JP2002519015A - Antisense modulation of TNFR1 expression - Google Patents

Abstract

  (57) [Summary]   Provided are antisense compounds, compositions and methods that modulate TNFR1 expression. The composition comprises an antisense compound, particularly an antisense oligonucleotide targeting a nucleic acid encoding TNFR1. Methods for using these compounds to modulate TNFR1 expression and for treating diseases associated with TNFR1 expression are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION

The present invention provides compositions and methods that modulate TNFR1 expression.
In particular, the present invention relates to antisense compounds, particularly oligonucleotides, that specifically hybridize to nucleic acids encoding human TNFR1. Such oligonucleotides have been shown to modulate TNFR1 expression.

[0002]

BACKGROUND OF THE INVENTION

One of the major mechanisms by which cellular regulation is effected is by transmitting extracellular signals to intracellular signals that modulate biochemical pathways. Examples of such extracellular signaling molecules include growth hormones, cytokines and chemokines. Thus, the cell surface receptors for these molecules and their associated signaling pathways are one of the primary means by which cell behavior is regulated. Because the phenotype of cells is greatly influenced by the activity of these pathways, there are now numerous pathologies and / or
Or, the disorder is thought to be the result of either abnormal activation or a functional mutation in a molecular component of the signaling pathway.

[0003] For example, the tumor cytokine necrosis factor (TNF), a polypeptide cytokine,
It is usually produced at the time of injury or invasion and serves as a tumor mediator of the inflammatory response. Recently, a number of in vivo animal and human studies have shown that overexpression of TNF by the host in response to the disease and the infection itself may be responsible for the pathological consequences associated with the underlying disease. For example, septic shock as a result of high bacterial infections has been attributed to infection-induced expression of TNF. Thus, systemic exposure to levels of TNF comparable to that following large amounts of bacterial infection results in a wide range of symptoms (shock, tissue damage, capillary leakage, hypoxia, pulmonary edema, multiple organ failure). , And high mortality), which are almost indistinguishable from sepsis syndrome [Tracey, 19
94 # 7415]. Additional evidence provided from animal models of septic shock showed that systemic exposure to anti-TNF neutralizing antibodies prevented bacterial-induced sepsis [Tracey, 1994 # 7415]. In addition to these acute effects, chronic exposure to low doses of TNF causes a cachectic syndrome characterized by anorexia, weight loss, dehydration, and a deficiency of systemic proteins and fats. Bring. Chronic production of TNF has been linked to a number of diseases, including AIDS and cancer [Tracey, 1994 # 7415]. To date, TNFR1 and T
Two different TNF cell surface receptors, known as NFR2, have been described. Molecular analysis of TNFR1 and TNFR2 indicated that the two receptors share little homology in their intracellular domains and appear to activate distinct intracellular pathways [Tracey, 1994 # 7415].

In recent studies using transgenic TNFR1 knockout mice, TNFR1-mediated signaling plays an important role in the clearance of low levels of bacterial infection, as well as TNF-induced septic shock following high levels of bacterial infection. [Lotz, 1996 # 7436]. These findings suggest that TNFR1
We show that compositions of substances that can inhibit receptor-mediated signaling can serve as useful targets for inhibiting TNF-induced deleterious effects such as septic shock.

[0005] Antisense oligonucleotide inhibition of TNFR1 has proven to be a useful tool in understanding the role of TNFR1 stimulation in cytokine induction and cell proliferation. Ojwang et. Al. Disclose partially phosphorothioate antisense oligodeoxynucleotides containing a C-5-propynyl or hexyl derivative of 2'-deoxyuridine, which is a TNFR1 mRNA.
And attenuated proteins and inhibited TNF-α-induced IL-6 expression in MRC-cells [Ojwang, 1997 # 5863]. These oligonucleotides targeted the poly (A) signal site of TNFR1 mRNA. Homogeneous phosphorothioate oligonucleotides targeting the translation initiation codon of TNFR1 were found to be ineffective. There remains a need for improved compositions and methods of inhibiting TNFR1 gene expression.

[0006]

[Summary of the Invention]

The present invention relates to oligonucleotides that modulate TNFR1 expression targeting antisense compounds, particularly nucleic acids encoding TNFR1. Pharmaceutical and other compositions comprising the antisense compounds of the present invention are also provided. Further provided is a method of modulating TNFR1 expression in a cell or tissue comprising contacting the cell or tissue with one or more antisense compounds or compositions of the invention. . Further provided is whether a patient has a disease or condition associated with TNFR1 expression by administering a therapeutically or prophylactically effective amount of one or more antisense compounds or compositions of the present invention. Or a method of treating an animal suspected of having the same, particularly a human.

[0007]

DESCRIPTION OF THE INVENTION

The present invention utilizes oligomeric antisense compounds, particularly oligonucleotides, to modulate the function of a nucleic acid molecule encoding TNFR1 and ultimately modulate the amount of TNFR1 produced. This is achieved by providing an antisense compound that specifically hybridizes to one or more nucleic acids encoding TNFR1. As used herein, the terms "target nucleic acid" and "TNFR1-encoding nucleic acid" refer to TNFR1-encoding DNA, RNA transcribed from such DNA (pre-mRNA and mRNA).
And cDNAs derived from such RNAs. When an oligomeric compound specifically hybridizes to its target nucleic acid, it interferes with the normal function of the nucleic acid. Modulating its function with a compound that specifically hybridizes to the target nucleic acid in this way is commonly referred to as "antisense." Functions of the DNA to be interfered with include replication and transcription. The function of the interfering RNA may involve or involve, for example, translocation of the RNA to a protein translation site, translation of the protein from the RNA, splicing of the RNA to produce one or more mRNA species, and RNA. Includes all critical functions such as catalytic activity that can be promoted by The overall effect of such interference on the function of the target nucleic acid will be the modulation of TNFR1 expression. In the context of the present invention, "modulation" means either an increase (enhancement) or a decrease (inhibition) in gene expression. In the context of the present invention, the preferred form of gene expression modulation is inhibition, and the preferred target is mRNA.

It is preferred that certain nucleic acids be targeted for antisense. "Targeting" an antisense compound to a particular nucleic acid is a multi-step method in the context of the present invention. The method usually begins with identifying the nucleic acid sequence whose function is to be modulated. This can be, for example, a cellular gene whose expression is associated with a particular disorder or condition (or mRNA transcribed from that gene), or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding TNFR1. Methods of targeting also include determining the site within the gene where the antisense interaction occurs such that a desired effect occurs, for example, detection or modulation of expression of the protein. In the context of the present invention, a preferred intragenic site is a region spanning the translational start or stop codon of the open reading frame (ORF) of the gene. As is known in the art, the translation initiation codon is also 5'-AUG (in the transcribed mRNA molecule; 5'-ATG in the corresponding DNA molecule), so that the translation initiation codon is also "AUG". Also referred to as "codon", "start codon" or "AUG start codon". A few genes have translation initiation codons whose RNA sequence is 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG function in vivo. It is shown to be. Thus, the terms "translation initiation codon" and "initiation codon" refer to a number of codon sequences, even though in each case the starting amino acid is usually methionine (eukaryote) or formylmethionine (prokaryote). May be included. In the art, eukaryotic and prokaryotic genes have two or more alternative start codons, one of which, in a particular cell type or tissue, or under a particular set of conditions, It is also known that it can be used preferentially for translation initiation. In the context of the present invention, "initiation codon" and "translation initiation codon" refer to the translation of an mRNA molecule transcribed from the gene encoding TNFR1, regardless of the sequence of such codons.
It also refers to the codons used to initiate in vivo.

[0009] In the art, the translation stop codon (or "stop codon") of a gene has three sequences: 5'-UAA, 5'-UAG, and 5'-TGA (the corresponding DNA sequence is 5'-TAA, 5'-TAG and 5'-TGA). The terms "initiation codon region" and "translation initiation codon region" are defined as approximately two orientations (ie, 5 'or 3') from the translation initiation codon.
A portion of an mRNA or gene that contains from 5 to about 50 contiguous nucleotides. Similarly, the terms "stop codon region" and "translation stop codon region" refer to mRNA as comprising about 25 to about 50 contiguous nucleotides in both directions (ie, 5 'or 3') from the translation stop codon. Or a part of a gene.

[0010] An open reading frame (ORF) or "coding region" is known in the art to mean a region located between a translation initiation codon and a translation termination codon, but can be effectively targeted. But also. Other target regions include 5 '
Untranslated region (5'-UTR) [in the art, the portion of the mRNA that is 5 'from the translation initiation codon, and thus the nucleotide, or gene, between the 5' cap site and the mRNA translation initiation codon. And the 3 'untranslated region (3'-UTR) [in the art, the portion of the mRNA that is in the 3' direction from the translation stop codon, and thus the mRNA Or the portion encompassing the nucleotide between the translation termination codon and the 3 'end of the gene, or the corresponding nucleotide on the gene]. The 5 'cap of the mRNA contains an N7 methylated guanosine residue linked to the 5' terminal residue of the mRNA via a 5'-5 'triphosphate linkage. It is believed that the 5 'cap region of the mRNA includes the 5' cap structure itself, as well as the first 50 nucleotides adjacent to the cap. The 5 'cap region can also be a preferred target region.

Although some eukaryotic mRNA transcripts are translated directly, many
Contains one or more regions known as "introns" that are excised from the transcript before being translated. The remaining (and therefore translated) region is an “exon”
And are spliced together to form a continuous mRNA sequence. mR
Since the splice site of NA, the intron-exon junction, may also be a preferred target region, aberrant splicing may be associated with the disease, or overproduction of certain mRNA splice products may be associated with the disease. This is especially useful in situations where you may be Abnormal fusion junctions caused by various rearrangements and deletions are also preferred targets. It may also be desirable to target specific exons of alternatively spliced mRNA. Introns also include, for example, DNA
Alternatively, it has been found that it is effective for antisense compounds targeted to pre-mRNA, and thus may be preferred. Once one or more target sites have been identified, oligonucleotides are selected that are sufficiently complementary to the target, ie, hybridize with sufficient strength and sufficient specificity, to produce the desired effect.

In the context of the present invention, “hybridization” means a hydrogen bond between complementary nucleosides or nucleotide bases, which may be a Watson-Crick, Hoogsteen or reverse Hoogsteen type hydrogen bond. For example, adenine and thymine are complementary nucleobases that pair via hydrogen bond formation. As used herein, "complementary" refers to the ability of exact pairing between two nucleotides. For example, if the nucleotide at a position in the oligonucleotide is DN
If the A or RNA molecule can hydrogen bond to a nucleotide at the same position, the oligonucleotide and DNA or RNA are considered to be complementary to each other at that position. Oligonucleotides and DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary"
Is used to demonstrate a sufficient degree of complementarity or precise pairing that stable and specific binding between the oligonucleotide and the DNA or RNA target occurs.
It is understood in the art that the sequence of an antisense compound need not be 100% complementary to the sequence of its target nucleic acid to be able to specifically hybridize. The antisense compound can specifically hybridize to the target DNA
Or when binding of the compound to an RNA molecule interferes with the normal function of the target DNA or RNA, causing loss of utility, and under conditions where specific binding is desired,
That is, under physiological conditions for an in vivo assay or therapeutic treatment, or under the conditions of performing the assay for an in vitro assay, a sufficient degree of avoidance of non-specific binding of the antisense compound to a non-target sequence. This is when there is complementarity.

[0013] Antisense compounds are commonly used as research reagents and diagnostics. For example,
Antisense oligonucleotides that can inhibit gene expression with strong specificity are often used by those skilled in the art to elucidate the function of a particular gene. This is often referred to as "target validation". Antisense compounds are also used, for example, to identify the function of various members of a biological pathway. Thus, antisense modulation has been exploited for research use.

The specificity and sensitivity of antisense has also been exploited by those skilled in the art for therapeutic use. Antisense oligonucleotides have been used as therapeutic moieties in the treatment of animal and human conditions. Antisense oligonucleotides have been safely and effectively administered to humans, and numerous clinical trials are currently underway.
Thus, it has been demonstrated that oligonucleotides can be useful therapeutic modalities that can be constructed to be useful in therapeutic modalities for the treatment of cells, tissues and animals, particularly humans.

In the context of the present invention, the term “oligonucleotide” refers to ribonucleic acid (RNA
) Or deoxyribonucleic acid (DNA) or mimetics thereof. The term also includes oligonucleotides consisting of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring portions that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms, for example, because of enhanced cell uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases For such desirable properties.

Although the preferred form of the antisense compound is an antisense oligonucleotide, the present invention also includes other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics as set forth below. Antisense compounds according to the present invention preferably contain from about 8 to about 30 nucleobases. Particularly preferred are antisense oligonucleotides comprising about 8 to about 30 nucleobases (ie, about 8 to about 30 linked nucleosides). As is known in the art, a nucleoside is a base-sugar conjugate. The base portion of the nucleoside is usually a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides that include a pentofuranosyl sugar, a phosphate group can be linked to either the 2 ', 3' or 5 'hydroxyl moiety of the sugar. When forming oligonucleotides, adjacent nucleosides are linked together via a phosphate group to form a linear polymer compound. Further, both ends of this linear structure may be joined to form a circular structure, but generally an open linear structure is preferred. Within the oligonucleotide structure, it is commonly said that the phosphate groups form the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is 3
'-5' phosphodiester linkage.

[0017] Particular examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or unnatural internucleoside linkages. As defined herein, oligonucleotides having a modified backbone include:
Those having a phosphorus atom in the skeleton and those having no phosphorus atom in the skeleton are included. For the purposes of this specification, as sometimes referred to in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3′-alkylene phosphonates having the usual 3′-5 ′ linkage. Methyl and other alkyl phosphonates, including chiral phosphonates, phosphinates, phosphoramidites, including 3'-aminophosphoramidites and aminoalkyl phosphoramidites, thionophosphoramidites, thionoalkylphosphonates, thionoalkylphosphonates Triesters and boranophosphates, 2'-5 'linked analogs of the above, and adjacent pairs of nucleoside units having a 3'-5' to 5'-3 'or 2'-5' to 5'-2 ' With opposite polarity connected to Nitai is included. Various salts, mixed salts, and free acid forms are also included.

Representative US patents that teach the manufacture of the above phosphorus-containing linkages include, but are not limited to, US Pat. Nos. 3,687,808; 4,469,863;
No. 1, No. 5,023,243; No. 5,177,196; No. 5,188,89
No. 7, No. 5,264,423; No. 5,276,019; No. 5,278,30
No. 2, No. 5,286,717; No. 5,321,131; No. 5,399,67
No. 6,5,405,939; 5,453,496; 5,455,23
No. 3,5,466,677; 5,476,925; 5,519,12
No. 6, No. 5,536,821; No. 5,541,306; No. 5,550, 11
No. 5,563,253; 5,571,799; 5,587,36
No. 5,625,050; and 5,697,248, some of which are owned by the present application, all of which are incorporated herein by reference.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom include short chain alkyl or cycloalkyl internucleoside linkages, mixed heteronucleotide and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroaryl It has therein a skeleton formed by atomic or heterocyclic internucleoside linkages. These include skeletons with morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane skeletons; sulfide, sulfoxide and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons. ; alkene containing backbones; sulfamate backbones; Mechiren'imino and methylene hydrazino skeleton; sulfonate and sulfonamide backbones; amide backbones; and N, O, contains other scaffolds with mixing section S and CH ₂ component.

Representative US patents that teach the production of the oligonucleotides include, but are not limited to, US Pat. Nos. 5,034,506; 5,166,315; 5,185,
No. 444; 5,214,134; 5,216,141; 5,235.
No. 033; 5,264,562; 5,264,564; 5,405.
No. 938; 5,434,257; 5,466,677; 5,470,
No. 967; 5,489,677; 5,541,307; 5,561.
No. 225; 5,596,086; 5,602,240; 5,610,
No. 289; 5,602,240; 5,608,046; 5,610,
No. 289; 5,618,704; 5,623,070; 5,663.
No. 3,12; 5,633,360; 5,677,437; and 5,67.
No. 7,439, of which some are owned by the present application, all of which are incorporated herein by reference.

In another preferred oligonucleotide mimetic, both the sugar and the internucleoside linkage of the nucleotide unit, ie, the backbone, are replaced with a novel group. This base unit is
Maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound is an oligonucleotide mimetic that has been shown to have excellent hybridization properties, including peptide nucleic acids (PNA).
). In the PNA compound, the sugar skeleton of the oligonucleotide is substituted with an amide-containing skeleton, particularly an aminoethylglycine skeleton. The nucleobase is retained and is directly or indirectly attached to the aza nitrogen atom of the amide portion of the backbone. PNA
Representative U.S. patents that teach the preparation of compounds include, but are not limited to, U.S. Pat.
Nos. 9,082; 5,714,331; and 5,719,262, all of which are incorporated herein by reference. Further teachings on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

The most preferred embodiments of the present invention are oligonucleotides having a phosphorothioate backbone and nucleosides having a heteroatom backbone, particularly -CH ₂ -NH-O- of US Pat. No. 5,489,677, referenced above. CH ₂ —, CH ₂ —N (C
_{H 3) -O-CH 2 -} [ known as a methylene (methylimino) or MMI _{backbone], - CH 2 -O-N} (CH 3) -CH 2 -, - CH 2 -N (CH 3) -N (
CH ₃ ) —CH ₂ — and —ON (CH ₃ ) —CH ₂ —CH ₂ — skeletons (where the natural phosphodiester skeleton is represented as —OP—O—CH ₂ —), And the amide skeleton of U.S. Pat. No. 5,602,240 referred to above.
Also preferred are the oligonucleotides having a morpholino backbone structure of US Pat. No. 5,034,506, referenced above.

[0024] The modified oligonucleotide also contains one or more substituted sugar moieties.
Can have. Preferred oligonucleotides include at the 2 ′ position one of the following: OH;
F; O-, S- or N-alkyl, O-, S- or N-alkenyl, O-, S-
Or N-alkynyl wherein alkyl, alkenyl and alkynyl are substituted
Substituted or unsubstituted C₁~ C_TenAlkyl, C_Two~ C_TenAlkenyl
And alkynyl). Particularly preferred is O [(CH_Two)_nO]_mCH_Three,
O (CH_Two)_nOCH_Three, O (CH_Two)_nNH_Two, O (CH_Two)_nCH_Three, O (CH_Two) _n ONH_TwoAnd O (CH_Two)_nON [(CH_Two)_nCH_Three]_Two(Where n and
m is 1 to about 10). Other preferred oligonucleotides have the following at the 2 ′ position:
Including one: C₁~ C_TenLower alkyl, substituted lower alkyl, alkaryl
, Aralkyl, O-alkaryl or O-aralkyl, SH, SCH_Three, OCN
, Cl, Br, CN, CF_Three, OCF_Three, SOCH_Three, SO_TwoCH_Three, ONO_Two , NO_Two, N_Three, NH_Two, Heterocycloalkyl, heterocycloalkaryl,
Aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group
, Reporter groups, intercalators, groups that improve the pharmacodynamic properties of oligonucleotides,
Groups that improve the pharmacokinetic properties of gonucleotides, and other substitutions with similar properties
Base. Preferred modifications include 2'-methoxyethoxy (2'-O-CH_TwoCH_TwoOC
H_Three, Also known as 2'-O- (2-methoxyethyl) or 2'-MOE)
(Martin et al., Helv. Chim. Acta, 1995, 78, 486-504).
It contains a lucoxy group. More preferred modifications include 2'-dimethylaminooxy
Ethoxy, O (CH also known as 2'-DMAOE_Two)_TwoON (CH_Three )_TwoWhich are described in U.S. Patent Application No.
No. 09 / 016,520, which is owned with the present application and
Is incorporated herein by reference.

[0025] Other preferred modifications include 2 '- methoxy (2' -O-CH _3), 2 '- aminopropoxy (2' -OCH ₂ CH ₂ CH ₂ NH ₂₎ and 2 '- fluoro (2' −
F). Similar modifications may be made at other sites on the oligonucleotide, especially the 3 'and 5' positions of the sugar on the 3 'terminal nucleotide or in the 2'-5' linked oligonucleotide.
This can be done at the 5 'position of the terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents that teach the production of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,427; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639. 5,646,265; 5,658,873; 5,670,633 and 5,700,920 (some of which are owned with the present application, Each of which is incorporated herein by reference), and Filed on January 5, 1995, it authorized the United States patent application 08/46
No. 8,037 (U.S. Patent Nos. X, XXX, XXX; this patent is owned with the present application and incorporated herein by reference).

Oligonucleotides can also include modifications or substitutions of nucleobases (referred to in the art simply as “bases”). As used herein, "unmodified" or "natural" nucleobases include purine bases adenine (A) and guanine (G), pyrimidine bases thymine (T), cytosine (C) And uracil (U)
Is included. Modified nucleobases include 5-methylcytosine (5-me-C
), 5-hydroxymethylcytosine, 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,
-Thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8- Thiols, 8-thioalkyls, 8-hydroxyls and other 8-substituted adenines and guanines, 5-halo, especially 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 other synthetic and natural nucleobases such as 3-deazaguanine and 3-deazaadenine. Additional nucleobases include US Pat. No. 3,687,8.
08, Concise Encyclopedia Of Polymer Science And Engi
neering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Editio.
n, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antis
ense Research and Applications, pages 289-302, Crooke, ST and Lebleu,
B., ed., CRC Press, 1993. Some 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, O-6 positions, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. Includes substituted purines. 5
-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C (Sanghvi, YS, Crooke, ST, and Lebleu, B. eds., Antisense Re
search and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), especially when combined with 2'-O-methoxyethyl sugar modification, is currently the preferred base substitution.

Representative US patents that teach the preparation of some of the above noted modified nucleobases, as well as some other modified nucleobases, include, but are not limited to, US Pat. 808, and U.S. Patent Nos. 4,845,205; 5,
No. 130,302; No. 5,134,066; No. 5,175,273;
567,066; 5,432,272; 5,457,187; 5,
No. 4,559,255; No. 5,484,908; No. 5,502,177;
No. 525,711; 5,552,540; 5,587,469;
Nos. 5,594,121; 5,596,091; 5,614,617 and 5th.
No., 681,941 (of which some are owned with the present application, all of which are incorporated herein by reference), as well as U.S. patent applications filed Dec. 10, 1996 and granted. 08 / 762,488 (U.S. Pat.
XXX, XXX; this patent is owned with the present application and is incorporated herein by reference).

[0028] Other modifications to the oligonucleotides of the invention include one or more moieties or conjugates chemically linked to the oligonucleotide that enhance the activity, intracellular distribution or uptake of the oligonucleotide. . Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci., US
A, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bi
oorg. Med. Chem. Lett., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 276.
5-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,
20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Sa
ison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FE
BS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49
-54), a phospholipid such as di-hexadecyl-rac-glycerol or 1,2.
-Di-O-hexadecyl-rac-glycero-3-H-triethylammonium phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea e
tal., Nucl. Acids Res., 1990, 18 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 9).
69-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36, 3651-3654), 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 US patents that teach the preparation of the above-described oligonucleotide conjugates include, but are not limited to, US Pat. Nos. 4,828,979; 4,948,882;
No. 218,105; 5,525,465; 5,541,313; 5,
No. 545,730; No. 5,552,538; No. 5,578,717;
No. 580,731; No. 5,591,584; No. 5,109,124;
No. 118,802; 5,138,045; 5,414,077;
No. 486,603; No. 5,512,439; No. 5,578,718;
No. 608,046; No. 4,587,044; No. 4,605,735;
No. 667,025; No. 4,762,779; No. 4,789,737;
No. 824,941; No. 4,835,263; No. 4,876,335;
No. 904,582; No. 4,958,013; No. 5,082,830;
No. 112,963; 5,214,136; 5,082,830; 5,
No. 112,963; 5,214,136; 5,245,022; 5,
No. 5,254,469; No. 5,258,506; No. 5,262,536;
No. 272,250; No. 5,292,873; No. 5,317,098;
No. 371,241; No. 5,391,723; No. 5,416,203;
No. 451,463; 5,510,475; 5,512,667; 5,
No. 514,785; 5,565,552; 5,567,810;
No. 574,142; 5,585,481; 5,587,371; 5,
No. 595,726; 5,597,696; 5,599,923; 5,
No. 599,928 and No. 5,688,941, some of which are owned with the present application, both of which are incorporated herein by reference.

Not all positions of a particular compound need be uniformly modified;
One or more of the above modifications may be incorporated into a single compound or even a single nucleoside in a given oligonucleotide. The invention also includes antisense compounds that are chimeric compounds. A “chimera” antisense compound or “chimera”
In the context of the present invention, antisense compounds, in particular oligonucleotides, containing two or more chemically distinct regions, each consisting of at least one monomer unit, ie a nucleotide in the case of an oligonucleotide compound. Typically, these oligonucleotides are at least modified such that the oligonucleotides are modified to provide them with increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. 1
Contains two regions. Additional regions of the oligonucleotide can serve as substrates for enzymes that can cleave RNA: DNA or RNA: RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA: DNA duplex, and thus activation of RNase H results in cleavage of the RNA target, thereby inhibiting oligonucleotide inhibition of gene expression. Increase efficiency. Therefore,
Using shorter oligonucleotides when using chimeric oligonucleotides often gives equivalent results as when phosphorothioate deoxyoligonucleotides hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis, if necessary, in combination with related nucleic acid hybridization techniques known in the art.

The chimeric antisense compounds of the present invention can be formed as a composite structure 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 U.S. Patents that teach the manufacture of such hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5,220.
No. 5,007; No. 5,256,775; No. 5,366,878; No. 5,403.
No. 5,491,133; No. 5,565,350; No. 5,623.
5,652,355; 5,652,356 and 5,70
No. 0,922 (some of which are owned by the present application, both of which are incorporated herein by reference) and US patent applications filed Jun. 6, 1995 and granted. 08 / 465,880 (U.S. Patent Nos. X, XXX, X
XX; this patent is owned with the present application and is incorporated herein by reference).

The antisense compounds used according to the present invention can be conveniently and routinely produced via known techniques of solid phase synthesis. An apparatus for such synthesis is, for example, App
It is marketed by several vendors, including led Biosystems (Foster City, CA). Other means for such synthesis known in the art may additionally or alternatively be utilized. It is also known to use similar techniques for producing oligonucleotides such as phosphorothioates and alkylated derivatives. The antisense compounds of the present invention are synthesized in vitro and do not include antisense compositions of biological origin or gene vector constructs designed to direct in vivo synthesis of antisense molecules.

The compounds of the present invention may also include other molecules, molecular structures, such as liposomes, receptor targeting molecules, oral, rectal, topical administration or other formulations, to enhance uptake, distribution and / or absorption. Alternatively, it may be mixed, encapsulated, conjugated or otherwise combined with a mixture of compounds. Representative U.S. patents that teach the manufacture of formulations that facilitate such uptake, distribution and / or absorption include, but are not limited to, U.S. Pat. No. 5,108,92.
No. 5,354,844; 5,416,016; 5,459,12
No. 7, No. 5,521, 291; No. 5,543,158; No. 5,547, 93
No. 5,583,020; 5,591,721; 4,426,33
No. 0; 4,534,899; 5,013,556; 5,108,92
No. 1, No. 5,213,804; No. 5,227,170; No. 5,264,22
No. 5,356,633; 5,395,619; 5,416,01
No. 6, No. 5,417,978; No. 5,462,854; No. 5,469,85.
No. 4, No. 5,512,295; No. 5,527,528; No. 5,534,25
No. 9, No. 5,543, 152; No. 5,556,948; No. 5,580,57
And No. 5,595,756, both of which are incorporated herein by reference.

The antisense compounds of the present invention are pharmaceutically acceptable, which can provide (either directly or indirectly) biologically active metabolites or residues when administered to animals, including humans. Salts, esters or salts of such esters, or other compounds. Thus, for example, the disclosure of the present invention also covers prodrugs and pharmaceutically acceptable salts of the compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Quoted.

The term “prodrug” is an inactive form that is converted into an active form (ie, a drug) within a living organism or its cells by the action of endogenous enzymes and other chemicals and / or conditions. Shows the therapeutic agent being manufactured. In particular, prodrug versions of the oligonucleotides of the invention are described in Gosselin et al. WO93 / 24510 (1
Published December 9, 993) or Imbach et al. SATE [(S-acetyl-2-thioethyl) phosphate] by the method disclosed in WO 94/26764 to
Manufactured as a derivative. The term "pharmaceutically acceptable salt" refers to a physiologically and pharmaceutically acceptable salt of a compound of the present invention, i.e., that retains the desired biological activity of the parent compound and exhibits undesired toxicological effects. It is the salt that is not given to it.

[0036] 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, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-
Methylglucamine and procaine (see, for example, Berge et al., "Pharmaceuti
cal Salts, "J. of Pharma Sci. 1977, 66, 1-19). The base addition salt of the acidic compound is converted into a sufficient amount of the desired base to produce the salt in the conventional manner. Prepared by contacting the free acid form, which can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. Although slightly different from the respective salt forms in certain physical properties, such as solubility in polar solvents, the salts are otherwise equivalent to the respective free acids for the purposes of the present invention, as used herein. In addition, "medicine addition salts" include pharmaceutically acceptable salts of one of the ingredients that make up the compositions of the present invention. This includes the organic or inorganic acid salts of the above amines. Preferred acid salts are hydrochloride,
Acetate, salicylate, nitrate and phosphate. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, inorganic acids such as hydrochloric, hydrobromic, sulfuric or phosphoric acid; for example, acetic, propionic, glycolic, succinic, maleic,
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-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid;
Amino acids such as 20 α-amino acids involved in natural protein synthesis such as glutamic acid or aspartic acid; furthermore, 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- A wide variety of inorganic and organic acids, such as organic carboxylic acids such as cyclohexyl sulfamic acid (along with the formation of cyclamate), sulfonic acids, sulfo or phospho acids or N-substituted sulfamic acids; or other acid organic compounds such as ascorbic acid. Includes basic salts. Pharmaceutically acceptable salts of the compounds may also be formed with pharmaceutically acceptable cations. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkali, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or bicarbonates are also possible.

For oligonucleotides, examples of preferred pharmaceutically acceptable salts include, but are not limited to, (a) formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, and the like. (B) acid addition salts formed with inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, etc .; (c) 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, etc. Salts formed with such organic acids; and (d) chlorine, bromine And salts formed from elemental anions such as iodine.

The antisense compounds of the present invention can be used as diagnostics, therapeutics, prophylaxis, and as research reagents and kits. With respect to therapeutics, animals, preferably humans, suspected of having a disease or disorder that can be treated by modulating TNFR1 expression can be treated by administering an antisense compound according to the present invention. The compounds of the present invention can be utilized in the form of a pharmaceutical composition by adding an effective amount of the antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Uses and methods of the antisense compounds of the present invention may also be useful prophylactically, for example, to prevent or delay infection, inflammation or tumor formation.

The antisense compounds of the present invention are useful for research and diagnosis because they hybridize to a nucleic acid encoding TNFR1 to facilitate the construction of a sandwich method or other assay. Is possible. Hybridization between an antisense oligonucleotide of the invention and a nucleic acid encoding TNFR1 can be detected by means known in the art. Such means may include coupling an enzyme to the oligonucleotide, radiolabeling the oligonucleotide, or other suitable means of detection. Kits for detecting TNFR1 levels in a sample using such means can also be made.

The present invention includes pharmaceutical compositions and formulations that include the antisense compounds of the present invention. The pharmaceutical compositions of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration is
Topical administration (including mucous membranes including the eye and vagina, and rectal delivery), including pulmonary administration by inhalation or insufflation of powders or aerosols, including nebulizers, intratracheal,
It can be intranasal, epithelial or transdermal, oral or parenteral. For parenteral administration,
Intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial administration, for example, intrathecal or intraventricular administration. Oligonucleotides having 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, solutions and powders. Conventional pharmaceutical carriers, aqueous powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, grabs, etc. 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, fragrances, diluents, emulsifiers, dispersing aids or binders may also be desired. Compositions and formulations for parenteral, intrathecal, or intraventricular administration include buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers. Things can also be included.

[0042] Pharmaceutical compositions and / or formulations comprising the oligonucleotides of the invention also include:
Penetration enhancers may also be included to enhance gastrointestinal delivery of the oligonucleotide. Penetration enhancers can be classified as belonging to one of five broad categories: fatty acids, bile salts, chelators, surfactants and non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems). ,
1991, 8, 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33). One or more penetration enhancers from one or more of the above broad categories may be included. For penetration enhancers, see U.S. patent application Ser. No. 08 / 886,8, filed Jul. 1, 1997.
No. 29, U.S. Patent Nos. X, XXX, XXX, and U.S. Patent Application No. 08 / 961,469, filed October 31, 1997, U.S. Patent Nos. X, XXX, XX
No. X, both of which are owned with the present application, both of which are incorporated herein by reference.

Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, zicaplate, tricaplate, resinlate,
Monoolein (also known as 1-monooleoyl-rac-glycerol)
, Dilaurin, caprylic acid, arachidonic acid, glyceryl-1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- and diglycerides, and their physiologically acceptable salts (i.e., oleate, laurate, caprate, myristate, palmitate, stearate,
Linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic
Drug Carrier Systems, 1991, 8: 2, 91-192; Muranishi, Critical Reviews in
Therapeutic Drug Carrier Systems, 1990, 7: 1, 1-33; El-Hariri et al., J.
Pharm. Pharmacol., 1992, 44, 651-654). Examples of currently preferred fatty acids are sodium caprate and sodium laurate, used alone or together at a concentration of 0.5-5%.

Preferred penetration enhancers are disclosed in co-pending US patent application Ser. No. 08 / 886,829, filed Jul. 1, 1997, US Pat. No. X, XXX, XXX, which is incorporated herein by reference. And is incorporated herein by reference. The physiological role of bile includes the promotion of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman &Gilman's The Pharmacological Bass).
is of Therapeutics, 9th Ed., Hardman et al., eds., McGraw-Hill, New York
, NY, 1996, pages 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus, the term "bile salt" encompasses the naturally occurring components of bile, as well as any synthetic derivatives thereof. Preferred bile salts are described in co-pending US patent application Ser. No. 08/886, filed Jul. 1, 1997.
No. 829, U.S. Patent No. X, XXX, XXX, which is owned with the present application and incorporated herein by reference. An example of a currently preferred bile salt is chenodeoxycholic acid (CDCA) (Sigma Chemical Company, St. Louis, MO), which is typically used at a concentration of 0.5-2%.

[0045] Complex formulations comprising one or more penetration enhancers may be used. For example, bile salts can be used in combination with fatty acids to provide a combined preparation. Preferred complex preparations include those obtained by complexing sodium caprate or sodium laurate with CDCA (usually 0.5 to 5%). Chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (ED
TA), citric acid, salicylates (eg, sodium salicylate, 5-methoxysalicylate and homovanillate), N-acyl derivatives of collagen, N-aminoacyl derivatives of laureth-9 and β-diketone (enamine) (Leamine).
e et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8:
2, 92-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Syste
ms, 1990, 7: 1, 1-33; Buur et al., J. Control Rel., 1990, 1443-51). Chelating agents have the added advantage of also serving as DNase inhibitors.

Examples of the surfactant include sodium lauryl sulfate and polyoxyethylene-9.
-Lauryl ether and polyoxyethylene-20-cetyl ether (Lee et a
l., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, 8: 2, 92-
191); and perfluorochemical emulsions such as FC-43 (Takahashi et al.
, J. Pharm. Pharmacol., 1988, 40: 252-257). Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therap).
eutic Drug Carrier Systems, 1991, 8: 2, 92-191); and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39). , 621-626).

A “carrier compound” as used herein is inert (ie, does not itself possess biological activity), but for example, degrades biologically active nucleic acids or A nucleic acid or analog thereof that is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of the nucleic acid with biological activity by promoting its removal from the circulation. Co-administration of the nucleic acid and the carrier compound, generally in excess of the latter substance, substantially reduces the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoir. This is probably due to competition of the nucleic acid with the carrier compound for a common receptor. For example, recovery of a partially phosphorothioated oligonucleotide in liver tissue is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid. Then decrease (Miyao et al
., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & N
ucl. Acid Drug Dev., 1996, 6, 177-183).

[0048] In contrast to a carrier compound, a "pharmaceutically acceptable carrier" (excipient) is a pharmaceutically acceptable solvent, suspension, or one or more nucleic acids to an animal. Other pharmacologically inert media for delivery. Pharmaceutically acceptable carriers can be liquid or solid and are pre-planned to provide the desired size, consistency, etc. when combined with the nucleic acids and other components of a given pharmaceutical composition. The choice is made using the method of administration. Typical pharmaceutically acceptable carriers include, but are not limited to, a binder (eg, gelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); a filler (eg, lactose and other sugars,
Microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate, etc .; lubricants (eg, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogen) Additional vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (eg, starch, sodium starch glycolate, etc.); or wetting agents (sodium lauryl sulfate, etc.). For sustained release oral delivery systems and / or enteric coatings in oral dosage forms, see US Patent Nos. 4,704,295; 4,556,552;
No. 309,406; and 4,309,404.

The compositions of the present invention may additionally contain other adjuvant components conventionally found in pharmaceutical compositions at the levels of use established in the state of the art. Thus, for example, the compositions of the present invention may contain compatible additional pharmaceutically active substances such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain coloring agents, fragrances Contains additional substances useful in physically formulating various dosage forms of the composition of the present invention, such as preservatives, antioxidants, opacifiers, thickeners and stabilizers. obtain. However, such substances, when added, must not unduly interfere with the biological activities of the components of the compositions of the present invention.

Whatever the method of introducing an antisense compound of the present invention into a patient, the colloidal dispersion enhances the in vivo stability of the compound and / or targets a particular organ, tissue or cell type to the compound. As a delivery carrier for Colloidal dispersions include, but are not limited to, polymer complexes, nanocapsules, microspheres,
Beads, beads and lipid-based systems, including oil-in-water emulsifiers, micelles,
Mixed micelles, liposomes, and unspecified lipid: oligonucleotide complexes are included. A preferred colloidal dispersion is a plurality of liposomes. Liposomes are microscopic spheres whose aqueous nucleus is surrounded by one or more outer layers of lipids arranged in a bilayer structure (generally, Chonn et al., Current
Op. Biotech., 1995, 6, 698-708). For the production of liposomes, see US patent application Ser. No. 08 / 961,4, filed Oct. 31, 1997.
No. 69, U.S. Patent No. X, XXX, XXX, which is owned with the present application and incorporated herein by reference.

One embodiment of the present invention relates to (a) one or more antisense compounds, and (
b) Provide liposomes and other compositions containing one or more other chemotherapeutic agents that function by a non-antisense mechanism. Examples of such chemotherapeutic agents are
Without limitation, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5- FU), floxyuridine (5-
FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol (DES). In general terms, The Merck Manu
al of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway
, NJ, pages 1206-1228. Non-steroidal anti-inflammatory agents and anti-inflammatory agents, including but not limited to corticosteroids, and anti-viral agents, including but not limited to ribavirin, vidarabine, acyclovir and ganciclovir, can also be used with the compositions of the present invention (generalization in general). Is the Merck Manual of Diagnosis
and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, NJ, pages 2
499-2505 and 46-49, respectively). Other non-antisense chemotherapeutic agents are within the scope of the present invention. Two or more composite compounds can be used together or sequentially.

[0052] In another related aspect, the composition of the present invention comprises a first nucleic acid that targets the first nucleic acid.
It may contain one or more antisense compounds, especially oligonucleotides, and one or more additional antisense compounds targeting a second nucleic acid. Examples of antisense oligonucleotides include, but are not limited to, the following U.S. patents or pending U.S. patent applications, which are owned with the present application and incorporated herein by reference, or the published PCT shown below. Includes those directed to targets as disclosed in the application: raf (WO 96/39415, WO 95/3
No. 2987, and U.S. Pat. Nos. 5,563,255 issued on Oct. 8, 1996 and 5,656,612 issued on Aug. 12, 1997), p120 nucleolar antigen (WO93 / 17125, And U.S. Patent No. 5, issued August 12, 1997.
656,743), protein kinase C (WO95 / 02069, WO9
5/03833 and WO 93/19203), a multidrug resistance-related protein (W
No. O95 / 10938 and U.S. Pat. No. 5,510, issued Mar. 23, 1996.
239), a subunit of the transcription factor AP-1 (U.S. patent application Ser. No. 08 / 837,201, filed Apr. 14, 1997), Jun kinase (Aug. 1, 1997).
U.S. patent application Ser. No. 08 / 910,629 filed on the third day, MDR-1 (multidrug resistant glycoprotein; U.S. patent application Ser. No. 08 / 731,199, filed on Sep. 30, 1997).
), HIV (US Patent No. 5,166,195 issued November 24, 1992 and 5,591,600 issued January 7, 1997), herpes virus (
U.S. Pat. No. 5,248,670 issued Sep. 28, 1993 and U.S. Pat. No. 5,514,577 issued May 7, 1996), cytomegalovirus (1)
U.S. Pat. No. 5,442,049 issued Aug. 15, 995, and Jan. 1, 1997
No. 5,591,720 issued on May 7, 2009), papilloma virus (October 1995)
U.S. Pat. No. 5,457,189, issued on Oct. 10), intercellular adsorption molecule-1 (IC
AM-1) (U.S. Pat. No. 5,514,788 issued May 7, 1996), 5
-Lipoxygenase (U.S. Patent No. 5,530,11 issued June 25, 1996)
No. 4) and influenza virus (US Pat. No. 5, issued Dec. 3, 1996).
580,767). Two or more composite compounds can be used together or sequentially.

The formulation and subsequent administration of a therapeutic composition is considered to be within the skill of those in the art. Dosage will depend on the severity and responsiveness of the condition being treated, and the duration of treatment will last from days to months, or until the treatment is effective or the condition has attenuated. Optimal dosing schedules can be calculated from measurements of drug accumulation in the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimal dosages may vary with the relative potency of the individual oligonucleotides, but generally will vary in vitro and in vitro.
may be estimated based on EC ₅₀ is the effective in vivo animal models. Generally, dosages will be from 0.01 μg 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.
One skilled in the art can readily estimate repetition rates for administration based on the residence time and concentration of the drug in bodily fluids or tissues. If treatment is successful, it may be desirable to administer maintenance therapy to the patient to prevent recurrence of the condition. Here, the oligonucleotide is administered at a maintenance dose in the range of 0.01 μg to 100 g per kg of body weight, once a day or several times to once every 20 years. While the invention has been particularly described with reference to certain preferred embodiments thereof, the following examples only illustrate the invention, but do not limit it.

[0054]

【Example】

Example 1 Nucleoside phosphoramidite deoxy and 2'-alkoxyamidite for oligonucleotide synthesis 2'-deoxy and 2'-methoxy-β-cyanoethyldiisopropyl phosphoramidite were purchased from commercial sources (eg Chemgenes, Needham, M
A, or Glen Research, Stirling, VA). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in US Pat. No. 5,506,351, which is incorporated herein by reference. For oligonucleotides synthesized using 2'-alkoxyamidites, a standard cycle of unmodified oligonucleotides was used, but the waiting step after pulse delivery of tetrazole and base was increased to 360 seconds. Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C) nucleotides are known in the art using commercially available phosphoramidites (Glen Research, Sterling, VA, or Chemgenes, Needham, MA). It was synthesized by a method (Sanghvi, et. Al., Nucleic Acids Research, 1993, 21, 3197-3203).

The 2′-fluoroamidite 2′-fluorodeoxyadenosine amidite 2′-fluoro-oligonucleotide can be obtained by known methods (Kawasaki et al., J. Med. Chem. 1993, 36, 831-841) and U.S. Pat. No. 5,670,633. Simply put, commercially available 9
Using β-D-arabinofuranosyladenine as a starting material and modifying the literature method by introducing a 2′-α-fluoro atom by _SN 2 substitution of the 2′-β-trityl group, A protected nucleoside, N6-benzoyl-2'-deoxy-2'-fluoroadenosine, was synthesized. Thus, N6-benzoyl-
9-β-D-arabinofuranosyladenine was selectively protected in a moderate yield as a 3 ′, 5′-ditetrahydropyranyl (THP) intermediate. THP and N
Deprotection of the 6-benzoyl group was performed using standard methods and 5 using standard methods.
'-Dimethoxytrityl- (DMT) and 5'-DMT-3'-phosphoramidite intermediates were obtained.

2′-Fluorodeoxyguanosine The synthesis of 2′-deoxy-2′-fluoroguanosine uses tetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanine as a starting material. And converted to the intermediate diisobutyrylarabinofuranosylguanosine. After deprotection of the TPDS group, the hydroxyl group was protected with THP to give diisobutyryldi-THP protected arabinofuranosylguanine. The crude product was treated with fluoride following selective O-deacylation and trifurylation, and then the THP group was deprotected. Using standard methods, 5'-DMT- and 5'-
DMT-3'-phosphoramidite was obtained.

The synthesis of 2′-fluorouridine 2′-deoxy-2′-fluorouridine is based on 2,2′-anhydro-
Achieved by literature change, treating 1-β-D-arabinofuranosyluracil with 70% hydrogen fluoride / pyridine. 5'-DMT and 5'-D using standard methods
MT-3 'phosphoramidite was obtained. Amination of 2'-fluorodeoxycytidine 2'-deoxy-2'-fluorouridine results in 2'-deoxy-
After synthesis of 2'-fluorocytidine, selective protection afforded N4-benzoyl-2'-deoxy-2'-fluorocytidine. 5'-D using standard method
MT and 5'-DMT-3 'phosphoramidites were obtained.

2′-O- (2-methoxyethyl) -Modified Amidite Nucleoside amidites substituted with 2′-O-methoxyethyl are described below in Martin, P., Helvetica Chimica Acta, 1995, 78,486. Same as -504 or manufactured in other ways. 2,2′-Anhydro [1- (β-D-arabinofuranosyl) -5-methyluridine] 5-methyluridine (commercially available from ribosylthymine, Yamasa, Choshi, Japan) (72)
. 0g, 0.279M), diphenyl carbonate (90.0g, 0.420M)
) And sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated under reflux with stirring to release the generated carbon dioxide gas in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure.
The resulting syrup was poured into diethyl ether (2.5 L) with stirring.
The product formed a rubber. The ether is decanted and the residue is treated with a minimum amount of methanol (
(About 400 mL). Pour this solution into fresh ether (2.5 L),
This produced a hard rubber. The ether is decanted and this rubber is vacuum oven (60 ° C,
(1 mmHg, 24 hours) to give a solid which was ground to a light brown powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure and phenol was present as a sodium salt (approximately 5%). This material is used as is in subsequent reactions (or can be further purified by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid with a melting point of 222-4 ° C.). .

2′-O-methoxyethyl-5-methyluridine 2,2′-anhydro-5-methyluridine (195 g, 0.81 M), tris (2-methoxyethyl) boric acid (231 g, 0.98 M) And 2-methoxyethanol (1.2 L) are added to a 2 L stainless steel pressure vessel, and 160 ° C.
In a heated oil bath. After heating at 155-160 ° C. for 48 hours, the vessel was opened, the solution was evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in warm acetone (1 L). The insoluble salts are filtered off and acetone (150 m
L) and the filtrate was evaporated. The residue (280 g) was added to CH ₃ CN (600 mL).
) And evaporated. A silica gel column (3 kg) was packed with CH ₂ Cl ₂ / acetone / MeOH (20: 5: 3) containing 0.5% Et ₃ NH. The residue was dissolved in CH ₂ Cl ₂ (250 mL) and adsorbed on silica (150 g) before loading on the column. The product was eluted with the filling solvent, and 160 g of the product (63%
)Obtained. The low purity fraction was reprocessed to give more material.

2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine 2′-O-methoxyethyl-5-methyluridine (160 g, 0.506M)
Was azeotroped with pyridine (250 mL) and the dried residue was dissolved in pyridine (1.3 L). First aliquot of dimethoxytrityl chloride (94.3 g, 0.278)
M) was added and the mixture was stirred at room temperature for 1 hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction was stirred for another hour. Then the reaction was stopped by adding methanol (170 mL). HPLC indicated the presence of about 70% product. The solvent was evaporated and CH ₃ CN (200
mL) and triturated. The residue was dissolved in CHCl ₃ (1.5 L) and 2 × 500
Extracted with mL of saturated NaHCO ₃ and 2 × 500 mL of saturated NaCl. The organic phase was dried over Na ₂ SO _4, filtered and evaporated. 275 g of residue were obtained. 0.5
The residue was purified on a 3.5 kg silica gel column packed and eluted with EtOAc / hexane / acetone (5: 5: 1) containing% Et ₃ NH. Evaporation of the pure fractions gave 164 g of product. About 20 g more was obtained from the low purity fraction, giving a total yield of 183 g (57%).

3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytri
Tyl-5-methyluridine 2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluri
Gin (106 g, 0.167 M), DMF / pyridine (562 mL of DMF and
750 mL of a 3: 1 mixture prepared from 188 mL of gin) and acetic anhydride (24.
(38 mL, 0.258 M) and stirred at room temperature for 24 hours. First MeOH
The reaction was monitored by tlc, which quenched the tlc sample by adding. tlc
MeOH (50 mL) was added at the end of the reaction, as judged by
Evaporated. The residue is CHCl_Three(800mL), 2x200mL saturated
Extracted with sodium bicarbonate and 2 × 200 mL of saturated NaCl. CHCl _Three (200 mL). The combined organic layers were dried over sodium sulfate,
Evaporation gave 122 g of residue (about 90% product). 3.5 kg of this residue
Purify on silica gel column and elute using EtOAc / hexane (4: 1)
Was. Evaporation of the pure product fractions yielded 96 g (84%). From later fractions
Further, 1.5 g was recovered.

3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytri
Tyl-5-methyl-4-triazoleuridine 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytri
Cyl-5-methyluridine (96 g, 0.144 M) was added to CH_ThreeCN (700 mL
) To prepare a first solution and set aside. Triazole (90 g, 1.
3M) CH_ThreeTriethylamine (189 mL, 1.44 M) was added to a CN (1 L) solution.
), Cooled to −5 ° C., and stirred for 0.5 hour using an overhead stirrer.
Was. While maintaining the solution at 0 to 10 ° C and stirring, the POCl_Three30 minutes for each drop
And the resulting mixture was stirred for an additional 2 hours. The first solution is added to this mixture.
The solution was added dropwise over 45 minutes. The resulting reaction mixture was kept in the cold room overnight
. The salts were filtered from the reaction mixture and the solution was evaporated. Residue in EtOAc (1 L)
Melted and insoluble solids were removed by filtration. The filtrate was washed with 1 x 300 mL of NaHCO _Three And 2 × 300 mL of saturated NaCl, dried over sodium sulfate and evaporated.
I let you. The residue was triturated with EtOAc to give the title compound.

2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl- 4- triazole uridine (103g, 0.141M) in dioxane _{(500mL) / NH 4 OH (} 30mL) solution was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue was azeotroped with MeOH (2 × 200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) filled with NH ₃ gas was added, and the
Heated to 100 ° C. over time (tic showed complete conversion). The contents of the vessel were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and saturated Na
Washed once with Cl (200 mL). The organic layer was dried over sodium sulfate and the solvent was evaporated, yielding 85 g (95%) of the title compound.

N4-benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine 2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tl
c indicated that the reaction was about 95% complete. The solvent was evaporated and the residue was evaporated in MeOH (
(200 mL). The residue was dissolved in CHCl ₃ (700 mL) and saturated
aHCO ₃ (2 × 300 mL) and saturated NaCl (2 × 300 mL)
Dried over MgSO ₄ and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica gel column using EtOAc / hexane (1: 1) containing 0.5% Et ₃ NH as the elution solvent. Evaporation of the pure product fractions provided 90 g (90%) of the title compound.

N4-benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite N4-benzoyl-2′-O-methoxyethyl-5′-O-dimethoxy trityl-5- methylcytidine (74 g, 0.10 M) was dissolved in CH ₂ Cl ₂ (1L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-
Tetra (isopropyl) phosphite (40.5 mL, 0.123 M) was added under a nitrogen environment with stirring. The resulting mixture was stirred at room temperature for 20 hours (tic indicated that the reaction was approximately 95% complete). The reaction mixture was washed with saturated NaHCO ₃ (1 ×
(300 mL) and saturated NaCl (3 × 300 mL). Aqueous cleaning solution C
Re-extract with H ₂ Cl ₂ (300 mL), combine the extracts, dry over MgSO ₄ ,
Concentrated. 1.5k using EtOAc / hexane (3: 1) as elution solvent
g The resulting residue was chromatographed on a silica gel column. Pure fractions were combined to give 90.6 g (87%) of the title compound.

2 ′-(aminooxyethyl) nucleoside amidite and 2 ′-(dimethylaminooxyethyl) nucleoside amidite Aminooxyethyl and dimethylaminooxyethyl amidite are 1998
U.S. Patent Application Nos. 10 / 037,143 and 1998 filed on February 14, 1998.
It is manufactured according to the method of U.S. patent application Ser. Each of which is owned with the present application and incorporated herein by reference.

Example 2 Oligonucleotide Synthesis Unsubstituted and substituted phosphodiester (P = O) oligonucleotides were synthesized on an automated DNA synthesizer (Ap) using standard phosphoramidite chemistry with iodine oxidation.
combined biosystems, model 380B). Phosphorothioates (P = S) are synthesized similarly to phosphodiester oligonucleotides, but standard oxidation bottles are used for 3H-1,2-benzodithiol-3-one-1,1 used for the stepwise thioation of phosphite linkages. -Replaced with a 0.2 M solution of the dioxide in acetonitrile. The waiting step for thioation was increased to 68 seconds and the capping step continued. The oligonucleotide is purified from a 0.5 M NaCl solution by separating from a CPG column, deprotecting with concentrated ammonium hydroxide at 55 ° C. (18 hours), and then precipitating twice with 2.5 volumes of ethanol. did.
Phosphinate oligonucleotides are prepared as described in US Pat. No. 5,508,270, which is incorporated herein by reference.

The alkyl phosphonate oligonucleotides are prepared as described in US Pat. No. 4,469,863, which is incorporated herein by reference. Oligonucleotides of 3'-deoxy-3'-methylenephosphonate are disclosed in U.S. Patent No. 5,610,289 or 5,6,8, incorporated herein by reference.
Produced as described in U.S. Pat. Phosphoramidite oligonucleotides are prepared as described in US Pat. Nos. 5,256,775 or 5,366,878, which are incorporated herein by reference.

[0069] Alkyl phosphonothioate oligonucleotides are known from PCT Application Nos. PCT / US94 / 00902 and PCT, which are hereby incorporated by reference.
T / US93 / 06976 (WO94 / 17093 and WO94, respectively)
/ 02499). The 3′-deoxy-3′-amino phosphoramidite oligonucleotide is
Manufactured as described in US Pat. No. 5,476,925, which is incorporated herein by reference. Phosphotriester oligonucleotides are prepared as described in US Pat. No. 5,023,243, which is incorporated herein by reference. Boranophosphate oligonucleotides are prepared as described in US Pat. Nos. 5,130,302 and 5,177,196, both of which are incorporated herein by reference.

Example 3 Oligonucleoside Synthesis Methylenemethylimino-linked oligonucleoside (MMI-linked oligonucleoside), methylenedimethylhydrazo-linked oligonucleoside (MDH-linked oligonucleoside) and methylenecarbonylamino-linked oligonucleoside (amide-
3-linked oligonucleosides) and methyleneaminocarbonyl-linked oligonucleosides (amide-4-linked oligonucleosides), as well as, for example, compounds of mixed backbones having an MMI and a P = O or P = S linkage alternately are all by reference. U.S. Patent Nos. 5,378,825 and 5,386,02, which are incorporated herein.
No. 3, No. 5,489,677, No. 5,602,240 and No. 5,610,28
Prepared as described in No. 9. Formacetal and thioformacetal-linked oligonucleosides are described in US Pat. Nos. 5,264,562 and 5,26, incorporated herein by reference.
It is prepared as described in US Pat. Ethylene oxide linked oligonucleosides are prepared as described in US Pat. No. 5,223,618, which is incorporated herein by reference.

Example 4 PNA Synthesis Peptide nucleic acid (PNA) was obtained from Peptide Nucleic Acids (PNA): Synthesis, Pro.
perties and Potential Applications, Bioorganic & Medicinal Chemistry, 19
96, 4, 5-23. They are also disclosed in U.S. Patent Nos. 5,539,082, 5,700, which are incorporated herein by reference.
, 922, and 5,719,262.

Example 5 Synthesis of Chimeric Oligonucleotides The chimeric oligonucleotides, oligonucleosides or oligonucleotide / oligonucleoside mixtures of the present invention can be of several different types. This involves the "gap" segment of the connecting nucleoside being the 5 'of the connecting nucleoside.
And a 3 '"wing" segment, a first type located between the "gap" segments, and a second "open end" type, wherein the "gap" segment is located at either the 3' or 5 'end of the oligomeric compound. The first type of oligonucleotide is
Also known in the art as "gapmers" or gapped oligonucleotides. The second type of oligonucleotide is also known in the art as "hemimer" or "wingmer".

[2'-O-Me]-[2'-deoxy]-[2'-O-Me] Chimeric Phosphorothioate Oligonucleotide Segments of 2'-O-alkyl phosphorothioate and 2'-deoxyphosphorothioate oligonucleotides The chimeric oligonucleotide having the following sequence was used as described above in Applied Biosystems' automatic DNA synthesizer, model 3
It is synthesized using 80B. The DNA portion is 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite, 5 'and 3' wings are 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoro. An amidite is used to synthesize an oligonucleotide using an automatic synthesizer. The standard synthesis cycle is modified to increase the wait step after delivery of the tetrazole and base to 600 seconds and repeat four times for RNA and two times for 2'-O-methyl. The fully protected oligonucleotide is separated from the support, deprotected from phosphate groups in 3: 1 aqueous ammonia / ethanol at room temperature overnight, and then lyophilized to dryness. After treatment in aqueous methanolic ammonia at room temperature for 24 hours to deprotect all bases, the samples are again lyophilized to dryness. The pellet is left at room temperature for 24 hours,
Resuspend in M TBAF / THF and deprotect 2 'position. The reaction is then quenched with 1 M TEAA, then concentrated in vacuo to half volume and desalted on a G25 size exclusion column. The yield and purity of the recovered oligonucleotides are analyzed spectrophotometrically by capillary electrophoresis and mass spectrometry.

[2′-O- (2-methoxyethyl)] —— [2′-deoxy] — [2′-
O- (methoxyethyl)] chimeric phosphorothioate oligonucleotide [2'-O- (2-methoxyethyl)]-[2'-deoxy]-[2'-
O- (methoxyethyl)] chimeric phosphorothioate oligonucleotides are
2'-O-methylamidite was replaced with 2'-O- (methoxyethyl) amidite, and a 2'-O-methyl chimeric oligonucleotide was prepared in the same manner as described above.

[2′-O- (2-methoxyethyl) phosphodiester] --- [2′-deoxyphosphorothioate] — [2′-O- (2-methoxyethyl) phosphodiester] chimeric oligonucleotide [2 ′ -O- (2-methoxyethyl) phosphodiester]-[2'-deoxyphosphorothioate]-[2'-O- (2-methoxyethyl) phosphodiester] chimeric oligonucleotide is 2'-O-methylamidite To 2'-O-
A 2'-O-methyl chimeric oligonucleotide is prepared in the same manner as described above, substituting (methoxyethyl) amidite. Oxidation with iodine to create phosphodiester internucleotide linkages within the wing portion of the chimeric structure,
-1,2-benzodithiol-3-one-1,1-dioxide (Beaucag
e-reagent) to form phosphorothioate internucleotide linkages in the central gap. Other chimeric oligonucleotides, chimeric oligonucleosides, and chimeric oligonucleotide / oligonucleoside mixtures are synthesized according to US Pat. No. 5,623,065, which is incorporated herein by reference.

Example 6 Isolation of Oligonucleotides Separated from a controlled pore glass column (Applied Biosystems)
After deblocking with concentrated ammonium hydroxide at 55 ° C. for 18 hours, the oligonucleotide or oligonucleoside was diluted with 2.5 volumes of ethanol in 0.5 M NaC.
Precipitate twice from l and purify. The synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on a non-denaturing gel and determined to be at least 85% full-length material. The relative amounts of the phosphorothioate and phosphodiester linkages obtained in the synthesis are checked at regular intervals by ³¹ P nuclear magnetic resonance spectroscopy, and in some tests Chiang et al. , J. Biol. Purified by HPLC as described in Chem. 1991, 266, 18162-18171. The results obtained for the material purified by HPLC were similar to those obtained for the material not purified by HPLC.

Example 7 Oligonucleotide Synthesis-96-Well Plate Format Oligonucleotides were synthesized by solid phase P (III) phosphoramidite chemistry on an automated synthesizer that can assemble 96 sequences simultaneously in a standard 96-well format. Oxidation with aqueous iodine provided the internucleotide linkage of the phosphodiester. The phosphorothioate internucleotide linkage was generated by sulfation using a solution of 3, H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) in anhydrous acetonitrile. Standards of base-protected β-cyanoethyldiisopropyl phosphoramidite are available from commercial vendors (eg, P
E-Applied Biosystems, Foster City, CA or Pharmacia, Piscataway, NJ). Non-standard nucleosides are synthesized according to known literature or patent methods. They are utilized as base-protected β-cyanoethyldiisopropyl phosphoramidites. The oligonucleotide was separated from the support and left at elevated temperature (55-60
Deprotection with conc. NH ₄ OH) and the liberated product was dried in vacuo. Resuspend the dried product in sterile water and place on a master plate. This is diluted using a robot pipettor to provide plate samples for any analysis and testing.

Example 8 Oligonucleotide analysis-96-well plate format The oligonucleotide concentration in each well was evaluated by sample dilution and UV absorption spectroscopy. For each product, the full-length extent was in a 96-well format (Beckman P / ACE ^™ MDQ), or for individually manufactured samples, a commercially available CE device (eg, Beckman P / ACE ^™ 5000, ABI 270). Either was assessed by capillary electrophoresis (CE). The composition of the base and the skeleton was confirmed by mass spectrometry of the compound using electrospray mass spectrometry. All assay test samples were diluted from the master plate using a single / multi-channel robotic pipettor. At least 8 of the compounds on the plate
If 5% was at least 85% full length, the plate was considered valid.

Example 9 Cell Culture and Oligonucleotide Treatment The effect of an antisense compound on a target nucleic acid can be tested in a wide variety of cell types if the target nucleic acid is present at measurable levels. This can be routinely quantified using, for example, PCR or Northern blot analysis. The following four cell types are provided for illustration, and other cell types may be used as is.

T-24 cells: The transitional cell bladder cancer cell line, T-24, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). 10% fetal bovine serum (Gibco / Life Technologies, Gaithersburg, MD), 100 units / mL penicillin and 100 microgram / mL streptomycin
Complete McCoy's 5A basal medium supplemented with (Gibco / Life Technologies, Gaithersburg, MD) (Gibco / Life Technologies, Gaithersburg, MD)
In), T-24 cells were routinely cultured. When cells reached 90% confluence, they were routinely passaged by trypsinization and dilution. For use in RT-PCR analysis, cells were plated at a density of 7000 cells / well in 96-well plates (Falcon-
Primaria # 3872). For Northern blots and other analyses, cells can be plated on 100 mm or other standard tissue culture plates and treated similarly using appropriate volumes of media and oligonucleotides.

A549 cells: The human lung cancer cell line, A549, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). DMEM basal medium (Gibco / Gibco / Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (Gibco / Life Technologies, Gaithersburg, MD), penicillin 100 units / mL, and streptomycin 100 μg / mL (Gibco / Life Technologies, Gaithersburg, MD). A549 cells were routinely cultured at Life Technologies, Gaithersburg, MD). When cells reached 90% confluence, they were routinely passaged by trypsinization and dilution.

NHDF cells: Human neonatal dermal fibroblasts (NHDF) were obtained from Clontics Corporation (Walkersville, MD). NHDF was routinely maintained in fibroblast growth medium (Clonics Corporation, Walkersville, MD) as recommended by the supplier. Cells were maintained for up to 10 subcultures as recommended by the supplier.

HEK cells: Human embryonic keratinocytes (HEK) are derived from the Clontics Corporation (
Walkersville, MD). HEK was routinely maintained in keratinocyte growth media (Clontics Corporation, Walkersville, MD) prepared as recommended by the supplier. Cells were routinely maintained for up to 10 subcultures as recommended by the supplier.

Treatment with antisense compounds: After cells reached 80% confluence, they were treated with oligonucleotide. For cells grown in a 96-well plate, the wells were washed once with 200 μL of Opti-MEM ^™ -1 reduced serum medium (Gibco BRL), and then LIPOFECTIN ^™ (
Gibco BRL) 130 of Opti-MEM ^™ -1 containing 3.75 μg / mL.
μl, and 150 nM final concentration of the desired oligonucleotide. 4
After the time treatment, the original medium was replaced with fresh medium. Cells were harvested 16 hours after oligonucleotide treatment.

Example 10 Antisense Inhibition of TNFR1 Expression by Phosphorothioate Oligodeoxynucleotides According to the present invention, a known sequence (GenBank accession number: X55313, incorporated herein as SEQ ID NO: 1) using human known TNFR1 ・ R
A series of oligonucleotides targeting different regions of NA were designed. This oligonucleotide is shown in Table 1. The target site is indicated by the nucleotide number to which the oligonucleotide binds, as shown in the reference sequence (GenBank accession number: X55313). The compounds in Table 1 are all oligodeoxynucleotides having a phosphorothioate backbone (internucleoside linkage). TNFR1 ・ m
The effect of these compounds on RNA levels was analyzed by real-time PCR quantification as described in the examples below. Data are the average of three experiments.

[0086]

[Table 1]

[0087]

[Table 2]

As shown in Table 1, SEQ ID NOs 11, 15, 16, 17, 22,
5, 30, 33, 42, 45, 62, 70, 74, 76, 78, 82 and 90 show at least 35% inhibition of TNFR1 expression in this assay and are therefore preferred.

Example 11 Analysis of Oligonucleotide Inhibition of TNFR1 Expression Antisense modulation of TNFR1 expression can be assayed in a variety of ways known in the art. For example, TNFR1 mRNA levels can be quantified by Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Presently preferred is real-time quantitative PCR. RNA analysis is based on total RNA or poly (A) + mR
It can be implemented based on NA. RNA isolation methods are described, for example, in Ausubel, FM e
t al., Current Protocols in Molecular Biology, Volume 1, pp. 4. 1. 1-4.
2.9 and pp. 4. 5. 1-4. 5. 3, John Wiley & Sons, Inc., 1993. Northern blot analysis is a routine task in the art, for example, Ausu
bel, FM et al., Current Protocols in Molecular Biology, Volume 1, pp.
4. 2. 1-4. 2. 9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using a commercially available ABI PRISM ^™ 7700 sequence detection system supplied by PE-Applied Biosystems, Foster City, CA and used by the manufacturer's instructions. . Other PCR methods are also known in the art.

The levels of TNFR1 protein can be determined by immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).
) Can be quantified by various methods well known in the art. TNFR1
Antibodies against MSRS are available in the MSRS antibody catalog (Aerie Corporation).
n, Birmingham, MI) or may be obtained from various sources, or may be produced by conventional antibody production methods. Methods for preparing polyclonal antisera are described, for example, in Ausubel, FM et al., Current Protocols in Molecular Biology, Volum.
e 2, pp. 11. 12. 1-11. 12. 9, taught by John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is described, for example, in Ausubel, FM et al., Current
Protocols in Molecular Biology, Volume 2, pp. 11.4.1-1-11.11, 5, John
Wiley & Sons, Inc., 1997.

Immunoprecipitation is a standard method in the art and is described, for example, in Ausubel, FM et al., C
urrent Protocols in Molecular Biology, Volume 2, pp. 10. 16. 1-10. 16. 1
1, John Wiley & Sons, Inc., 1998. Western blot (immunoblot) analysis is a standard method in the art and is described, for example, in Ausubel, FM et al.,
Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-1-10.8.2
1, John Wiley & Sons, Inc., 1997. Enzyme-linked immunosorbent assay (
ELISA) is a standard method in the art and is described, for example, in Ausubel, FM et al., C
urrent Protocols in Molecular Biology, Volume 2, pp. 11. 2. 1-11. 2. 22,
John Wiley & Sons, Inc., 1991.

Example 12 Isolation of Poly (A) + mRNA Poly (A) + mRNA was obtained from Miura et al., Clin. Chem., 1996, 42, 1758-1764.
Was isolated. Other methods for isolating poly (A) + mRNA are described, for example, in Ausube
l, FM et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.
5. 1-4. 5. 3, John Wiley & Sons, Inc., 1993. Briefly, for cells grown in 96-well plates, the growth medium was removed from the cells and cold P
Each well was washed with 200 μl of BS. Lysis buffer (10 mM Tris-HCl, p
H7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 2
60 μl (0 mM vanadyl-ribonucleoside conjugate) was added to each well, the plate was rocked gently, and then incubated at room temperature for 5 minutes. Oligo d (T
Solution 55 into 96-well plate (AGCT, Irvine, CA) coated with
The μl was transferred. Plates are incubated for 60 minutes at room temperature and washed with wash buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.3 M NaCl)
Washed three times with 200 μl. After the final wash, the plate was blotted on a paper towel to remove excess wash buffer and then air dried for 5 minutes. Add 60 μl of elution buffer (5 mM Tris-HCl, pH 7.6) previously heated to 70 ° C. to each well, incubate the plate on a hot plate at 90 ° C. for 5 minutes, then add the eluate to a fresh 96 well. Transferred to plate. Cells grown on 100 mm or other standard plates can be treated similarly, using appropriate amounts of all solutions.

Example 13 Total RNA Isolation Total mRNA was isolated using RNEASY 96 ^™ kit and buffer purchased from Qiagen (Valencia, CA) according to the manufacturer's recommendations.
Briefly, for cells grown in 96-well plates, growth medium was removed from the cells and each well was washed with 200 μl of cold PBS. 100 μL of RLT buffer was added to each well and the plate was shaken vigorously for 20 seconds. Then, add 70% ethanol 1 to each hole.
00 μL was added, and the contents were mixed by three times of upper and lower pipetting operations. Then, a QIAVAC ^™ manifold with a waste collection tray and a R with a vacuum source
This sample was transferred to a NEASY 96 ^{™ well} plate. Vacuum was applied for 15 seconds. Add 1 mL of RW1 buffer to each well of the RNEASY 96 ^™ plate,
Vacuum was applied for 5 seconds. Next, RPE is inserted into each hole of the RNEASY 96 ^™ plate.
1 mL of buffer was added and vacuum was applied for 15 seconds. The washing of the RPE buffer was then repeated and vacuum was applied for another 10 minutes. The plate was then removed from the QIAVAC ^™ manifold, blotted on a paper towel and dried. The plate was reattached to a QIAVAC ^™ manifold with a collection tube rack containing 1.2 mL collection tubes. RNA was eluted by pipetting 60 μL of water into each well, incubating for 1 minute, and then applying vacuum for 30 seconds. Further, 60 μL of water was added, and this elution step was repeated.

Example 14 Real Time Quantitative PCR Analysis of TNFR1 mRNA Levels ABI PRISM ^™ 7700 Sequence Detection System (PE-Applied B
real-time quantitative P using iosystems, Foster City, CA)
TNFR1 mRNA levels were quantified by CR according to the manufacturer's instructions. This is a sealed tube, non-gel based, fluorescence detection system that allows high-throughput quantification of polymerase chain reaction (PCR) products in real time. Unlike standard PCR, which quantifies amplification products after PCR is complete, the products of real-time quantitative PCR are quantified with accumulation. This is achieved by specifically annealing between the front and back PCR primers and including in the PCR reaction an oligonucleotide probe containing two fluorescent dyes. A reporter dye (eg, JOE or FAM, PE-Ap)
plied Biosystems, Foster City, Calif.) and a quencher dye (eg, TAMRA, P
E-Applied Biosystems, Foster City, CA). If the probe and the dye are intact, the release of the reporter dye is suppressed because the 3 'quencher dye is nearby. During amplification, annealing of the probe to the target sequence produces a substrate that can be cleaved by the 5'-exonuclease activity of Taq polymerase. In the extension phase of the PCR amplification cycle, Taq
Cleavage of the probe by the polymerase releases the reporter dye from the remainder of the probe (and simultaneously from the quencher portion), producing a sequence-specific fluorescent signal. At each cycle, an additional reporter dye molecule is cleaved from each probe, and the fluorescence intensity is monitored at regular (6 second) intervals by laser light incorporated into the ABI PRISM ^™ 7700 sequence detection system. For each assay, a standard curve was generated from a series of parallel reactions containing serial dilutions of mRNA from an untreated control sample and used to quantify the percent inhibition of test samples following treatment with antisense oligonucleotides. I do.

[0095] PCR reagents were obtained from PE-Applied Biosystems, Foster City, CA. PCR cocktail (1 × Taqman ^™ buffer A, 5.5 mM MgC) was added to a 96-well plate containing 25 μl of poly (A) mRNA solution.
l ₂ , dATP, dCTP and dGTP, each 300 μM, 600 μM dUTP
, Forward primer, backward primer and probe, 100 nM each, RNAse inhibitor 20 units, AmpliTaq Gold ^™ 1.25 units, and M
uLV reverse transcriptase (12.5 units) by adding 25 μl, RT-PC
An R reaction was performed. The RT reaction was performed by incubation at 48 ° C. for 30 minutes followed by 95 ° C. for 10 minutes to activate the AmpliTaq Gold ^™ , and a 40 cycle two-step PCR protocol was performed as follows: 95 ° C. 15 seconds (denaturation), then 60 ° C for 1 minute (annealing /
Stretch).

The TNFR1 PCR primers were as follows: Front primer: GCTCTCAAAAACCACCTCCAGACA (SEQ ID No. 2), Rear primer: CCGGTCCCTGTGCAAGAA (SEQ ID N)
o. 3) and PCR probe: FAM-TCAGCTGCTCCAAATGCCGAAAGG
-TAMRA (SEQ ID No. 4). Here, FAM (PE-Appli
ed Biosystems, Foster City, CA) is a fluorescent reporter dye, and TAMRA (PE-Applied Biosystems, Foster City, CA) is a quencher dye. The GAPDH PCR primers were as follows: Forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID N
o. 5), Back primer: GAAGATGGTGATGGGATTTC (SEQ ID No. 6) and PCR probe: 5 ′ JOE-CAAGCTTCCCGTTCTCAGCC-T
AMRA3 '(SEQ ID No. 7). Here, JOE (PE-Appli
ed Biosystems, Foster City, CA) is a fluorescent reporter dye, and TAMRA (PE-Applied Biosystems, Foster City, CA) is a quencher dye.

Example 15 Northern Blot Analysis of TNFR1 mRNA Levels Eighteen hours after antisense treatment, the cell monolayer was washed twice with cold PBS and RN
AZOL ^™ (TEL-TEST “B”, Friendswood, TX) was dissolved in 1 mL. Total RNA was prepared according to the manufacturer's recommended protocol. 20 μg of total RNA was fractionated by electrophoresis through a 1.2% agarose gel containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Solon, OH). Northern / Southern transfer buffer system (TEL-TEST “B”,
Capillary transfer using Friendswood, TX) was performed overnight to transfer RNA from the gel to Hybond ^™ -N + nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ). RNA transfer was confirmed by UV visualization. STRATALINKER ^™ UV Crosslinker 240
The membrane was immobilized by UV crosslinking using 0 (Stratagene, La Jolla, CA).

The forward primer GCTTCAGAAAACCACCCTCAGACA (SEQ
ID No. 2) and the back primer CCGGTCCACTGTGCAAG
QUICKH under stringent conditions recommended by the manufacturer using a TNFR1-specific probe produced by PCR using AA (SEQ ID No. 3)
The membrane was probed using a YB ^™ hybridization solution (Stratagene, La Jolla, CA). To normalize for variations in loading and transfer efficiency, membranes were cut and glyceraldehyde-3-phosphate dehydrogenase (
G3PDH) RNA (Clontech, Palo Alto, CA). PHOSPHORIMAGER ^™ and IMAGEQUANT ^™ software V3
. 3 (Molecular Dynamics, Sanivert, CA) was used to visualize and quantify the hybridized membrane. Data was normalized to G3PDH levels in untreated controls.

Example 16 Western Blot Analysis of TNFR1 Protein Levels Western blot analysis (immunoblot analysis) is performed using standard methods.
Cells are harvested 16-20 hours after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 μl / well), boiled for 5 minutes,
Load on a 6% SDS-PAGE gel. Run the gel at 150V for 1.5 hours,
Transfer to membrane for western blotting. A suitable first antibody against TNFR1;
In addition, a second antibody that is radioactively or fluorescently labeled with respect to the first antibody is used. The bands are visualized using PHOSPHORIMAGER ^™ (Molecular Dynamics, Sanivert, CA).

Example 17 TN with phosphorothioate 2′-MOE gapmer oligonucleotide
Antisense Inhibition of FR1 Expression According to the present invention, a second series of oligonucleotides targeting human TNFR1 was synthesized. The oligonucleotide sequence is shown in Table 2. The target site is indicated by the nucleotide number shown in the reference sequence (GenBank accession number: X55313) to which the oligonucleotide binds. Each of the compounds in Table 2 has a chimeric oligonucleotide (18 nucleotides in length)
"Gapmer"), which consists of a central "gap" region consisting of 10 2'-deoxynucleotides and four adjacent nucleotides "wings" on both sides (5 'and 3' directions). Be composed. The wing consists of 2'-methoxyethyl (2'-MOE) nucleotides. This internucleoside (backbone) linkage is phosphorothioate (P = S) throughout the oligonucleotide. The cytidine residue of the 2'-MOE wing is 5-methylcytidine. Data is obtained by real-time quantitative PCR as described in the previous example and averaged over three experiments.

[0101]

[Table 3]

[0102]

[Table 4]

As shown in Table 2, this experiment showed that expression of TNFR1 was inhibited by at least 75%, and therefore, preferred is SEQ ID NO: 11, 15, 1
6, 17, 21, 22, 23, 28, 29, 30, 31, 33, 35, 36, 3,
7, 38, 39, 41, 43, 46, 47, 48, 50, 59, 67, 70, 7
1, 74, 76, 77, 78, 81, 84 and 91.

[Sequence list]

[Procedure amendment]

[Submission date] December 27, 2000 (2000.12.27)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

17. The method of claim 16, wherein the disease or condition is an inflammatory condition.

[Procedure amendment]

[Submission date] January 23, 2001 (2001.1.23)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 43/00 111 C12Q 1/68 Z C12Q 1/68 C12N 15/00 ZNAA (81) Designated country EP (AT) , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ) , BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C , CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ , TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW. AA01 AA11 CA06 CA12 DA03 GA11 HA17 4B063 QA05 QQ08 QQ53 QR14 QR32 QR56 QR62 QR66 QS25 QS34 QX02 4C084 AA06 AA13 BA35 MA52 MA63 NA14 ZB111 4C086 AA01 AA02 EA16 MA52 MA63 NA14 ZB11

Claims (17)

[Claims] Claims: 1. A nucleic acid molecule encoding human TNFR1 having a length of 8 to
An antisense compound of 30 nucleotides, which inhibits expression of human TNFR1. 2. The antisense compound according to claim 1, which is an antisense oligonucleotide. 3. SEQ ID NOs: 11, 15, 16, 17, 21, 22
, 23, 25, 28, 29, 30, 31, 33, 35, 36, 37, 38, 39
, 41, 42, 43, 45, 46, 47, 48, 50, 59, 62, 67, 70
, 71, 74, 76, 77, 78, 81, 82, 84, 90 and 91. 4. SEQ ID NO: 11, 15, 16, 17, 22, 30
33. The oligonucleotide of claim 2, comprising 33, 70, 74, 76 and 78. 5. The oligonucleotide according to claim 2, comprising at least one modified internucleoside linkage. 6. The oligonucleotide of claim 5, wherein the modified internucleoside linkage is a phosphorothioate linkage. 7. The oligonucleotide of claim 2, comprising at least one modified sugar moiety. 8. The oligonucleotide according to claim 7, wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety. 9. The oligonucleotide according to claim 2, comprising at least one modified nucleobase. 10. The oligonucleotide according to claim 9, wherein the modified nucleobase is 5-methylcytosine. 11. The oligonucleotide according to claim 2, which is a chimeric oligonucleotide. 12. A pharmaceutical composition comprising the antisense compound according to claim 1 and a pharmaceutically acceptable carrier or diluent. 13. The pharmaceutical composition according to claim 12, comprising a colloidal dispersion system. 14. The pharmaceutical composition according to claim 12, wherein the antisense compound is an antisense oligonucleotide. 15. A method for inhibiting the expression of TNFR1 in a human cell or tissue, wherein the cell or tissue is contacted with the antisense compound according to claim 1 so that the expression of TNFR1 is inhibited. A method comprising: 16. A method for treating a human having a disease or condition related to TNFR1, wherein the antisense compound according to claim 1 is in a therapeutically or prophylactically effective amount so that the expression of TNFR1 is inhibited. Administering to the animal. 17. The method of claim 16, wherein the disease or condition is an inflammatory condition.