Oligonucleotides conjugated to protein-binding drugs

Details Oligonucleotides conjugated to protein-binding drugs Oligonucleotides conjugated to protein-binding drugs

1999-06-15

2003-12-02

LeGuyader, John L. (Department: 1635)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500

Reexamination Certificate

active

06656730

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to ligand-conjugated oligomeric compounds which bind to protein molecules and possess enhanced pharmacokinetic properties. The present invention further relates to methods for increasing the concentration of oligomeric compounds in serum and methods for promoting the cellular uptake of oligomeric compounds in cells.
BACKGROUND OF THE INVENTION
Protein synthesis is directed by nucleic acids through the intermediacy of messenger RNA (mRNA). Antisense methodology is the complementary hybridization of relatively short oligonucleotides to mRNA or DNA such that the normal, essential functions, such as protein synthesis, of these intracellular nucleic acids are disrupted. Hybridization is the sequence-specific hydrogen bonding via Watson-Crick base pairs of oligonucleotides to RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
The naturally-occurring events that provide the disruption of the nucleic acid function, discussed by Cohen (
Oligonucleotides: Antisense Inhibitors of Gene Expression,
CRC Press, Inc., 1989, Boca Raton, Fla.) are thought to be of two types. The first, hybridization arrest, describes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (Miller et al. (1987)
Anti
-
Cancer Drug Design,
2:117-128), and &agr;-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
Another means by which antisense oligonucleotides disrupt nucleic acid function is by hybridization to a target mRNA, followed by enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Considerable research is being directed to the application of oligonucleotides and oligonucleotide analogs as antisense agents for diagnostics, research applications and potential therapeutic purposes. One of the major hurdles that has only partially been overcome in vivo is efficient cellular uptake which is severely hampered by the rapid degradation and excretion of oligonucleotides. The generally accepted process of cellular uptake is by receptor-mediated endocytosis which is dependent on the temperature and concentration of the oligonucleotides in serum and extra vascular fluids.
Efforts aimed at improving the transmembrane delivery of nucleic acids and oligonucleotides have utilized protein carriers, antibody carriers, liposomal delivery systems, electroporation, direct injection, cell fusion, viral vectors, and calcium phosphate-mediated transformation. However, many of these techniques are limited by the types of cells in which transmembrane transport is enabled and by the conditions needed for achieving such transport. An alternative that is particularly attractive for the transmembrane delivery of oligonucleotides is modification of the physicochemical properties of oligonucleotides via conjugation to a molecule that facilitates transport. Another alternative is to increase the stability of oligonucleotides in serum, thereby increasing their concentration and distribution.
It has been previously reported that oligonucleotides modified with a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine reactive functionality at a 5′-phosphate position react with albumin and immunoglobulins M and G (Yu et al.,
FEBS Letters,
1994, 334:96-98). Binding to albumin was weak at about 20 &mgr;M with immunoglobulin binding stronger at about 4 to 6 &mgr;M. This study further reported that oligonucleotides conjugated to steroids had increased affinity for blood cells and thus changed their distribution and increased their lifetime in serum.
One method for increasing membrane or cellular transport of oligonucleotides is the attachment of a pendant lipophilic group. Ramirez et al. (
J. Am. Chem. Soc.,
1982, 104:5483) introduced the phospholipid group 5′-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl) into the dimer TpT independently at the 3′ and 5′ positions. Subsequently Shea et al. (
Nuc. Acids Res.,
1990, 18:3777) disclosed oligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linked to a 5′-phosphate on the 5′-terminus of the oligonucleotide. Certain of the Shea et al. authors also disclosed these and other compounds in patent application PCT/US90/01002. A further glucosyl phospholipid was disclosed by Guerra et al.,
Tetrahedron Letters,
1987, 28:3581.
In other work, a cholesteryl group was attached to the internucleotide linkage between the first and second nucleotides (from the 3′ terminus) of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013 and further in Letsinger et al.,
Proc. Natl. Acad. Sci. USA,
1989, 86:6553. Additional approaches to the delivery and study of oligonucleotides have involved the conjugation of a variety of other molecules and reporter groups. The aromatic intercalating agent anthraquinone was attached to the 2′ position of a sugar fragment of an oligonucleotide as reported by Yamana et al. (
Bioconjugate Chem.,
1990, 1:319), Lemairte et al. (
Proc. Natl. Acad. Sci. USA,
1986, 84:648) and Leonetti et al. (
Bioconjugate Chem.,
1990, 1:149).
Lysine and polylysines have also been conjugated to oligonucleotides to improve their charge-size characteristics. The poly(
L
-lysine) was linked to the oligonucleotide via periodate oxidation of the 3′-terminal ribose followed by reduction and coupling through a N-morpholine ring. Oligonucleotide-poly(
L
-lysine) conjugates are described in European Patent application 87109348.0. In this instance, the lysine residue was coupled to a 5′ or 3′ phosphate of the 5′ or 3′ terminal nucleotide of the oligonucleotide. A disulfide linkage has also been utilized at the 3′ terminus of an oligonucleotide to link a peptide to the oligonucleotide. See, Corey and Schultz,
Science,
1987, 238:1401; Zuckermann et al.,
J. Am. Chem. Soc.,
1988, 110:1614; and Corey et al.,
J. Am. Chem. Soc.,
1989, 111:8524.
A linking reagent for attaching biotin to the 3′-terminus of an oligonucleotide has also been described. Nelson et al.,
Nuc. Acids Res.,
1989, 17:7187. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commercially available from Clontech Laboratories (Palo Alto, Calif.) under the name 3′-Amine on. It is also commercially available under the name 3′-Amino-Modifier reagent from Glen Research Corporation (Sterling, Va.). This reagent was also utilized to link a peptide to an oligonucleotide as reported by Judy et al. (
Tetrahedron Letters,
1991, 32:879). A similar commercial reagent (actually a series of such linkers having various lengths of polymethylene connectors) for linking to the 5′-terminus of an oligonucleotide is 5′-Amino-Modifier C6. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or similar ones were utilized by Krieg et al. (
Antisense Research and Development,
1991, 1:161) to link fluorescein to the 5′-terminus of an oligonucleotide. Other compounds of interest have also been linked to the 3′-terminus of an oligonucleotide. Asseline et al. (
Proc. Natl. Acad. Sci. USA,
1984, 81:3297) describe linking acridine on the 3′-terminal phosphate group of an poly (Tp) oligonucleotide via a polymethylene linkage. Haralambidis et al. (
Tetrahedron Letters,
1987, 28:5199) report building a peptide on a solid state support and then linking an oligonucleotide to that peptide via the 3′ h

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