The research of liver-targeted drugs based on asialoglycoprotein receptor / 药学学报

Asialoglycoprotein receptor (ASGPR) is highly expressed on the surface of parenchymal liver cells. It can specifically recognize and bind to desialylated glycoproteins which contain terminal galactose or N-acetylgalactosamine residues, and endocytosed by clathrin-mediated endocytosis, transported and then degraded in lysosome. Based on this character, ASGPR mediated liver-targeted drug delivery is likely to increase drug distribution, reduce potential side effects and lower dose. This article reviewed the expression, structure, ligand binding and endocytosis of ASGPR, and summarized the design and optimization of ASGPR ligands and the release strategies. Finally, we put forward some expects about the clinical drug development for ASGPR.

A small molecule UPR modulator for diabetes identified by high throughput screening

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and

Expression of ASBT and ASGPR mediated receptors for oral liver-targeting preparations in a rat model of hepatic alveolar echinococcosis / 临床肝胆病杂志

ObjectiveTo investigate the feasibility of apical sodium-dependent bile salt transporter (ASBT) and asialoglycoprotein receptor (ASGPR) in the design of oral liver-targeting preparations for the treatment of hepatic alveolar echinococcosis (HAE) by measuring the expression of ASBT and ASGPR. MethodsA total of 18 male Sprague-Dawley rats were selected, among which 10 were used to establish a model of HAE (HAE group) and 8 were used as controls (normal group). Immunofluorescence assay, Western blotting, and quantitative real-time PCR were used to measure the expression distribution, protein expression level, and mRNA expression level of ASBT in the ileal tissue of HAE model rats and normal rats; the same methods were used to measure the expression level of ASGPR in the non-diseased liver tissue and the marginal zone of liver tissue lesion of HAE model rats and the liver tissue of normal rats. The t-test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between three groups, and the least significant difference t-test was used for comparison between two groups. ResultsThe results of immunofluorescence assay, Western blotting, and quantitative real-time PCR showed that compared with the normal group, the HAE group had significantly upregulated expression of ASBT in the ileal tissue (t=5309, 4.110, and 28.060, all P＜0.05) and a significantly higher expression level of ASGPR (the closer to the lesion, the higher the expression) (F=110666, 128.201, and 143.879, all P＜0.001). ConclusionASBT and ASGPR can be used as potential mediated receptors for oral liver-targeting preparations for HAE, which provides a theoretical basis for the design of oral liver-targeting preparations for the treatment of HAE.

Enzymatic synthesis of three kinds of galactose-cholesterol ligands and their structure-activity relationship with liver targeting / 中草药

Objective To construct three kinds of doxorubicin liposomes modified with cholesterol-galactose ligand by lipase-catalyzed method and compare their characteristic of pharmacokinetics and tissue distribution in vivo. Methods Three types of cholesterol-galactose ligands, CHS-C8-GalNAc, CHS-C8-GAL, and CHS-C8-LA were synthetized by lipase-catalyzed method in nonaqueous phase. The structure characterizations of products were obtained by MS and NMR. Conventional liposomes (CL DOX) and ligand-coupled liposomes (NGal-LP DOX, Gal-LP DOX, and LA-LP DOX) were prepared by thin film dispersion-ammonium sulphate gradient method. Structure-activity relationship between asialoglycoprotein receptor (ASGPr) and the chemical structure of the glycolipids was explored through the pharmacokinetics and tissue distribution parameters of ligand-coupled liposomes in vivo. Results The desired compounds with a high yield of above 90% were confirmed by MS and NMR. The liposomes average size was lower than 90 nm, polymer dispersity index was lower than 0.1, encapsulation efficiency was greater than 99%, leakage rat was lower than 5% with 24 h, and zeta potential closed to zero. The affinity of the three ligand molecules to liver was the following order: CHS-C8-GalNAc > CHS-C8-LA > CHS-C8-Gal. However, only the liposomes modified with CHS-C8-GalNAc could significantly be inhibited by the preinjection of asialofetuin for hepatic uptake rate (P 0.05). Conclusion The ligand with N-acetylgalactosamine residue showed high targeting efficiency for hepatocytes, while the ligand with D-galactose (Gal) or lactitol residue could competitive bind with Gal particle receptor on kupffer cells.

Establishment and Clinical Evaluation of sH2a Subunit from ASGPR in Diagnosis of Hepatic Injury / 现代检验医学杂志

Objective To diagnose the process of hepatic injury,a method of quantitating the concentration of sH2a subunit from asialoglycoprotein receptor (ASGPR) in serum by enzyme-linked immunosorbent assay was established and clinical evaluated.Methods 210 serum samples were collected in Suzhou Kowloon Hospital.Among them,70 subjects with cirrhosis,viral hepatitis and fatty liver disease were as hepatic injury group and 140 subjects of healthy and high fat,hemolysis,jaundice and with autoimmune disease were as control group.The serum sH2a of two group were measured by ELISA kit.The results of sH2a ELISA were analyzed by four table chi-square test and SPSS20.0 software.Results sH2a protein level in liver injury group and control group were 105.92+ 53.41 ng/ml and 69.25+27.45 ng/ml,respectively.The difference between the control group and the liver injury group was statistically significant (F=14.375,t=5.397,P=0.000).The sensitivity and specificity of the sH2a ELISA kit were 68.57％ (95％CI:56.37～79.15％) and 82.86％ (95％CI:75.58％～88.70％),and the total compliance rate was 78.10％ (95％CI:71.88％～83.49％) with KAPPA coefficient:0.510 6 (95％ CI:0.3877～0.6336).Conclusion SH2a serum ELISA kit with positive and negative coincidence rate between SH2a serum level and meet the clinical requirement,which could be used as new marker for diagnosis of heptieal injury related diseases.

Advances in asialoglycoprotein receptor-mediated liver cancer targeted drug delivery system / 中国药科大学学报

@#Asialoglycoprotein receptor(ASGPR)is a receptor expressed mainly on the surface of liver sinusoidal and basolateral cells. It can exclusively identity, combine and clear desialylated glycoproteins with exposed non-reducing D-galactose(Gal)or nacetylgalactosamine(GalNAc)as end groups. Based on this characteristic, ASGPR-mediated targeted liver cancer therapy has drawn extensive attention. The present review details the latest research progress of this field in three aspects, glycosylated prodrug, small molecular nanocarriers, and glycosylated gene complex therapy system.

Verification of the interaction between ASGPR and HBV preS1 protein / 西安交通大学学报(医学版)

Objective To verify the interaction between asialoglycoprotein receptor (ASGPR)and hepatitis B virus (HBV)preS1 protein in vivo and in vitro ,and identify ASGPR as a cell-surface receptor for HBV,which could elucidate the molecular mechanism of HBV infection.Methods The preS1-ASGPR interaction was examined in mammalian two-hybrid and coimmunoprecipitation system by strictly following the manufacturer’s instructions.Results ASGPR interacted specifically and directly with the preS1 domain of HBV in vivo and in vitro .Conclusion ASGPR may be a candidate receptor for HBV that mediates further step of HBV entry.

Optimization of enzymatic synthesis of asialoglycoprotein receptor ligand cholesterol-vinyl sebacate-lactitol / 中草药

Objective: To synthesize asialoglycoprotein receptor (ASGPR) ligand cholesterol-vinyl sebacate-lactitol (CH-VS-LA) by using enzymatic reaction in organic phase and to optimize its synthesis process. Methods: MS and 13C-NMR were used to identify the structure of product, enzymatic synthesis conditions were optimized via single-factor test and orthogonal experimental design. Results: The enzymatic optimum conditions were as following: the molar ratio of cholesterol-vinyl sebacate (CH-VS) and lactitol (LA) was 4:1, the amount of lipase Novozym 435 was 25 mg, reactional time was 32 h, and productive rate was 92%. Conclusion: The method is highly effecient, the reaction conditions are reasonable, and the process produces no by-product.

Preparation of mouse anti-human asialoglycoprotein receptor monoclonal antibody and its preliminary application / 第二军医大学学报

Objective To prepare a mouse monoclonal antibody against human asialoglycoprotein receptor (ASGPR), and to apply it for detecting ASGPR expression in cell lines and tissues. Methods The structure of ASGPR HI major subunit was analyzed and the full length of ASGPR1 was selected to synthesize immunizing peptide. cDNA was amplified by RT-PCR and then subcloned into prokaryotic vector pGEX-4T-1. The recombinant protein was expressed by E. coli BL21 and purified for subsequent immunization. The conventional hybridoma technique was used to generate mouse monoclonal antibody. The isotype and the titer were regularly tested. Inhibition experiment was conducted to identify the specific binding of the antibody to ASGPR. Finally, the expression of ASGPR was detected in various intra-hepatic and extra-hepatic cell lines by flow cytometry and in different liver tissues by immunohistochemistry method. Results Monoclonal antibody against human ASGPR was successfully prepared and was identified as IgG1, with the titer reaching 1: 12 800. Inhibition experiment indicated a satisfactory specific binding of the antibody to ASGPR and that the recognition epitope was located in the extracellular domain of ASGPR. Flow cytometric analysis showed various levels of ASGPR expression in intra-hepatic cell lines, but not in extra-hepatic cell lines. Immunohistochemistry detection showed that ASGPR was specifically expressed in the normll liver tissues and hepatocellular carcinoma (HCC) tissues, and the expression in HCC tissues was associated with the differentiation degree, with the expression being significantly higher in well-differentiated HCC than that in the poorly-differentiated HCC (75. 0% vs 28. 6%, P<0. 05). Conclusion We have successfully prepared the monoclonal antibody against human ASGPR with high specificity; the antibody can be used for flow cytometric analsis and immunohistochemistry detection of ASGPR and for clinical distinguish of primary or metastatic liver cancer.

Lipase-catalyzed synthesis of cholesteryl vinyl hemi-sebacate for selective targeting of liposomes in organic media / 中草药

Objective: To synthesize cholesteryl vinyl hemi-sebacate from cholesterol and divinyl sebacate in non aqueous phase. Methods: TLC, MS, and NMR were used to identify the structure of production; the technological conditions of enzymatic esterification were determined through orthogonal test. Results: The best condition: isooctane 5 mL, reaction temperature 35°C, reaction time 24 h, Candida rugosa Lipase 10 mg/mL, molar ratio of cholesterol to divinyl sebacate 1:6. Conclusion: The highest esterification rate is above 95%.

Study on the enzyme-catalyzed synthesis of ligands for ASGPR-mediated liver targeting liposomes / 中国药学杂志

OBJECTIVE: To synthesize asialoglycoprotein receptor ligand-targeted modifier which is used to insert the surface of liposome by enzyme-catalyzed amidation of lactobionic acid and stearamine. METHODS: The structure of the product was confirmed by IR, ESI-MS and H1-NMR. The effects of types and quantity of enzyme, organic solvents, molar ratio of substrate and temperature of reaction were studied. RESULTS: When using DMSO as reaction medium, Novozym 435 immobilized lipase at 400 U · mL-1, molar ratio of lactobionic acid to stearamine at 2:1, and reacting at 40°C for 24 h, the transformation of stearamine reached more than 99%. CONCLUSION: The enzyme catalysis is useful for synthesizing liver targeting liposomes.

Study on the role of asialoglycoprotein receptor for human bone marrow mesenchymal stem cells against hepatitis B virus infection / 中华传染病杂志

Objective To investigate the susceptibility of bone marrow mesenchymal stem cell (BMSC) to hepatitis B virus (HBV) infection during induction toward hepatocyte and the role of asialoglycoprotein receptor (ASGPR) in BMSC HBV infection. Methods BMSC obtained from hepatitis B patients were tested for HBV infection and then cultured with HBV infectious serum in vitro and induced to differentiate into hepatocyte through exposure to hepatocyte growth factor (HGF), fibroblast growth factor-4(FGF-4), and epidermal growth factor(EGF). Subsequently these cells were determined for the presence of hepatitis B virus e antigen( HBeAg), hepatitis B virus surface antigen(HBsAg) and ASGPR. All experiments were repeated for 3 times in 5 different samples. The results were analyzed by non-parametric test. Results After 6 days of exposure, BMSC-derived hepatocyte-like cells expressed hepatic special genes and proteins, including alpha fetoprotein(AFP),cytokeratin18 (CK18), albumin (Alb), and manifested hepatocyte functions, including glycogen synthesis, urea secretion and albumin synthesis. Expressions of CK18 and Alb were increased, and AFP was decreased with time of induction. The BMSC were resistant to HBV infection both in vitro and in vivo or after induction toward hepatocyte. ASGPR expression level was low in BMSC, which was increased in the induced BMSC but still lower than that of the control HepG2 cells. Conclusions BMSC are resistant to HBV infection both in vitro and in vivo. The low level expression of ASGPR may be a reason for this.

Cloning and expression of the human asialoglycoprotein receptor H1 subunit and preliminary application / 中华检验医学杂志

Objective To clone and express the human asialoglycoprotein receptor(ASGPR) H1 subunit, purify and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay (ELISA) to detect anti-ASGPR antibodies in diagnosis of autoimmune hepatitis. Methods The CRDHI cDNA (435 bp) was subcloned into eukaryotic vector PEGH, and the recombinant protein expression was induced by D (+)-Galactose. The recombinant CRDH1 was purified with Glutathione Sepharose 4B, and its immunoreactivity was identified by SDS-PAGE and western blot as well as MALDI-TOF. ELISA was established to detect the anti-ASGPR antibodies in serum samples of 45 patients with AIH, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequencing of recombinant plasmid showed the CRDH1 gene was successfully inserted to the eukaryotic expression vector with correct sequence and open reading frame. The fusion protein showed a molecular weight of 42 500 Da on SDS-PAGE gel and confirmed to be the human ASGPR by MALDI-MS through peptide mass fingerprint analysis with Mascot in human protein database. It shared 98. 34% homology with ASGPR H1 subunit. Western blot analysis showed that the fusion protein had the same immunoreactivity as human ASGPR. The results of ELISA indicated that the positive rate of anti-ASGPR was 35.6% ( 16/45 ), but the ELISA was negative in other control. There was significant difference of positivity of the autoantibodies between AIH and non-AIH controls (χ2 = 31.85,P < 0. 01 ). Conclusions The human plasmid containing ASGPR is successfully clone into Saccharomyces cerevisiae Y258. The recombinant autoantigen owns good antigenicity and specificity. ELISA established with the purified protein shows good specificity for diagnosis of AIH.

Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan / 华中科技大学学报（医学）（英德文版）

The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E. coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

Liver asialoglycoprotein receptor scintigraphy:a new prospect of segmentational three-dimensional evaluation of liver function / 基础医学与临床

Asialoglycoprotein receptor(ASGPR) is a specific receptor of mammalian hepatocytes.Reduction in ASGPR concentration has been proven to appear in liver cirrhosis and liver cancer.By liver ASGPR scintigraphy with 99mTcGSA,some indexes such as HH15、LHL15、[R]_0、R_0 can be obtained,which is useful for the evaluation of liver function.When combining with the techniques of functional scintigraphy and single photon emission computed tomography(SPECT),it becomes possible to functionally simulate the extension of hepatic resection and predict to some extent postoperative outcomes related to liver function.It is still a new professional field internationally,and almost absent domestically.It could be an important tool for quantificational evaluation of the risk of liver surgery and help determining the surgical procedures.We have here,with some of our research experiences in this field,written a review of ASGPR scintigraphy.

Biodistribution of (99m) Tc-Lactosylated Serum Albumin in Mice with Diethylnitrosamine or Thiacetamide Induced Liver Injury / 대한핵의학회잡지

PURPOSE: Tc-99m labeled diethylenetriaminepentaacetic acid (DTPA) -coupled galactosylated human serum albumin (GSA) is a currently used imaging agent for asialoglycoprotein receptor (ASGPR) of the liver, but, it has several shortcomings. Recently a new ASGPR imaging agent, (99m) Tc-lactosylated human serum albumin (LSA), with simple labeling procedure, high labeling efficiency, high stability was developed. In order to assess the feasibility of the (99m) Tc-LSA as a ASGPR imaging radiopharmaceuticals, we performed biodistribution study of the tracer in liver injured mice model and the results were compared with histolgic data. MATERIALS AND METHODS: To induce hepatic damage in ICR mice, diethylnitrosamine (DEN) (60 mg/kg/week X 5 time, low dose or 180 mg/kg/week X 2 times, high dose) and thioacetamide (TAA) (50 mg/kg X 1 time) were administrated intraperitoneally. Degree of liver damage was evaluated by tissue hematoxilin-eosin stain, and expression of asialoglycoprotein receptor (ASGPR) was assessed by immunohistochemistry using ASGPR antibody. (99m) Tc-LSA was intravenously administrated via tail vein in DEN or TAA treated mice, and biodistribution study of the tracer was also performed. RESULTS: DEN treated mice showed ballooning of hepatocyte and inflammatory cell infiltration in low dose group and severe hapatocyte necrosis in high dose group, and low dose group showed higher ASGPR staining than control mice in immunohistochemical staining. TAA treated mice showed severe hepatic necrosis. (99m) Tc-LSA Biodistribution study showed that mice with hepatic necrosis induced by high dose DEN or TAA revealed higher blood activity and lower liver activity than control mice, due to slow clearance of the tracer by the liver. The degree of liver uptake was inversely correlated with the degree of histologic liver damage. But low dose DEN treated mice with mild hepatic injury showed normal blood clearance and hepatic activity, partly due to overexpression of ASGPR in mice with mild degree hepatic injury. CONCLUSION: Liver uptake of (99m) Tc-LSA was inversely correlated with degree of histologic hepatic injury in DEN and TAA treated mice. These results support that (99m) Tc-LSA can be used to evaluate the liver status in liver disease patients.

Feasibility on Systemic Delivery of Asialoorosomucoid Complex to Hepatic Origin Cells Mediated by Asialoglycoprotein Receptor / 华中科技大学学报（医学）（英德文版）

Summary: Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After intravenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.

Evaluation of Liver Function Using 99mTc-Lactosylated Serum Albumin Liver Scintigraphy in Rat with Acute Hepatic Injury Induced by Dimethylnitrosamine / 대한핵의학회잡지

OBJECTS: 99mTc-lactosylated human serum albumin (LSA) is a newly synthesized radiopharmaceutical that binds to asialoglycoprotein receptors, which are specifically presented on the hepatocyte membrane. Hepatic uptake and blood clearance of LSA were evaluated in rat with acute hepatic injury induced by dimethylnitrosamine (DMN) and results were compared with corresponding findings of liver enzyme profile and these of histologic changes. MATERIALS AND METHODS: DMN (27 mg/kg) was injected intraperitoneally in Sprague-Dawley rat to induce acute hepatic injury. At 3 (DMN-3), 8 (DMN-8), and 21 (DMN-21) days after injection of DMN, LSA injected intravenously, and dynamic images of the liver and heart were recorded for 30 minutes. Time-activity curves of the heart and liver were generated from regions of interest drawn over liver and heart area. Degree of hepatic uptake and blood clearance of LSA were evaluated with visual interpretation and semiquantitative analysis using parameters (receptor index : LHL3 and index of blood clearance : HH3), analysis of time-activity curve was also performed with curve fitting using Prism program. RESULTS: Visual assessment of LSA images revealed decreased hepatic uptake in DMN treated rat, compared to control group. In semiquantitative analysis, LHL3 was significantly lower in DMN treated rat group than control rat group (DMN-3: 0.842, DMN-8: 0.898, DMN-21: 0.91, Control: 0.96, p< 0.05), whereas HH3 was significantly higher than control rat group (DMN-3: 0.731, DMN-8: 0.654, DMN-21: 0.604, Control: 0.473, p< 0.05). AST and ALT were significantly higher in DMN-3 group than those of control group. Centrilobular necrosis and infiltration of inflammatory cells were most prominent in DMN-3 group, and were decreased over time. CONCLUSION: The degree of hepatic uptake of LSA was inversely correlated with liver transaminase and degree of histologic liver injury in rat with acute hepatic injury.

Quantitative Evaluation of Liver Function with Hepatic Receptor Scintigraphy using Tc-99m Galactosylated Serum Albumin ( GSA ) / 대한핵의학회잡지

The reduction in the amount of asialoglycoprotein (ASGP) receptor, which resides exclusively on the plasma membrane of functioning mammalian hepatocytes, as a consequence of hepato-cellular damage has been demonstrated in various pathologic conditions of the liver. Galac tosylated human serum albumin (GSA) is a newly developed receptor-binding agent, specific for the ASGP receptor. Tc-99m GSA binds quantitatively to liver ASGP receptors and the rate of accumulation in the liver is dependent on hepatic function represented as the amount of receptor, as well as the amount of ligand injected, its affinity to the receptor and the hepatic blood flow. The findings of Tc-99m GSA scintigraphy were reported to reflect the hepatic function of the patients with large hepatic tumors, obstructive jauniice, acute and chronic liver disease. Tc-99m GSA scintigraphy is an easy and reliable test and has the clinical potentials to evaluate the liver function in the patients with hepatic disorders.