18F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model

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Abstract

Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-18F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide (18F-FBHGal), a new 18F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P <0.0001) was observed using confocal microscopy when co-incubated with 0.5 μM of asialofetuin, a well known ASGPR blocking agent. Animal studies showed the accumulation of 18F-FBHGal in fibrosis liver (14.84 ± 1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50 ± 1.51 %ID/g, P <0.01) at 30 min post-injection. The receptor indexes (liver/liver-plus-heart ratio at 30 min post-injection) of hepatic fibrosis mice derived from both microPET imaging and biodistribution study were significantly lower (P <0.01) than those of normal mice. The pharmacokinetic parameters (T1/2α, T1/2β, AUC and Cl) derived from microPET images revealed prolonged systemic circulation of 18F-FBHGal in hepatic fibrosis mice compared to that in normal mice. The findings in biological characterizations suggest that 18F-FBHGal is a feasible agent for PET imaging of hepatic fibrosis in mice and may provide new insights into ASGPR-related liver dysfunction.

Introduction

The chronic liver disease and cirrhosis, not including hepatoma, were the 8th leading cause of death for men and the 10th for women in Taiwan in 2010.1 Liver fibrosis is a dynamic and complicatedly regulated wound-healing response, resulting from chronic liver damage due to accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver diseases.2 Hepatitis B and C are the main causes of liver fibrosis. In addition to viral-associated hepatitis, alcohol abuse, non-alcoholic steatohepatitis and autoimmune disease also contribute to the pathogenesis of liver fibrosis.3 The fibrotic response underlies virtually all the complications of end-stage liver disease, including portal hypertension, ascites, encephalopathy, synthetic dysfunction, and impaired metabolic capacity. Liver fibrosis is reversible,2, 4 whereas advanced cirrhosis may not be completely reversible, and recovery depends on the etiology and stage of the disease. Thus, early detection of liver fibrosis in patients has become an important clinical issue for predicting the prognosis and early treatment to prevent disease progression.

Traditionally, liver biopsy and histological examination is considered the ‘gold standard’ for identifying the cause of liver disease and assessing the stage of fibrosis. However, several limitations were recognized for this invasive procedure, such as the possibility of sampling error, intra- and inter-observer variation, and only provides a static data.5 In recent years, several imaging approaches have been adapted to detect liver fibrosis6 including ultrasonography (FibroScan7 and acoustic radiation force impulse8), CT and MRI. These morphologic imaging techniques mainly provide information about structural aspects of liver fibrosis, such as the changing in liver echogenicity and nodularity, degree of liver stiffness as well as signs of portal hypertension. Molecular imaging is a new biomedical discipline that enables the visualization, characterization, and quantification of biologic processes at the cellular and molecular levels within living subjects. Thus, efforts to diagnose liver fibrosis down to the molecular level using non-invasive strategy have direct clinical implications.

The asialoglycoprotein receptors (ASGPR) are renowned for existing in the mammalian liver and located on the surface of hepatocytes which contain 100,000–500,000 binding sites per cell.9, 10 The receptors play an essential role in maintaining serum glycoprotein homeostasis by the recognition, binding, and endocytosis of glycoprotein owning galactose residues on the terminal position of saccharide chain, such as asialoglycoproteins.11, 12 After internalized via clathrin-coated pits and fused with endosomes, the asialoglycoproteins are released in the acidic environment of the endosome and transported to lysosomes for degradation, while the receptor is recycled back to the cell surface. In the previous reports,13, 14 Sawamura et al. demonstrated that the asialoglycoprotein receptor activity in the cirrhotic liver was about 28% of the control value in patients. Since 1985, 99mTc-labeled neoglycoalbumin (99mTc-NGA)15, 16 and 99mTc-labeled galactosyl-human serum albumin (99mTc-GSA)17, 18, 19 were developed as single photon emission computed tomography (SPECT) imaging probes for acquiring the asialoglycoprotein receptor scintigraphy to evaluate the liver function. Fluorine-18 labeled galactosyl-neoglycoalbumin (18F-FNGA)20 was presented as a radiotracer for positron emission tomography (PET) scans. In recent years, polymer backbone radiotracers were also reported as ASGPR targeting agents, such as chitosan21 and poly(vinylbenzyl-O-β-d-galactopyranosyl-d-gluconamide) (PVLA).22 Although the exact physiological role of ASGPR has not been elucidated, previous studies suggested that an abnormal cell-surface distribution of this receptor might occur in the liver dysfunction such as cirrhosis or liver cancer.12, 23 Evaluation of ASGPR expression based on 99mTc-NGA scintigraphic imaging has been employed for distinguishing and monitoring the graft and native liver functions in patients.24

Although previous studies had suggested that the in vitro ASGPRs binding affinity of multi- and di-valent galactoside were 104 to 105-fold and 10-fold higher than the monovalent ligand,25, 26 a 18F-labled monoantennary galactoside may still be useful for noninvasive evaluation of ASGPR-related hepatic fibrosis owing to the much rapid pharmacokinetics than that of multivalent macromolecules in vivo. This study reported for the first time a monoantennary galactose derivative 4-18F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide (18F-FBHGal) as a ASGPR-specific PET probe. The biological evaluations demonstrate that 18F-FBHGal PET can characterize the reduction of ASGPR in liver fibrosis in a dimethylnitrosamine (DMN)-treated mouse model and may have the potential to evaluate ASGPR-related liver disease.

Section snippets

Synthesis of β-d-galactosyl precursor for 18F labeling and the authentic standard of FBHGal

The synthetic scheme is shown in Figure 1. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer or Varian 500 MHz NMR spectrometer and referenced to CDCl3 (1H NMR: CDCl3 at 7.24 ppm and 13C NMR: CDCl3 at 77.0 ppm), DMSO-d6 (1H NMR: DMSO-d6 at 2.49 ppm and 13C NMR: DMSO-d6 at 39.5 ppm) and methanol-d4 (1H NMR: methanol-d4 at 3.30 ppm and 13C NMR: methanol-d4 at 49.0 ppm).

Chemistry and radiochemistry

Starting from d-galactose, the β-d-galactosyl precursor (7) suitable for 18F labeling can be obtained via a four-step synthesis with an overall yield of 25% (Fig. 1). The authentic FBHGal (4a) was prepared similarly with an overall yield of 22%. All reaction products were characterized by 1H NMR, 13C NMR and ESI-MS analysis. After coupling the precursor (7) with 18F-SFB, 18F-FBHGal (4b) was obtained with a high radiochemical purity (>98%, Fig. 2). The specific activity was 0.23 GBq/μmol as

Discussion

Measurement of hepatic function in liver fibrosis patients has been linked to several complications, such as ascites, encephalopathy, and portal hypertension. Noninvasive quantification of the ASGPR density on the hepatocytes in vivo provided unambiguous evaluation of hepatic function. 99mTc-labeled galactosyl-neoglycoalbumin (99mTc-NGA) and galactosyl-human serum albumin (99mTc-GSA) have been used as ASGPR-binding agents in clinical studies. However, using albumin-based detection probe may

Conclusion

In this study, an ASGPR-specific probe conjugated with FITC or labeled with fluorine-18 was developed and characterized with HepG2 hepatoma cells (high ASGPR expression) and a hepatic fibrosis mouse model. The in vitro studies demonstrated that GalH-FITC can be specifically bound and internalized into HepG2 cells through ASGP receptor-mediated endocytosis. 18F-FBHGal, with its highly specific ASGPR-targeting ability, good in vivo stability, and the exhibition of significantly different

Acknowledgements

This study was supported by grants from the Institute of Nuclear Energy Research of Taiwan (982001INER071 and 992001INER081). We thank the staff of the Molecular and Genetic Imaging Core/National Research Program for Genomic Medicine (NSC100-2319-B010-003) who kindly provided the radioisotope and excellent technical assistance. The technical support in part by the Imaging Core Facility of Nanotechnology in National Yang-Ming University and the Division of Experiment Surgery of the Department of

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