.
2.10. Ex vivo biodistribution
the radiochemical purity was above 95%. Stability in murine plas-
ma was also very high, [18F]FNGA was still intact after 4 h at 37 °C.
HPLC analysis results of in vitro stability are shown in Figure 2.
Ex vivo biodistribution study of [18F]FNGA was carried out in
normal mice. [18F]FNGA (about 0.185 MBq in 100
lL solution con-
3.2. Metabolic stability
tained about 2
lg NGA) was injected through the tail vein. At se-
lected time points (5 and 30 min), mice (n = 5 at each time point)
were sacrificed, and the tissues and organs of interest were col-
lected, wet weighed and counted in a c-counter. The percentage
The metabolic stability of [18F]FNGA was determined in mouse
blood, liver and urine samples. HPLC analysis results of blood, liver
and urine samples are shown in Figure 2. After 5 min injection of
the tracer, HPLC analysis results of blood indicated that a small
amount of [18F]FNGA which not uptake by liver were still stable
in blood. After 30 min, most of [18F]FNGA were degraded in lyso-
some, and the metabolites were eliminated from liver. When ana-
lyzed by HPLC, the liver supernatant at 30 min depicted a major
radioactivity peak at a retention time close to that of [18F]FB-Lys,
while the peak of [18F]FNGA was disappeared. The HPLC analysis
results of urine sample showed same results. The retention time
of urinary metabolite was 4.9 min. The results indicated that the
major radiolabeled metabolite in liver coincided with chromato-
graphic behaviors of [18F]FB-Lys.
of injected dose per gram (%ID/g) for each sample was calculated
by comparing its activity with appropriate standard of injected
dose (ID), the values are expressed as mean SD.
In order to further confirm that the [18F]FNGA had specific
receptor-binding, blocking study was performed by conducting
the biodistribution experiment in the presence of free NGA
(10 mg/kg body weight) as blocking agent. Five minutes after the
first injection of free NGA, [18F]FNGA was intravenously injected
(about 0.185 MBq in 100 lL solution). Mice were sacrificed at
5 min post-injection time (n = 5). Results were expressed as the
percentage of the injected dose per gram tissue (%ID/g). Averages
and standard deviations were calculated.

W. Yang et al. / Bioorg. Med. Chem. 17 (2009) 7510–7516
7513
18F
NGA
O
NH
18F
NGA
m
18F
18F
OTf
N
[
18F]F-
NaOH
HCl
TSTU
O
O
O
N
18F
O
CO2Et
CO2Et
CO2H
Lysine
NH
NH2
OH
O
O
Scheme 1. Radiolabeling of [18F]FNGA through active ester intermediate [18F]SFB to coupling the -amide of lysine residue of NGA (m is less than 1). The [18F]FB-Lys was
e
radiosynthesised with same process. It can predict [18F]FB-Lys mainly conjugate
e
-amide due to excess of lysine.
radioactivity was eliminated from the liver after 15 min, the out-
line of liver was still clear. There were no significant uptakes in kid-
ney, spleen and other organs in the abdomen. On the contrary, the
liver accumulation was significant lower after inhibition, and the
outline of liver was vague. The cardiac levels of activity were high
and reduced at later time points when nonspecific activity accu-
mulation in the blood after blocking had been gradually cleared.
We can also find the significant uptake in the kidney, and gradually
increase over time. After 15 min the high radioactivity concentra-
tion in kidney and urinary bladder indicated rapid clearance of
the unbounded tracer through the kidney.
[19F]FNGA 12.1min
[18F]FB-Lys 4.3min
[18F]FNGA 12.3min
[18F]SFB 10.2min
[18F]FB 7.2min
4. Discussion
It has been more than 17 years since the 99mTc-NGA/GSA clini-
cal use as ASGP receptor-binding radiopharmaceutical. It provided
a unique way to evaluate hepatic function and diagnose disease by
a non-invasive method. There are over 10,000 99mTc-GSA tests per-
formed annually in Japan.1 Positron emission tomography (PET) is
one of the most sensitive molecular imaging techniques. Of all the
positron nuclides employed in PET, fluorine-18 labeled PET radio-
pharmaceuticals have the most favorable physical properties,13
and it is easily produced in the small biomedical cyclotrons. The in-
creased sensitivity and unique spatial resolution that can be
achieved with PET imaging allows for optimal quantitative imag-
ing. The objective of this study was to get a novel 18F labeled PET
tracer for ASGP receptor imaging. N-Succinimidyl-4-[18F]fluor-
obenzoate ([18F]SFB), an agent widely used for labeling proteins
and peptides with the positron-emitting radionuclide 18F. It has
good conjugation yields and metabolic stability.11 We choose
NGA as targeted molecular, and labeled 18F via [18F]SFB.
0
5
10
15
20
25
30
35
Time (min)
Figure 1. The radio-HPLC radiochromatograms of the [18F]FB, [18F]SFB, [18F]FNGA
and [18F]FB-Lys. The retention time was 7.2, 10.2, 12.3 and 4.3 min, respectively, in
our gradient system. [19F]FNGA was analyzed in same gradient with UV detection at
220 nm. The retention time of [19F]FNGA was 12.1 min which is similar with
[
18F]FNGA.
3.3. Ex vivo biodistribution
To evaluate ex vivo tissue distribution characteristics of
[
18F]FNGA, the biodistribution studies were performed using Kun-
ming normal mice. The results were shown in Figure 3. For
[
18F]FNGA, the liver uptake was 79.18 7.17% of the injected dose
per gram at 5 min after injection, which decreased to
18.55 2.63%ID/g in the presence of a blocking dose of free NGA
(10 mg/kg mice body weight). The different of kidney and blood
accumulation between control and blocking groups were also statis-
tically significant (P <0.01). The ratio of liver/blood was also
decreasedfrom 23.2to 0.44afterblocking. In normalmice, theradio-
activity was rapid eliminated from the liver by both hepatobiliary
and renal excretion. At 30 min after injection, the radioactivity accu-
mulation of liver was only 13.85 3.10%ID/g. The radioactivity in
kidney and small intestine was increased significantly.
Starting with [18F]Fꢀ, the [18F]SFB was synthesized by two-step
reaction. The NGA was reacted with activated ester [18F]SFB
through e-amino groups of lysine residues under basic condition.
In Scheme 1, the ‘m’ in the structure of [18F]FNGA is less than 1.
That is because the amount of NGA is far excess compared with
[
18F]SFB. Specific activity results also confirmed that. We choose
HiTrap desalting column to purify [18F]FNGA. The HiTrap desalting
column is packed with Sephadex G25. The column is easy to per-
form separation of protein with low molecular weight materials.
The time required for purification is less than using traditional
size-exclusion chromatographic column. The radiochemical purity
of [18F]FNGA was above 99% after purification. The overall radio-
chemical yield with decay correction was about 8–10%, including
final purification. The coupling yield for NGA is relatively lower
than other proteins reported in the literature using [18F]SFB. That
3.4. MicroPET imaging
We also evaluated [18F]FNGA in vivo with microPET scans. The
representative microPET images were displayed in transaxial, coro-
nal and sagittal orientation in Figure 4. High liver activity accumu-
lation was observed at 5 min after injection. A small portion of

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W. Yang et al. / Bioorg. Med. Chem. 17 (2009) 7510–7516
12.3 min
4.5 min
5 min p.i. in blood
30 min p.i. in liver
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Retention time (min)
Retention time (min)
12.4 min
4.9 min
30 min p.i. in urine
incubated in serum
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Retention time (min)
Retention time (min)
Figure 2. HPLC profiles of metabolic stability of [18F]FNGA. Blood was collected at 5 min after injection of [18F]FNGA to a female mice, liver and urine were collected at 30 min
pi. The retention time of blood sample of 5 min pi was 12.3 min, and that of liver and urine sample of 30 min pi was 4.5 and 4.9 min, respectively. In vitro stability incubated
in murine plasma at 37 °C for 4 h, the [18F]FNGA still intact. The retention time was 12.4 min.
might be due to about 38 of 56 lysine groups in NGA molecule have
been derivatized with galactose groups, which leads to more steric
also confirmed the initial stability in blood after injection. The re-
sults of metabolic analysis of liver showed that the [18F]FNGA was
metabolized in the liver and generated to hydrophilic compounds
after 30 min injection. According to the results of biodistribution
and metabolic stability study, we speculate the intact [18F]FNGA
would be rapidly taken up by hepatocyte via receptor-mediated
endocytosis and then degraded in lysosome. The metabolites
excreted rapidly mainly through the kidney to the urinary bladder.
That is quite different with 99mTc-NGA/GSA, which has a longer
retention time in the liver and mainly eliminated through intes-
tine. The difference in biodistribution between [18F]FNGA and
99mTc-NGA/GSA can be explained by the different ways of chemical
conjugation with the radionuclide. Gore et al.14 found after injec-
tion of 99mTc-NGA, 40% of the radioactivity was secreted into the
hindrance to acylate the residual e-amino. When we use lysine to
couple with [18F]SFB, the labeling yield is high because the amount
of ligand is far excess.
In the study of ex vivo biodistribution, [18F]FNGA displayed a
rapid and high uptake in the liver. While the liver accumulation
decreased obviously by 30 min, the uptake in kidney and small
intestine increased significantly (P <0.01). Higher radioactivity
levels in the kidney indicated that radiometabolites excreted pri-
marily through the kidneys, and increased radioactivity in small
intestine indicated that small amount of radiometabolites excreted
through the intestinal tract. [18F]FNGA showed good plasma stabil-
ity in vitro, and the result of in vivo metabolic stability analysis

W. Yang et al. / Bioorg. Med. Chem. 17 (2009) 7510–7516
7515
gated polypeptides or proteins such as GSA (DTPA-NGA) were
delivered to the lysosome when internalized by hepatocyte. The
backbone of the GSA can be degraded to yield DTPA coupled lysine
(DTPA-lysine) in lysosome. These metabolites remain within the
lysosome and are only slowly released from the cell. Arano
et al.16 made similar findings in their study. The research of
111In-DTPA-labeled NGA and NMA has indicated that lysine-
DTPA-111In is the final radiolabeled metabolite after lysosomal pro-
teolysis in liver, and the metabolite is retained in the lysosomal
fraction of murine liver for relatively long time. The biological
characteristics of radiolabeled metabolites play a critical role in
radioactivity elimination from liver cells.7,16 The similar result
was also found when using HYNIC as bifunctional coupling agent.
The [99mTc](HYNIC-NGA)(tricine)2 showed persistent localization
of radioactivity in the liver, which was attributed to the slow elim-
ination rate of the final radiometabolite, [99mTc](HYNIC-lysine)(tri-
cine)2 from the lysosomes.17 In our study the 18F was covalently
substituted into the 4-fluorobenzoic acid and attached to the
90
75
60
45
30
15
0
A
5min p.i.
5min p.i. block
*
*
*
*
*
*
*
*
90
75
60
45
30
15
0
B
5 min p.i.
30 min p.i.
e-amino groups of lysine residues in the NGA through activated es-
ter intermediate. Since [18F]FB was also conjugated to the lysine
residues of protein backbone, we can surmise that after metaboliz-
ing in liver lysosome the protein backbone of [18F]FNGA is de-
graded, but the amido linkage between [18F]FB and lysine does
not break, so the major radiometabolite is fluorine-18 conjugated
lysine, [18F]FB-Lys. [18F]FB-Lys was prepared as a standard com-
pound for analysis of the radioactivity metabolism. The in vivo
metabolic stability analysis of the liver and urine sample 30 min
after injection indicated the major metabolite was [18F]FB-Lys (
Fig. 2). The [18F]FB-Lys has high hydrophilicity, which can be
quickly cleared from liver and rapidly excreted to bladder through
kidneys, and eliminated from the body finally. This result confirms
our previous speculation.
*
*
*
Figure 3. The biodistribution of the [18F]FNGA in normal mice: (A) Mice were
injected intravenously with 185 kBq of radiotracer with or without the pre-
injecting of free NGA at 10 mg/kg mice body weight and executed at 5 min after
injection. (B) The biodistribution at 5 and 30 min pi. Data are expressed as mean
%ID/g SD, n = 5. *Statistically significant, P <0.01, unpaired two-tail t-test.
The ex vivo receptor blocking studies were investigated by
using 10 mg/kg of free NGA as the inhibitor. There have significant
difference of biodistribution before and after inhibition, which was
shown in Figure 3A. The most of radioactivity retained in the blood
and the uptake in the kidney was also increased, while liver uptake
declined significantly. The blocking and non-blocking results show
significant difference in many other organs, such as heart, lung,
spleen, bone and small intestine. That might be mainly due to
the high radioactivity in blood which makes the increase of the
radioactivity concentration in other non-target organs. When
excess inhibitor was preinjected, most receptor situated on the
bile within 1 h and postulated this may result from active transport
of the 99mTcO4ꢀ. The 99mTc was electrostatic bonding to the NGA
with low affinity. Rapid lysosomal degradation of 99mTc-NGA in
hepatocyte would release as 99mTcO4 which will persist in the
ꢀ
liver for sufficient time. Duncan and Welch15 reported DTPA conju-
Figure 4. microPET images of rats. The upper is the control group: the liver is visualized as transaxial, coronal and sagittal sections at 5 and 15 min after injection of
18F]FNGA (3.7 MBq [100
Ci]) (decay corrected to time of tracer injection). The lower is the blocking group: transaxial, coronal and sagittal images were collected at 5 and
15 min after injection of [18F]FNGA (3.7 MBq [100
Ci]) with free NGA as blocking agent (10 mg/kg rats body weight). The liver (L) uptake decreased significantly comparing
with control group. Radioactivity concentration was also seen in the heart (H) and kidney (K).
[
l
l

7516
W. Yang et al. / Bioorg. Med. Chem. 17 (2009) 7510–7516
surface of hepatocyte membrane was occupied. The followed
radiotracers would not uptake by the liver through receptor-med-
iated, and circulated in the blood. The tracers were absorbed by
other organs slowly, metabolized and excreted eventually. The re-
sult of metabolic analysis and ex vivo biodistribution show that the
the risk of liver surgery and help determining the surgical
procedures.
Acknowledgments
[
18F]FNGA has high affinity with the ASGP receptor, and its uptake
The Project was sponsored by the Scientific Research Founda-
tion for the Returned Overseas Chinese Scholars, State Education
Ministry, and partly by the National Natural Science Foundation
of China (20871020) and Beijing Natural Science Foundation
(2092018). The authors will also thank Professor Boli Liu for his
valuable discussion and guidance.
in the liver via receptor-mediated.
The in vivo microPET evaluation of [18F]FNGA was observed
with high liver accumulation and a certain retention. The liver up-
take was decreased significantly after blocking with free NGA. The
block group shows high kidney uptake, and the high radioactivity
concentration in urinary bladder can also be seen after 15 min
(not show in Fig. 4). The result of microPET coincides with
ex vivo biodistribution. Both of the blocking experiments results
indicated high affinity of [18F]FNGA with the ASGP receptor.
It has been reported several models for quantitative evaluation
of liver function using 99mTc-GSA. Recently 99mTc-GSA has been
applied to quantitative evaluation of regional liver function and
estimation of residual liver function in candidates for hepatec-
tomy.18–20 Regional evaluation requires more accuracy and finer
resolution. Comparing with SPECT, PET has higher resolution, espe-
cially when combining with CT technologies which improves the
reconstruction and visualization of data. The novel PET tracer
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5. Conclusion
This study demonstrates the successful coupling of galactosyl-
neoglycoalbumin with positron-emitting radionuclide 18F through
the prosthetic labeling group [18F]SFB. [18F]FNGA has initial high
activity accumulation in liver via ASGP receptor-mediated. The
other tissues show low radioactivity accumulation. High liver/
background ratio affords promising biological properties to get
clear images. PET imaging with [18F]FNGA may provide quantita-
tive information and better resolution. That is the first report of
18F labeled probe for ASGP receptor PET imaging so far as we know.
That could be an important tool for quantificational evaluation of