Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

Article abstract of DOI:10.1016/j.bmcl.2020.127187

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein recept

Full text of DOI:10.1016/j.bmcl.2020.127187

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose
1
8
derivative [ F]FPGal for imaging the hepatic asialoglycoprotein receptor
a,⁎
b
a
a
a
a
Penghui Sun , Yun Zhu , Yanjiang Han , Kongzhen Hu , Shun Huang , Meng Wang ,
a
a
Hubing Wu , Ganghua Tang
a
b
Nanfang PET Center, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China
Liver Tumor Center, Department of Infectious Diseases and Hepatology Unit, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong Province, China
A R T I C L E I N F O
Keywords:
A B S T R A C T
The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it re-
cognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic
distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function
1
8
[
F]FPGal
ASGPR
Liver function imaging agent
PET imaging
18
using PET imaging. In this study, the positron imaging agent [ F]FPGal was designed to specifically target
hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radio-
Click chemistry
1
8
18
18
synthesis of [ F]FPGal required 50 min with total radiochemical yields of [ F]FPGal from [ F]fluoride as 10%
1
8
(
corrected radiochemical yield). The K
d
of [ F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake
values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by
1
8
2
00 mM D(+)-galactose (< 0.1%). In vivo biodistribution analysis showed that the liver accumulation of [ F]
FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic
mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images
at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was sig-
1
8
nificantly lower when assessed through dynamic data collection for 1 h. Therefore, [ F]FPGal is a feasible PET
probe that provide insight into ASGPR related liver disease.
Hepatitis, cirrhosis and liver cancer are common liver diseases in
discovered by Ashwell and Morell during the assessment of mammalian
1
10
China. Liver fibrosis is an injury repair response that proceeds chronic
plasma glycoprotein metabolism. ASGPR is a transmembrane protein
liver injury. Chronic liver disease eventually develops into cirrhosis, the
present on the surface of hepatocytes that contains 100,000–500,000
binding sites per cell. Its two major receptor subtypes include ASGPR1
and ASGPR2. ASGPR1 specifically recognizes and binds to glycopro-
teins with galactosyl and N-acetylgalactosamine groups leading to their
2
complications of which are life threatening. Liver fibrosis leads to
hepatic portal hypertension, hepatic ascites, synthetic dysfunction, and
impaired metabolic capacity. In-depth studies of liver fibrosis have
important clinical significance in relieving liver fibrosis. To-date, the
clinical methods for the assessment of liver function include serum
biochemical indicators, the Child-Pugh scoring system, a model for end-
stage liver disease (MELD) scoring system, indocyanine green (ICG)
excretion tests, and imaging examinations, such as single photon
emission computed tomography (SPECT) and positron emission tomo-
1
1
metabolism. ASGPR1 has utility as a drug target, with anticancer and
antiviral drugs that bind to ASGPR1 being specifically endocytosed into
liver cells, enhancing their liver targeting. Natural ligands of ASGPR
include galactose, lactose, N-acetylgalactosamine, asialofetuin and
1
2
asialo-serum mucin.
Over the past four decades, several studies have reported the de-
3
–5
13–20
3,15,19 99m
graphy (PET).
Whilst effective, these tests only reflect preoperative
velopment of radiolabeled ASGPR ligands,
but only a few have
1
liver function, whilst the function of the resected or residual liver is not
been successfully imaged by PET or SPECT.
[
Tc]-NGA was the
13
6
assessed prior to surgery. Recently, the development of 3D-imaging
first imaging agent used to evaluate liver function. In 1983, it was
techniques such as SPECT and PET/CT make it possible to evaluate the
employed as a single photon imaging agent to acquire human SPECT
7
–9
14
function of preoperative liver segments.
images.
To simplify the labeling stages, Kubota and colleagues
The asialoglycoprotein receptor (ASGPR) is a liver lectin that was
covalently linked three to four DTPA molecules to the backbone of NGA
⁎
Corresponding author at: Nanfang PET Center, Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guangzhou, Guangdong Province
5
10515, China.
E-mail address: sunsam2014@163.com (P. Sun).
https://doi.org/10.1016/j.bmcl.2020.127187
Received 25 February 2020; Received in revised form 9 April 2020; Accepted 9 April 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: Penghui Sun, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2020.127187

P. Sun, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
1
9
albumin to obtain diethylenetriamine pentaacetate galactosyl human
time of the cold reference [ F]FPGal was 11.05 min (Fig. 1A) and the
9
9m
18
18
serum albumin (GSA) and used [ Tc] for radiochemical labeling to
[
[
F]FPGal’s retention time was 11.15 min (Fig. 1B), indicating that
F]FPGal was the target product. The radiochemical purity was ≥
9
9m
15
obtain [ Tc]-GSA, which was the first commercially available re-
ceptor-binding radio- pharmaceutical. The current clinical use rates of
1
8
99% and the specific activity of [ F]FPGal was 6.85 MBq/μmol.
9
9m
9
18
[
Tc]-GSA in Japan are high. In 2009, Yang and colleagues labeled
The octanol–water partition coefficient for [ F]FPGal was de-
1
8
18
16
NGA with [ F]fluoride through N-succinimidyl-4- F-fluoro-benzoate
termined through the assessment of its distribution in n-octanol and
1
1
8
8
18
18
(
[
F]SFB) to obtain a novel PET tracer [ F]FNGA. The PET tracer
F]FNGA showed favorable biological properties highlighting its po-
water (pH = 7.4). The lipophilicity logP value of [ F]FPGal at pH 7.4
[
was −1.45 ± 0.07 (n = 3), indicating that the compound was hy-
drophilic.
tential application for the assessment of hepatocyte function with PET/
CT. However, the relatively low radiochemical yields proved an ob-
stacle to its clinical application. In 2010, Yang et al. labeled galactosyl
1
8
The cellular uptake of [ F]FPGal was evaluated in hepatocellular
carcinoma (HCC) HepG2 cells (Fig. 2A). HepG2 cells express high levels
1
8
18
18
18
chitosan with F through [ F]SFB to obtain a novel PET tracer [ F]
of ASGPR and the uptake of [ F]FPGal was rapid and moderately high,
1
7
18
FB-GC. Despite its promising biological properties, [ F]FB-GC was
limited for clinical applications due to its complex labeling process and
low final radiolabeling yield. In 2013, Kao et al. reported a novel PET
reaching 0.55% within 30 min of incubation. Incubation with 200 mM
of D(+)-galactose blocked HepG2 cellular uptake (< 0.1%), indicating
1
8
18
that the binding of [ F]FPGal was ASGPR-specific. The K
d
[
of [ F]
1
8
18
18
18
probe [ F]FBHGal. The biological characterization of [ F]FBHGal
suggested that it was a feasible tracer for PET imaging in hepatic fi-
brosis mouse models which may provide new insight into ASGPR-re-
lated liver dysfunction. In the same year, Haubner et al. reported a
FPGal was assessed at different concentrations of
(6.85 MBq/μmol) to HepG2 cells in 24-well plates. The K
F]FPGal
1
8
d
value of [ F]
FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM (Fig. 2B), which
1
18
31
was much lower than the
(K = 0.1 μM). By comparing the K values of [ F]FPGal and I-YEEE
18
K
d
of
I-YEEE(α-ah-GalNAc)
3
6
8
68
19
131
more clinically feasible Ga labeled probe, [ Ga]GSA. The ease of
preparation based on commercial GSA kits provided a promising pro-
d
d
(α-ah-GalNAc) , we can know that the binding affinity of [ F]FPGal
3
6
8
spective for [ Ga]GSA during liver function imaging with PET. Re-
cently, Gupta et al. reported three IDA radiopharmaceuticals for clinical
was very low, thus leading to its low uptake in HepG2 cells.
Twelve Kunming 4-week old mice weighing 20 g were purchased
from the Animal Experimental Center of Southern Medical University
(Guangzhou China). Animal models of liver fibrosis were produced
2
0
99m
99m
use.
These included
[
Tc]lidofenin (HIDA),
[
Tc]disofenin
9
9m
99m
(
DISIDA) and [ Tc]mebrofenin (BrIDA). Of these agents, [ Tc]
2
4
Mebrofenin displayed the highest levels of hepatic extraction, blood
clearance and lowest renal excretion. Whilst these studies have im-
using 20% carbon tetrachloride induction. Briefly, carbon tetra-
chloride/peanut oil at a volume ratio of 20% was administered to the
abdominal cavity at a dose of 2 mL/kg twice a week for 6 weeks. Mice
were sacrificed and liver function was assessed through pathological HE
staining and blood biochemical indicators. The results of pathological
HE staining are shown in Fig. 3C and D.
1
3,15,16,18,19 18
proved liver function assays,
F-labeled monoantagonal
galactoside shows promise for more accurate liver assessments.
In this study, we designed and radiosynthesized a fluorine-18 la-
1
8
beled galactose derivative 4-(2-[ F]fluoropropyl)-1-β-D-galactopyr-
1
8
anosyl-1,2,3-triazole ([ F]FPGal). We used the “click reaction” to label
The results of serum biochemical indicators showed that alanine
aminotransferase (ALT) values were 12-fold higher in the model group
compared to the control group, and the aspartate aminotransferase
(AST) detection values in the model group were 7-fold higher than the
control group (Fig. 3B). In most cases, the elevated levels of ALT and
AST were consistent with the degree of hepatocyte damage. Further
assessments of body weight, liver morphology, HE staining and blood
serum biochemical indicators indicated the successful production of the
1
8
1
-deoxy-β-D-galactopyranosyl azide with 5-[ F]fluoro-1-pentyne. The
,2,3-triazole scaffold was featured in a vast number of bioactive mo-
1
lecules which have exhibited considerable biological and pharmaceu-
2
1–23 19
tical activities.
[
F]FPGal was synthesized in four steps from β-D-
galactose pentaacetate (Scheme 1). The synthetic process was relatively
1
9
complex and the cold reference compound [ F]FPGal had a purity ≥
0% and a yield ≥ 34% (See Supporting information for all product
9
1
19
data including H NMR, F NMR and mass spectrometry).
liver fibrosis model.
1
8
18
The radiosynthesis of [ F]FPGal was initiated with fluorine-18 and
[
F]FPGal displayed good stability in PBS for up to 2 h, as analyzed
1
8
5
-(p-toluenesulfonyl)-1-yne to obtain 5-[ F]fluoro-1-pentyne in a PET-
by a reserve-phase HPLC. The result showed that the percentage of
intact probe remains > 95% after 2 h incubation at 37 °C (Fig. 4B).
MF-2V-IT-I synthesizer module (Scheme 2). This product was then re-
acted with 1-deoxy-β-D-galactopyranosyl azide using “click chemistry”
1
8
[
F]FPGal also has good stability in liver at 1 h (Fig. 4C). Metabolite
1
8
18
to obtain the final product. The radiosynthesis yield of [ F]FNGA was
analysis revealed that [ F]FPGal was slowly metabolized in vivo, with
30% of intact probe in plasma at 1 h after injection, and defluorination
was not observed (Fig. 4D).
1
8
8
–10% and the total reaction time was 150 min. Compared to [ F]
18
FNGA, the radiosynthesis yields of [ F]FPGal were comparable, but
the overall synthesis times were over 3-three times longer than those of
1
8
Biodistribution of [ F]FPGal was assessed in Kunming mice at 5
and 30 min to evaluate the distribution pattern of the radiofluorinated
compound in vivo. Table 1 shows the data obtained expressed as the
percentage of the total injected dose per gram of tissue (% ID/g). At
30 min, the liver uptake was 4.47 ± 0.97 %ID/g in normal mice, while
that in the hepatic fibrosis mice was significantly reduced
1
8
18
[
F]FPGal. Therefore, the advantage of [ F]FPGal is that comparable
yields can be obtained in a shorter synthesis time.
1
8
The [ F]FPGal was a colorless and clear solution of pH 7.0 and a
1
8
radiochemical purity of ≥ 99%. Typical [ F]FPGal spectra were de-
termined through analytical radioactivity HPLC (Fig. 1). The retention
Scheme 1. Synthesis of [19F]FPGal.
2

P. Sun, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
OAc
O
OAc
O
OH
O
OAc
OAc
OH
TMSN3,SnCl
4
MeOH,NaOMe,2h
Amberlite 732
AcO
OAc
AcO
N
OH
N
3
3
MS4A,DCM,RT
OAc
OAc
OH
OH
OH
MeCN, H
2
O
N
N
1
2
5
3
O
N
18F
OH
CuSO
4
,Sodium Ascorbate
OH
O
S
TsCl,TEA,DMAP
18F,K222
MeCN
[18F]FPGal
OH
O
18F
DCM,RT,overnight
O
4
6
Scheme 2. Synthesis of [18F]FPGal.
Fig. 1. Analytical HPLC of [18F]FPGal (RT = 11.15 min) (A) and standard references of [19F]FPGal (RT = 11.05 min) (B). RT = retention time.
A
B
30
60
Fig. 2. Cell uptake studies of [18F]FPGal in 30 and 60 min using HepG2 tumor cells (ASGP receptor-positive) (n = 3). Blocking studies in 30 and 60 min with 200 mM
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8
D(+)-galactose confirmed the receptor-specific uptake (A). Binding saturation curve of [ F]FPGal to ASGPR in HepG2 cell lines (B). Values are represented as
means ± SD, n = 3.
(
1.33 ± 0.07 %ID/g, P < 0.01). Besides, the L/M ratio in normal
was noticed, which leads to a close liver/blood ratio in model group
and control group (1.95 vs 1.96, P > 0.01). The reason may be that
lower binding affinity and faster in vivo metabolism result in a lower
liver/ background ratio, which is bad for a good probe. The kidney
uptake in control group increased from 10.33 ± 1.99 %ID/g (5 min) to
15.68 ± 4.3 %ID/g (30 min), while that in model group decreased
mice and in hepatic fibrisis mice at 30 min was 22.35 and 4.59, re-
spectively. The differences in liver uptakes between normal and hepatic
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8
fibrosis mice, though not remarkable in [ F]FPGal PET imaging, still
reached statistical significance in biodistribution study (P < 0.01).
Besides, rapid blood clearance of [18F]FPGal in the hepatic fibrosis mice
3

P. Sun, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
CC
200 ×
200 ×
D
Fig. 3. Body weight changes in model and control groups (A). Comparison of serum ALT and AST (B). HE staining in the model (200×) (C) and control groups
200×) (D). Values are represented as means ± SD, n = 3.
(
1
8
were not high. Besides, the liver uptake value of [ F]FPGal in normal
1
8
mice was 4.47 ± 0.97% ID/g, while the uptake value of [ F]FB-GC in
normal mice was > 10% ID/g (P < 0.01), the reason for the difference
1
8
may be that [ F]FPGal is a strong hydrophilic tracer, which leads to its
relatively rapid metabolism in the body. Furthermore, it also may be
1
8
that the binding affinity of [ F]FPGal for ASGPR is not as high as that
1
8
of [ F]FB-GC.
1
8
Dynamic micro PET/CT studies were performed with [ F]FPGal.
PET images at 5 and 30 min are shown in Fig. 5A-B, and blocking PET
images of hepatic fibrosis mice at 30 and 60 min are shown in Fig. 5C.
From the PET images at 30 min (Fig. 5A-B), the liver uptake of hepatic
fibrosis mice were visually lower than normal mice. High kidney and
bladder uptake were observed in both groups. Rapid kidney uptake was
visualized in the first 1520 min of the model group. Maximum kidney
uptake was achieved within the first 20 min after injection, then slowly
decreased throughout the 60 min scan time. While in the control group,
maximum kidney uptake was reached within the first 30 min post-in-
jection, then slowly decreased throughout the 60 min scan time, sug-
gesting that the metabolism of the kidneys was affected when liver
function was reduced. The speed of absorption of the tracer was also
affected.
1
8
18
Fig. 4. Radioactive HPLC analysis of [ F]FPGal (A), [ F]FPGal in PBS at 2 h
B), [ F]FPGal in liver at 1 h (C) and the metabolite (peak 1) of [ F]FPGal at
h (D).
1
8
18
(
1
from 13.13 ± 1.24 %ID/g (5 min) to 3.92 ± 1.67 %ID/g (30 min).
In this study, we used a semi-quantitative indicator standard uptake
value (SUV) to compare the liver of the model group and the control
group. At 5 min, the mean SUVs of liver in model group and control
group were 0.81 ± 0.14 and 0.90 ± 0.15, respectively, P < 0.01.
While at 30 min, the mean SUVs of liver in model group and control
group were 0.80 ± 0.10 and 0.98 ± 0.11, respectively, P < 0.01
1
8
Moreover, higher radioactivity levels in the kidney indicated that [ F]
FPGal were primarily excreted through the kidneys. In addition, the
1
8
levels of [ F]FPGal uptake in other organs of interest (bone, muscle,
brain and gallbladder) were relatively low. Moreover, the biodistribu-
1
8
18
17
tion of [ F]FPGal is similar to that of [ F]FB-GC. At 30 min, the
radioactivity mainly accumulated in the urinary bladder and kidney,
followed by the liver, while the radioactive uptakes of other organs
(
Fig. 5D). The SUV results of liver in both groups had significant sta-
tistical difference, which can be considered as a useful diagnostic
4

P. Sun, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Table 1
Biodistribution in model and control groups at 5 min and 30 min after the intravenous injection of [18F]FPGal. Values are represented as means ± SD, n = 3.
Control group
Model group
5
min
30 min
5 min
30 min
Bone
0.74 ± 0.21
0.53 ± 0.22
1.86 ± 0.76
0.13 ± 0.05
1.31 ± 0.93
2.75 ± 0.70
10.33 ± 1.99
0.80 ± 0.41
0.82 ± 0.71
0.46 ± 0.18
1.72 ± 0.52
1.36 ± 0.24
2.10 ± 1.15
5.19
0.75 ± 0.27
0.20 ± 0.10
0.76 ± 0.25
0.17 ± 0.13
0.60 ± 0.38
4.47 ± 0.97
15.69 ± 4.30
2.08 ± 0.59
0.44 ± 0.18
0.90 ± 0.20
2.42 ± 0.21
99.81 ± 21.73
2.28 ± 0.41
22.35
0.35 ± 0.26
0.55 ± 0.06
2.52 ± 0.07
0.15 ± 0.03
1.08 ± 0.07
2.67 ± 0.10
13.13 ± 1.24
1.11 ± 0.04
0.74 ± 0.10
0.58 ± 0.31
2.1 ± 0.69
7.64 ± 1.87
2.45 ± 0.22
4.85
0.27 ± 0.06
0.29 ± 0.15
0.76 ± 0.06
0.05 ± 0.03
0.37 ± 0.08
1.33 ± 0.07
3.92 ± 1.67
0.86 ± 0.43
0.39 ± 0.27
0.3 ± 0.17
0.82 ± 0.06
133.45 ± 25.06
0.68 ± 0.13
4.59
Muscle
Lung
Brain
Heart
Liver
Kidney
Spleen
Gallbladder
Stomach
Intestine
Urinary bladder
Blood
Liver/Muscle
Liver/Heart
Liver/Blood
2.10
7.45
2.47
3.59
1.31
1.96
1.09
1.95
A
B
1
.0 SUV
0
SUV
5
min
30 min
5 min
30 min
C
1.0 SUV
D
0
SUV
3
0 min
60 min
Fig. 5. Micro-PET/CT images of [18F]FPGal in hepatic fibrosis mice (model group) 5, 30 min after intravenous injection (A). Micro-PET/CT images of [ F]FPGal in
18
Kunming mice (control group) at 5, 30 min intravenous injection (B). Micro PET/CT images of the blocking experiment in hepatic fibrosis mice (model group) at 30,
6
0 min post-intravenous injection (C). The SUV of liver derived from micro PET images (D). (n = 3 per group; bars represent means ± SD).
5

P. Sun, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Table 2
Appendix A. Supplementary data
The SUV of liver derived from micro PET in control group, model group and
1
8
blocking group at 30 and 60 min after the intravenous injection of [ F]FPGal.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127187.
(
n = 3 per group; values represent means ± SD.)
Time (min)
Model group
Blocking group
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0.66 ± 0.11
0.64 ± 0.09
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Declaration of Competing Interest
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galactosyl human serum albumin for liver function imaging. Eur J Nucl Med Mol
Imaging. 2013;40:1245–1255.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
20. Gupta Manoj, Choudhury Partha Sarathi, Singh Shivendra, Hazarika Dibyamohan. .
Liver functional volumetry by Tc-99m mebrofenin hepatobiliary scintigraphy before
major liver resection: a game changer. Indian J Nucl Med. 2018;33:277–283.
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This work was supported by grants from President Foundation
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China (2014A020212175), and President Foundation Project of
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