Erik T. Rump

Low-density lipoprotein receptor-mediated delivery of a lipophilic daunorubicin derivative to B16 tumours in mice using apolipoprotein E-enriched liposomes.

Many tumours express relatively high levels of low-density lipoprotein (LDL) receptors on their membranes. The LDL receptor is, therefore, an attractive target for the selective delivery of antineoplastic drugs to tumour cells. We reported previously on the synthesis of small apolipoprotein E (apoE)-containing liposomes that behave in vivo in a very similar way to native LDL. In this study, we examined the interaction of this liposomal carrier with cultured B16 melanoma cells. Binding of apoE liposomes to the cells is saturable, with a maximum binding of approximately 90000 particles per cell. Cross-competition studies indicated that apoE liposomes are bound by the LDL receptor. Association of apoE liposomes to B16 cells is strictly Ca2+ dependent, which forms additional evidence for a role of the LDL receptor. The affinity of apoE liposomes for the LDL receptor on B16 cells is 15-fold higher than that of LDL (0.77 vs 11.5 nM respectively). ApoE is essential for the LDL receptor recognition because liposomes lacking apoE were, in competition studies, 20- to 50-fold less effective than apoE-containing liposomes. We examined in B16 tumour-bearing mice the tumour-localizing properties of apoE liposomes and the disposition of an incorporated lipophilic derivative of daunorubicin (LAD). Tissue distribution studies showed that LAD-loaded apoE liposomes were taken up and processed by the major LDL receptor-expressing organs (i.e. adrenals, liver and spleen). Of all other tissues, the tumour showed the highest uptake. The distribution patterns of LAD-loaded apoE liposomes and native LDL in the tumour-bearing mice were very similar, which supports the role of the LDL receptor in the disposition of the prodrug-loaded particles. The disposition of LAD followed the pattern of the liposomal carrier. We conclude that apoE liposomes enable LDL receptor-mediated specific delivery of antineoplastic (pro)drugs to tumours, and, therefore, constitute an attractive novel option for anti-tumour chemotherapy.

Synthesis of a lipophilic daunorubicin derivative and its incorporation into lipidic carriers developed for LDL receptor-mediated tumor therapy.

AbstractPurpose. Many tumors express elevated levels of LDL receptors (apoB, E receptors) on their membranes. Selective delivery of anti-neoplastic drugs to tumors by incorporation of these drugs into LDL or LDL-resembling particles should improve the efficacy of tumor therapy and minimize the severe side-effects. Since the apolipoproteins on the particles are essential for the LDL receptor recognition, drugs should preferably be incorporated into the lipid moiety. Most anti-tumor agents are too hydrophilic for incorporation into these carriers. Methods. We synthesized LAD, a lipophilic prodrug of daunorubicin, by coupling the drug via a lysosomally degradable peptide spacer to a cholesteryl oleate analog. Results. The overall yield of the synthesis was 50% with a purity of >90%. Radioactively ([3H]) labeled LAD was obtained via a slightly modified procedure (yield 40%). The octanol/water partition coeffient of LAD is 30-fold higher than that of daunorubicin. LAD could be incorporated into triglyceride-rich lipid emulsions and small liposomes, which, if provided with apoE, have been demonstrated earlier to be cleared in vivo via the LDL receptor. The liposomes contained approximately 10 molecules of LAD per liposomal particle. Analysis of differently sized LAD-containing emulsions suggests that LAD associates with the surface of lipidic particles. In the presence of human serum, LAD did not dissociate from the emulsion particles, indicating a firm association of LAD with the carrier. Conclusions. The coupling of a cholesterol ester analog to daunorubicin results in a lipophilic prodrug that can be firmly anchored into lipidic carriers. LAD-loaded emulsions and liposomes provided with recombinant apoE will be tested in the near future for their ability to deliver LAD to tumor tissue in vivo via the LDL receptor.

[23] Targeted delivery of antisense oligonucleotides to parenchymal liver cells in vivo

Publisher Summary Oligodeoxynucleotides (ODNs) inhibit gene expression at various levels both in vitro and in vivo. In vivo , the efficacy of ODN-induced regulation of genes in specific cell types may be suboptimal owing to poor accumulation of ODNs in these cells. In addition, untimely elimination of ODNs through renal clearance, degradation, and scavenger receptor-mediated uptake may further impair their therapeutic activity. These hurdles can be at least partly overcome by targeted delivery of the ODNs to the desired site of action. As short (and in particular uncharged) oligothymidinylates are poor substrates for hepatic scavenger receptors that are responsible for the rapid elimination of ODNs by cells of the reticuloendothelial system, the results leave unanswered whether longer, charged, and miscellaneous ODN sequences can also be redirected to the aimed target cell in vivo. In this respect, it is crucial to analyze the tissue distribution and identify the cellular uptake sites within the liver, using full-length antisense sequences. In the study described in the chapter, in vivo evidence is provided that untimely elimination of a miscellaneous 20-mer ODN by the preceding scavenger pathways can be circumvented and, concomitantly, accumulation by parenchymal liver cells can be enhanced after derivatization with a small-sized synthetic galactoside with high affinity for the asialoglycoprotein receptor.

Carrier-Mediated Delivery of 9-(2-Phosphonylmethoxyethyl)Adenine to Parenchymal Liver Cells: a Novel Therapeutic Approach for Hepatitis B

ABSTRACT 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. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3α-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [3H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% ± 7.7% of the dose (free [3H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [3H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% ± 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.

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. When the complexes were incubated with a mixture of high-density lipoprotein and albumin, or with rat plasma, the oleoyl steroid-conjugated ODNs appeared to be more stably associated with LDL than the cholesteryl-conjugated ODN. Intravenously injected free lipid-ODNs were very rapidly cleared from the circulation of rats. The area under the curve (AUC) of the lipid-ODNs in plasma was <0.4 microg x min/mL. After complexation with LDL, plasma clearance of the lipid-ODNs was delayed. This was most evident for ODN-5, the ODN conjugated with the oleoyl ester of lithocholic acid (AUC = 6.82 +/- 1.34 microg x min/mL). The AUC of ODN-4, a cholesteryl-conjugated ODN, was 1.49 +/- 0.37 microg x min/mL. In addition, the liver uptake of the LDL-complexed lipid-ODNs was reduced. The lipid-ODNs were also administered as a complex with lactosylated LDL, a modified LDL particle that is selectively taken up by the liver. A high proportion of ODN-5 was transported to the liver along with lactosylated LDL (69.1 +/- 8.1% of the dose at 15 min after injection), whereas much less ODN-4 was transported (36.6 +/- 0.1% of the dose at 15 min after injection). We conclude that the oleoyl ester of lithocholic acid is a more potent lipid anchor than the other steroid lipid anchors. Because of the stable association, the oleoyl ester of lithocholic acid is an interesting candidate for tumor targeting of anticancer ODNs with lipoproteins.

Synthesis of a Lipophilic Prodrug of 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) and Its Incorporation into a Hepatocyte-Specific Lipidic Carrier

AbstractPurpose. 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-3α-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-3α-oleate results in a lipophilic prodrug that readily associates with LacNeoHDL. The association of the prodrug does not affect the physico-chemical 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.

Conjugation of Oligonucleotides with Steroid Structures and Their Interaction with the Low-Density Lipoprotein

Abstract To induce association with low-density lipoprotein (LDL), antisense oligonucleotides were derivatized at their 3′ terminus with several steroid structures. It was found that only highly lipidic steroids can complex the oligonucleotide with LDL.

Lipophilic Derivatization of the Antiviral Drug 9-(2-Phosphonylmethoxyethyl)adenine and Its Incorporation into a Lactosylated Lipid Carrier to Improve Its Liver Uptake

Abstract Lipophilic derivatization of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) with lithocholic acid-3-oleate and its subsequent incorporation into a lactosylated lipid carrier was found to substantially increase uptake of the drug by the liver. Competition experiments with asialofetuin point to a major role of the parenchymal liver cell, the main site of hepatitis B virus infection.

Modification of a Phosphorothioate Oligonucleotide with Cholesterol Induces Association of the Oligonucleotide to Serum Lipoproteins and Affects Its Biological Fate

Abstract A cholesterol-conjugated phosphorothioate ICAM-1 antisense oligo-nucleotide was evaluated for its binding to lipoproteins and its biodistribution. Our study indicates that the conjugate behaves differently from the parent compound.

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). The phosphorothioate analog is the most extensively studied, and phosphorothioate oligodeoxynucleotides have been shown to be potent inhibitors of the expression of their target genes in vitro and in vivo (1,3). However, phosphorothioate oligodeoxynucleotides also bind avidly and nonspecifically to proteins, thus provoking a variety of non-antisense effects (4). Oligonucleotide analogs that do not bind to proteins are therefore expected to display less nonantisense side effects. However, protein binding also affects the in vivo disposition of oligonucleotides. Nonphosphorothioate oligonucelotide analogs generally do not bind to serum proteins, and are therefore rapidly cleared from the circulation, protein-bound phosphorothioate oligodeoxynucelotides circulate much longer (5,6).