Design and synthesis of a multivalent homing device for targeting to murine CD22.

CD22 is a cell-surface glycoprotein uniquely located on mature B-cells and B-cell derived tumour cells. Current evidence suggests that binding of endogenous ligands to CD22 leads to modulation of B-cell activation by antigen. Incidentally, however, B-cell activation may derail.

Modification of the plasma clearance and liver uptake of steroid ester-conjugated oligodeoxynucleotides by association with (lactosylated) low-density lipoprotein.

Low-density lipoprotein (LDL) has been proposed as carrier for the selective delivery of anticancer drugs to tumor cells. We reported earlier the association of several lipidic steroid-conjugated anticancer oligodeoxynucleotides (ODNs) with LDL. In the present study, we determined the stability of these complexes.

A structure-function study of ligand recognition by CD22beta.

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation.

Interactions of lipid-oligonucleotide conjugates with low-density lipoprotein.

The ability of antisense oligonucleotides to interdict, sequence-specifically, the expression of pathogenic genes affords an exciting new strategy for therapeutic intervention (1-3). Oligonucleotides with physiological phosphodiester internucleotide bonds are rapidly degraded, predominantly by exonucleases. Numerous oligonucleotide analogs have therefore been synthesized to confer resistance toward nuclease activity (3).

alpha3-galactosylated glycoproteins can bind to the hepatic asialoglycoprotein receptor.

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose-specific hepatic lectin, the 'asialoglycoprotein receptor'. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galalpha1-->3Galbeta1-->4R is present on glycoprotein glycans.

Novel hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine with improved pharmacokinetics and antiviral activity.

The device of new hepatotrophic prodrugs of the antiviral nucleoside 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with specificity for the asialoglycoprotein receptor on parenchymal liver cells is described. PMEA was conjugated to bi- and trivalent cluster glycosides (K(GN)(2) and K(2)(GN)(3), respectively) with nanomolar affinity for the asialoglycoprotein receptor. The liver uptake of the PMEA prodrugs was more than 10-fold higher than that of the parent drug (52+/-6% and 62+/-3% vs.

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation.

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.

Apolipoprotein E is resistant to intracellular degradation in vitro and in vivo. Evidence for retroendocytosis.

Apolipoprotein E (apoE) is an important determinant for the uptake of triglyceride-rich lipoproteins and emulsions by the liver, but the intracellular pathway of apoE following particle internalization is poorly defined. In the present study, we investigated whether retroendocytosis is a unique feature of apoE as compared with apoB by studying the intracellular fate of very low density lipoprotein-sized apoE-containing triglyceride-rich emulsion particles and LDL after LDLr-mediated uptake. Incubation of HepG2 cells with [(3)H]cholesteryl oleate-labeled particles at 37 degrees C led to a rapid release of [(3)H]cholesterol within 30 min for both LDL and emulsion particles.

Carrier-mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to parenchymal liver cells: a novel therapeutic approach for hepatitis B.

Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier.

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.

Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo.

Anti-sense oligodeoxynucleotides (ODNs) hold great promise for correcting the biosynthesis of clinically relevant proteins. The potential of ODNs for modulating liver-specific genes might be increased by preventing untimely elimination and by improving the local bioavailability of ODNs in the target tissue. In the present study we have assessed whether the local ODN concentration can be enhanced by the targeted delivery of ODNs through conjugation to a ligand for the parenchymal liver cell-specific asialoglycoprotein receptor.

Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor.

A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells.

Preparation of conjugates of oligodeoxynucleotides and lipid structures and their interaction with low-density lipoprotein.

The high expression level of receptors for low-density lipoprotein (LDL) on tumor cells makes LDL an attractive carrier for selective delivery of drugs to these cells. The aim of this study is to allow incorporation of oncogene-directed antisense oligodeoxynucleotides (ODNs) into the lipid moiety of LDL. Therefore, ODNs were conjugated with oleic acid, cholesterol, and several other steroid lipids.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.