Dissertations / Theses on the topic 'Asialoglycoproteine'

Le récepteur asialoglycoproteine (ASGPR) a attiré particulièrement l'attention pour concentrer les médicaments dans le foie, notamment parce que ce récepteur membranaire est exprimé presque exclusivement et avec une abondance élevée à la surface des hépatocytes. Dans cette thèse, un anticorps anti-ASGPR (Ac ASGPR) nouvellement développé ainsi que l'utilisation des techniques de modélisation mathématique sont utilisés pour déduire la cinétique d'absorption du récepteur in-vivo chez la souris, information cruciale pour optimiser le ciblage hépatique des médicaments. Une fois déduites ces informations quantitatives peuvent ensuite être utilisées pour informer et affiner le protocole d'administration de n'importe quelle entité ciblant l'ASGPR. Avec un protocole optimisé, la saturation du récepteur peut être évitée et ainsi obtenir un ciblage maximal des hépatocytes en minimisant la probabilité de survenue d'effets secondaires. Nous avons d'abord effectué une étude pharmacocinétique (PK) en utilisant l'Ac ASGPR pour estimer les paramètres cinétiques d'absorption via l'ASGPR en se focalisant sur son expression, sa vitesse de renouvellement et d'internalisation. L'expression du récepteur a été estimé autour d'un 1.8 million de molécules par cellule, ce qui confirme la forte abondance de récepteurs à la surface des hépatocytes. La demi-vie de dégradation du récepteur a été estimée à environ 15 heures and le complexe ligand-récepteur est internalisé avec une demi-vie de 5 jours. Cette lente internalisation est un avantage pour le ciblage des médicaments puisque cela permet de lier le médicament libre dans la circulation plasmatique et ensuite de l'absorber lentement dans les hépatocytes et cela même si sa clairance (non liée à l'ASGPR) est élevée dans le plasma. La cinétique de l'ASGPR montre que la saturation de l'endocytose est possible à des concentrations thérapeutiques, cependant la modélisation mathématique et l'utilisation de simulations permettent d'optimiser le protocole d'administration. Pour confirmer l'absorption spécifique dans le foie de l'Ac ASGPR mais aussi la description quantitative de son absorption, une étude de biodistribution a été entreprise. L'Ac ASGPR a été radio-marqué pour pouvoir mesurer les concentrations d'anticorps dans le foie et quantifier sa distribution dans les autres tissues. Une large distribution de l'Ac ASGPR a été détectée dans le foie et faiblement dans les autres tissues, confirmant la liaison importante et rapide de l'anticorps au foie. Dans le but de différencier l'absorption spécifique de l'anticorps, i.e. liée à l'ASGPR, de celle de la clairance générale des anticorps par le foie, un anticorps non-spécifique (l'Ac IL17) a été utilisé comme contrôle. En comparaison avec l'Ac ASGPR, l'Ac IL17 est beaucoup moins absorbé par le foie. Les données de l'étude de biodistribution ont permis de conclure que la liaison de l'anticorps à son récepteur a lieu uniquement dans le foie confirmant la description quantitative de l'absorption par l'ASGPR. Le modèle mathématique peut être utilisé à plusieurs fins. D'abord, le modèle peut être appliqué à d'autre molécules que l'Ac ASGPR à condition de changer les paramètres PK qui ne sont pas liés à l'ASGPR, comme par exemple le volume de distribution. Le but final est d'extrapoler le modèle PK construit à partir de données chez la souris à l'homme pour prédire un protocole d'administration chez les patients. Chez l'homme, certains paramètres sont déjà connus comme l'expression du récepteur mais d'autres processus de l'endocytose médiée par l'ASGPR doivent encore être investigués comme par exemple la vitesse de synthèse et de dégradation du récepteur. Une fois le modèle défini chez l'homme, le modèle sera applicable et utile pour estimer l'expression du récepteur chez les patients et investiguer l'impact de la plus faible expression du récepteur chez ces patients sur le protocole d'administration
The asialoglycoprotein receptor (ASGPR) has drawn particular attention to enhance drug delivery to hepatocytes, notably because this membrane endocytic receptor is expressed almost exclusively and with high abundance on hepatocytes making this receptor a target of choice for hepatic delivery. In this thesis we take advantage of a newly developed anti-ASGPR antibody (ASGPR Ab) and mathematical modeling to infer the uptake properties of the receptor in vivo in mice, crucial information to optimize drug delivery to hepatocyte. This quantitative knowledge can then be leveraged to inform the protocol of administration of any molecular entity targeting the ASGPR. With an optimal dosing regimen, receptor saturation can be avoided to obtain a maximal delivery into hepatocytes while minimizing the likelihood for systemic adverse effects. To estimate the ASGPR mediated uptake parameters, focusing on its expression, turnover and internalization rates, we performed a mouse pharmacokinetic (PK) study with the ASGPR Ab. The ASGPR expression level was found to be about 1.8 million molecules per hepatocyte, which confirms the high abundance of receptors expressed at the hepatocytes cell surface. The half-life of the degradation of the receptor was estimated to be about 15 hours and the formed ligand-receptor complex is internalized with a half-life of about 5 days. This slow internalization is an advantage for drug targeting as it allows to capture the free drug from the plasma by binding and then delivers the drug slowly into the hepatocytes even if the targeting drug as a fast non-ASGPR related PK in the plasma. The kinetics of the ASGPR shows that saturation of the shuttle at therapeutic concentrations is possible; however, modeling and simulation allows the dosing protocol to be optimized. Then, to confirm both the specific liver uptake of the ASGPR Ab and the quantitative description of the ASGPR mediated uptake we performed a biodistribution study. To measure the uptake of the ASGPR Ab in the liver and the distribution in other tissues, the antibody was radiolabeled and tissue radioactivity was quantified. A large distribution of the ASGPR Ab was detected in liver and minor distribution was noted in other tissues, confirming the rapid and extensive binding of the ASGPR Ab in the liver. In order to differentiate the specific uptake of the ASGPR Ab from the general liver clearance of antibodies, a radiolabeled non-targeting antibody (IL17 Ab) was used as a control. In comparison to the ASGPR Ab, the IL17 Ab distributes much less in liver confirming the specific distribution of the ASGPR Ab into the liver. From the biodistribution data it was possible to conclude that all the target mediated uptake of the ASGPR Ab happens solely in the liver and therefore confirm the quantitative description of the ASGPR mediated uptake. We suggest the following use of the ASGPR-mediated disposition model. First, it is applicable to any ASGPR targeting drugs by changing the PK properties which are non-ASGPR related, e.g. volume of distribution, non-ASGPR related clearance... etc. Second, the ASGPR mediated drug disposition model supports the selection of an optimal dosing regimen by maximizing liver uptake while minimizing non targeted organs distribution. Extrapolation of the mouse model to human is the final goal in order to predict optimal dosing regimen of ASGPR targeting drugs in patients. In human, some parameters are already known such as the receptor expression but other processes of the receptor mediated endocytosis must however be investigated such as the synthesis and degradation rate. Once defined in human, the model will be applicable and used for two purposes 1) estimate the receptor number in patients as suggested in the manuscript of the second paper 2) investigate the impact of decreased receptor number in the patients on the dosing regimen

Two galactose-binding receptors, the hepatic asialoglycoprotein receptor (ASGPR) and the macrophage galactose lectin (MGL) have been investigated. The ASGPR is believed to function in glycoprotein clearance from serum while MGL is involved in recognition of pathogens and tumours and in signalling and immunomodulation. This work describes the analysis of the specificity, structure and organisation of both receptors in humans and the two MGLs in mice. The ligand-binding properties of the two subunits of the ASGPR as well as MGL have been separately tested in glycan array analysis. The results show that primary binding to ligands in the human ASGPR occurs via the ASGPR-1 subunit. MGLs have different specificities even though they are highly similar in sequence and the two mouse MGLs differ markedly from the single MGL in humans and in rats. One of the mouse MGLs has a similar specificity to ASGPR-1 that evolved independently. Hydrodynamic studies of ASGPR-1 revealed that it can form homo-oligomers and circular dichroism analysis of the neck fragment showed that it has a coiled-coil structure. Hetero-oligomer formation was monitored using a mutant version of ASGPR1 that allows purification of the complex using double-affinity chromatography on galactose and mannose. Hetero-oligomers containing both types of subunits are more stable than homo-oligomers. The results suggest a model that can account for the variable subunit stoichiometries observed by various investigators. Hydrodynamic studies and circular dichroism of MGL suggest that the extracellular domain of the human protein is an oligomer not as stable as previously thought, and that its neck is a coiled-coil structure. For both receptors, transmembrane and cytoplasmic domains as well as glycosylation may have a role in their stability. The ability of MGL to recognise pathogen glycans was demonstrated using Trichinella spiralis secretions. It was found that similar glycoproteins are bound by the human and mouse receptors.

The primary objective of this study was to identify, develop and characterise a novel bioactive surface capable of binding hepatocytes and enabling the retention of hepatocyte-specific cell function during in-vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) found on hepatocytes. Hydrogels were created by modifying a commercially available block co-polymer of polyethylene glycol (PEG) and acrylamide, (PEGA) with galactose moieties contained within lactobionic acid (LA), producing a unique bioactive sugar-based gel. A control sugar, D-glucuronic acid (GA), was used as a non-ASGP-R binding control. Monomers used were mono- and bis-acryloamido PEG (Mw=1900 gmol-1), and dimethylacrylamide. The pendant PEGA amine groups were used as ligands to bind to the sugars. The resultant gels were characterised using Fourier Transform Infrared Spectroscopy (FT-IR), protein adsorption, Fmoc-Phe and dansyl chloride labelling. The biocompatibility of the gel surfaces was evaluated using a hepatocyte cell line and the degree of attachment, proliferation, and morphology was characterised using light microscopy, live/dead assays, DNA assays, immunochemical staining, flow cytometry and reverse-transcription polymerase chain reaction (RT-PCR).FT-IR analysis of LA revealed a distinctive band at approximately 1740cm-1 corresponding to carbonyl stretching (C=O) of carboxylic acid. This unique peak disappeared as the galactose moieties within the LA were incorporated into the PEGA gel. A similar trend was also observed with the control GA sugar within the PEGA gel, confirming that the sugars had been integrated into the material. Protein adsorption assays confirmed the non-fouling nature of PEGA. Cell culture experiments showed that hepatocytes attached preferentially to the sugar surfaces, with few cells seen on the PEGA surfaces. It was observed that cells on the PEGA with LA surface were more metabolically active, than the controls and proliferated to a monolayer by day 7 in culture. Immunocytochemical staining of the cells for actin, vinculin and phosphorylated focal adhesion kinase illustrated differences in cell morphology between cells grown on different surfaces. It was determined that the sugar PEGA surfaces maintained some characteristics of hepatocyte functionality e.g. urea synthesis over the course of 7 days. To improve the reproducibility of the surfaces generated, a preliminary investigation of two-dimensional PEG monolayer surfaces as a well defined platform for surface reactions was conducted. These were chemically functionalised in a stepwise manner with the sugars. The number of coupling steps and the choice of solvent were shown to affect the efficiency of the reaction. Further more, the need for careful sample preparation was highlighted as contamination could potentially inhibit the interpretation of the surface chemistry.The overall conclusion of this work is that saccharides within non-fouling surfaces composed of thin layers of PEG-acrylamide hydrogels are able to support hepatocyte attachment and the retention of cell type specific functions in culture. However, this preliminary work has shown that much further research is necessary to elucidate the role that the surface chemistry plays in the attachment of hepatocytes.

Understanding the biological mechanisms of polymeric DNA delivery is essential to develop vehicles that perform optimally. In this work, the cellular internalization mechanisms of poly(glycoamidoamine) (PGAA) DNA delivery polymers were investigated. Polymer:DNA complexes interact with cell-surface glycosaminoglycans (GAGs) in a manner independent of electrostatic interactions. Desulfation and GAG removal leads to decreased uptake. Individual polyplexes appear to have differing affinities for specific GAGs, as polyplex dissociation occurs in a charge-independent manner, and may influence binding. Internalization occurs through close interactions with GAGs, as GAGs accumulate on polyplex surfaces, resulting in negatively-charged polyplexes and decompaction of intact polyplexes is observed upon interaction with GAG. PGAA polyplexes enter cells via a complex, multifaceted internalization route. Pharmacological inhibition of endocytosis and visualization by confocal microscopy reveal that internalization occurs primarily through an actin and dynamin-dependent mechanism. Caveolae/raft-mediated endocytosis appears to be the predominant internalization mechanism, with clathrin-mediated endocytosis also significantly involved. Internalization occurs to a smaller degree via macropinocytosis and direct membrane penetration. Caveolae-mediated, but not clathrin-mediated, internalization leads to transgene expression, suggesting a targeting opportunity based on uptake mechanisms. PEGylation of PGAA polyplexes was achieved to minimize polyplex aggregation in serum. Polyplex size increased in serum, but PEGylation prevented further polyplex growth over time compared to non-PEGylated polymers. Specific targeting of hepatocytes through end-modification of PEG with galactose was unsuccessful, likely due to inaccessibility of targeting groups. Further hepatocyte targeting efforts focused on malonate-based polymers with clickable linkages for facile linkage of targeting groups. Despite favorable surface presentation of galactose, receptor-specific internalization of polyplexes was unsuccessful, as competitive inhibition in HepG2 cells resulted in significant polyplex internalization derived from nonspecific membrane interactions. Chemical modification of vehicles allows systematic study of structure-function properties leading to efficient intracellular delivery. Increasing G4 molecular weight generally increases toxicity and decreases transgene expression in HeLa cells. Incorporating galactose into a lanthanide-chelating polymer facilitated efficient cellular internalization that was visualized by two-photon microscopy. Increased gene expression was observed that correlated to increasing galactose, suggesting that polymer degradation increases gene expression. Also studied were branched peptides targeted to HIV-1 TAR, which displayed high biocompatibility and favorable internalization profiles in mammalian cells.
Ph. D.

The current thesis presents work on the structure and dynamics of oligosaccharides and polysaccharides as well as the free energetics of carbohydrate-protein interactions. By applying various computational tools such as molecular dynamics simulation, our in-house fast sugar structure prediction software, replica exchange molecular dynamics, homology modeling, umbrella sampling, steered molecular dynamics as well as the thermodynamic integration formalism, we have been able to study the role of water on the surface of homopolysaccharides as well as complex oligosachharides, we have been able to produce a prediction of the bound structure of triantennary oligosaccride on the asialoglycoprotein receptor, we have been able to estimate the free energy of binding of ManΑ1→2Man to the HIV-1 inactivating protein, Cyanovirin-N as well as the relative binding free energies of mutants of Cyanovirin-N to the same ligand.

碩士
國立臺灣大學
藥學研究所
84
Asialofetein (Afs), a glycoprotein having triantennary galactose terminated sugar chains, is selectively recognized by the specific receptor on the plasma meembrane of hepatocytes. In general liposome bearing different surface charges give different extent in cellular uptake. The purpose of this experiment is to find an optimal formulation of asialofetuin -liposome (AF-liposome) by modifying the lipid compisitions and surface charge of liposomes for targeeting to liver cells. Physicalchemical properties of liposomes, including size and Zate potential etc., were deterimented. In vitro cellular uptake of liposomes was studied, using four kinds of human hepatoma cell linees (SK-Hep1、HepG2、Hep3B and HuH7 cells) and one kind of mouse fibroblastoma cell (NIH 3T3 cells). In vivo tissue distribution of liposome in rats was analyzed after i.v. injection. 6-Carboxyflurosecein(6-CF)liposome was also prepared , and its distrition in liver was observed by flurorescent microscopy. Among the positive, negative,and neutral AF- liposome , the formulation was 523 AF (PC/PA/Chol=5/2/3, molar ratio), resulted better celllar uptake in vitro and tissue distribution in rats. After liver perfusion of extruted liposome, the uptake uptake of 523AF was significantly higher than N-liposome.

博士
國立陽明大學
生物醫學影像暨放射科學系
102
Part I Objective: Six radio-iodinated cytosine analogues, 123/131I-FIAC, 123/131I-ICdR, 131I-FIVAC, 131I-IVdC, 131I-FIEAC and 131I-IEdC were synthesized and evaluated their biological characteristic as novel gene expression probe or novel proliferation probe. Methods: Six precursors ( FTAC, TCdR, FTVAC, TVdC, FTEAC and TEdC) can be prepared via halo-metal coupling reaction from FIAC and ICdR. The radiolabelling of 131I-FIAC, 131I- ICdR, 131I-FIVAC , 131I-IVdC, 131I-FIEAC and 131I-IEdC were prepared form the organotin or organosilyl precursor with sodium iodide-131 (or iodide-123) under oxidative condition. The cellular uptake studies of nucleoside analogues 131I-FIAC, 131I-ICdR, 131I-FIVAC, 131I-IVdC, 131I-FIEAC and 131I-IEdC in NG4TL4-tk(+) and NG4TL4-tk(-) sarcoma cell lines were conducted. FVB/N mice bearing NG4TL4-tk(+) and NG4TL4-tk(-) sarcoma in the right and left flank were enrolled for animal studies. The biodistribution study and planar gamma imaging of 131I-FIAC and 131I-ICdR were performed in animals bearing NG4TL4-tk(+) and NG4TL4-tk(-) sarcoma. Results: Six radio-iodinated cytosine analogues, 123/131I-FIAC, 123/131I-ICdR, 131I-FIVAC, 131I-IVdC, 131I-FIEAC and 131I-IEdC were synthesized successfully, the radiochemical yield was about 93% and the radiochemical purities were both ≧ 98%. The cellular uptake of 131I-FIAC, 131I-ICdR and 131I-IVdC in NG4TL4-tk(+) sarcoma cells kept increasing with time. The cell-to-medium (C/M) ratio of 131I-FIAC, 131I-ICdR and 131I-IVdC reached 569.4, 229.6 and 60.8 after 8 h incubation. In the NG4TL4-tk(+) sarcoma-bearing mice model , The significantly high uptake of 131I-FIAC was observed in NG4TL4-tk(+) tumor mice at 8 h p.i. (10.87 %ID/g), and then declined slowly till 24 h p.i.. The tk(+) tumor-to- tk(-) tumor ratio kept increasing with time (10.02 at 1 h p.i.), and reached as high as 71.24 at 24 h p.i.. The results of biodistribution were consistent with those observed in scintigraphic imaging. The cellular uptake of 131I-ICdR in NG4TL4 sarcoma cells kept increasing with time. The cell-to-medium (C/M) ratio of 131I-ICdR reached 75.37 after 8 h incubation. The radioactivity distribution in NG4TL4 sarcoma-bearing mice after administration of 131I-ICdR were showed significantly high uptake in organs with fast proliferation (tumor, small intestine, and bone marrow) and the tumor uptake peaked at 4 h p.i. (4.09 %ID/g), and then declined slowly till 8 h p.i.. The tumor-to-muscle (T/M) ratio kept increasing with time (4.78 at 1 h p.i.), and reached as high as 29.56 at 8 h p.i.. Conclusion: Six radio-iodinated cytosine analogues, 123/131I-FIAC, 123/131I-ICdR, 131I-FIVAC, 131I-IVdC, 131I-FIEAC and 131I-IEdC are synthesized successfully and 123/131I-FIAC is revealed potential as a thymidine kinase gene expression probe. The biological characteristics of 123/131I-FIAC was similar with 131I-FIAU and even more 123/131I-FIAC was showed faster and higher accumulation in NG4TL4-tk(+) tumor at early time points p.i.. The study of 123ICdR is also showed as a more promising probe than 123IUdR for evaluation of tissue proliferation in asarcoma-bearing mouse model. 123ICdR SPECT may provide information on tumor biology and to monitor tumor response during and after cancer treatment in the future. Part II Two galactose derivatives, a monovalent 99mTc-MAMA-MGal galactoside and a divalent 99mTc-MAMA-DGal galactoside, were synthesized and radiolabeled in high radiochemical purity (>98%). Dynamic microSPECT imaging and biodistribution study of two traces in normal and liver fibrosis mice showed that the 99mTc-MAMA-DGal revealed higher specific binding to asialoglycoprotein receptors in liver and then rapidly excreted via both hepatobiliary system and renal clearance. The results suggest that 99mTc-MAMA-DGal may be used as SPECT probes for noninvasive evaluation of asialoglycoprotein receptor-related liver dysfunction.