Antisense inhibition of C/EBP alpha expression

Antisense compounds, compositions and methods are provided for modulating the expression of C/EBP alpha. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding C/EBP alpha. Methods of using these compounds for modulation of C/EBP alpha expression and for treatment of diseases associated with expression of C/EBP alpha are provided.

FIELD OF THE INVENTION
 The present invention provides compositions and methods for modulating the
 expression of C/EBP alpha. In particular, this invention relates to
 antisense compounds, particularly oligonucleotides, specifically
 hybridizable with nucleic acids encoding C/EBP alpha. Such
 oligonucleotides have been shown to modulate the expression of C/EBP
 alpha.
 BACKGROUND OF THE INVENTION
 Transcription factors represent a group of molecules within the cell that
 function to connect the pathways from extracellular signals to
 intracellular responses. Immediately after an environmental stimulus,
 these proteins which reside predominantly in the cytosol are translocated
 to the nucleus where they bind to specific DNA sequences in the promoter
 elements of target genes and activate the transcription of these target
 genes. One family of transcription factors, CCAAT/Enhancer-binding
 proteins (C/EBPs), regulates the expression of an extensive panel of genes
 that control normal tissue development and cellular function, cellular
 proliferation and functional differentiation. Six members of this family
 have been identified to date all of which form both homo- and heterodimers
 with other C/EBP family members as well as with members of the NFkB and
 Fos/Jun families of transcription factors (Lekstrom-Himes and
 Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548). While all of the
 members of the C/EBP family have a similar modular protein structure,
 expression levels and tissue distributions vary widely leading to a
 diversity of roles (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998,
 273, 28545-28548).
 The first member, originally isolated from soluble extracts of rat liver
 nuclei, is C/EBP alpha, also known as CEBPA and simply C/EBP (Graves et
 al., Cell, 1986, 44, 565-576; Johnson et al., Genes Dev., 1987, 1,
 133-146). Studies of tissue distribution and developmental expression
 patterns showed that C/EBP alpha is found primarily in tissues involved in
 energy metabolism, with a capacity to metabolize lipids, cholesterol and
 other sterols (Birkenmeier et al., Genes Dev., 1989, 3, 1146-1156). The
 highest levels of expression were found in the liver, adipose and
 placental tissue, while lower levels were seen in lung and small
 intestine.
 In studies of the rat, C/EBP alpha has been shown to be involved in the
 regulation of adipocyte differentiation (Samuelsson et al., Embo J., 1991,
 10, 3787-3793; Shao and Lazar, J. Biol. Chem., 1997, 272, 21473-21478).
 Using vector-directed expression of antisens;e C/EBP RNA, the authors
 showed reduced expression of C/EBP alpha transcripts as well as reduced
 cytoplasmic triglyceride accumulation, indicating the necessity of the
 alpha isoform in adipocyte differentiation (Lin and Lane, Genes Dev.,
 1992, 6, 533-544). C/EBP alpha has also been shown to regulate
 chondrogenic differentiation (Vidal et al., J. Endocrinol., 1997, 155,
 433-441), follicular development and ovulation (Piontkewitz et al., Dev.
 Biol., 1996, 179, 288-296), steroid-induced cell cylce arrest in the liver
 (Ramos et al., Mol. Cell. Biol., 1996, 16, 5288-5301), and controling
 GLUT2, a glucose transporter, promotor activity (Kim and Ahn, Biochem. J.,
 1998, 336, 83-90). It is also expressed in activated microglial cells
 after brain injury (Walton et al., Brain Res. Mol. Brain Res., 1998, 61,
 11-22).
 These findings are futher supported by studies in humans showing C/EBP
 alpha to be involved in the hormonal regulation of metabolism and in
 granulocyte development (Radomska et al., Mol. Cell. Biol., 1998, 18,
 4301-4314; Roesler et al., J. Biol. Chem., 1998, 273, 14950-14957).
 C/EBP-deficient mice have been generated for five of the six members of the
 C/EBP family and these have been characterized for system-specific
 phenotypic abnormalities. C/EBP alpha is the primary signal controlling
 hepatic terminal differentiation, and mice lacking C/EBP alpha have
 profound derangement of liver structure and function and the majority die
 soon after birth due to hypoglycemia. In addition, mice lacking the C/EBP
 alpha gene exhibit deficient granulopoiesis (Zhang et al., J. Exp. Med.,
 1998, 188, 1173-1184), liver development (Timchenko et al., Mol. Cell.
 Biol., 1997, 17, 7353-7361; Tomizawa et al., Biochem. Biophys. Res.
 Commun., 1998, 249, 1-5) and differentiation of myeloid precursors (Zhang
 et al., Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 569-574).
 In addition to stage-specific expression level variations, the C/EBP
 members also undergo multiple isoform expression arising from alternative
 start positions, for the alpha and beta isoforms, in 5' upstream open
 reading frames (Geballe and Morris, Trends Biochem. Sci., 1994, 19,
 159-164; Lincoln et al., J. Biol. Chem., 1998, 273, 9552-9560). The
 steady-state level of the various pools of transcripts also changes as a
 function of age and stress challenges (Hsieh et al., Mol. Biol. Cell,
 1998, 9, 1479-1494). In mice the expression of certain transcripts of one
 isoform has also been shown to regulate the expression of other C/EBP
 isoforms (Burgess-Beusse et al., Hepatology, 1999, 29, 597-601).
 C/EBP alpha occurs as two isoforms in the cell, a full-length 42-kDa form
 and a shorter 30-kDa form. The shorter form displays alternative
 transactivation potential compared to the full-length protein
 (Lekstrom-Himes and Xanthopoulos, J. Biol. Chem., 1998, 273, 28545-28548).
 Disclosed in U.S. Pat. No. 5,545,563 and in the PCT application, WO
 94/20113, are the DNA sequence of the entire human C/EBP alpha gene and
 vectors for its expression (Darlington et al., 1994; Darlington et al.,
 1996). Also disclosed in WO 94/20113 is the use of the C/EBP alpha gene in
 methods to treat cancer and other diseases. Antisense agonists of C/EBP
 alpha are also generally disclosed.
 The pharmacological modulation of C/EBP alpha activity and/or expression
 may therefore be an appropriate point of therapeutic intervention in
 pathological conditions.
 Currently, there are no known therapeutic agents which effectively inhibit
 the synthesis of C/EBP alpha and to date, investigative strategies aimed
 at modulating C/EBP alpha function have involved the use of antibodies,
 antisense expression vectors, and gene knock-outs in mice. However, these
 strategies are untested as therapeutic protocols and consequently there
 remains a long felt need for agents capable of effectively inhibiting
 C/EBP alpha function.
 Antisense technology is emerging as an effective means for reducing the
 expression of specific gene products and may therefore prove to be
 uniquely useful in a number of therapeutic, diagnostic, and research
 applications for the modulation of C/EBP alpha expression.
 The present invention provides compositions and methods for modulating
 C/EBP alpha expression, including modulation of both the long and short
 isoforms of C/EBP alpha.
 SUMMARY OF THE INVENTION
 The present invention is directed to antisense compounds, particularly
 oligonucleotides, which are targeted to a nucleic acid encoding C/EBP
 alpha, and which modulate the expression of C/EBP alpha. Pharmaceutical
 and other compositions comprising the antisense compounds of the invention
 are also provided. Further provided are methods of modulating the
 expression of C/EBP alpha in cells or tissues comprising contacting said
 cells or tissues with one or more of the antisense compounds or
 compositions of the invention. Further provided are methods of treating an
 animal, particularly a human, suspected of having or being prone to a
 disease or condition associated with expression of C/EBP alpha by
 administering a therapeutically or prophylactically effective amount of
 one or more of the antisense compounds or compositions of the invention.
 DETAILED DESCRIPTION OF THE INVENTION
 The present invention employs oligomeric antisense compounds, particularly
 oligonucleotides, for use in modulating the function of nucleic acid
 molecules encoding C/EBP alpha, ultimately modulating the amount of C/EBP
 alpha produced. This is accomplished by providing antisense compounds
 which specifically hybridize with one or more nucleic acids encoding C/EBP
 alpha. As used herein, the terms "target nucleic acid" and "nucleic acid
 encoding C/EBP alpha" encompass DNA encoding C/EBP alpha, RNA (including
 pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from
 such RNA. The specific hybridization of an oligomeric compound with its
 target nucleic acid interferes with the normal function of the nucleic
 acid. This modulation of function of a target nucleic acid by compounds
 which specifically hybridize to it is generally referred to as
 "antisense". The functions of DNA to be interfered with include
 replication and transcription. The functions of RNA to be interfered with
 include all vital functions such as, for example, translocation of the RNA
 to the site of protein translation, translation of protein from the RNA,
 splicing of the RNA to yield one or more mRNA species, and catalytic
 activity which may be engaged in or facilitated by the RNA. The overall
 effect of such interference with target nucleic acid function is
 modulation of the expression of C/EBP alpha. In the context of the present
 invention, "modulation" means either an increase (stimulation) or a
 decrease (inhibition) in the expression of a gene. In the context of the
 present invention, inhibition is the preferred form of modulation of gene
 expression and mRNA is a preferred target.
 It is preferred to target specific nucleic acids for antisense. "Targeting"
 an antisense compound to a particular nucleic acid, in the context of this
 invention, is a multistep process. The process usually begins with the
 identification of a nucleic acid sequence whose function is to be
 modulated. This may be, for example, a cellular gene (or mRNA transcribed
 from the gene) whose expression is associated with a particular disorder
 or disease state, or a nucleic acid molecule from an infectious agent. In
 the present invention, the target is a nucleic acid molecule encoding
 C/EBP alpha. The targeting process also includes determination of a site
 or sites within this gene for the antisense interaction to occur such that
 the desired effect, e.g., detection or modulation of expression of the
 protein, will result. Within the context of the present invention, a
 preferred intragenic site is the region encompassing the translation
 initiation or termination codon of the open reading frame (ORF) of the
 gene. Since, as is known in the art, the translation initiation codon is
 typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the
 corresponding DNA molecule), the translation initiation codon is also
 referred to as the "AUG codon," the "start codon" or the "AUG start
 codon". A minority of genes have a translation initiation codon having the
 RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have
 been shown to function in vivo. Thus, the terms "translation initiation
 codon" and "start codon" can encompass many codon sequences, even though
 the initiator amino acid in each instance is typically methionine (in
 eukaryotes) or formylmethionine (in prokaryotes). It is also known in the
 art that eukaryotic and prokaryotic genes may have two or more alternative
 start codons, any one of which may be preferentially utilized for
 translation initiation in a particular cell type or tissue, or under a
 particular set of conditions. In the context of the invention, "start
 codon" and "translation initiation codon" refer to the codon or codons
 that are used in vivo to initiate translation of an mRNA molecule
 transcribed from a gene encoding C/EBP alpha, regardless of the
 sequence(s) of such codons.
 It is also known in the art that a translation termination codon (or "stop
 codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG
 and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA,
 respectively). The terms "start codon region" and "translation initiation
 codon region" refer to a portion of such an mRNA or gene that encompasses
 from about 25 to about 50 contiguous nucleotides in either direction
 (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms
 "stop codon region" and "translation termination codon region" refer to a
 portion of such an mRNA or gene that encompasses from about 25 to about 50
 contiguous nucleotides in either direction (i.e., 5' or 3') from a
 translation termination codon.
 The open reading frame (ORF) or "coding region," which is known in the art
 to refer to the region between the translation initiation codon and the
 translation termination codon, is also a region which may be targeted
 effectively. Other target regions include the 5' untranslated region
 (5'UTR), known in the art to refer to the portion of an mRNA in the 5'
 direction from the translation initiation codon, and thus including
 nucleotides between the 5' cap site and the translation initiation codon
 of an mRNA or corresponding nucleotides on the gene, and the 3'
 untranslated region (3'UTR), known in the art to refer to the portion of
 an mRNA in the 3' direction from the translation termination codon, and
 thus including nucleotides between the translation termination codon and
 3' end of an mRNA or corresponding nucleotides on the gene. The 5' cap of
 an mRNA comprises an N7-methylated guanosine residue joined to the 5'-most
 residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap region of
 an mRNA is considered to include the 5' cap structure itself as well as
 the first 50 nucleotides adjacent to the cap. The 5' cap region may also
 be a preferred target region.
 Although some eukaryotic mRNA transcripts are directly translated, many
 contain one or more regions, known as "introns," which are excised from a
 transcript before it is translated. The remaining (and therefore
 translated) regions are known as "exons" and are spliced together to form
 a continuous mRNA sequence. mRNA splice sites, i.e., intronexon junctions,
 may also be preferred target regions, and are particularly useful in
 situations where aberrant splicing is implicated in disease, or where an
 overproduction of a particular mRNA splice product is implicated in
 disease. Aberrant fusion junctions due to rearrangements or deletions are
 also preferred targets. It has also been found that introns can also be
 effective, and therefore preferred, target regions for antisense compounds
 targeted, for example, to DNA or pre-mRNA.
 Once one or more target sites have been identified, oligonucleotides are
 chosen which are sufficiently complementary to the target, i.e., hybridize
 sufficiently well and with sufficient specificity, to give the desired
 effect.
 In the context of this invention, "hybridization" means hydrogen bonding,
 which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen
 bonding, between complementary nucleoside or nucleotide bases. For
 example, adenine and thymine are complementary nucleobases which pair
 through the formation of hydrogen bonds. "Complementary," as used herein,
 refers to the capacity for precise pairing between two nucleotides. For
 example, if a nucleotide at a certain position of an oligonucleotide is
 capable of hydrogen bonding with a nucleotide at the same position of a
 DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are
 considered to be complementary to each other at that position. The
 oligonucleotide and the DNA or RNA are complementary to each other when a
 sufficient number of corresponding positions in each molecule are occupied
 by nucleotides which can hydrogen bond with each other. Thus,
 "specifically hybridizable" and "complementary" are terms which are used
 to indicate a sufficient degree of complementarity or precise pairing such
 that stable and specific binding occurs between the oligonucleotide and
 the DNA or RNA target. It is understood in the art that the sequence of an
 antisense compound need not be 100% complementary to that of its target
 nucleic acid to be specifically hybridizable. An antisense compound is
 specifically hybridizable when binding of the compound to the target DNA
 or RNA molecule interferes with the normal function of the target DNA or
 RNA to cause a loss of utility, and there is a sufficient degree of
 complementarity to avoid non-specific binding of the antisense compound to
 non-target sequences under conditions in which specific binding is
 desired, i.e., under physiological conditions in the case of in vivo
 assays or therapeutic treatment, and in the case of in vitro assays, under
 conditions in which the assays are performed.
 Antisense compounds are commonly used as research reagents and diagnostics.
 For example, antisense oligonucleotides, which are able to inhibit gene
 expression with exquisite specificity, are often used by those of ordinary
 skill to elucidate the function of particular genes. Antisense compounds
 are also used, for example, to distinguish between functions of various
 members of a biological pathway. Antisense modulation has, therefore, been
 harnessed for research use.
 The specificity and sensitivity of antisense is also harnessed by those of
 skill in the art for therapeutic uses. Antisense oligonucleotides have
 been employed as therapeutic moieties in the treatment of disease states
 in animals and man. Antisense oligonucleotides have been safely and
 effectively administered to humans and numerous clinical trials are
 presently underway. It is thus established that oligonucleotides can be
 useful therapeutic modalities that can be configured to be useful in
 treatment regimes for treatment of cells, tissues and animals, especially
 humans. In the context of this invention, the term "oligonucleotide"
 refers to an oligomer or polymer of ribonucleic acid (RNA) or
 deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
 oligonucleotides composed of naturally-occurring nucleobases, sugars and
 covalent internucleoside (backbone) linkages as well as oligonucleotides
 having non-naturally-occurring portions which function similarly. Such
 modified or substituted oligonucleotides are often preferred over native
 forms because of desirable properties such as, for example, enhanced
 cellular uptake, enhanced affinity for nucleic acid target and increased
 stability in the presence of nucleases.
 While antisense oligonucleotides are a preferred form of antisense
 compound, the present invention comprehends other oligomeric antisense
 compounds, including but not limited to oligonucleotide mimetics such as
 are described below. The antisense compounds in accordance with this
 invention preferably comprise from about 8 to about 30 nucleobases (i.e.
 from about 8 to about 30 linked nucleosides). Particularly preferred
 antisense compounds are antisense oligonucleotides, even more preferably
 those comprising from about 12 to about 25 nucleobases. As is known in the
 art, a nucleoside is a base-sugar combination. The base portion of the
 nucleoside is normally a heterocyclic base. The two most common classes of
 such heterocyclic bases are the purines and the pyrimidines. Nucleotides
 are nucleosides that further include a phosphate group covalently linked
 to the sugar portion of the nucleoside. For those nucleosides that include
 a pentofuranosyl sugar, the phosphate group can be linked to either the
 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides,
 the phosphate groups covalently link adjacent nucleosides to one another
 to form a linear polymeric compound. In turn the respective ends of this
 linear polymeric structure can be further joined to form a circular
 structure, however, open linear structures are generally preferred. Within
 the oligonucleotide structure, the phosphate groups are commonly referred
 to as forming the internucleoside backbone of the oligonucleotide. The
 normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester
 linkage.
 Specific examples of preferred antisense compounds useful in this invention
 include oligonucleotides containing modified backbones or non-natural
 internucleoside linkages. As defined in this specification,
 oligonucleotides having modified backbones include those that retain a
 phosphorus atom in the backbone and those that do not have a phosphorus
 atom in the backbone. For the purposes of this specification, and as
 sometimes referenced in the art, modified oligonucleotides that do not
 have a phosphorus atom in their internucleoside backbone can also be
 considered to be oligonucleosides.
 Preferred modified oligonucleotide backbones include, for example,
 phosphorothioates, chiral phosphorothioates, phosphorodithioates,
 phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl
 phosphonates including 3'-alkylene phosphonates and chiral phosphonates,
 phosphinates, phosphoramidates including 3'-amino phosphoramidate and
 aminoalkylphosphoramidates, thionophosphoramidates,
 thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates
 having normal 3'-5' linkages, 2'-5' linked analogs of these, and those
 having inverted polarity wherein the adjacent pairs of nucleoside units
 are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts
 and free acid forms are also included.
 Representative United States patients that teach the preparation of the
 above phosphorus-containing linkages include, but are not limited to, U.S.
 Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;
 5,183,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;
 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;
 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;
 5,587,361; and 5,625,050, certain of which are commonly owned with this
 application, and each of which is herein incorporated by reference.
 Preferred modified oligonucleotide backbones that do not include a
 phosphorus atom therein have backbones that are formed by short chain
 alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl
 or cycloalkyl internucleoside linkages, or one or more short chain
 heteroatomic or heterocyclic internucleoside linkages. These include those
 having morpholino linkages (formed in part from the sugar portion of a
 nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;
 formacetyl and thioformacetyl backbones; methylene formacetyl and
 thioformacetyl backbones; alkene containing backbones; sulfamate
 backbones; methyleneimino and methylenehydrazino backbones; sulfonate and
 sulfonamide backbones; amide backbones; and others having mixed N, O, S
 and CH.sub.2 component parts.
 Representative United States patents that teach the preparation of the
 above oligonucleosides include, but are not limited to, U.S. Pat. Nos.
 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;
 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;
 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;
 5,633,360; 5,677,437; and 5,677,439, certain of which are commonly owned
 with this application, and each of which is herein incorporated by
 reference.
 In other preferred oligonucleotide mimetics, both the sugar and the
 internucleoside linkage, i.e., the backbone, of the nucleotide units are
 replaced with novel groups. The base units are maintained for
 hybridization with an appropriate nucleic acid target compound. One such
 oligomeric compound, an oligonucleotide mimetic that has been shown to
 have excellent hybridization properties, is referred to as a peptide
 nucleic acid (PNA). In PNA compounds, the sugar-backbone of an
 oligonucleotide is replaced with an amide containing backbone, in
 particular an aminoethylglycine backbone. The nucleobases are retained and
 are bound directly or indirectly to aza nitrogen atoms of the amide
 portion of the backbone. Representative United States patents that teach
 the preparation of PNA compounds include, but are not limited to, U.S.
 Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein
 incorporated by reference. Further teaching of PNA compounds can be found
 in Nielsen et al., Science, 1991, 254, 1497-1500.
 Most preferred embodiments of the invention are oligonucleotides with
 phosphorothioate backbones and oligonucleosides with heteroatom backbones,
 and in particular --CH.sub.2 --NH--O--CH.sub.2 --, --CH.sub.2
 --N(CH.sub.3)--O--CH.sub.2 -- [known as a methylene (methylimino) or MMI
 backbone], --CH.sub.2 --O--N(CH.sub.3)--CH.sub.2 --, --CH.sub.2
 --N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 -- and --O--N(CH.sub.3)--CH.sub.2
 --CH.sub.2 -- [wherein the native phosphodiester backbone is represented
 as --O--P--O--CH.sub.2 --] of the above referenced U.S. Pat. No.
 5,489,677, and the amide backbones of the above referenced U.S. Pat. No.
 5,602,240. Also preferred are oligonucleotides having morpholino backbone
 structures of the above-referenced U.S. Pat. No. 5,034,506.
 Modified oligonucleotides may also contain one or more substituted sugar
 moieties. Preferred oligonucleotides comprise one of the following at the
 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; O--, S--
 or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl
 may be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2
 to C.sub.10 alkenyl and alkynyl. Particularly preferred are
 O[(CH.sub.2).sub.n O].sub.m CH.sub.3, O(CH.sub.2).sub.n OCH.sub.3,
 O(CH.sub.2).sub.n NH.sub.2, O(CH.sub.2).sub.n CH.sub.3, O(CH.sub.2).sub.n
 ONH.sub.2, and O(CH.sub.2).sub.n ON [(CH.sub.2).sub.n CH.sub.3)].sub.2,
 where n and m are from 1 to about 10. Other preferred oligonucleotides
 comprise one of the following at the 2' position: C.sub.1 to C.sub.10
 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
 O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
 SOCH.sub.3, SO.sub.2 CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
 heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,
 substituted silyl, an RNA cleaving group, a reporter group, an
 intercalator, a group for improving the pharmacokinetic properties of an
 oligonucleotide, or a group for improving the pharmacodynamic properties
 of an oligonucleotide, and other substituents having similar properties. A
 preferred modification includes 2'-methoxyethoxy (2'--O--CH.sub.2 CH.sub.2
 OCH.sub.3, also known as 2'--O--(2-methoxyethyl) or 2'-MOE) (Martin et
 al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A
 further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a
 O(CH.sub.2).sub.2 ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as
 described in examples hereinbelow, and 2'-dimethylamino-ethoxyethoxy (also
 known in the art as 2'-O-dimethylamino-ethoxyethyl or 2'-DMAEOE), i.e.,
 2'--O--CH.sub.2 --O--CH.sub.2 --N (CH.sub.2).sub.2, also described in
 examples hereinbelow.
 Other preferred modifications include 2'-methoxy (2'--O--CH.sub.3),
 2'-aminopropoxy (2'--OCH.sub.2 CH.sub.2 CH.sub.2 NH.sub.2) and 2'-fluoro
 (2'--F). Similar modifications may also be made at other positions on the
 oligonucleotide, particularly the 3' position of the sugar on the 3'
 terminal nucleotide or in 2'-5' linked oligonucleotides and the 5'
 position of 5' terminal nucleotide. Oligonucleotides may also have sugar
 mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
 Representative United States patents that teach the preparation of such
 modified sugar structures include, but are not limited to, U.S. Pat. Nos.
 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;
 5,456,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;
 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;
 5,670,633; and 5,700,920, certain of which are commonly owned with the
 instant application, and each of which is herein incorporated by reference
 in its entirety.
 Oligonucleotides may also include nucleobase (often referred to in the art
 simply as "base") modifications or substitutions. As used herein,
 "unmodified" or "natural" nucleobases include the purine bases adenine (A)
 and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and
 uracil (U). Modified nucleobases include other synthetic and natural
 nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,
 xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl
 derivatives of adenine and guanine, 2-propyl and other alkyl derivatives
 of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,
 cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,
 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
 and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other
 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine,
 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and
 3-deazaguanine and 3-deazaadenine. Further nucleobases include those
 disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise
 Encyclopedia Of Polymer Science And Engineering, pages 858-859,
 Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by
 Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613,
 and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
 Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press,
 1993. Certain of these nucleobases are particularly useful for increasing
 the binding affinity of the oligomeric compounds of the invention. These
 include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6
 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and
 5-propynylcytosine. 5-methylcytosine substitutions have been shown to
 increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y.
 S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and
 Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently
 preferred base substitutions, even more particularly when combined with
 2'-O-methoxyethyl sugar modifications.
 Representative United States patents that teach the preparation of certain
 of the above noted modified nucleobases as well as other modified
 nucleobases include, but are not limited to, the above noted U.S. Pat. No.
 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;
 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;
 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;
 5,614,617; and 5,681,941, certain of which are commonly owned with the
 instant application, and each of which is herein incorporated by
 reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the
 instant application and also herein incorporated by reference.
 Another modification of the oligonucleotides of the invention involves
 chemically linking to the oligonucleotide one or more moieties or
 conjugates which enhance the activity, cellular distribution or cellular
 uptake of the oligonucleotide. Such moieties include but are not limited
 to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.
 Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,
 Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
 hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,
 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a
 thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538),
 an aliphatic chain, e.g., dodecandiol or undecyl residues
 (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,
 FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,
 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
 triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
 (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,
 Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene
 glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.,
 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys.
 Acta, 1995, 1264, 229-237), or an octadecylamine or
 hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.
 Exp. Ther., 1996, 277, 923-937.
 Representative United States patents that teach the preparation of such
 oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.
 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;
 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;
 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;
 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;
 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;
 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;
 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;
 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;
 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;
 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which
 are commonly owned with the instant application, and each of which is
 herein incorporated by reference.
 It is not necessary for all positions in a given compound to be uniformly
 modified, and in fact more than one of the aforementioned modifications
 may be incorporated in a single compound or even at a single nucleoside
 within an oligonucleotide. The present invention also includes antisense
 compounds which are chimeric compounds. "Chimeric" antisense compounds or
 "chimeras," in the context of this invention, are antisense compounds,
 particularly oligonucleotides, which contain two or more chemically
 distinct regions, each made up of at least one monomer unit, i.e., a
 nucleotide in the case of an oligonucleotide compound. These
 oligonucleotides typically contain at least one region wherein the
 oligonucleotide is modified so as to confer upon the oligonucleotide
 increased resistance to nuclease degradation, increased cellular uptake,
 and/or increased binding affinity for the target nucleic acid. An
 additional region of the oligonucleotide may serve as a substrate for
 enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example,
 RNase H is a cellular endonuclease which cleaves the RNA strand of an
 RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of
 the RNA target, thereby greatly enhancing the efficiency of
 oligonucleotide inhibition of gene expression. Consequently, comparable
 results can often be obtained with shorter oligonucleotides when chimeric
 oligonucleotides are used, compared to phosphorothioate
 deoxyoligonucleotides hybridizing to the same target region. Cleavage of
 the RNA target can be routinely detected by gel electrophoresis and, if
 necessary, associated nucleic acid hybridization techniques known in the
 art.
 Chimeric antisense compounds of the invention may be formed as composite
 structures of two or more oligonucleotides, modified oligonucleotides,
 oligonucleosides and/or oligonucleotide mimetics as described above. Such
 compounds have also been referred to in the art as hybrids or gapmers.
 Representative United States patents that teach the preparation of such
 hybrid structures include, but are not limited to, U.S. Pat. Nos.
 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;
 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,
 certain of which are commonly owned with the instant application, and each
 of which is herein incorporated by reference in its entirety.
 The antisense compounds used in accordance with this invention may be
 conveniently and routinely made through the well-known technique of solid
 phase synthesis. Equipment for such synthesis is sold by several vendors
 including, for example, Applied Biosystems (Foster City, Calif.). Any
 other means for such synthesis known in the art may additionally or
 alternatively be employed. It is well known to use similar techniques to
 prepare oligonucleotides such as the phosphorothioates and alkylated
 derivatives.
 The antisense compounds of the invention are synthesized in vitro and do
 not include antisense compositions of biological origin, or genetic vector
 constructs designed to direct the in vivo synthesis of antisense
 molecules. The compounds of the invention may also be admixed,
 encapsulated, conjugated or otherwise associated with other molecules,
 molecule structures or mixtures of compounds, as for example, liposomes,
 receptor targeted molecules, oral, rectal, topical or other formulations,
 for assisting in uptake, distribution and/or absorption. Representative
 United States patents that teach the preparation of such uptake,
 distribution and/or absorption assisting formulations include, but are not
 limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127;
 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330;
 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221;
 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854;
 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and
 5,595,756, each of which is herein incorporated by reference.
 The antisense compounds of the invention encompass any pharmaceutically
 acceptable salts, esters, or salts of such esters, or any other compound
 which, upon administration to an animal including a human, is capable of
 providing (directly or indirectly) the biologically active metabolite or
 residue thereof. Accordingly, for example, the disclosure is also drawn to
 prodrugs and pharmaceutically acceptable salts of the compounds of the
 invention, pharmaceutically acceptable salts of such prodrugs, and other
 bioequivalents.
 The term "prodrug" indicates a therapeutic agent that is prepared in an
 inactive form that is converted to an active form (i.e., drug) within the
 body or cells thereof by the action of endogenous enzymes or other
 chemicals and/or conditions. In particular, prodrug versions of the
 oligonucleotides of the invention are prepared as SATE
 [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods
 disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in
 WO 91/26764 to Imbach et al.
 The term "pharmaceutically acceptable salts" refers to physiologically and
 pharmaceutically acceptable salts of the compounds of the invention: i.e.,
 salts that retain the desired biological activity of the parent compound
 and do not impart undesired toxicological effects thereto.
 Pharmaceutically acceptable base addition salts are formed with metals or
 amines, such as alkali and alkaline earth metals or organic amines.
 Examples of metals used as cations are sodium, potassium, magnesium,
 calcium, and the like. Examples of suitable amines are
 N,N'-dibenzylethylenediamine, chloropocaine, choline, diethanolamine,
 dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see,
 for example, Berge et al., "Pharmaceutical Salts," J. of Pharma Sci.,
 1977, 66, 1-19). The base addition salts of said acidic compounds are
 prepared by contacting the free acid form with a sufficient amount of the
 desired base to produce the salt in the conventional manner. The free acid
 form may be regenerated by contacting the salt form with an acid and
 isolating the free acid in the conventional manner. The free acid forms
 differ from their respective salt forms somewhat in certain physical
 properties such as solubility in polar solvents, but otherwise the salts
 are equivalent to their respective free acid for purposes of the present
 invention. As used herein, a "pharmaceutical addition salt" includes a
 pharmaceutically acceptable salt of an acid form of one of the components
 of the compositions of the invention. These include organic or inorganic
 acid salts of the amines. Preferred acid salts are the hydrochlorides,
 acetates, salicylates, nitrates and phosphates. Other suitable
 pharmaceutically acceptable salts are well known to those skilled in the
 art and include basic salts of a variety of inorganic and organic acids,
 such as, for example, with inorganic acids, such as for example
 hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid;
 with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted
 sulfamic acids, for example acetic acid, propionic acid, glycolic acid,
 succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
 acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid,
 glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid,
 mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzo.ic
 acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic
 acid; and with amino acids, such as the 20 alpha-amino acids involved in
 the synthesis of proteins in nature, for example glutamic acid or aspartic
 acid, and also with phenylacetic acid, methanesulfonic acid,
 ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic
 acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid,
 naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with
 the formation of cyclamates), or with other acid organic compounds, such
 as ascorbic acid. Pharmaceutically acceptable salts of compounds may also
 be prepared with a pharmaceutically acceptable cation. Suitable
 pharmaceutically acceptable cations are well known to those skilled in the
 art and include alkaline, alkaline earth, ammonium and quaternary ammonium
 cations. Carbonates or hydrogen carbonates are also possible.
 For oligonucleotides, preferred examples of pharmaceutically acceptable
 salts include but are not limited to (a) salts formed with cations such as
 sodium, potassium, ammonium, magnesium, calcium, polyamines such as
 spermine and spermidine, etc.; (b) acid addition salts formed with
 inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric
 acid, phosphoric acid, nitric acid and the like; (c) salts formed with
 organic acids such as, for example, acetic acid, oxalic acid, tartaric
 acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric
 acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid,
 alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic
 acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic
 acid, and the like; and (d) salts formed from elemental anions such as
 chlorine, bromine, and iodine.
 The antisense compounds of the present invention can be utilized for
 diagnostics, therapeutics, prophylaxis and as research reagents and kits.
 For therapeutics, an animal, preferably a human, suspected of having a
 disease or disorder which can be treated by modulating the expression of
 C/EBP alpha is treated by administering antisense compounds in accordance
 with this invention. The compounds of the invention can be utilized in
 pharmaceutical compositions by adding an effective amount of an antisense
 compound to a suitable pharmaceutically acceptable diluent or carrier. Use
 of the antisense compounds and methods of the invention may also be useful
 prophylactically, e.g., to prevent or delay infection, inflammation or
 tumor formation, for example.
 The antisense compounds of the invention are useful for research and
 diagnostics, because these compounds hybridize to nucleic acids encoding
 C/EBP alpha, enabling sandwich and other assays to easily be constructed
 to exploit this fact. Hybridization of the antisense oligonucleotides of
 the invention with a nucleic acid encoding C/EBP alpha can be detected by
 means known in the art such means may include conjugation of an enzyme to
 the oligonucleotide, radiolabelling of the oligonucleotide or any other
 suitable detection means. Kits using such detection means for detecting
 the level of C/EBP alpha in a sample may also be prepared.
 The present invention also includes pharmaceutical compositions and
 formulations which include the antisense compounds of the invention. The
 pharmaceutical compositions of the present invention may be administered
 in a number of ways depending upon whether local or systemic treatment is
 desired and upon the area to be treated. Administration may be topical
 (including ophthalmic and to mucous membranes including vaginal and rectal
 delivery, pulmonary, e.g., by inhalation or insufflation of powders or
 aerosols, including by nebulizer; intratracheal, intranasal, epidermal and
 transdermal), oral or parenteral. Parenteral administration includes
 intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular
 injection or infusion; or intracranial, e.g., intrathecal or
 intraventricular, administration. Oligonucleotides with at least one
 2'-O-methoxyethyl modification are believed to be particularly useful for
 oral administration.
 Pharmaceutical compositions and formulations for topical administration may
 include transdermal patches, ointments, lotions, creams, gels, drops,
 suppositories, sprays, liquids and powders. Conventional pharmaceutical
 carriers, aqueous, powder or oily bases, thickeners and the like may be
 necessary or desirable. Coated condoms, gloves and the like may also be
 useful.
 Compositions and formulations for oral administration include powders or
 granules, suspensions or solutions in water or non-aqueous media,
 capsules, sachets or tablets. Thickeners, flavoring agents, diluents,
 emulsifiers, dispersing aids or binders may be desirable.
 Compositions and formulations for parenteral, intrathecal or
 intraventricular administration may include sterile aqueous solutions
 which may also contain buffers, diluents and other suitable additives such
 as, but not limited to, penetration enhancers, carrier compounds and other
 pharmaceutically acceptable carriers or excipients.
 Pharmaceutical compositions of the present invention include, but are not
 limited to, solutions, emulsions, and liposome-containing formulations.
 These compositions may be generated from a variety of components that
 include, but are not limited to, preformed liquids, self-emulsifying
 solids and self-emulsifying semisolids.
 The pharmaceutical formulations of the present invention, which may
 conveniently be presented in unit dosage form, may be prepared according
 to conventional techniques well known in the pharmaceutical industry. Such
 techniques include the step of bringing into association the active
 ingredients with the pharmaceutical carrier(s) or excipient(s). In general
 the formulations are prepared by uniformly and intimately bringing into
 association the active ingredients with liquid carriers or finely divided
 solid carriers or both, and then, if necessary, shaping the product.
 The compositions of the present invention may be formulated into any of
 many possible dosage forms such as, but not limited to, tablets, capsules,
 liquid syrups, soft gels, suppositories, and enemas. The compositions of
 the present invention may also be formulated as suspensions in aqueous,
 non-aqueous or mixed media. Aqueous suspensions may further contain
 substances which increase the viscosity of the suspension including, for
 example sodium carboxymethylcellulose, sorbitol and/or dextran. The
 suspension may also contain stabilizers.
 In one embodiment of the present invention the pharmaceutical compositions
 may be formulated and used as foams. Pharmaceutical foams include
 formulations such as, but not limited to, emulsions, microemulsions,
 creams, jellies and liposomes. While basically similar in nature these
 formulations vary in the components and the consistency of the final
 product. The preparation of such compositions and formulations is
 generally known to those skilled in the pharmaceutical and formulation
 arts and may be applied to the formulation of the compositions of the
 present invention.
 Emulsions
 The compositions of the present invention may be prepared and formulated as
 emulsions. Emulsions are typically heterogenous systems of one liquid
 dispersed in another in the form of droplets usually exceeding 0.1 .mu.m
 in diameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
 Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
 (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
 in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,
 Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in
 Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton. Pa.,
 1985, p. 301). Emulsions are often biphasic systems comprising of two
 immiscible liquid phases intimately mixed and dispersed with each other.
 In general, emulsions may be either water-in-oil (w/o) or of the
 oil-in-water (o/w) variety. When an aqueous phase is finely divided into
 and dispersed as minute droplets into a bulk oily phase the resulting
 composition is called a water-in-oil (w/o) emulsion. Alternatively, when
 an oily phase is finely divided into and dispersed as minute droplets into
 a bulk aqueous phase the resulting composition is called an oil-in-water
 (o/w) emulsion. Emulsions may contain additional components in addition to
 the dispersed phases and the active drug which may be present as a
 solution in either the aqueous phase, oily phase or itself as a separate
 phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes,
 and anti-oxidants may also be present in emulsions as needed.
 Pharmaceutical emulsions may also be multiple emulsions that are comprised
 of more than two phases such as, for example, in the case of
 oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
 Such complex formulations often provide certain advantages that simple
 binary emulsions do not. Multiple emulsions in which individual oil
 droplets of an o/w emulsion enclose small water droplets constitute a
 w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of
 water stabilized in an oily continuous provides an o/w/o emulsion.
 Emulsions are characterized by little or no thermodynamic stability. Often,
 the dispersed or discontinuous phase of the emulsion is well dispersed
 into the external or continuous phase and maintained in this form through
 the means of emulsifiers or the viscosity of the formulation. Either of
 the phases of the emulsion may be a semisolid or a solid, as is the case
 of emulsion-style ointment bases and creams. Other means of stabilizing
 emulsions entail the use of emulsifiers that may be incorporated into
 either phase of the emulsion. Emulsifiers may broadly be classified into
 four categories: synthetic surfactants, naturally occurring emulsifiers,
 absorption bases, and finely dispersed solids (Idson, in Pharmaceutical
 Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
 Inc., New York, N.Y., volume 1, p. 199).
 Synthetic surfactants, also known as surface active agents, have found wide
 applicability in the formulation of emulsions and have been reviewed in
 the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger
 and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
 (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
 Surfactants are typically amphiphilic and comprise a hydrophilic and a
 hydrophobic portion. The ratio of the hydrophilic to the hydrophobic
 nature of the surfactant has been termed the hydrophile/lipophile balance
 (HLB) and is a valuable tool in categorizing and selecting surfactants in
 the preparation of formulations. Surfactants may be classified into
 different classes based on the nature of the hydrophilic group: nonionic,
 anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
 Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
 N.Y., volume 1, p. 285).
 Naturally occurring emulsifiers used in emulsion formulations include
 lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases
 possess hydrophilic properties such that they can soak up water to form
 w/o emulsions yet retain their semisolid consistencies, such as anhydrous
 lanolin and hydrophilic petrolatum. Finely divided solids have also been
 used as good emulsifiers especially in combination with surfactants and in
 viscous preparations. These include polar inorganic solids, such as heavy
 metal hydroxides, nonswelling clays such as bentonite, attapulgite,
 hectorite, kaolin, montmorillonite, colloidal aluminum silicate and
 colloidal magnesium aluminum silicate, pigments and nonpolar solids such
 as carbon or glyceryl tristearate.
 A large variety of non-emulsifying materials are also included in emulsion
 formulations and contribute to the properties of emulsions. These include
 fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants,
 hydrophilic colloids, preservatives and antioxidants (Block, in
 Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,
 Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in
 Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,
 MarceL Dekker, Inc., New York, N.Y., volume 1, p. 199).
 Hydrophilic colloids or hydrocolloids include naturally occurring gums and
 synthetic polymers such as polysaccharides (for example, acacia, agar,
 alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),
 cellulose derivatives (for example, carboxymethylcellulose and
 carboxypropylcellulose), and synthetic polymers (for example, carbomers,
 cellulose ethers, and carboxyvinyl polymers). These disperse or swell in
 water to form colloidal solutions that stabilize emulsions by forming
 strong interfacial films around the dispersed-phase droplets and by
 increasing the viscosity of the external phase.
 Since emulsions often contain a number of ingredients such as
 carbohydrates, proteins, sterols and phosphatides that may readily support
 the growth of microbes, these formulations often incorporate
 preservatives. Commonly used preservatives included in emulsion
 formulations include methyl paraben, propyl paraben, quaternary ammonium
 salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric
 acid. Antioxidants are also commonly added to emulsion formulations to
 prevent deterioration of the formulation. Antioxidants used may be free
 radical scavengers such as tocopherols, alkyl gallates, butylated
 hydroxyanisole, butylated hydroxytoluene, or reducing agents such as
 ascorbic acid and sodium metabisulfite, and antioxidant synergists such as
 citric acid, tartaric acid, and lecithin.
 The application of emulsion formulations via dermatological, oral and
 parenteral routes and methods for their manufacture have been reviewed in
 the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger
 and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
 199). Emulsion formulations for oral delivery have been very widely used
 because of reasons of ease of formulation, efficacy from an absorption and
 bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms,
 Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
 N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman,
 Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
 volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and
 high fat nutritive preparations are among the materials that have commonly
 been administered orally as o/w emulsions.
 In one embodiment of the present invention, the compositions of
 oligonucleotides and nucleic acids are formulated as microemulsions. A
 microemulsions may be defined as a system of water, oil and amphiphile
 which is a single optically isotropic and thermodynamically stable liquid
 solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
 Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
 245). Typically microemulsions are systems that are prepared by first
 dispersing an oil in an aqueous surfactant solution and then adding a
 sufficient amount of a fourth component, generally an intermediate
 chain-length alcohol to form a transparent system. Therefore,
 microemulsions have also been described as thermodynamically stable,
 isotropically clear dispersions of two immiscible liquids that are
 stabilized by interfacial films of surface-active molecules (Leung and
 Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,
 Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).
 Microemulsions commonly are prepared via a combination of three to five
 components that include oil, water, surfactant, cosurfactant and
 electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an
 oil-in-water (o/w) type is dependent on the properties of the oil and
 surfactant used and on the structure and geometric packing of the polar
 heads and hydrocarbon tails of the surfactant molecules (Schott, in
 Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,
 1985, p. 271).
 The phenomenological approach utilizing phase diagrams has been extensively
 studied and has yielded a comprehensive knowledge, to one skilled in the
 art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage
 Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
 York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,
 Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
 N.Y., volume 1, p. 335). Compared to conventional emulsions,
 microemulsions offer the advantage of solubilizing water-insoluble drugs
 in a formulation of thermodynamically stable droplets that are formed
 spontaneously.
 Surfactants used in the preparation of microemulsions include, but are not
 limited to, ionic surfactants, non-ionic surfactants, Brij 96,
 polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,
 tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),
 hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),
 decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),
 decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone
 or in combination with cosurfactants. The cosurfactant, usually a
 short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to
 increase the interfacial fluidity by penetrating into the surfactant film
 and consequently creating a disordered film because of the void space
 generated among surfactant molecules. Microemulsions may, however, be
 prepared without the use of cosurfactants and alcohol-free
 self-emulsifying microemulsion systems are known in the art. The aqueous
 phase may typically be, but is not limited to, water, an aqueous solution
 of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols,
 and derivatives of ethylene glycol. The oil phase may include, but is not
 limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty
 acid esters, medium chain (C8-C12) mono, di, and tri-glycerides,
 polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
 polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides,
 vegetable oils and silicone oil.
 Microemulsions are particularly of interest from the standpoint of drug
 solubilization and the enhanced absorption of drugs. Lipid based
 microemulsions (both o/w and w/o) have been proposed to enhance the oral
 bioavailability of drugs, including peptides (Constantinides et al.,
 Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.
 Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of
 improved drug solubilization, protection of drug from enzymatic
 hydrolysis, possible enhancement of drug absorption due to
 surfactant-induced alterations in membrane fluidity and permeability, ease
 of preparation, ease of oral administration over solid dosage forms,
 improved clinical potency, and decreased toxicity (Constantinides et al.,
 Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996,
 85, 138-143). Often micrcemulsions may form spontaneously when their
 components are brought together at ambient temperature. This may be
 particularly advantageous when formulating thermolabile drugs, peptides or
 oligonucleotides. Microemulsions have also been effective in the
 transdermal delivery of active components in both cosmetic and
 pharmaceutical applications. It is expected that the microemulsion
 compositions and formulations of the present invention will facilitate the
 increased systemic absorption of oligonucleotides and nucleic acids from
 the gastrointestinal tract, as well as improve the local cellular uptake
 of oligonucleotides and nucleic acids within the gastrointestinal tract,
 vagina, buccal cavity and other areas of administration.
 Microemulsions of the present invention may also contain additional
 components and additives such as sorbitan monostearate (Grill 3),
 Labrasol, and penetration enhancers to improve the properties of the
 formulation and to enhance the absorption of the oligonucleotides and
 nucleic acids of the present invention. Penetration enhancers used in the
 microemulsions of the present invention may be classified as belonging to
 one of five broad categories--surfactants, fatty acids, bile salts,
 chelating agents, and non-chelating non-surfactants (Lee et al., Critical
 Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
 classes has been discussed above.
 Liposomes
 There are many organized surfactant structures besides microemulsions that
 have been studied and used for the formulation of drugs. These include
 monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes,
 have attracted great interest because of their specificity and the
 duration of action they offer from the standpoint of drug delivery. As
 used in the present invention, the term "liposome" means a vesicle
 composed of amphiphilic lipids arranged in a spherical bilayer or
 bilayers.
 Liposomes are unilamellar or multilamellar vesicles which have a membrane
 formed from a lipophilic material and an aqueous interior. The aqueous
 portion contains the composition to be delivered. Cationic liposomes
 possess the advantage of being able to fuse to the cell wall. Non-cationic
 liposomes, although not able to fuse as efficiently with the cell wall,
 are taken up by macrophages in vivo.
 In order to cross intact mammalian skin, lipid vesicles must pass through a
 series of fine pores, each with a diameter less than 50 nm, under the
 influence of a suitable transdermal gradient. Therefore, it is desirable
 to use a liposome which is highly deformable and able to pass through such
 fine pores.
 Further advantages of liposomes include; liposomes obtained from natural
 phospholipids are biocompatible and biodegradable; liposomes can
 incorporate a wide range of water and lipid soluble drugs; liposomes can
 protect encapsulated drugs in their internal compartments from metabolism
 and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger
 and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
 245). Important considerations in the preparation of liposome formulations
 are the lipid surface charge, vesicle size and the aqueous volume of the
 liposomes.
 Liposomes are useful for the transfer and delivery of active ingredients to
 the site of action. Because the liposomal membrane is structurally similar
 to biological membranes, when liposomes are applied to a tissue, the
 liposomes start to merge with the cellular membranes. As the merging of
 the liposome and cell progresses, the liposomal contents are emptied into
 the cell where the active agent may act.
 Liposomal formulations have been the focus of extensive investigation as
 the mode of delivery for many drugs. There is growing evidence that for
 topical administration, liposomes present several advantages over other
 formulations. Such advantages include reduced side-effects related to high
 systemic absorption of the administered drug, increased accumulation of
 the administered drug at the desired target, and the ability to administer
 a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
 Several reports have detailed the ability of liposomes to deliver agents
 including high-molecular weight DNA into the skin. Compounds including
 analgesics, antibodies, hormones and high-molecular weight DNAs have been
 administered to the skin. The majority of applications resulted in the
 targeting of the upper epidermis.
 Liposomes fall into two broad classes. Cationic liposomes are positively
 charged liposomes which interact with the negatively charged DNA molecules
 to form a stable complex. The positively charged DNA/liposome complex
 binds to the negatively charged cell surface and is internalized in an
 endosome. Due to the acidic pH within the endosome, the liposomes are
 ruptured, releasing their contents into the cell cytoplasm (Wang et al.,
 Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
 Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather
 than complex with it. Since both the DNA and the lipid are similarly
 charged, repulsion rather than complex formation occurs. Nevertheless,
 some DNA is entrapped within the aqueous interior of these liposomes.
 pH-sensitive liposomes have been used to deliver DNA encoding the
 thymidine kinase gene to cell monolayers in culture. Expression of the
 exogenous gene was detected in the target cells (Zhou et al., Journal of
 Controlled Release, 1992, 19, 269-274).
 One major type of liposomal composition includes phospholipids other than
 naturally-derived phosphatidylcholine. Neutral liposome compositions, for
 example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or
 dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions
 generally are formed from dimyristoyl phosphatidylglycerol, while anionic
 fusogenic liposomes are formed primarily from dioleoyl
 phosphatidylethanolamine (DOPE). Another type of liposomal composition is
 formed from phosphatidylcholine (PC) such as, for example, soybean PC, and
 egg PC. Another type is formed from mixtures of phospholipid and/or
 phosphatidylcholine and/or cholesterol.
 Several studies have assessed the topical delivery of liposomal drug
 formulations to the skin. Application of liposomes containing interferon
 to guinea pig skin resulted in a reduction of skin herpes sores while
 delivery of interferon via other means (e.g. as a solution or as an
 emulsion) were ineffective (Weiner et al., Journal of Drug Targeting,
 1992, 2, 405-410). Further, an additional study tested the efficacy of
 interferon administered as part of a liposomal formulation to the
 administration of interferon using an aqueous system, and concluded that
 the liposomal formulation was superior to aqueous administration (du
 Plessis et al., Antiviral Research, 1992, 18, 259-265).
 Non-ionic liposomal systems have also been examined to determine their
 utility in the delivery of drugs to the skin, in particular systems
 comprising non-ionic surfactant and cholesterol. Non-ionic liposomal
 formulations comprising Novasome.TM. I (glyceryl
 dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome.TM.
 II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were
 used to deliver cyclosporin-A into the dermis of mouse skin. Results
 indicated that such non-ionic liposomal systems were effective in
 facilitating the deposition of cyclosporin-A into different layers of the
 skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
 Liposomes also include "sterically stabilized" liposomes, a term which, as
 used herein, refers to liposomes comprising one or more specialized lipids
 that, when incorporated into liposomes, result in enhanced circulation
 lifetimes relative to liposomes lacking such specialized lipids. Examples
 of sterically stabilized liposomes are those in which part of the
 vesicle-forming lipid portion of the liposome (A) comprises one or more
 glycolipids, such as monosialoganglioside G.sub.M1, or (B) is derivatized
 with one or more hydrophilic polymers, such as a polyethylene glycol (PEG)
 moiety. While not wishing to be bound by any particular theory, it is
 thought in the art that, at least for sterically stabilized liposomes
 containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the
 enhanced circulation half-life of these sterically stabilized liposomes
 derives from a reduced uptake into cells of the reticuloendothelial system
 (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer
 Research, 1993, 53, 3765).
 Various liposomes comprising one or more glycolipids are known in the art.
 Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the
 ability of monosialoganglioside G.sub.M1, galactocerebroside sulfate and
 phosphatidylinositol to improve blood half-lives of liposomes. These
 findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci.
 U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
 Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the
 ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S. Pat. No.
 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin.
 Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed
 in WO 97/13499 (Lim et al.).
 Many liposomes comprising lipids derivatized with one or more hydrophilic
 polymers, and methods of preparation thereof, are known in the art.
 Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described
 liposomes comprising a nonionic detergent, 2C.sub.12 15G, that contains a
 PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that
 hydrophilic coating of polystyrene particles with polymeric glycols
 results in significantly enhanced blood half-lives. Synthetic
 phospholipids modified by the attachment of carboxylic groups of
 polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.
 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)
 described experiments demonstrating that liposomes comprising
 phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have
 significant increases in blood circulation half-lives. Blume et al.
 (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations
 to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the
 combination of distearoylphosphatidylethanolamine (DSPE) and PEG.
 Liposomes having covalently bound PEG moieties on their external surface
 are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to
 Fisher. Liposome compositions containing 1-20 mole percent of PE
 derivatized with PEG, and methods of use thereof, are described by Woodle
 et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S.
 Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes
 comprising a number of other lipid-polymer conjugates are disclosed in WO
 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO
 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide
 lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos.
 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe
 PEG-containing liposomes that can be further derivatized with functional
 moieties on their surfaces.
 A limited number of liposomes comprising nucleic acids are known in the
 art. WO 96/40062 to Thierry et al. discloses methods for encapsulating
 high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221
 to Tagawa et al. discloses protein-bonded liposomes and asserts that the
 contents of such liposomes may include an antisense RNA. U.S. Pat. No.
 5,665,710 to Rahman et al. describes certain methods of encapsulating
 oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses
 liposomes comprising antisense oligonucleotides targeted to the raf gene.
 Transfersomes are yet another type of liposomes, and are highly deformable
 lipid aggregates which are attractive candidates for drug delivery
 vehicles. Transfersomes may be described as lipid droplets which are so
 highly deformable that they are easily able to penetrate through pores
 which are smaller than the droplet. Transfersomes are adaptable to the
 environment in which they are used, e.g. they are self-optimizing
 (adaptive to the shape of pores in the skin), self-repairing, frequently
 reach their targets without fragmenting, and often self-loading. To make
 transfersomes it is possible to add surface edge-activators, usually
 surfactants, to a standard liposomal composition. Transfersomes have been
 used to deliver serum albumin to the skin. The transfersome-mediated
 delivery of serum albumin has been shown to be as effective as
 subcutaneous injection of a solution containing serum albumin.
 Surfactants find wide application in formulations such as emulsions
 (including microemulsions) and liposomes. The most common way of
 classifying and ranking the properties of the many different types of
 surfactants, both natural and synthetic, is by the use of the
 hydrophile/lipophile balance (HLB). The nature of the hydrophilic group
 (also known as the "head") provides the most useful means for categorizing
 the different surfactants used in formulations (Rieger, in Pharmaceutical
 Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
 If the surfactant molecule is not ionized, it is classified as a nonionic
 surfactant. Nonionic surfactants find wide application in pharmaceutical
 and cosmetic products and are usable over a wide range of pH values. In
 general their HLB values range from 2 to about 18 depending on their
 structure. Nonionic surfactants include nonionic esters such as ethylene
 glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl
 esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
 alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated
 alcohols, and ethoxylated/propoxylated block polymers are also included in
 this class. The polyoxyethylene surfactants are the most popular members
 of the nonionic surfactant class.
 If the surfactant molecule carries a negative charge when it is dissolved
 or dispersed in water, the surfactant is classified as anionic. Anionic
 surfactants include carboxylates such as soaps, acyl lactylates, acyl
 amides of amino acids, esters of sulfuric acid such as alkyl sulfates and
 ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates,
 acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The
 most important members of the anionic surfactant class are the alkyl
 sulfates and the soaps.
 If the surfactant molecule carries a positive charge when it is dissolved
 or dispersed in water, the surfactant is classified as cationic. Cationic
 surfactants include quaternary ammonium salts and ethoxylated amines. The
 quaternary ammonium salts are the most used members of this class.
 If the surfactant molecule has the ability to carry either a positive or
 negative charge, the surfactant is classified as amphoteric. Amphoteric
 surfactants include acrylic acid derivatives, substituted alkylamides,
 N-alkylbetaines and phosphatides.
 The use of surfactants in drug products, formulations and in emulsions has
 been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker,
 Inc., New York, N.Y., 1988, p. 30 285).
 Penetration Enhancers
 In one embodiment, the present invention employs various penetration
 enhancers to effect the efficient delivery of nucleic acids, particularly
 oligonucleotides, to the skin of animals. Most drugs are present in
 solution in both ionized and nonionized forms. However, usually only lipid
 soluble or lipophilic drugs readily cross cell membranes. It has been
 discovered that even non-lipophilic drugs may cross cell membranes if the
 membrane to be crossed is treated with a penetration enhancer. In addition
 to aiding the diffusion of non-lipophilic drugs across cell membranes,
 penetration enhancers also enhance the permeability of lipophilic drugs.
 Penetration enhancers may be classified as belonging to one of five broad
 categories, i.e., surfactants, fatty acids, bile salts, chelating agents,
 and non-chelating non-surfactants (Lee et al., Critical Reviews in
 Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned
 classes of penetration enhancers are described below in greater detail.
 Surfactants: In connection with the present invention, surfactants (or
 "surface-active agents") are chemical entities which, when dissolved in an
 aqueous solution, reduce the surface tension of the solution or the
 interfacial tension between the aqueous solution and another liquid, with
 the result that absorption of oligonucleotides through the mucosa is
 enhanced. In addition to bile salts and fatty acids, these penetration
 enhancers include, for example, sodium lauryl sulfate,
 polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et
 al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92);
 and perfluorochemical emulsions, such as FC-43. Takahashi et al., J.
 Pharm. Pharmacol., 1988, 40, 252).
 Fatty acids: Various fatty acids and their derivatives which act as
 penetration enhancers include, for example, oleic acid, lauric acid,
 capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid,
 linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein
 (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,
 glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,
 acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
 t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,
 caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,
 Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;
 Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7,
 1-33; E1 Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
 Bile salts: The physiological role of bile includes the facilitation of
 dispersion and absorption of lipids and fat-soluble vitamins (Brunton,
 Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of
 Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,
 pp. 934-935). Various natural bile salts, and their synthetic derivatives,
 act as penetration enhancers. Thus the term "bile salts" includes any of
 the naturally occurring components of bile as well as any of their
 synthetic derivatives. The bile salts of the invention include, for
 example, cholic acid (or its pharmaceutically acceptable sodium salt.,
 sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic
 acid (sodium deoxycholate), glucholic acid, sodium glucholate), glycholic
 acid (sodium glycocholate), glycodeoxycholic acid (sodium
 glycodeoxycholate), taurocholic acid (sodium taurocholate),
 taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid
 (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium
 tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and
 polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in
 Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:
 Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack
 Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical
 Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et
 al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.
 Sci., 1990, 79, 579-583).
 Chelating Agents: Chelating agents, as used in connection with the present
 invention, can be defined as compounds that remove metallic ions from
 solution by forming complexes therewith, with the result that absorption
 of oligonucleotides through the mucosa is enhanced. With regards to their
 use as penetration enhancers in the present invention, chelating agents
 have the added advantage of also serving as DNase inhibitors, as most
 characterized DNA nucleases require a divalent metal ion for catalysis and
 are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993,
 618, 315-339). Chelating agents of the invention include but are not
 limited to disodium ethylenediaminetetraacetate (EDTA), citric acid,
 salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
 homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl
 derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in
 Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical
 Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,
 J. Control Rel., 1990, 14, 43-51).
 Non-chelating non-surfactants: As used herein, non-chelating non-surfactant
 penetration enhancing compounds can be defined as compounds that
 demonstrate insignificant activity as chelating agents or as surfactants
 but that nonetheless enhance absorption of oligonucleotides through the
 alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier
 Systems, 1990, 7, 1-33). This class of penetration enhancers include, for
 example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone
 derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier
 Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such
 as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,
 J. Pharm. Pharmacol., 1987, 39, 621-626).
 Agents that enhance uptake of oligonucleotides at the cellular level may
 also be added to the pharmaceutical and other compositions of the present
 invention. For example, cationic lipids, such as lipofectin (Junichi et
 al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and
 polycationic molecules, such as polylysine (Lollo et al., PCT Application
 WO 97/30731), are also known to enhance the cellular uptake of
 oligonucleotides.
 Other agents may be utilized to enhance the penetration of the administered
 nucleic acids, including glycols such as ethylene glycol and propylene
 glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene
 and menthone.
 Carriers
 Certain compositions of the present invention also incorporate carrier
 compounds in the formulation. As used herein, "carrier compound" or
 "carrier" can refer to a nucleic acid, or analog thereof, which is inert
 (i.e., does not possess biological activity per se) but is recognized as a
 nucleic acid by in vivo processes that reduce the bioavailability of a
 nucleic acid having biological activity by, for example, degrading the
 biologically active nucleic acid or promoting its removal from
 circulation. The coadministration of a nucleic acid and a carrier
 compound, typically with an excess of the latter substance, can result in
 a substantial reduction of the amount of nucleic acid recovered in the
 liver, kidney or other extracirculatory reservoirs, presumably due to
 competition between the carrier compound and the nucleic acid for a common
 receptor. For example, the recovery of a partially phosphorothioate
 oligonucleotide in hepatic tissue can be reduced when it is coadministered
 with polyinosinic acid, dextran sulfate, polycytidic acid or
 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al.,
 Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl.
 Acid Drug Dev., 1996, 6, 177-183).
 Excipients
 In contrast to a carrier compound, a "pharmaceutical carrier" or
 "excipient" is a pharmaceutically acceptable solvent, suspending agent or
 any other pharmacologically inert vehicle for delivering one or more
 nucleic acids to an animal. The excipient may be liquid or solid and is
 selected, with the planned manner of administration in mind, so as to
 provide for the desired bulk, consistency, etc., when combined with a
 nucleic acid and the other components of a given pharmaceutical
 composition. Typical pharmaceutical carriers include, but are not limited
 to, binding agents (e.g., pregelatinized maize starch,
 polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers
 (e.g., lactose and other sugars, microcrystalline cellulose, pectin,
 gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium
 hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
 silica, colloidal silicon dioxide, stearic acid, metallic stearates,
 hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium
 benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium
 starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate,
 etc.).
 Pharmaceutically acceptable organic or inorganic excipient suitable for
 non-parenteral administration which do not deleteriously react with
 nucleic acids can also be used to formulate the compositions of the
 present invention. Suitable pharmaceutically acceptable carriers include,
 but are not limited to, water, salt solutions, alcohols, polyethylene
 glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic
 acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and
 the like.
 Formulations for topical administration of nucleic acids may include
 sterile and non-sterile aqueous solutions, non-aqueous solutions in common
 solvents such as alcohols, or solutions of the nucleic acids in liquid or
 solid oil bases. The solutions may also contain buffers, diluents and
 other suitable additives. Pharmaceutically acceptable organic or inorganic
 excipients suitable for non-parenteral administration which do not
 deleteriously react with nucleic acids can be used.
 Suitable pharmaceutically acceptable excipients include, but are not
 limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin,
 lactose, amylose, magnesium stearate, talc, silicic acid, viscous
 paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
 Other Components
 The compositions of the present invention may additionally contain other
 adjunct components conventionally found in pharmaceutical compositions, at
 their art-established usage levels. Thus, for example, the compositions
 may contain additional, compatible, pharmaceutically-active materials such
 as, for example, antipruritics, astringents, local anesthetics or
 anti-inflammatory agents, or may contain additional materials useful in
 physically formulating various dosage forms of the compositions of the
 present invention, such as dyes, flavoring agents, preservatives,
 antioxidants, opacifiers, thickening agents and stabilizers. However, such
 materials, when added, should not unduly interfere with the biological
 activities of the components of the compositions of the present invention.
 The formulations can be sterilized and, if desired, mixed with auxiliary
 agents, e.g., lubricants, preservatives, stabilizers, wetting agents,
 emulsifiers, salts for influencing osmotic pressure, buffers, colorings,
 flavorings and/or aromatic substances and the like which do not
 deleteriously interact with the nucleic acid(s) of the formulation.
 Aqueous suspensions may contain substances which increase the viscosity of
 the suspension including, for example, sodium carboxymethylcellulose,
 sorbitol and/or dextran. The suspension may also contain stabilizers.
 Certain embodiments of the invention provide pharmaceutical compositions
 containing (a) one or more antisense compounds and (b) one or more other
 chemotherapeutic agents which function by a non-antisense mechanism.
 Examples of such chemotherapeutic agents include, but are not limited to,
 anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,
 bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,
 cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),
 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),
 colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and
 diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis
 and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages
 1206-1228). Anti-inflammatory drugs, including but not limited to
 nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral
 drugs, including but not limited to ribivirin, vidarabine, acyclovir and
 ganciclovir, may also be combined in compositions of the invention. See,
 generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et
 al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
 Other non-antisense chemotherapeutic agents are also within the scope of
 this invention. Two or more combined compounds may be used together or
 sequentially.
 In another related embodiment, compositions of the invention may contain
 one or more antisense compounds, particularly oligonucleotides, targeted
 to a first nucleic acid and one or more additional antisense compounds
 targeted to a second nucleic acid target. Numerous examples of antisense
 compounds are known in the art. Two or more combined compounds may be used
 together or sequentially.
 The formulation of therapeutic compositions and their subsequent
 administration is believed to be within the skill of those in the art.
 Dosing is dependent on severity and responsiveness of the disease state to
 be treated, with the course of treatment lasting from several days to
 several months, or until a cure is effected or a diminution of the disease
 state is achieved. Optimal dosing schedules can be calculated from
 measurements of drug accumulation in the body of the patient. Persons of
 ordinary skill can easily determine optimum dosages, dosing methodologies
 and repetition rates. Optimum dosages may vary depending on the relative
 potency of individual oligonucleotides, and can generally be estimated
 based on EC.sub.50 s found to be effective in in vitro and in vivo animal
 models. In general, dosage is from 0.01 ug to 100 g per kg of body weight,
 and may be given once or more daily, weekly, monthly or yearly, or even
 once every 2 to 20 years. Persons of ordinary skill in the art can easily
 estimate repetition rates for dosing based on measured residence times and
 concentrations of the drug in bodily fluids or tissues. Following
 successful treatment, it may be desirable to have the patient undergo
 maintenance therapy to prevent the recurrence of the disease state,
 wherein the oligonucleotide is administered in maintenance doses, ranging
 from 0.01 ug to 100 g per kg of body weight, once or more daily, to once
 every 20 years.
 While the present invention has been described with specificity in
 accordance with certain of its preferred embodiments, the following
 examples serve only to illustrate the invention and are not intended to
 limit the same.