CA2191777A1 - Cyclodextrin cellular delivery system for oligonucleotides - Google Patents

Abstract

Disclosed is a composition including an oligonucleotide complexed with a cyclodextrin. The oligonucleotide may be noncovalently associated with the cyclodextrin. Alternatively, the oligonucleotide may be covalently complexed with adamantane which is noncovalently associated with the cyclodextrin. Also disclosed are methods of enhancing the cellular uptake and intracellular concentration of oligonucleotides, methods of increasing the solubility of an oligonucleotide in a cell, and methods of treating a cell for viral infection or to prevent viral infection.

Description

~ wo 9sl32739 2 1 q 1 7 7 7 ~ f~16 cyrT~ ~RT.T.m.~T DELIVERY

BACKGR0U~P OF ~HE INVENTI0N

This invention relates to antisense therapy.
More particularly, this invention relates to COmpOsitiQns and methods for Pnh~n~l ng- the ~Pl 1 nl ~r uptake of antisense oligonucleotides.

New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression. These agents, called antisense oligonucleotides, are sI~gle-stranded oligonucleotides which bind to a target nucleic acid molecules according to the Watson-Crick or Hoogsteen rule of base pairing, and in doing so, disrupt the function of the target by one of several r ~~An;,G~~ by preventing the binding of factors re~uired for normal transcription, splicing, or translation; by triggering the enzymatic destruction of RNA by RNase H, or by destroying the target via reactive groups attached directly to the AntiGtpn~G~e oligonucleotide. Thus, they have become widely used research tools for inhibiting gene expression sequence 8pecifically, and are under investigation for possible use as therapeutic agents (see, e.g., Agrawal et al.
( Proc . Natl. Acad. Sci. (USA) (1993) 90:3860-3884); Bayever et al. (1992) Antisense Res. DG~

2:109-110).

In order for antisense molecules to have therapeutic value, they must have the ability to W095/32739 21 9 ~ '7 7~ : F~~ 6916 ~

enter a cell and coL~act target endogenous nucleic acids. Furthermore, they must be able to withstand the rigors of the highly nucleolytic environment of the cell.
Recent studies have shown that oligonucleotides with certain modifications, such as artificial ;ntprnncleotide linkages, not only render the oligonucleotides resistant to nucleolytic degradation (see, e g., Agrawal et al.
(1988) Proc. Natl. Acad. Sci. (USAJ 85:7079-7083;
Agrawal et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:7790-7794; Gao et al. (1990) Antimicrob. Agents Chem. 34:808; and Storey et al. ~1991) Nucleic Acids Res. 19:4109), but also may increase ~Pllnl~r uptake of the oligonucleotide. For example, oligonucleotides with phosphorothioate or methyl~hnsrhnn~te ;ntprnnrleotide linkages have been found to bind to, and to be taken up by cells more readily than phosphodiester-linked olig~n-l~l~ntides (Zhao et a. (1993) Anti~ense Res.
De~. 3:53-56).

Oligonucleotide uptake i8 saturable, sequence-;n~p~n~nt, and temperature and energy dependent. While there is some eviaence to suggest that such uptake may occur through a 80,000 dalton membrane protein (Loke et al. (1989) Proc. Natl . Acad. Sci. (USA) 86:3474; Yakubov et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:6454), the gene for this protein has not yet been cloned or characterized. One study suggests int~rn~l;7~t;nn of the oligonucleotide is by a caveolar, protocytotic I ~h~n;~m rather than by ~ ~ossl32739 2 19 1 ~7 7 7 r~ 916 endocytosis ~Zamecnick (199~) Proc. Natl. Acad.
Sci. (USA) 91:3156). Whether oligonucleotides are ;n~rn~l; 7ed via a receptor-mediated endocytotic pathway, a pinocytic m~h~ni~-, or a combination of both remain~ poorly understood.

To improve on the cellular uptake of oligonucleotides, the oligonucleotides have been modified in ways other than those described above.
Eor example, WO 9323570 discloses an oligonucleotide with improved cellular uptake having at least one nucleotide residue covalently linked at its 2' position with various molecules including an amino acid, polypeptide, protein, sugar, sugar phosphate, neurotransmitter, hormone, cyclodextrin, starch, steroid, or vltamin.
Enhanced cellular uptake of biotinylated oligonucleotide in the presence of avidin has also been demonstrated (Partridge et al. (1991) FEPS ~ett. 288:30-32).

In addition, phosphodiester-linked oligodeoxy-nucleotides have been introduced into cells by t~e pore-forming agent streptolysin O
(Barry et al. (1993) Biotechnigues 15:1016-1018), and a liposomal preparation including cationic lipid has been shown to enhance the cellular uptake of antisense molecules targeted to a portion of the human intercellular adhesion molecule (Bennett et al. (1992) ~ol. Pharmacol.
41:1023-1033). Phosphodiester-linked oligonucleotides bearing a 5'-cholesteryl modification show increased cellular uptake and biological effects (Krieg et al. (1993) Proc.

WO 95/32739 ~ 1 9 1 7 7 7 r~ c 916 Natl . Acad. Sci . (USA) 90:1048). Antibody-targeted liposomes have also been used to enhance the cellular uptake of ol;r,nnnrlrotide~ targeted to H~A class I molecules expressed by HIV-infected cells (Zelphati et al. (1993) An~isense ~e~. Dev.

3:323-338).

However, insufficient uptake of modified and unmodified oligonucleotides remains a problem both 10 in vitro and in vivo. There is therefore a need for improved composltions and methodg for ~nh~nr; nr~
the cPl 1 nl ~r uptake of antisense oligonucleotides.
Such ~n~n~ ' would ultimately result in an increased efficacy of antisense olirinnllrl~ntides and a reduction in the dose administered.
Ideally, such compositions and methods wil~ also be useful for increasing the general solubility of nl; rJnnnrl eotide9.

SUMMARY OF THE INV~ION

It has been discovered that the uptake of antisense oligonucleotides into cells can be ~nh~nre~ by noncovalently aggociating such oligonucleotides with a cyclodOEtrin. This discovery has been exploited to produce the various compositions of the invention.

In one embodiment, the oligonucleotide is noncovalently associated directly to the cyclodOEtrin, which preferably is a beta (~
cyclodOEtrin, a gamma (~) -2-cyrl n~P~trinl a methyl substituted cyclodextrin, or a derivative thereof.
Preferred ~rl v~t; ve~ include 2-hydroxypropyl-~-~ Wogsl32739 ~ 77 7 ~ P~ 916 ~5-cyclodextrin, hydroxypropyl-~-cyclodextrin, hydroxyethyl-3-cyclodextrin, ~-cyrlo~trin ¢ polysulfate, trimethyl ~-cyclodextrin, and ~-cyclodextrin polysulfate, and methyl substituted ~ 5 cyclodextrins.

In some Pmho~; nt~ of the invention, the oligonucleotide to which the cyclodextrin is complexed contains at leagt one deoxyr;h~nnrl~otide, one ribonucleotide, or both deoxyribonucleotides and ribonucleotides (i.e., a chimeric oligonucleotide). The oligonucleotide is interconnected with phosphodiester ;ntrrmlrleotide linkages in some rmho~;m~nt~ while in others, the oligonucleotide is modified.

The term "modified oligonucleotide" i8 used herein as an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester between the 5' end of one nucleotide and the 3~ end of another nucleotide in which the 5~ nucleotide phosphate has been replaced with any number of chemical groups.
Preferable synthetic linkages include alkylphosphonates, rh~srh~te esters, alkylphosphonates, phosphorothioates, phosphorodithioates, 2-0-methyl carbonates, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidate, and carboxymethyl esters. In one preferred embodiment of the invention, the oligonucleotide comprises at least one phosphorothioate and/or one alkylphosphonate linkage.

~ ~m! ~ -The term "modified oligonucleotide~alsornrrmrcc~eg oligonucleotide3 with a modified base and/or sugar. ~or example, a 3', 5'-substituted oligonucleotide is a modified oligonucleotide having a sugar which, at both its 3~ and 5' positions is attached to a chemical group other than a hydroxyl group (at its 3~ position) and other than a phosphate group (at its 5' position).
A modified oligonucleotide may also be a capped species. In addition, unoxidized or partially r~ ; 7e~ oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides. Also rrnc;~Pred ag r-a;fi~
oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3' and~or 5~ end(s) and/or various other structural modifications not found invivo without human intervention are also considered herein as modified.

In another preferred embodiment, the oligonucleotide of the composition of the invention is covalently bonded to ~alcm~nt~np which is noncovalently associated with the cyclodextrin The covalent association is between the 3'-hydroxyl or the 5'-amino of the oligonucleotide and the adamantane. In other embodiments where the oligrnllrl~ntide r~ntc;nc a ribonucleotide, the 3~ ~alcr-nt~n~ ig covalently associated with the 2'-hydroxyl of the ribonucleotide.

This invention also provides a pharmaceutical formulation including the cyclodextrin-compIexed ~ W095l32739 ~ 7~ P~l/u ~ 6 oligonucleotide composition, preferably in a physiologically acceptable carrier. Such a formulation :iB useful in a method of increasing the cellular uptake, and thus, of ~nh~nr1ng the intracellular rnnr~ntrationl of an exogenous oligonucleotide. The formulation i5 also used in a method of treating a cell, for example, for viral infection, or to prevent a viral infection.

Also provided by this invention are methods of increasing the in vivo availability of an oligonucleotide by complexing it to a cyclodextrin.

In another aspect of the invention, pharmaceutical formulations are provided which contain the oligonucleotide composition described above. These formulations are used in another aspect of the invention, namely, methods of increasing the c~l 1 nl ~r uptake and intracellular cnnr~n~ration of an exogenous oligonucleotide. In these methods, a cell is treated with the pharmaceutical f~L, l5tion~

In yet another aspect of the invention, a method of treating a cell for viral infection, or for the prevention of viral infection, is provided. In this method, a cell is contacted with a pharmaceutical formulation cnnt~lnlng an oligonucleotide having a nucleotide sequence complementary to a portion of the nucleic acid of a virus. Thus, the invention provides a useful composition for treating inadvertently infected cell culture lines. rnnt~m;r~tion of cell lines W09~32739 ~1 91 7 7 7 r~ 6916 with mycoplasma or viruses can be eliminated by using the compositions according to the invention =

The invention also provides methods of ~
increasing the solubility of an oligonucleotide in vlvo, including the step of noncovalently complexing a cyrl o~ rin to an ol; r~nnrl eotide.
In some embodiments, the oligonucleotide is covalently complexed with ~r-nt~n~, and 10 ~r~n~n~ is noncovalently complexed to the cyclodextrin.

~ W095132739 ~ :2 l 9 ! 7 7 7~ . ", ~ r~sl6 pl~TI;!~ IJ~3~ , OF ~T~ n~wTr~R

The foregoing and other objects of the present invention, the various ~eature6 thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

FIG. lA is a schematic repres~nt~t;cn of 2-11YdLU~Y~ u~ -cyclodextrin (C42H~0O3s);

FIG. lB is a schematic repreg~nt~t; nn Of ~-cyclodextrin (C~8H80O40);
FIG. 2 is a flow cytometry data output record showing the fluorescent intensity of cell cultures treated with (A) no oligonucleotides; (B) PS
oligonucleotide; (C) cyclodextrin-complexed PS
oligonucleotide at 4~C over night; or (D) cyclodextrin-complexed PS oligonucleotide at 25~C
for 1 hour;

FIG. 3A is a photograph of a fluorescent micrograph showing cells treated with FITC-linked 20mer PS-oligonucleotide;

FIG. 3B is a photograph of a fluorescent micrograph showing cells with FITC-linked 20mer complexed with cyclodext~in;

FIG. 3C is a photograph of a fluorescent micrograph showing cells treated with FITC-linked 42mer PS-oligonucleotide;

W09~32739 ' ~ 7 7 7 ~ l6 FIG. 3D is a photograph of a fluorescent micrograph showing cells with FITC-linked 42mer complexed with cyclodextrin;

FIG. 4 ib an autoradiograph of an SDS gel of cells extracted at various times after treatment with 32p_1 ~oh~ o1 (A) uncomplexed or ~B) cyclodextrin-complexed P0- and PS-oligonucleotides;
FIG. 5 is an autoradiograph of an SDS gel comparing the relative mobilities of 3'P-labelled uncomplexed and cy~lo~o~trin-complexed P0- and PS-oligonucleotides; and FIG. 6 i8 a graphic representation of the fluorescent intensity of cell cultures treated with FITC-labelled uncomplexed, cyclodextrin-complexed, and cyclodextrin-~r-ntAno complexed PS- oli~nnnclentides.

FIG. 7 is a schematic representation of the preparation of o~o~~nt~on~-linked cyclodextrin;

FIG. 8 is a schematic repreg~nt~tinn Of fluorescein rhnsphnramidite;

FIG. 9A is a g~h t;n repregentation of a phosphodiester linked oligonucleotide covalently linked to o~or-ntonp;

FIG. 9B i6 a schematic represenFation of a PS-oligonucleotide covalently linked to ntonloi and ~ W09~32739 ' 21 9~ 77i r~ s ~

'11 -FIG. 9C is a ~chematic repre~nt~ n of a FITC-conjugated PS-oligonucleotide covalently - linked to ~r-n~n~. -1. .2l.9.l~777 ==' D~T~TT.Tm V~S~KI~Ll~N OF T~E ~K~KK~ EM3ODIMENTS

The patent and scientific literature referred to herein estAhl;qh~q the knowledge that is available to those with skill in the art.

This invention provides oligonucleotide compositions which enhance the uptake of oligonucleotides into cells, thereby increasing the efficacy of the treatment and reducing the dose required. The compositions include an oligonucleotide complexed with a cyclodextrin or other polysaccharide.

i5 Cyclodextrins, also known as cycloamyloses, are a group of cyclic polysaccharides consisting of six to eight naturally occurring D(+)-glucopyranose units in ~-tl,4) linkage. They are classified by the number of the glucose units they contain: alpha (a)-cyclodextrin has six glucose units; beta (3)-cy~lo~tr;n has seven, and gamma (~)-cyclodextrin has eight tBrewster et al. (1989) J. Pare~eral Sci.
Technol. 43:231-240). FIGS. lA-lB show representative cyclo~P~tr; nC of these classes. Cyclodextrins as a group are cone-shaped molecules have a slightly apolar internal cavity which can accommodate the inclusion of various other molecules. Their peripheral structure co~tAinq a large number of hydroxyl groups which provide water solubility.

'P

~ wossm73s Z 1 91 7 77 ~ r~ 5~l6 50me cyclodextrins and various substituted derivatives thereof, such as hydL~y~Lu~yl-, - hydroxyethyl-, methyl-, o~ sulfate-substituted cyclodextrins, have the ability to enhance the ~ 5 solubility and availability of a variety of pharmacological agents. For example, 2-hydroxypropyl ~-cyclodextrin (HPCD) subgt~nt;~l1y enhance solubility and uptake of some sparingly soluble drugs auch as hydrophobic protein c~nt~;n;ng drugs (Brewster et al. (1991) r1~
Res. 8:792-795; Yaksh et al. (1991) LifeSci. 48:623-633) such as inaulin (Merkus et al. (1991) ri~ Res. 8:588-592), bovine growth hormone (Simpkins et al. (1991) ~. Parenteral Sci.
Technol. 45:266-269), and methyltestosterone (Muller et al. (1991) J. rl~ Sci. 80:599-604).
In addition, ethylated-~-cyclodextrin has been used as slow-release type carriers for hydrophilic drugs such as diltiazem (Horiuchi et al. (199) J.
r~ sci. 79:128-132).

Other cyclodextrins have uni~ue biological features. For example, cyclodextrin sulfates have anti-;nfl t~ry, antilipemic, and antiviral activity, and have been found to inhibit replication of HIV by either prevention of viral absorption or budding (Pitha et al. (1991) J.
r~ . Res. 8:1151-1154; Anand et al. (199) Antiviral Chem. Chemother. 1:41-46); Moriya et al.
- 30 (1991) J. Med. Chem. 34:2301-2304; Weiner et al.
(1992) Pathooiol. 60:206-212). In addition, cyclodextrin sulfates have protective e_fects on W09~32739 21 9i 777 _ ~ P~ 'C69l6 the gentamicin-induced nephrotoxicity (Uekama et al. (1993) ~. Pharm. Pharmacol. 45:745-747) and on hemolysis of erythrocytes (Weisz et=al. (1993) Biochem. Pharmacol. 45:1011-1016).
Since cyclodextrins are biocompatible polymers composed of naturally occurring D-glucose subunits, their therapeutic~application has been regarded are relatively safe. Indeed, in vivo administration of cyclodextrin ~oncentrations of 5 to 10~ has been generally used to enhance adsorption of drugs in animal studies, and no significant cytotoxic effects have been reported.
(Gerloczy et al. (1994) J. ph~r--c~ut. Sci.
83:193-196).

Besides standard intravenous administration, cyclodextrins can be easily absorbed through nasal (Merkus et al. (1991) Pharm. Res. 8:588-592; Shao et al_ (1992) Pharm. Res. 9:1157-1163), intestinal (~k~ni ~h; et al. (1992) Chem. Pharm. BU11.
40:1252-1256), corneal (Jansen et al. (1990) ~ens Eye Tox. Res. 7:459-468), rectal epithelium (Arima et al. (1992) ~. Pharm. Soc. ~apan 112:65-72), and by transdermal injection (Yoshida et al. (1990) Chem. Pharm. Bull . 3 8:176-179).

In addition, cyclodextrins have also been found to ~l;m;n~te some of the undesirable side-effects of the drugs to which they have been complexed. For example, wh~en used as a vehicle in~
orhth~lm;c eye-drop formulations, 2-hydroxypropyl-$-cyclodextrin can suppress the immune reaction to ~ W09~32739 ~~ l' 9 1 7 7 7 ,~ ql6 -~15-a corneal graft ~Arima et al. (1992) J. r~
SoC. Japan 112:65-72) ~ and i8 not toxic to the corneal epithelium.

Cy~lod~trins can be prepared~by methods known in the art (see. e.g., Moriya et al. (1993) ~. ~ed. Chem. 36:1674-1677) and are c ~ ~ially available.

The oligonucleotides to which the cyclodextrin i8 complexed are composed of deoxyribonucleotides, ribonucleotides, or a combination of both, with the 5' end of one nucleotide and the 3' end of another nucleotide : being covalently linked. These oligonucleotides are at least 6 nucleotides in length, but are preferably 10 to 50 nucleotides long, with 15 to 25mers being the most common. Oligonucleotides can be prepared by the art recognized methods such as rh~sph~amidate or X-phosphonate chemistry which can be carried out manually or by an Allt~ t~ 8ynthesizer as deccribed by Brown in A
~3rief History of Oligonucleotide Synthesis.
Protocols for Oligonucleotides and Analogs, ~ethods in ~olecular Biology (1994) 20:1-8).

The oligonucleotides of the composition may also be modified in a number of ways without ~ ising their ability to hybridize to the target nucleic acid and to complex with A~Ar-nt~n~
and/or cyclodextrin. For example, the oligonucleotides may contains other than phosphodiester ;nt~mlrleotide linkages between the 5' end of one nucleotide and the 3' end of WO95132739 - r~ 5~1 777 r~l,u~ ~916 another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups. Examples of such chemical groups include alkylphosphonates, phosphorothioates, ~hn5phnrodith;oAtp~, alkylphosphonoth;oatpcl alkylrhnsrhnnAtPc~ 2-O-methyls phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triester6. Oligonucleotides with these linkages can be prepared according to known methods (see, e.g., Sonveaux "Protecting Groups in Oligonucleotides Synthesis" in Agrawal (1994) Methods in Molecular ~iology 26:1-72; Uhlmann et al. (199C) Chem. Rev. 90: 543-583).
Other modifications include those which are inter~al or at the end(s) of the oligonucleotide molecule and include A~;t;nn~ to the molecule of the ;ntPrnncleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which blnd to the viral genome.
Examples of such modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide having a sugar which, at both its 3' and 5' positions is attached to a chemical group other than a hydroxyl group ~at its 3~ position) and other than a phosphate group (at its 5' position). Other modified oligonucleotides are capped~with a nuclease WO 95132739 . ~~ 16 ~ '7 7 7 resistance-conferring bulky substituent at their 3' and/or 5~ end(s) , or have a substitution in one nonbridging oxygen per nucleotide. Such modifications can be at some or all of the intPrnnrleogide linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule.

Oligonucleotides which are self-etabilized are also r~n~;~Pred to be modified oligonucleotides useful in the methods of the invention (Tang et al. (1993) Nucleic Acids Res.
20:2729-2735). These cligonucleotides comprise two regions: a target hybridizing region; and a self-complementary region having an oligonucleotide sequence complementary to a nucleic acid sequence that is within the self-st~h;l;7ed oligonucleotide.

The oligonucleotides complexed to the cyclodextrin can have any nucleotide sequence desired and are able to hybridize to R~A or DNA
under normal physiological conditions existing within a cell harboring the target nucleic acid.
Such conditions include pH, temperature, and ionic conditions.

The preparation of these unmodified and modified oligonucleotides is well known in the art (reviewed in Agrawal et al. (1992) TrendsPio~o ~' 10:152-158). For example, nucleotides can be covalently linked using art-recognized techniques such as phosphoramidate, H-phosphonate chemistry, or methylphosphoramidate chemistry (see, e.g., Uhlmann et al. (1990) Che~. Rev. 90:543-584; Agrawal et WO95/32739 ~ 2 .l 9 1 7 7 7 . ~ . 9~6 al. (1987) Tetrahedron. Lett. 28:(31):3539-3542);
Caruthers et al. (1987) Meth. Enzymol. 154:287-313;
U.S. Patent 5,149,798). Oligomeric phosphorothioate analogs can be prepared usi~g methods well known in the field such as methoxyphosphoramidite (see, e.g., Agrawal (1988) Proc. Natl. Acad. Scl. (USA) 85:7079-7073) or ~-phosphonate (see, e.g., Froehler, "Oligonucleotide Synthesis: X-phosphonate Approach" in Agrawal (1994) MethodsinMolecularBiology 20:63-80) chemistry.

An ~l1g~nllrleotide can be noncovalently complexed to a cyclo~tr- n by mixing them together in an aqueous solution such as a cellular growth medium or various buffers.

Alternatively, an olig~nll~ tide can be covalently linked to an ~r-nt~n~ molecule which is then noncovalently linked to the cyclodextrin.
~r-nt~n~ enters into the cavity of a cyclodextrin and forms a stable, noncovalent complex with it (Brinker et al. (1993) Angew.
Chem., Int. Ed. Engl. 32:1344-1345, Ueno et al.
(1993) J. Am. Chem. Soc. 115:12575-12576).
~ inkage oi the ~-~~'nt~n~ molecule can be accomplished at the 3~-hydroxyl or 5' amino terminus of the oligonucleotide having a (or both) deoxyribonucleotide terminal residue(s) termini.
Alternatively, A~r-nt~n~ can be covalently c~ with the 2'-hydroxyl of a ribonucleotide residue. This can be accomplished with a linker phosphoramidite or X-phosphonate as the final ~ wossl32739 ~ '91l1~77 ~ ~ r~,u~5 ~6 coupling step in machine-aided assembly of an ol; gnnl~cl eotide, as has been used for the - attachment of single reporter group3 to a synthetic oligonucleotide (see, e.g., Agrawal et al. (1986) Nucleic Acid~ Res. 14:6229-6245;
Misiura et al (1990) Nucleic Acids Res. 18:4345-4354; Nelson et al. (1992) Nucleic Acids Res.
20:6253-6259).

Covalent linkage of adamantane to the oligonucleotide can also be accomplished with the aid of an amino linker as described by Misiura et al. (J. Nucleic Acids Res. (1990) 18:4345-4353).
The A~Ar~ntAn~-linked ~ligon~ eotide is then noncovalently associated with the cyclodextrin by mixing the two in an aqueous medium or buffer (see, e.g., Simpkins et al. (1991) ~. Parental sci. & ~echnol. 45:266).

The oligonucleotide composition or therapeutic formulation including the composition iB useful in methods of increasing the cellular uptake and ~n~Anc;ng the intraceLlular concentration of an ~y~ us olig~nll~leotide, in methods of increasing the 801ubility of an oligonucleotide in vivo, and in methods of treating a cell, for example, for viral infection, or to prevent a viral infection.

That cyclodextrin-complexed oligonucleotides are taken up by cells was confirmed as follows.
Fluorescein (FITC)-conjugated phosphorothioate (PS) oligonucleotides were complexed with cyclodextrin either at 4~C, overnight or at 25~C.

W09~32739 ' ~7 t~qI 777 ~ c~916 Eluoreecein ~FITC)-conjugated phosphorothioate (PS) oligonucleotides were complexed with cyclodextrin either at 4~C, overnight or at 25~C
for 1 hour. A cultured T cell l~nk~m;~ cell line (OEM) was then contacted with the treated oligonucleotides. The fluorescent intensity of the CEM cells was measured by computer-analyzed flow cytometry Ag shown in the computer-generated scans in FIG. 2, the fluorescent intensity i8 greatly increased when the oligonucleotide is complexed with cyclodextrin, indicating that compl~Y~t; on greatly increases cellular uptake.
To ~t~rm; n~ whether the size of oligonucleotide affects the~ability of the cyclodextrin to affect cellular uptake, 20mer and 42mer FITC-conjugated PS-oligonucleotides were contacted with cyclo~ytrin at 25~C for 1 hour and then were added to CEM cells The cells were ~m; n~d under a fluorescent microscope.

As shown in the fluu~esc~llce micrographs in FIGS. 3A-3D, both the 20mer and the 42mer oligonucleotides which were complexed with cyclodextrin were taken up by the cells.
Furthermore, more fluorescent cells were ~t~ct~d after treatment with oligonucleotides complexed with cyclodextrin than after treatment with uncomplexed oligonucleotides. This indicates that oligonucleotide uptake is enhanced by complexing with cyclodextrin.

~ W095/32739 '~ '2 1 9 1 7 7 7 ~ si6 To determine if cyclodextrin complexation has an effect o~ the stability of an oligonucleotide administered to a cell, cyclodextrin-complexed 3'P-labelled PS- and PO-oligonucleotides administered to cell cultures were P~mlr~ at different times after administration. The oligonucleotides were extracted from the cells and analyzed by electrophoresis and autoradiography.

As shown in FIG. 4, PO-oligonucleotides are rapidly degraded even after 1~ minutes, while PS-oligonucleotides remain mostly intact at all times studied. Thus, PS-oligonucleotides are more stable in.cells than PO-~l;g~nllrleotides. There is no 8ign; f j r~nt difference between olig~nllrler,tides that have been complexed with cyclodextrin and uncomplexed oligonucleotides, indicating that,complexation does not affect the stability of the ol;gonllrle~tides.
To determine the effect of cyclodextrin complexation on the relative mobility of olig~nnr1entides, 32P-labelled uncomplexed or cyclodextrin-complexed PO- and PS-olig~nllrl~otides were analyzed by electrophoresis and autoradiography.

As shown in FIG. 5, there is no marked difference ~etween the mobility of oligonucleotides complexed with cyclodextrin and the mobility of uncomplexed oligonucleotides;
both complexed and uncomplexed PO oligonucleotides migrate more quickly than complexed and uncomplexed PS-oligonucleotides.

W09S132i39 ~i~1777 ~ ~ y~ c~ 6 ~

To determine whether linkage of the cy~lo~Ytrin-associated oli~nnnrl~tide to ~r-nt~n~ had an effect on th~1n nptake into cells, cells were treated for varying amounts of time with fluorescently (FITC) l~h~
oligonucleotide, fluorescently labelled, cyclodextrin-~so~tP~ oligonucleotide, or fluorescently labelled, covalently-linked r-nt:~nf~/OligOnUCleOtide AC80~ ted with cyclodextrin. The fluorescence intensity of the cells was then analyzed by flow cytometry.

A8 shown in FIG. 6, FITC oligonucleotide uptake into cells increased gradually during the time course studied. In the presence of cyclodextrin, the increase is much more dramatic, with the incrcase being the greatest with ~-~-nt~nP-linked olig~nll~l~tides (oligonucleotide-A). Thus, covalent linkage of oligonucleotides to ~r-nt~n~ Pnh~n~P~ the cellular uptake of cyrlo~trin-associated oligonucleotides.

To administer the pharmaceutical formulation of the invention, the cycloxdetrin-associated or adamantane-linked, cy~lo~trin-associated oligonucleotide is mixed with a physiologically acceptable carrier and then injected intravenously, intramuscularly, intraperitoneally, or by intranasal (Merkus et al. (1991) r~
Res. 8:588-592; Shao et al. (1992) Fl2~r - Res.
9:1157-1163), oral, transdermal, or subcutaneous administration. CyclodextrinA can also be easily ~h8~rh~ through intestinal (Nakanishi et al.

~ ~ 1 9 1 7 7 7 (1992) Chem.Pharm.Bull. 40:1252-1256), corneal (Jansen et al. (199Q) Lens Eye ~oxicity Res. 7:459-468), and rectal (Arima et al. (1992) J. r~ La~cu~ Soc. Japan 112:65-72) epithelium, and by transdermal injection (Yoshida et al. (1990) Chem. rl~/ 7~~-~ Bull. 38:176-179). Effective dosages of the oligonucleotide and - modes of its administration in the treatment of the particular disorder for which the oligonucleotide is being administered can be determined by routine experimentation. The pharmaceutical forms suitable for injectable or other use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile. It must be stable under the conditions of manufacture and storage and may be preserved against the c~nt~m; n~ting action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents. Prolonged absorption of the injectable therapeutic agents can be brought about by the use of the compositions of agents delaying absorption.

The following examples illustrate the preferred modes of making and practicing the present invention.

AMENI~
' ~ Wos~/32739 Z 1 9 1 7 7 7 r~ r sr~6 EXAMPLES

1. Preparation of Oligonucleotides PO- and PS-oligonucleotides were synthesized on an automated synthesizer (Miilipore 8700, Millipore Corp., Bedford, MA) using phosphoramidate chemistry (see Agrawal et al.
(1989) Proc. Natl. Acad. Sci. (USA) 86:7790-7794;
McBride et al. (1983) Tetrahedron Lett. 24:245).
The o~idation reagents used in the syntheses were standard solution of iodine, for phosphodiester linkages, and 3H-1,2-benzodithiole-3-one-1,1-dioxide as a solution of 1 g iu ioo ml of acetonitrile, for phosphorothioate linkages formation. Methylphosphonates were prepared according to the method of Beaucage, "Oligonucleotide Synthesis: Phosphramidite Approach" in Protocols for Oligonucleotides and Analogs, ~ehtods in ~olecular ~ology (1994) 20 :33-62). Oligonucleotide concentrations were determined by absuLb~lce at 260 nm, taking into account the molar extinction coefficient of the nucleotides present in each sequence (Ausubel et al. (eds.~ (1987) CurrentProtoco~inMolecularBiolo~
(~iley, New York)).

2. FITC-Labelling of Oligonucleotides Fluorescein (FITC) was conjugated to the oligonucleotides through the 5~-hydroxyl using a fluorescein amidite (Clontech Laboratories, Inc., Palo Alto, CA) according to the method of Schubert (Nucleic Acids ~es. (1990) 18:3427). All ~ W095132739 ~ ~ 2 1 ~9~ 7.~ P~ C .I6 oligonucleotides were deprotected by treatment with concentrated ammonia at 55~C for 12 hours.
The oligonucleotides were purified by polyacrylamide gel electrophoresis (PAGE), desalted by Sep-Pak C18 cartridges (Waters, Milliford, MA) and lyophilized to dryness prior to use .

3. Preparation of Cyclodextrins 2-hydLu~y,uLu~yl-~-cyclodextrin (HPCD) was prepared according to the method of Pitha et al.
(Int. J. Pharm. (1986) 29:73-82) or obtained commercially ~rom, e.g., Sigma ~h~mic~l Co., St.
Louis, MO). Other cyclodextrins such as ~(C36H6003o) cyclodextrin and ~- (C~8H~004o) cyclnd~tr;n are also commercially available (from, e.g., Sigma Chemical Co., St. ~ouis, MO), and l1YdL~Y~LU~Y1 ~-cyclodextrin, hydroxyethyl ~-cyclodextrin, trimethyl ~-cyclodextri~, hydLu~y~Lu~yl cyclodextrin, and sul~ated ~-cyclodextrin available from Amai~o (Hammond, IN).

4. Preparation of FITC-~abelled, HPCD-As80ciated Oligonucleotide 1 ~g of fluorescent (FITC) conjugated PS-oligonucleotides were mixed with 5 - 10% HPCD in 75 ~1 RPMI medium. The mixtures were kept either at 4~C for overnight or at 25~C for 1 hour for noncovalent complexation. (Simpkins et al. (1991) J. Parental Sci. & Technol. 45:266).

W09~32739 ~ 9 1 7 7 7~ glc 5 32p-T~hpll;ng of Oligonucleotides nl; g~n~ P~tides were 5' end labelled by incubating 200 ng oligonucleotide (PO or PS~ with 2 ~l polynucleotide kinase (Pharmacia, Piscataway, NJ), and 2 ~l of (~-'3P)ATP (Amersham LIFE
Science, Arlington Heights, IL) in a final volume of 20 ~l at 37~C for 1 hour. The mixture was passed over a Sephadex G=25 column (5 Prime-3 Prime, Boulder, CO) to separate the 32P-labelled oligonucleotide irom the unlabelled oligonucleotide.

6. Preparation of 32P-Labelled Cyclodextrin-As60ciated Oligonucleotide Half of the 33P-labelled oligonucleotide (PO
or PS), together with 7 ~g of corresponding nnl~hPlled oligonucleotide (PO or PS) were mixed with 10~ HPCD in 175 ~l of plain RPMI medium and ~et at 4~C for overnight noncovalent conjugation.
Another half of the 33P-labelled oligonucleotide was set up the same way except without HPCD in the bolution (control).

7. Preparation of Covalently-Associated Oligonucleotide/~r-nt~nP Complex A. Linker~ r~nt~n~ Complex Synthesis of an amino linker was performed according to the method of Misiura et al. (~.
~ucleic Acidc Res. (1990) 18:4345-4354) (FIG. 7).
Briefly, reaction of readily available ~olkPt~l ~ W095l32739 i ~ t 9 1 7 7 7 ~ ,6 _ . , (compound 1) with acrylonitrile in the presence of sodium hydride in terahydolfurane (THF) resulted in the addition product 2-cyanoethyl solketal (compound 2). Reduction of nitrile (c ~.ulld 2) using sodium borohydride in the presence of cobalt (II) chloride in methanolic solution gave 3-aminopropyl solketal (compound 3) which was purified by fraction distillation.

Compound 3 was reacted with l-~A~-nt~n~-carbonyl chloride to give N-adamantoyl-3-aminopropyl solketal (compound 4). More sperif;r~lly/ 5.0 g (26.42 mmole) of compound 3 was dissolved in dry dichloromethane (50 ml) under inert ai ~h~re of N~. To a solution was added dry triethylamine (4.2 ml, 3.04 g, 30.0 mmole) via syringe, following by dropwise addition of a solution of ~r~ntAn~r~rh~nyl chloride (5.2 g, 260 mmole) in 10 ml dry dichloromethane. The solution was left to stir at room temperature for 1 hour and then concentrated. The residue was dissolved in 100 ml dichloromethane and washed with saturated sodium bir~rhrn~t~ solution (3 X 50 ml) and the organic extracts were combined, stirred, and evaporated to dryness. The oily residue was purified on 8ilica gel column (300 g) and eluted with a mixture of dichlo,l ~th~n~ th~nrl in ratio 19:1, to give 8.84 g (97~) , ulld 4.
To prepare ~u...~uulld 5 (1-0-(4,4'-dimethoxytrityl)-3-0-(N-adamantoyl-3-aminopropyl) glycerol), 8.56 g, (24.35 mmole) compound 4 was dissolved in a mixture of T~F (48.7 ml) and 1 M

W09~/32739 ~l,ql 777 r~ 69,6 aqueous HCl (48.7 ml). The solution was btirred at room temperature for 30 minutes. 50 ml absolute ethanol (50 ml) was then added. The solution was concentrated, the re3idue was redissolved in 50 ml absolute ethanol, and the solution concentrated again. The resultant product was dried by co-evaporation with pyridine (2 x 50 ml) to give an oil which was redissolved in dry pyridine (150 ml). 8.25 g (24.35 mmole) 4,4~-dimethoxytrityl chloride was then added in two portions with stirring for 15 minutes. The resulting solution was left for 1 hour_ 50 ml absolute ethanol was added and the solution was concentrated. The residue was dissolved in 200 ml dichl~ th~nP and then washed with saturated sodium bi~rh~n~t~ solution (2 x 60 ml). The aqueous layer was washed with dichloromethane (2 x 30 ml) and the organic extractg were c ';n~A, dried and evaporated. The residue was chromatographed on silica column (300 g) and eluted with a mixture of dichloromethane: methanol (19:1) to give a white foamy product (9.16 g (61.3~).

Compound 5 was further attached to long chain alkylamidopropanoic acid-controlled pore glass (CPG) beads, since the carboxyl moiety could be esterified with the free hydroxyl group of compound 5 in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, using standard procedures (Damha et al. (1990) Nucleic Acids ~es. 8:3813-3821) to give compound 6. ~oading was 22.1 ~mole~g CPG.

~ wossl32739 2 1 q 1 7 7 7 ~ I/U~ _ ~ S~16 Approximately 10 mg of compound 6 was placed in a 10 ml volumetric flask and treated with 0.2 ml of HCl04-EtOH (3:2) for 1 minute to release the dimethoxytrityl group Then, 9.8 ml of acetonitrile was added and the absorbance oi light at 498 nm was measured to determine loading eff~r~iPn~y according to the equation:

A498 x 10 x 14.3)/weight CPG (mg) = /lmole/g B. Linkage of Labelled Oligonucleotide to A~l;)r-ntAnP

The pho8phodiester-linked, arlAr-ntAnP_ associated oligonucleotide (SEQ ID NO:1) (FIG.
9A), the A-lAr-ntArP associated PS-oligonucleotide (SEQ ID NO:1) (FIG. 9B), and the Arl~r-n~Ane F~so~ tpd~ EITC-conjugated PS-oligonucleotide (EIG. 9C) were cleaved off the CPG and deprotected with cr~n~ trated ammonia at room temperature for 15 hrs and then for 6 additional hours at 55~C
The 5'-ODMT protected oligonucleotides were purified on a preparative C-18 reverse phase column by elution with linear gradient of solvent A (0.1 M ammonium acetate) and solvent B (2090 0.1 M ammonium acetate ~ 8090 of acetonitrile).
Detritylation was carried out by treatment with 8090 aqueous acetic acid for 30 minutes at room temperature. The resulting fully deprotected oligonucleotides were purified once again on the same column by eluting with the same gradient as at DMT stage W09~32739 2-l9l~T77 ~ r~ 6 8. 2reparation of PITC-T.~hP1lP~, ~PCD-Associated, Adamantane-~inked Olig~nnml~tide 1 ~g of fluorescent ~FITC) conjugated, ~r~ntAn~-li~ked PS-oligon~cleotides were mixed with 10~ 2-llydru~y~ru~yl-~-cyclodextrin (HPCD) in 75 ~l plain RPMI medium. The mixture3 were kept either at 4~C for overnight or at 25~C for ~ hour _.
for noncovalent complexation. (Simpkins et al.
(1991) J. Parental Sci. & Technol. 45:266).

9. Cell Culture CEM, a human T cell leukemia cell li~e (Foley et al. ~1965) Cancer 4:522~, and H9, a human T-cell lymphoma cell line ~American Type Culture Collection, Rockville, MD, ATCC No. HTB 176) were used in these studiea. They were cultured in RPMI
medium (JRH Biosciences, Lenexa, KS) supplemented with 10~ heat inactivated (56~C for 30 min.) fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin/streptomycin solution (JRH Biosciences, Lenexa, KS) at 37~C in a 5~ C02 - 95~ 0, humidified air ; n~nhat~r, 10. Uptake of FITC-Labelled, Cyclodextrin-Associated nl;g~nn~leotide CEM cells were grown to subconfluency before experiment and resu~pended in RPMI medium ~nt~ln;ng 20~ fetal calf serum (FCS) (also ~nt~;n~ penicillin streptomycin solution, glnt~m;n~ as described before). 1 ~g FITC
oligonucleotide that had been complexed with 10 ~ Wo9~l32739 2 1- 9~i ~7 7 7 , ~ 16 HPCD or ~ _lP~ (as control) (in 75 ~l of plain RPMI media) were added to 5 x 105 CEM cells in 75 ~1 of.RPMI media ron~ining 20~ FCS. The final mixture ~nnt~;nR 1 ~g of FITC oligonucleotide, 5%
HPCD, 5 x 105 CEM cells in 150 ~l of RPMI media c~n~;n;ng lOso FCS. The cells were cultured at 37~C for 4 hours and washed with Hank's balanced salt solution (HBSS) supplemented with l~ BSA, 1%
sodium azide.
The fluorescence of CEM cells were then analyzed by flow cytometry (FACScan, Beckman-Dickson, Mountain View, CA; or Epics XL, Coulter, Hialeah, FL), and analyzed with Lysis II software (when using FACScan) or Epics XL software, version 1.5 (when using Epics XB) (Zhao et al. (1993) Antisense ~es. & Dev. 3:55).

11. Fluorescent Microscopic Studies 20mer and 42mer fluorescent conjugated PS
oligonucleotide were contacted with 5~ HPCD at 25~C for 1 hour 1 ~g oligonucleotide was added to CEM cells (5 x 105 per tube) which were then cultured at 37~C for 4 hours. At the end of the 4 hour culture, the cells were washed with FACS
washing buffer (HBSS with 1~ BSA, 1~ sodium azide), and observed under a fluorescent microscope (LH50A, Olympus, Japan) (see FIGS. 3A-3D).

WO95r32739 ~ 7 7 7 . ~ 9l6 12. ~ptake of~32P-Labelled, Cyclodextrin-Associated Oligonucleotide and Effect of Cyclodextrin on ~l; rJnmlrl Pntide-stability~ -CEM cells were grown to subconfluency before the experiment and resuspended in RPMI medium with 20~ fetal calf serum (FCS). The complex mixture (in a volume of 175 ~l plain RPMI medium) was added to 106 CEM cells (in 175 ~l of RPMI medium rnntA;n;ng 20~ FCS. The final mixture rnntA;nP~ 7 ~g unlabeled oligonucleotide, about 80 ng of 32p labelled oligonucleotide ~the recovery efficiency of the Sephadex column is 80~), 5~ HPCD in 350 ~l of RPMI medium cnntA; n; nj 1~ ~ FCS). The treated cells were then cultured at 37~C. At different 15 minute time points, 50 ~l aliquots of cell culture mixture were taken, and the oligonucleotides were extracted from cell culture in a manner similar to that described by T~ ; et al. (Anti~ense ~es.
& Dev. ~1994) 4:35). Briefly, aliquots from cell culture mixture were treated with proteinase K (2 mg/ml) final cnnrpntration) in 0.5~ sodium dodecyl sulfate ~SDS) for 20 min. at 37CC, following by phenol-chloroform extraction and ethanol precipitation. The 32p_1 AhPl 1 P~ ol; gnnllrl eotides were analyzed by electrophoresis on 20 polyacrylamide gel and exposed for autoradiography. The extracts were run on denaturing polyacrylamide gels (see FIG. 4).

~ woss/32739 Zl 9 1 7 7 7 ~ V~ 6 13. Effect of Cyclodextrin on the Mobility of Oligonucleotides To determine the effect of cyrlo~t~in complexation on the relative mobility of oligonucleotides, both 33P-labelled (Amersham, Arlington Heights, IL) PO- and PS-oligonucleotides were purified through a spin column packed with ~ephadex, mixed with 55; cyclodextrin in plain RPMI
medium at 25~C for 1 hour, and then analyzed by electrophoresis on nondenaturing 15~
polyacrylamide gels which were exposed for autoradiography (FIG. 5).

14. Effect of Cyclodextrin on Cellular Uptake of Adamantane-Linked Olig~nnrleotides Fluorescently labelled Ps-oligrnnrlentidel or fluore 9 cently labelled, covalently-linked ~Ar-ntAn~/ps-oligonucleotide were mixed with 1.255~ EPCD in plain RMPI medium at 4~C for overnight complexing. 8 ~g FITC oligonucleotides that had been complexed with 1.23~ HPCD ~as described above) or uncomplexed were added to 8 x 105 H9 cells in a final volume of 1.2 ml RMPI
r~ntA;n~ng 10~ fetal bovine serum. The cells were set to culture at 37~C. At various time points, aliquots of the cell culture media were taken and washed with Hank' 9 RA1 Anrr~ Salt Solution (HBSS) supplemented with 1% BSA, 1~ sodium azide. The fluorescence intensity of E9 cells was then analyzed by flow cytometry (FIG. 6).

~ WO95/32739 ~ 2~:~1777 ~ r~l6 ~3 ~l r ~U~N~ LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HYBRIDON, INC.
(ii) TITLE OF INVEh-TION: Cyclodextrin ~ r Delivery System For Oligonucleotides (iii) N5MBER OF ~U~N~S: l (iv) CORRESPONDENCE ADD~ SS:
(A) AnnRF..~qTTN Lappin & Rusmer (B) STREET: 200 State Street (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: USA
(F) ZIP: 02109 (v) COMPUTER RFAnARTR FORM:
(A) MEDIUM TYPE: Floppy disk (B) Co.l~ul~: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORWEY/AGENT INFORMATION:
(A) NAME: Kerner, Ann-Louise (B) REGISTRATION NUMBER: 33,523 (C) ~K~N~/DOCRET XUMBER: HYZ-019PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-330-1300 (B) TELEFAX: 617-330-1311 (2) INFORMATION FOR SEQ ID NO:1:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) sTRA~n~n~q single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~ CAL: NO

W095/32739 ~1 91 7 7 7 ~ F~1/~ J/~6~16 (iii) HYPOT~ETICAL: NC - . _ ~iv) ANTT-SENSE: YES
~xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:l:

CTCTCGCACC CATCTCTCTC.CTTCT ~ 25

Claims (23)

What is claimed is:

1. A composition comprising an oligonucleotide noncovalently complexed with a cyclodextrin, wherein the oligonucleotide comprises at least one internucleotide linkage selected from the group consisting of phosphorothioate, alkylphosphonate, and combinations thereof.

2. The composition of claim 1 further comprising adamantane which is covalently linked to the oligonucleotide and noncovalently complexed with the cyclodextrin.

3. The composition of claim 2 wherein adamantane is covalently linked to the 3'-hydroxyl or the 5'-amino of the oligonucleotide.

4. The composition of claim 2 wherein the oligonucleotide comprises at least one internucleotide linkage selected from the group consisting of phosphorothioate, phosphodiester, alkylphosphonate, and combinations thereof.

5. The composition of claim 1 wherein the cyclodextrin is selected from the group consisting of a .beta.-cyclodextrin, a .gamma.-cyclodextrin, a methyl substituted cyclodextrin, and derivatives thereof.

6. The composition of claim 2 wherein the cyclodextrin is selected from the group consisting of a .beta.-cyclodextrin, a .gamma.-cyclodextrin, a methyl substituted cyclodextrin, and derivatives thereof.

7. The composition of claim 5 wherein the cyclodextrin is a 2-hydroxypropyl-.beta.-cyclodextrin, hydroxypropyl-.gamma.-cyclodextrin, hydroxyethyl-.beta.-cyclodextrin, 5-cyclodextrin polysulfate, .gamma.-cyclodextrin polysulfate, and mixtures thereof.

8. The composition of claim 6 wherein the cyclodextrin is a 2-hydroxypropyl-.beta.-cyclodextrin, hydroxypropyl-.gamma.-cyclodextrin, hydroxyethyl-.beta.-cyclodextrin, trimethyl .beta.-cyclodextrin, .beta.-cyclodextrin polysulfate, .gamma.-cyclodextrin polysulfate, and mixtures thereof.

9. The composition of claim 1 wherein the oligonucleotide comprises at least one deoxyribonucleotide.

10. The composition of claim 2 wherein the oligonucleotide comprises at least one deoxyribonucleotide.

11. The composition of claim 1 wherein the oligonucleotide comprises at least one ribonucleotide.

12. The composition of claim 2 wherein the oligonucleotide comprises at least one ribonucleotide.

13. The composition of claim 9 wherein the oligonucleotide comprises at least one ribonucleotide.

14. The composition of claim 10 wherein the oligonucleotide comprises at least one ribonucleotide.

15. The composition of claim 2 wherein the oligonucleotide comprises at least one ribonucleotide and adamantane is covalently linked to the 2'-hydroxyl of the ribonucleotide.

16. A pharmaceutical formulation comprising the oligonucleotide composition of claim 1.

17. A pharmaceutical formulation comprising the oligonucleotide composition of claim 2.

18. A method of increasing the cellular uptake and intracellular concentration of an exogenous oligonucleotide comprising the step of treating a cell with the pharmaceutical formulation of claim 16.

19. A method of increasing the cellular uptake and intracellular concentration of an exogenous oligonucleotide comprising the step of treating a cell with the pharmaceutical formulation of claim 17.

20. A method of treating a cell for viral infection, or for the prevention of viral infection, comprising contacting a cell with the pharmaceutical formulation of claim 16, the oligonucleotide having a nucleotide sequence complementary to a portion of the nucleic acid of a virus.

21. A method of treating a cell for viral infection, or for the prevention of viral infection, comprising contacting a cell with the pharmaceutical formulation of claim 17, the oligonucleotide having a nucleotide sequence complementary to a portion of the nucleic acid of a virus.

22. The use of a cyclodextrin to reduce a required dose of an antisense oligonucleotide, wherein the cyclodextrin is noncovalently complexed to the oligonucleotide.

23. The use according to claim 24, wherein the oligonucleotide is covalently complexed with adamantane, and adamantane is noncovalently complexed to the cyclodextrin.