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E A Biessen

Publications and source records attributed to E A Biessen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Synthesis of a lipophilic prodrug of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its incorporation into a hepatocyte-specific lipidic carrier.

PURPOSE: 9-(2-Phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of Hepatitis B virus replication, is in vivo hardly taken up by parenchymal liver cells (the site of infection). Our aim is to examine whether lactosylated reconstituted HDL (LacNeoHDL), a lipidic particle that is specifically internalized by parenchymal liver cells, is a suitable carrier for the selective delivery of PMEA to this cell type. METHODS: To incorporate PMEA into LacNeoHDL, we synthesized a lipophilic prodrug (PMEA-LO) by coupling PMEA via an acid-labile phosphonamidate bond to lithocholic acid-3alpha-oleate. RESULTS: The yield of the synthesis was 52% ([3H]PMEA-LO: 24%). [3H]PMEA-LO readily incorporated into LacNeoHDL (13 molecules/particle) without affecting the size and net negative charge of the carrier. Further, incubation studies at lysosomal pH showed [3H]PMEA was completely released from the carrier whereas, at neutral pH or in plasma, appreciable release was not observed. CONCLUSIONS: The conjugation of PMEA with lithocholic acid-3alpha-oleate results in a lipophilic prodrug that readily associates with Lac-NeoHDL. The association of the prodrug does not affect the physicochemical properties of the particle, and PMEA is released from the carrier at lysosomal pH. These findings indicate that by using the prodrug approach, LacNeoHDL is a suitable carrier to deliver PMEA to parenchymal liver cells.

Adenine↗
PubMed1998

Liver uptake of phosphodiester oligodeoxynucleotides is mediated by scavenger receptors.

The therapeutic activity of antisense oligodeoxynucleotides (ODNs) often is impaired due to premature degradation and poor ability to reach the (intra)cellular target. In this study, we addressed the in vivo fate of ODNs and characterized the major sites responsible for the clearance of intravenously injected phosphodiester ODN. On injection into rats, 32P-ODNs (miscellaneous sequences and GT-containing ODNs with variable G content) are rapidly cleared from the bloodstream (t1/2 = 0.6-0.7 min), with the liver being the main site of elimination. The contribution of the liver to ODN clearance depended on its sequence and varied considerably. Hepatic uptake tended to be lower for G-rich ODNs as a result of increased bone marrow uptake. Within the liver, both Kupffer cells (KC) and endothelial cells (EC) were responsible for 32P-ODN uptake. To elucidate the mechanism of liver uptake, 32P-ODN binding studies using isolated EC and KC were performed. Binding to both cell types seemed to be saturable, of moderate affinity, and mediated by a membrane-bound protein. The inhibition profiles of 32P-ODN binding to EC and KC by various (poly)anions were essentially equal and corresponded closely to those of 125I-acetylated low-density lipoprotein. In summary, the results indicate that scavenger receptors on nonparenchymal liver and bone marrow cells contribute to the elimination of ODNs from the bloodstream. Minor changes in ODN sequence markedly affect receptor recognition, resulting in considerable shifts in the biodistribution of antisense ODNs.

Animals↗
PubMed1997

In vivo fate of phosphorothioate antisense oligodeoxynucleotides: predominant uptake by scavenger receptors on endothelial liver cells.

Systemically administered phosphorothioate antisense oligodeoxynucleotides can specifically affect the expression of their target genes, which affords an exciting new strategy for therapeutic intervention. Earlier studies point to a major role of the liver in the disposition of these oligonucleotides. The aim of the present study was to identify the cell type(s) responsible for the liver uptake of phosphorothioate oligodeoxynucleotides and to examine the mechanisms involved. In our study we used ISIS-3082, a phosphorothioate antisense oligodeoxynucleotide specific for murine ICAM-1. Intravenously injected [3H]ISIS-3082 (dose: 1 mg/kg) was cleared from the circulation of rats with a half-life of 23.3+/-3.8 min. At 90 min after injection (>90% of [3H]ISIS-3082 cleared), the liver contained the most radioactivity, whereas the second-highest amount was recovered in the kidneys (40.5+/-1.4% and 17.9+/-1.3% of the dose, respectively). Of the remaining tissues, only spleen and bone marrow actively accumulated [3H]ISIS-3082. By injecting different doses of [3H]ISIS-3082, it was found that uptake by liver, spleen, bone marrow, and kidneys is saturable, which points to a receptor-mediated process. Subcellular fractionation of the liver indicates that ISIS-3082 is internalized and delivered to the lysosomes. Liver uptake occurs mainly (for 56.1+/-3.0%) by endothelial cells, whereas parenchymal and Kupffer cells account for 39.6+/-4.5 and 4.3+/-1.7% of the total liver uptake, respectively. Preinjection of polyinosinic acid substantially reduced uptake by liver and bone marrow, whereas polyadenylic acid was ineffective, which indicates that in these tissues scavenger receptors are involved in uptake. Polyadenylic acid, but not polyinosinic acid, reduced uptake by kidneys, which suggests renal uptake by scavenger receptors different from those in the liver. We conclude that scavenger receptors on rat liver endothelial cells play a predominant role in the plasma clearance of ISIS-3082. As scavenger receptors are also expressed on human endothelial liver cells, our findings are probably highly relevant for the therapeutic application of phosphorothioate oligodeoxynucleotides in humans. If the target gene is not localized in endothelial liver cells, the therapeutic effectiveness might be improved by developing delivery strategies that redirect the oligonucleotides to the actual target cells.

Animals↗
PubMed1997

Antagonists of the mannose receptor and the LDL receptor-related protein dramatically delay the clearance of tissue plasminogen activator.

BACKGROUND: Clinical application of tissue plasminogen activator (TPA) as a fibrinolytic agent is complicated by its rapid clearance from the bloodstream, which is caused by TPA liver uptake. The mannose receptor on endothelial liver cells and the LDL receptor-related protein (LRP) on parenchymal liver cells were reported to contribute to liver uptake. METHODS AND RESULTS: In this study, we addressed whether TPA clearance can be delayed by inhibiting receptor-mediated endocytosis of TPA. A series of cluster mannosides was synthesized, and their affinity for the mannose receptor was determined. A cluster mannoside carrying six mannose groups (M6L5) displayed a subnanomolar affinity for the mannose receptor (Ki = 0.41 +/- 0.09 nmol/L). Preinjection of M6L5 (1.2 mg/kg) reduced the clearance of 125I-TPA in rats by 60% because of specific inhibition of the endothelial cell uptake. The low toxicity of M6L5, combined with its accessible synthesis and high specificity for the mannose receptor, makes it a promising agent to improve the pharmacokinetics of TPA. Blockade of LRP by 39-kD receptor-associated protein (GST-RAP) also inhibited TPA clearance by 60%. Finally, combined preinjection of M6L5 and GST-RAP almost completely abolished reduced liver uptake of TPA and delayed its clearance by a factor of 10. CONCLUSIONS: It can be concluded that (1) the mannose receptor and LRP appear to be the sole major receptors responsible for TPA clearance and (2) therapeutic levels of TPA can be maintained for a prolonged time span by coadministration of the aforementioned receptor antagonists.

Animals↗
PubMed1997

Cluster mannosides can inhibit mannose receptor-mediated tissue-type plasminogen activator degradation by both rat and human cells.

Recently, we developed a series of cluster mannosides that were able to inhibit tissue-type plasminogen activator (t-PA) binding to the isolated mannose receptor. The mannoside with the highest affinity was able to inhibit t-PA clearance by the liver in the rat. To test whether these mannosides would also be efficient inhibitors in humans, we studied the expression of the mannose receptor in the human liver and determined the efficacy of the mannosides to inhibit mannose receptor-mediated t-PA degradation by both rat and human cells. Immunohistochemistry indicates that, like the rat, human liver endothelial cells and human Kupffer cells do express the mannose receptor. The mannosides do inhibit mannose receptor-mediated t-PA binding, association, and degradation by isolated rat liver endothelial cells and t-PA association and degradation by cultured human macrophages at similar concentrations. The cluster mannoside with six mannose residues connected with a backbone of five lysine groups (M6L5) was, like unlabeled t-PA, able to inhibit 125I-t-PA degradation in the nmol/L range, while the mannoside M5L4 inhibited 125I-t-PA degradation in the micromol/L range. The concentrations of mannoside necessary to inhibit 125I-t-PA degradation in vitro were comparable with the concentrations necessary to inhibit mannose receptor-mediated 125I-t-PA clearance in vivo. We conclude that there is no species difference between rat and humans with respect to the distribution of the mannose receptor in the liver and the affinity of the cluster mannosides, establishing the relevance of the inhibition of mannose receptor-mediated t-PA clearance by M6L5 as observed in the rat, for the human situation.

Animals↗
PubMed1996

Lysine-based cluster mannosides that inhibit ligand binding to the human mannose receptor at nanomolar concentration.

In search of synthetic high affinity ligands for the mannose receptor, we synthesized a series of lysine-based oligomannosides containing two (M2L) to six (M6L5) terminal alpha-D-mannose groups that are connected with the backbone by flexible elongated spacers (16 A). The synthesized cluster mannosides were all able to displace binding of biotinylated ribonuclease B and tissue-type plasminogen activator to isolated human mannose receptor. The affinity of these cluster mannosides for the mannose receptor was continuously enhanced from 18-23 microM to 0.5-2.6 nM, with mannose valencies increasing from two to six. On average, expansion of the cluster mannoside with an additional alpha-D-mannose group resulted in a 10-fold increase in its affinity for the mannose receptor. M3L2 to M6L5 displayed negative cooperative inhibition of ligand binding to the mannose receptor, suggesting that binding of these mannosides involves multiple binding sites. The nanomolar affinity of the most potent ligand, the hexamannoside M6L5 makes it the most potent synthetic cluster mannoside for the mannose receptor yet developed. As a result of its high affinity and accessible synthesis, M6L5 not only is a powerful tool to study the mechanism of ligand binding by the mannose receptor, but it is also a promising targeting device to accomplish cell-specific delivery of genes and drugs to liver endothelial cells or macrophages in bone marrow, lungs, spleen, and atherosclerotic plaques.

Binding, Competitive↗
PubMed1996

Increased selective uptake in vivo and in vitro of oxidized cholesteryl esters from high-density lipoprotein by rat liver parenchymal cells.

Oxidation of low-density lipoprotein (LDL) leads initially to the formation of LDL-associated cholesteryl ester hydroperoxides (CEOOH). LDL-associated CEOOH can be transferred to high-density lipoprotein (HDL), and HDL-associated CEOOH are rapidly reduced to the corresponding hydroxides (CEOH) by an intrinsic peroxidase-like activity. We have now performed in vivo experiments to quantify the clearance rates and to identify the uptake sites of HDL-associated [3H]Ch18:2-OH in rats. Upon injection into rats, HDL-associated [3H]Ch18:2-OH is removed more rapidly from the circulation than HDL-associated [3H]Ch18:2. Two minutes after administration of [3H]Ch18:2-OH-HDL, 19.6 +/- 2.6% (S.E.M.; n = 4) of the label was taken up by the liver as compared with 2.4 +/- 0.25% (S.E.M.; n = 4) for [3H]Ch18:2-HDL. Organ distribution studies indicated that only the liver and adrenals exhibited preferential uptake of [3H]Ch18:2-OH as compared with [3H]Ch18:2, with the liver as the major site of uptake. A cell-separation procedure, employed 10 min after injection of [3H]Ch18:2-OH-HDL or [3H]Ch18:2-HDL, demonstrated that within the liver only parenchymal cells take up HDL-CE by the selective uptake pathway. Selective uptake by parenchymal cells of [3H]Ch18:2-OH was 3-fold higher than that of [3H]Ch18:2, while Kupffer and endothelial cell uptake of the lipid tracers reflected HDL holoparticle uptake (as analysed with iodinated versus cholesteryl ester-labelled HDL). The efficient uptake of [3H]Ch18:2-OH by parenchymal cells was coupled to a 3-fold increase in rate of radioactive bile acid secretion from [3H]Ch18:2-OH-HDL as compared with [3H]Ch18:2-HDL. In vitro studies with freshly isolated parenchymal cells showed that the association of [3H]Ch18:2-OH-HDL at 37 degrees C exceeded [3H]Ch18:2-HDL uptake almost 4-fold. Our results indicate that HDL-associated CEOH are efficiently and selectively removed from the blood circulation by the liver in vivo. The selective liver uptake is specifically exerted by parenchymal cells and coupled to a rapid biliary secretion pathway. The liver uptake and biliary secretion route may allow HDL to function as an efficient protection system against potentially atherogenic CEOOH.

Animals↗
PubMed1996

Interaction of mutants of tissue-type plasminogen activator with liver cells: effect of domain deletions.

The fibrin-specific thrombolyticum tissue-type plasminogen activator (t-PA) has proven to be a potent drug in several clinical trials, but its clinical application is complicated by the rapid clearance of t-PA from the circulation. The rapid plasma clearance of t-PA results from the uptake of t-PA in the liver. t-PA consists of several domains which may be involved in the interaction with the liver. Three domain-deletion mutants, which were produced by the use of a cassette gene system, were studied in vivo and in vitro for their capacity to bind to the various types of rat liver cells. The three mutants lacked, in comparison to control t-PA, the epidermal growth factor (G) domain, the finger (F) domain or the G domain plus the first kringle (K1). The plasma clearance of the three mutants was slower than that of control t-PA. The slower plasma clearance resulted from a decreased liver uptake: 50 and 80% for t-PA mutants and control t-PA respectively. It was found that the K1 domain was of major importance for the uptake of t-PA by liver endothelial cells in vivo and in vitro. The high-affinity binding of t-PA (and t-PA mutants) to parenchymal liver cells depended largely on the presence of the G domain. Other domain(s), like the F, K2 or protease domain, may be responsible for low-affinity, t-PA-specific binding to rat parenchymal liver cells.

Animals↗
PubMed1996

Induction of hepatic uptake of lipoprotein(a) by cholesterol-derivatized cluster galactosides.

We have previously developed triantennary galactosides [TG(4A)C and TG(20A)C] that lower cholesterol levels by inducing liver uptake of lipoproteins via galactose-recognizing hepatic receptors. In this study, we have investigated whether this strategy could also be applied to reduce elevated serum levels of the atherogenic lipoprotein(a) [Lp(a)]. Both TG(4A)C and TG(20A)C could be incorporated into Lp(a). Incorporation of these glycolipids induced a rapid clearance of Lp(a). Concomitantly, the hepatic uptake of 125I-Lp(a) was enhanced from 4 +/- 1% to 80 +/- 4% of the injected dose for TG(4A)C (P < .0001) and to 17 +/- 4% of the injected dose for TG(20A)C (P < .006). TG(4A)C was apparently more effective in accelerating the serum decay of 125I-Lp(a), which may be caused by the higher hydrophobicity of this glycolipid relative to TG(20A)C. The TG(4A)C- and TG(20A)C-induced stimulation of the serum decay and liver uptake of 125I-Lp(a) could be significantly inhibited (> 85%) by preinjection of N-acetyl-galactosamine (150 mg), indicating that galactose-recognizing receptors are involved in the liver uptake of the glycolipid/Lp(a) complexes. The TG(4A)C-induced liver uptake of 125I-Lp(a) could be ascribed mainly to Kupffer cells (76 +/- 7%), whereas the parenchymal liver cell was the major site for liver uptake of TG(20A)C-laden 125I-Lp(a) (55 +/- 12%). In conclusion, both TG(4A)C and TG(20A)C stimulate the catabolism of 125I-Lp(a) by enhancing hepatic uptake. Because endocytosis of the substrate via galactose-recognizing receptors on Kupffer and parenchymal liver cells is followed by lysosomal degradation, we anticipate that both approaches for Lp(a) targeting may prove valuable as therapeutic modalities for lowering atherogenic levels of Lp(a).

Animals↗
PubMed1995

The cholesterol derivative of a triantennary galactoside with high affinity for hepatic asialoglycoprotein receptor: a potent cholesterol lowering agent.

Cholesterol-derivatized galactosides have been devised in order to induce liver uptake of lipoproteins via the galactose-recognizing asialoglycoprotein receptor in the liver. In this study we describe the derivatization of a newly developed triantennary cluster galactoside having high affinity for the asialoglycoprotein receptor, N-[[tris-O-(3,6,9-trioxaundecanyl-beta-D-galactopyranosyl)metho xym ethyl] -N alpha-[1-(6-methyladipyl)]glycinamide (TG(20A)) with cholesterol. Hereto, TG(20A) was coupled to glycine-(5-cholesten-3 beta-yl ester) in the presence of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, affording N-[[tris-O-(3,6,9-trioxaundecanyl-beta-D- galactopyranosyl)methoxymethyl]methyl]-N alpha-[1-(6-(5-cholesten-3 beta-yloxy)glycyl)adipyl]glycinamide (TG(20A)C) in 46% yield. This compound is an amphiphilic, water-soluble compound. In aqueous solution it readily formed small micelles (4.9 +/- 1.2 nm) consisting of approximately 20 molecules. Upon incubation with human serum, TG(20A)C spontaneously incorporated into the most prominent serum lipoproteins, i.e., low-density lipoprotein (LDL) and high-density lipoprotein (HDL), thereby inducing an increase in buoyant density of these lipoproteins. The integrity of HDL and LDL, as judged from particle size analysis of both lipoproteins, was not altered by incubation with up to 0.33% of TG(20A)C (w/v). Following intravenous bolus injection into rats, TG(20A)C induced a dose-dependent decrease in the serum cholesterol content of maximally 44%, at a dose of 1.9 mg kg-1. This makes TG(20A)C at least 30-fold more effective than the previously developed N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[4-(5- cholesten-3 beta-yloxy)succinyl]glycinamide (TG(4A)C), provided with a cluster galactoside that displayed a 2000-fold lower affinity for the asialoglycoprotein receptor than TG(20A). In conclusion, the hypocholesterolemic activity of a cholesterylated galactoside can be strongly enhanced by using a cluster galactoside with higher affinity for the asialoglycoprotein receptor.

Animals↗
PubMed1995

Synthesis of cluster galactosides with high affinity for the hepatic asialoglycoprotein receptor.

High-affinity ligands for the asialoglycoprotein receptor, which is uniquely localized on the parenchymal liver cell and recognizes oligoantennary galactosides, might be utilized as homing device to specifically target drugs or genes to parenchymal liver cells. In the present study, the synthesis of galactose-terminated triantennary glycosides, provided with various spacers between the beta-galactopyranosyl moieties and the branching point of the dendrite, is described. N-[Tris[[(methylthio)methoxy]methyl]methyl]-N alpha-[1-(6- methyladipy)]glycinamide (3b) was glycosylated with monogalactosyl derivatives, containing propanediol or ethylene glycol units as hydrophilic spacer moieties, to yield the corresponding cluster galactosides. To determine the affinity of the cluster galactosides for the asialoglycoprotein receptor, we have performed competition studies of [125I]ASOR binding, a specific ligand for the asialoglycoprotein receptor, to isolated parenchymal cells. The affinity for the asialoglycoprotein receptor significantly increased with increasing spacer length. N-[[[Tris-O-(beta-D-galactopyranosyl)-3,6,9-trioxaunde- canoxy]methoxy]methyl]-N-alpha-[1-(6-methyladipyl)]glycinami de (4e), a cluster galactoside provided with a 20 A spacer, possessed an at least 2000-fold higher affinity for the receptor than N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[1-(6- methyladipyl)]glycinamide (4a), a cluster galactoside lacking the spacer. It is concluded that vicinal galactosyl moieties within a cluster galactoside are more optimal recognized by the galactose binding sites of the asialoglycoprotein receptor upon proper spacing. The most potent galactoside, TG(20A), may constitute an attractive targeting device for the specific delivery of drugs and/or genes to the parenchymal liver cell.

Animals↗
PubMed1995

Cholesterol derivative of a new triantennary cluster galactoside lowers serum cholesterol levels and enhances secretion of bile acids in the rat.

BACKGROUND: Previous studies have demonstrated that cholesterol-derivatized galactosides exert a hypocholesterolemic effect by inducing hepatic uptake of atherogenic lipoproteins by means of galactose-recognizing receptors in the liver. However, a prolonged infusion of high concentrations of these compounds was required for this effect, possibly because of low affinity for the galactose-recognizing asialoglycoprotein receptor on the parenchymal liver cell. METHODS AND RESULTS: We have designed a new series of triantennary galactosides to optimize the affinity and specificity for this receptor. The affinity of a triantennary galactoside for the asialoglycoprotein receptor appeared to be dramatically enhanced by proper spacing of the three terminal galactose groups. In rats, a single injection of N-[tris-O-(3,6,9-trioxaundecanyl-beta-D-galacto- pyranosyl)methoxymethyl]methyl-N alpha-[1-(6-(5-cholesten-3 beta- yloxy)glycyl)adipyl]glycinamide [TG(20A)C], the cholesterol derivative of the most selective galactoside, causes a dose-dependent decrease of < or = 45% in the serum cholesterol concentration (P < .001). This decrease is mainly attributed to a decrease in the level of serum HDL (P = .0066) and, to a lesser extent, serum LDL (P = .036). In addition, TG(20A)C strongly enhances the bile-acid secretion in rats during the first 2 hours after administration, which indicates that TG(20A)C-induced clearance of cholesterol from the bloodstream is efficiently coupled to hepatic bile-acid secretion. CONCLUSIONS: We conclude that TG(20A)C efficiently directs lipoproteins that contain cholesterol to the liver at a 30-fold-lower concentration than previously developed cholesterol-derived cluster galactosides. This newly developed approach to lower cholesterol levels may prove valuable for familial hypercholesterolemic patients or those with familial defective apolipoprotein B-100 who do not respond or who respond insufficiently, respectively, to conventional therapies.

Animals↗
PubMed1994

Binding characteristics of scavenger receptors on liver endothelial and Kupffer cells for modified low-density lipoproteins.

Previous studies showed that both endothelial and Kupffer cells contain specific recognition sites of oxidized low-density lipoprotein (OxLDL), in addition to recognition sites which recognize OxLDL and acetylated LDL (AcLDL). We have determined the binding characteristics of the recognition sites for OxLDL on Kupffer cells and endothelial cells (OxLDL-specific binding-site) in comparison to the recognition site for AcLDL on endothelial cells, which recognizes both AcLDL and OxLDL (Ac/OxLDL binding site). The capacity of Kupffer cells to bind OxLDL (Bmax. = 779 ng of 125I-OxLDL/mg of cell protein; Kd = 6 micrograms/ml) was comparable to the binding-capacity of endothelial cells (Bmax. = 803 ng of 125I-OxLDL/mg of cell protein; Kd = 5 micrograms/ml). The effect of net charge of modified LDL on its affinity for the recognition sites on Kupffer and endothelial cells was evaluated using competition studies. The affinity of AcLDL for the Ac/OxLDL binding site was greatly increased from 460 micrograms/ml to 4 micrograms/ml with increasing extent of modification and thus net charge. The Ac/OxLDL binding-site on endothelial cells also displayed an increased affinity towards LDL with an increasing degree of oxidation. The affinity of OxLDL for the Ac/OxLDL binding-site appeared to be about 4-fold higher than that of AcLDL with a similar extent of modification. At higher degrees of oxidation of LDL, the affinity for the OxLDL-specific site on endothelial and Kupffer cells was also strongly enhanced; the OxLDL-specific binding-site possesses a higher affinity for mildly oxidized LDL as compared with the Ac/OxLDL binding-site. It is concluded that recognition of OxLDL by both the OxLDL-specific binding-site and the Ac/OxLDL binding-site on liver endothelial and Kupffer cells depends on the net negative charge of modified LDL. The similarity in binding pattern of these binding sites makes it likely that the newly described 95 kD OxLDL binding protein on Kupffer cells [Y. B. De Rijke and Th. J. C. van Berkel, J. Biol. Chem. (1994), 269, 824-827] contains a recognition site with similar structural elements as described earlier for scavenger receptors.

Binding Sites↗
PubMed1994

Cholesterol derivative of a new triantennary cluster galactoside directs low- and high-density lipoproteins to the parenchymal liver cell.

We have developed a new triantennary galactoside, in which the terminal galactose moieties are connected to the branching point of the cluster galactoside via a 20 A (2 nm) spacer [TG(20A)]. In vitro binding studies have demonstrated that introduction of a 20 A spacer resulted in avid and specific binding of the triantennary galactoside to the asialoglycoprotein receptor on the parenchymal liver cell. Derivatization of this galactoside with a cholesterol moiety afforded a compound [TG(20A)C] that lowered the serum cholesterol concentration when injected into rats. In the present study we have evaluated the direct effect of TG(20A)C on the in vivo fate of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). A direct association of TG(20A)C with HDL and LDL was observed on mixing these components. Incorporation of TG(20A)C into 125I-HDL and 125I-LDL significantly accelerated the serum decay and concomitantly stimulated the hepatic uptake of these lipoproteins in rats. The liver uptake of TG(20A)C-loaded 125I-HDL or 125I-LDL could be inhibited by 81% and 82% respectively by preinjection of 150 mg of N-acetylgalactosamine, indicating that the enhanced liver uptake proceeded via galactose-specific receptors. More than 96% of the hepatic uptake of TG(20A)C-loaded 125I-HDL could be attributed to the parenchymal cell. Surprisingly, the parenchymal cell also accounted for 93% of the liver association of TG(20A)C-loaded 125I-LDL, suggesting that TG(20A)C stimulates the uptake and processing of both lipoproteins by the asialoglycoprotein receptor on the parenchymal liver cell. This contrasts with earlier data indicating that a triantennary cluster galactoside provided with a 4 A spacer between the terminal galactose moieties and the branching point of the dendrite stimulated hepatic uptake of LDL via the Kupffer cells. The parenchymal cell is the only liver cell type that is capable of irreversibly removing cholesterol from the body in the form of bile acids. The above results imply that administration of TG(20A)C not only facilitates the hepatic uptake of lipoprotein-derived cholesterol (esters) but also their elimination from the body. In addition, it might be possible to utilize TG(20A)C as a targeting device to selectively deliver large drug carriers and possibly genes to the parenchymal liver cell.

Acetylgalactosamine↗
PubMed1994

Characterization of the interaction of galactose-exposing particles with rat Kupffer cells.

The characteristics of the recognition system involved in the binding of galactose-exposing particles to freshly isolated rat Kupffer cells were determined. For this purpose we used iodinated lactosylated low-density lipoprotein (125I-Lac-LDL) as a ligand for the galactose receptor on Kupffer cells. The affinity of the binding of 125I-Lac-LDL to Kupffer cells was saturable (23,500 galactose-specific binding sites per cell) and of high affinity (2.4 +/- 0.3 nM). The order of potency of various carbohydrates in inhibiting the association of 125I-Lac-LDL with Kupffer cells was as follows: N-acetylgalactosamine > L-fucose >> N-acetylglucosamine/mannan. Association of 125I-Lac-LDL with Kupffer cells in the absence of Ca2+ was at the same level as in the presence of 50 mM N-acetylgalactosamine. A polyclonal antibody raised against the rat asialoglycoprotein receptor inhibited the binding of 125I-Lac-LDL to Kupffer cells and reacted in a Western blot with two proteins (molecular mass 88 and 77 kDa), which correspond to the molecular mass of the fucose receptor [Lehrman, Haltiwanger and Hill (1986) J. Biol. Chem. 261, 7426-7432]. Furthermore, the ability of fucosylated neoglycoproteins to displace 125I-Lac-LDL from Kupffer cells was equally dependent on the extent of fucosylation as previously reported for the fucose receptor. We conclude that the fucose receptor and not the C-reactive protein, as recently proposed [Kempka, Roos and Kolb-Bachofen (1990) J. Immunol. 144, 1004-1009], functions as the galactose-particle receptor on the Kupffer cell. The binding of galactose-exposing particles to the fucose receptor is a previously unknown property of this receptor.

Acetylgalactosamine↗
PubMed1994

Ligand size is a major determinant of high-affinity binding of fucose- and galactose-exposing (lipo)proteins by the hepatic fucose receptor.

Previous in vivo studies have demonstrated that small galactose-exposing particles are preferentially internalized by the asialoglycoprotein receptor on the parenchymal liver cell and large particles by the galactose-particle receptor on the Kupffer cell. In this study, we have investigated using in vitro binding studies whether the affinity for either receptor is affected by the ligand size. The asialoglycoprotein receptor appeared to bind and process lactosylated proteins irrespective of their size. In contrast, recognition of galactose-exposing proteins by the galactose-particle receptor on the Kupffer cell was strongly dependent on size. The affinity increased 3000-fold with protein sizes increasing from 5 to 15 nm, reaching its maximum at approx. 1 nM for ligands larger than 15 nm. Apparently, the preferential in vivo uptake of large galactose-exposing ligands by Kupffer cells does not result from an inability of the parenchymal liver cells to internalize these ligands, but from the high affinity of large ligands for the galactose-particle receptor and the strategic anatomical localization of the Kupffer cells in the liver. In the preceding paper [Kuiper, Bakkeren, Biessen and Van Berkel (1994) Biochem. J. 299, 285-290] the galactose-particle receptor on the Kupffer cell was suggested to be identical with the fucose receptor. 125I-Lac-LDL-binding studies clearly showed that the galactose-particle receptor exhibited high-affinity binding of fucose-exposing proteins also. The affinity of fucosylated proteins for the galactose-particle receptor was greatly affected by ligand size. The above data strongly support the hypothesis that the galactose-particle receptor is identical with the fucose receptor. The size of neoglycoproteins can be appreciated as a new major determinant of affinity for the fucose receptor.

Animals↗
PubMed1994

Specific targeting of the antiviral drug 5-iodo 2'-deoxyuridine to the parenchymal liver cell using lactosylated poly-L-lysine.

In this study, we describe the development and characterization of lactosylated poly-L-lysine as a potential carrier for targeting anti-viral drugs to the parenchymal liver cell. Poly-L-lysine (M(r) 38,000) was modified with 2 to 130 lactose residues per molecule poly-L-lysine. In vitro competition studies for the asialoglycoprotein receptor on parenchymal liver cells using 125I-asialoorosomucoid as radioligand revealed that mild modification of poly-L-lysine with only five lactose residues was sufficient for high affinity competition. In vivo studies showed that, after injection of poly-L-lysine modified with at least five lactose residues, about 70-80% of the injected dose was taken up by the liver. Preinjection of N-acetyl galactosamine almost completely blocked the hepatic uptake of lactosylated poly-L-lysine, indicating that galactose-recognizing receptors are involved. At 10 min following injection, the contribution of the various liver cell types to the hepatic uptake of lactosylated poly-L-lysine was determined; the parenchymal cell appeared to be responsible for more than 98% of the total liver uptake. To assess the applicability of lactosylated poly-L-lysine as an anti-viral drug carrier, it was derivatized with 4 to 15 residues of the antiviral drug 5-iodo 2'-deoxyuridine, 5'-monophosphate per molecule poly-L-lysine (4-16% by weight) via an acid-labile phosphamide bond. Maximally 0.7% of the conjugated 5-iodo 2'-deoxyuridine 5'-monophosphate was released after 1 h incubation of the drug/carrier conjugate with serum at 37 degrees C, thus establishing the stability of the conjugate in serum. The drug-carrier conjugate was rapidly cleared from the bloodstream within 1 min. Approximately 90% of the injected dose could be recovered in the liver. The parenchymal liver cell was responsible for 97% of the hepatic uptake. In vitro studies on the kinetics of endocytosis of lactosylated poly-L-lysine, derivatized with 5-iodo 2'-deoxyuridine 5'-monophosphate, by parenchymal liver cells revealed that the ligand was immediately internalized and, after a 10-min lag phase, deacetylated. Internalization and degradation did not occur in the presence of 100 mM N-acetyl galactosamine. In conclusion, the bioavailability of 5-iodo 2'-deoxyuridine 5'-monophosphate to the parenchymal liver cell is dramatically enhanced as a result of the conjugation of the anti-viral drugs to lactosylated poly-L-lysine. Accordingly, lactosylated poly-L-lysine constitutes a suitable carrier for targeting anti-viral drugs to the parenchymal liver cell.(ABSTRACT TRUNCATED AT 400 WORDS)

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