Targeted oligonucleotide conjugates

Details Targeted oligonucleotide conjugates Targeted oligonucleotide conjugates

1998-06-16

2001-10-09

Riley, Jezia (Department: 1656)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S006120, C536S022100, C536S023100, C536S024500, C536S025300, C514S001000

Reexamination Certificate

active

06300319

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to complex compounds and methods for using such complex compounds. The compounds of the invention are preferably used in methods for targeting cellular receptors that facilitate endocytic processes. The present invention takes advantage of this receptor targeting to enhance the intracellular uptake of biologically active compounds for therapeutic purposes.
BACKGROUND OF THE INVENTION
The use of synthetic, short, single stranded oligonucleotide sequences to inhibit gene expression has evolved to the clinical stage in humans. It has been demonstrated that incorporation of chemically modified nucleoside monomers into oligonucleotides can produce antisense sequences which can form more stable duplexes and can have high selectivity towards RNA (Frier et al.,
Nucleic Acids Research,
1997, 25, 4429-4443). Two modifications that have routinely given high binding affinity together with high nuclease resistance are phosphorothioates and methylphosphonates.
There are a number of desirable properties such as specificity, affinity and nuclease resistance that oligonucleotides should possess in order to elicit good antisense activity. The ability to selectively target and be taken up by diseased cells is another important property that is desirable in therapeutic oligonucleotides. Natural oligonucleotides are polyanionic and are known to penetrate cells at very low concentrations. Neutral oligonucleotides, such as the methylphosphonates, are taken up by cells at much higher concentrations. Although the processes by which antisense oligonucleotides enter the cell membrane are not well understood, there is substantial evidence for distinct mechanisms of cell entry based on the electronic character of the antisense sequence.
Delivery of an antisense oligonucleotide to a specific, diseased cell is a very important area of active research. The majority of projected antisense therapies are for viral infections, inflammatory and genetic disorders, cardiovascular and autoimmune diseases and significantly, cancer. For example, in conventional chemotherapy, neoplasticity and virus-related infections are treated with high drug concentrations, leading to overall high systemic toxicity. This method of treatment does not distinguish between diseased cells and healthy ones.
In the treatment of cancers, the ability of antisense agents to down-regulate or inhibit the expression of oncogenes involved in tumor-transforming cells has been well documented in culture and animal models. For example, antisense inhibition of various expressed oncogenes has been demonstrated in mononuclear cells (Tortora et al.,
Proc. Natl. Acad. Sci.,
1990, 87, 705), in T-cells, in endothelial cells (Miller et al., P.O.P.,
Biochimie,
1985, 67, 769), in monocytes (Birchenall-Roberts et al., Suppl. 1989, 13 (P.t. C), 18), in reticulocytes (Jaskulski, et al., Science 1988, 240, 1544)and in many other cell types, as generally set forth in Table 1.
TABLE 1
INHIBITION OF MAMMALIAN GENE EXPRESSION
INHIBITION OF EXPRESSION
CELL TYPE
T cell receptor
T cells
Colony-stimulating factors
Endothelial cells
&bgr;-Globin
Reticulocytes
Multiple drug resistance
MCF-1 cells
cAMP kinase
HL-60 cells
bcl-2
L697 cells
c-myb
Mononuclear cells
c-myc
T-lymphocytes
Interleukins
Monocytes
Virally infected cell cultures and studies in animal models have demonstrated the great promise of antisense and other oligonucleotide therapeutic agents. Exemplary targets from such therapy include eukaryotic cells infected by human immunodeficiency viruses(Matsukura et al.,
Proc. Natl. Acad. Sci.,
1987, 84, 7706; Agrawal et al.,
Proc. Natl. Acad. Sci.,
1988, 85, 7079), by herpes simplex viruses (Smith et al., P.O.P.,
Proc. Natl. Acad. Sci.,
1986, 83, 2787), by influenza viruses (Zerial et al.,
Nucleic Acids Res.,
1987, 15, 9909) and by the human cytomegalovirus (Azad et al.,
Antimicrob. Agents Chemother.,
1993, 37, 1945). Many other therapeutic targets also are amenable to such therapeutic protocols.
The use of non-targeted drugs, to treat disease routinely causes undesirable interactions with non-diseased cells (Sidi et al.,
Br. J. Haematol.,
1985, 61, 125; Scharenberg et al.,
J. Immunol.,
1988, 28, 87; Vickers et al.,
Nucleic Acids Res.,
1991, 19, 3359; Ecker et al.,
Nucleic Acids Res.,
1993, 21, 1853). One example of this effect is seen with the administration of antisense oligonucleotide in hematopoietic cell cultures that exhibit non-specific toxicity due to degradative by-products.
Other research efforts suggest that antisense oligonucleotides possess more side effects in both in vitro and in vivo animal models. For example, non-complementary DNA sequences have been shown to interfere with cell proliferation and viral replication events through unknown mechanisms of action (Kitajima ibid) . This reinforces the desirability of oligonucleotides that are specifically targeted to diseased cells.
When phosphodiester oligonucleotides are administered to cell cultures, a concentration of typically about 1 mmol is required to see antisense effects. This is expected since local endonucleases and exonucleases cleave these strands effectively and only 1-2% of the total oligonucleotide concentration becomes cell-associated (Wickstorm et al.,
Proc. Natl. Acad. Sci.
1988, 85, 1028; Wu-Pong et al.,
Pharm. Res.
1992, 9, 1010). If chemically modified oligonucleotides, such as the phosphorothioates or methylphosphonates are used, the observed antisense effects are anywhere between 1 and 100 &mgr;M. This observed activity is primarily due to the relatively slow cellular uptake of oligonucleotides. There is evidence which suggests that a 80 kiloDalton (kDa) membrane receptor mediates the endocytic uptake of natural and phosphorothioate oligonucleotides in certain type of cells. Other data question the existence of such a link between receptor-mediated oligonucleotide uptake and internalization of oligonucleotides. For example, inhibitors of receptor-mediated endocytosis have no effect on the amount of oligonucleotide internalized in Rauscher cells (Wu-Pong et al.,
Pharm. Res.,
1992, 9, 1010). For uncharged methylphosphonates, it was previously believed that internalization of such agents occurred by passive diffusion (Miller et al.,
Biochemistry
1981, 20, 1874). These findings were disproved by studies showing that methylphosphonates take up to 4 days to cross phospholipid bilayers, which correlates well with the fate of internalization of natural oligonucleotides (Akhtar et al.,
Nucleic Acids Res.,
1991, 19, 5551).
Increased cellular uptake of antisense oligonucleotides by adsorptive endocytosis can be obtained by liposome encapsulation. In one study, researchers showed that a 21-mer complementary to the 3′-tat splice acceptor of the HIV-1 was able to markedly decrease the expression of a p24 protein while encapsulated into a liposome containing diastearoylphosphatidylethanol-amine (Sullivan et al.,
Antisense Res. Devel.,
1992, 2, 187). Many other examples have been reported, including pH-sensitive liposomes (Huang et al.,
Methods Enzymol.,
1987, 149, 88) which are well detailed in several good review articles (Felgner et al.,
Adv. Drug Delev. Rev.,
1990, 5, 163 and Farhood et al.,
N.Y. Acad. Sci.,
1994, 716, 23). When phosphorothioate oligonucleotides, that are complementary to the methionine initiation codon of human intracellular adhesion molecule-1, were encapsulated, a 1000-fold increase of antisense potency was seen relative to the non-encapsulated phosphorothioate oligonucleotide (Bennett et al.,
Mol. Pharmacol.,
1992, 41, 1023). The oligonucleotide delivery systems are good for in vitro cell systems, but have not been shown to be widely applicable to in vivo studies, due to rapid liposome destabilization and non-specific uptake by liver and spleen cells.
Other, non-specific oligonucleotide uptake enhancements attend attaching hydrophobic cholesterol (Letsinger et al.,
Proc. Natl. Acad. Sci.,
1989, 86, 6553) type or phospholipid type molecules (Shea et al.,
Nucleic Acids Re

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