KR20020007398A - Inhibition of histone deacetylase - Google Patents

Abstract

The present invention relates to the inhibition of the expression of histone deacetylase and the activity of the enzyme, and more particularly to the inhibition of specific histone deacetylase. The invention also relates to compositions comprising antisense oligonucleotides and methods of inhibiting histone deacetylases using the compositions. In addition, the present invention relates to a method for identifying histone deacetylase, which is involved in the induction of cell division, and a method for identifying a component that reduces the activity of the enzyme by interacting with the histone deacetylase.

Description

Inhibition of histone deacetylase {INHIBITION OF HISTONE DEACETYLASE}

This specification has priority to US provisional patent application 60 / 132,287, filed May 3, 1999.

Deacetylation of the core histones H1 to H4 is mediated by two related families of enzymes called histone deacetylases. The first histone deacetylase family includes HDAC-1, HDAC-2 and HDAC-3. The second histone deacetylase family includes HDAC-4 (formerly known as HDAC-A), HDAC-5 (formerly known as HDAC-B), HDAC-C, HDAC-D and HDAC-E. The activation of histone deacetylases is thought to regulate the access of transcription factors to enhancer and promoter sites. Indeed, abundant deacetylated histone H4 is found at genomic sites that are transcriptionally inactive (Taunton et al, Science , 1996 , 272, 408-411).

Functional histone deacetylases have been considered necessary for cell cycle progression in both normal and tumor cells. Trichostatin A (TCA), an antibiotic isolated from Streptomyces , is known to inhibit histone deacetylase activity, stopping cell cycle progression in the G1 and G2 phases (Yoshida et al, J. Biol. Chem. , 1990 , 265, 17174-17179; Yoshida et al., Exp. Cell Res. , 1988 , 177, 122-131). Other histone deacetylase inhibitors such as trichostatin C, trapoxin, depudecin, suberoylanilide hydroxamic acid (SAHA), FR901228 (Fujisawa Pharmaceutical Co.) and butyrate, Similarly, it is known to inhibit cell cycle progression in cells (Taunton et al., Science , 1996 , 272, 408-411; Kijima et al., J. Biol. Chem. , 1993 , 268 (30), 22429 -22435; Kwon et al., Porc. Natl. Acad. Sci. USA , 1998 , 95 (7), 3356-3361).

Known histone deacetylase inhibitors are all natural products and are small molecules that inhibit the activity of histone deacetylase at the protein level. Moreover, all known histone deacetylase inhibitors are non-specific for a particular histone deacetylase and to some extent inhibit all members of both histone deacetylase families to the same extent.

Therefore, there is a need to develop a substance that inhibits the expression of a specific histone deacetylase as well as a substance that inhibits histone deacetylase at the gene level. There is also a need to develop methods for using these materials to identify and inhibit specific histone deacetylases involved in tumor formation.

The present invention relates to the expression of histone deacetylase and the inhibition of enzymatic activity.

The present invention provides materials and methods for inhibiting histone deacetylase at the nucleic acid level, and also provides materials and methods for inhibiting expression of specific histone deacetylases by inhibiting expression at the nucleic acid level. The present invention provides for the identification and specific inhibition of specific histone deacetylases involved in tumorigenesis.

Thus, in a first aspect, the present invention provides antisense oligonucleotides that inhibit the expression of histone deacetylases. In some embodiments of the invention, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E. In another embodiment, the oligonucleotides inhibit one or more histone deacetylases or all histone deacetylases. Preferably, the oligonucleotides are chimeric oligonucleotides or hybrid oligonucleotides.

In certain preferred embodiments of the invention, the oligonucleotides inhibit the transcription of nucleic acid molecules encoding histone deacetylases. The nucleic acid molecule may be genomic DNA (eg gene), cDNA or RNA. In another embodiment, the oligonucleotides inhibit translation of histone deacetylases.

In various embodiments of the invention, the antisense oligonucleotides are phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphoester ( phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, branched at least one internucleotide linkage selected from the group consisting of branched phosphoramidates, branched methylene phosphonates, branched phosphorothioates and sulfone internucleotide linkages It has a substance. In some embodiments, the oligonucleotides comprise ribonucleotides or 2′-O-substituted ribonucleotide sites and deoxyribonucleotide sites.

In a second aspect, the invention provides a method of inhibiting histone deacetylase in a cell comprising contacting the cell with an antisense oligonucleotide of the first aspect of the invention. In certain preferred embodiments of the invention, the division of cells is inhibited by the cells in contact. In a preferred embodiment, the cells can be tumor cells of animals, including humans, and can also grow neoplastic. In certain preferred embodiments, the methods of the present invention further comprise contacting the cell with a histone deacetylase inhibitor that interacts with the histone deacetylase to reduce the activity of the enzyme. Preferably, histone deacetylase inhibitors are functionally linked with antisense oligonucleotides.

In a third aspect, the invention provides a therapeutically effective amount of an antisense oligonucleotide of the first aspect of the invention contained in a pharmaceutically acceptable carrier in an animal having at least one tumor cell in the body in a therapeutically effective amount. A method of inhibiting growth of neoplastic cells in an animal comprising administering for a period of time is provided.

In certain preferred embodiments of the present invention, the present invention interacts with histone deacetylases to reduce enzymatic activity and to treat a therapeutically effective amount of a histone deacetylase inhibitor contained in a pharmaceutically acceptable carrier. And further administration during the period of academic usefulness. Preferably, histone deacetylase inhibitors are functionally linked with antisense oligonucleotides.

In a fourth aspect, the present invention provides a method for identifying histone deacetylases involved in the induction of cell division comprising contacting cells with antisense oligonucleotides that inhibit expression of histone deacetylases. Here, histone deacetylase is identified as a histone deacetylase that is involved in the induction of cell division by inhibiting cell division in contacted cells. In some preferred embodiments, the cells are tumor cells and the induction of cell division is the formation of a tumor. In a preferred embodiment, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

In a fifth aspect, the invention comprises contacting a histone deacetylase identified by the method of the fourth aspect of the invention with a candidate compound and measuring the activity of the histone deacetylase that has been contacted. It provides a method for identifying a histone deacetylase inhibitor that inhibits histone deacetylase involved in the induction of cell division. Here, candidate compounds are identified as histone deacetylase inhibitors that inhibit histone deacetylases involved in the induction of cell division by inhibiting the activity of contacted histone deacetylases. In certain preferred embodiments, histone deacetylase inhibitors interact with fewer enzymes than all histone deacetylases to reduce the activity of the enzyme.

In a sixth aspect, the invention provides a method for identifying histone deacetylases involved in the induction of cell differentiation, comprising contacting a cell with an antisense oligonucleotide that inhibits expression of histone deacetylase. . Herein, histone deacetylase is identified as a histone deacetylase that is involved in inducing cell differentiation by inducing differentiation in contacted cells. In some preferred embodiments, the cells are tumor cells. In a preferred embodiment, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

In a seventh aspect, the invention comprises contacting a histone deacetylase identified by the method of the sixth aspect of the invention with a candidate compound and measuring the activity of the histone deacetylase that has been contacted. Provided are methods for identifying histone deacetylase inhibitors that inhibit histone deacetylases involved in induction. Here, candidate compounds are identified as histone deacetylase inhibitors that inhibit histone deacetylase, which is involved in cell differentiation by inhibiting the activity of contacted histone deacetylase. In certain preferred embodiments, histone deacetylase inhibitors interact with fewer enzymes than all histone deacetylases to reduce the activity of the enzyme.

In an eighth aspect, the present invention provides histone deacetylase inhibitors identified by the method of the fifth or seventh aspect of the present invention. Preferably, the histone deacetylase inhibitor is substantially purified.

In a ninth aspect, the invention provides an antisense oligonucleotide that inhibits histone deacetylase, a histone deacetylase inhibitor, a DNA methyltransferase, and a DNA methyltransferase inhibitor. Provided are methods for inhibiting cell division, including contacting cells with a substance. In one embodiment, the growth inhibition of cells in contact is greater than the growth inhibition of cells in contact with only one substance. In some embodiments, each of the materials selected from the group is sufficiently purified. In a preferred embodiment, the cell is a tumor cell. In another preferred embodiment, the materials selected from the group are functionally linked.

According to the present invention, substances known to specifically inhibit histone deacetylases present in tumors can be used as therapeutic agents to inhibit the growth of tumor cells in cancer patients. For example, antisense oligonucleotides that inhibit the expression of histone deacetylases suffer from neoplasia or hyperplasia with pharmaceutically acceptable carriers (eg, sterile aqueous saline solution). The patient may be administered via any route of administration to alleviate any consequent disease syndrome (eg death). Similarly, antisense oligonucleotides that inhibit the expression of histone deacetylases can be included in expression vectors for gene therapy (eg, adenovirus vectors with defects in replication), and phage particles carrying such vectors. ) May be delivered directly to cells showing neoplastic or hyperplastic growth with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and their formulations are well known and generally described, for example, in Remington's Pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990). .

Brief description of the drawings

1 is specifically directed to either nucleic acids encoding HDAC-1 or nucleic acids encoding HDAC-1 and HDAC-2 to inhibit expression of HDAV-1 mRNA or HDAC-1 mRNA and HDAC-2 mRNA, respectively. A graph showing Northern blot analysis showing the dose-dependent ability of the representative, nonlimiting synthetic oligonucleotides of the present invention to bind.

FIG. 2 shows Northern blot analysis showing the dose-dependent ability of a representative, non-limiting synthetic oligonucleotide of the present invention to specifically bind to nucleic acids encoding HDAC-2 to inhibit expression of HDAC-2 mRNA. It is a graph.

3 shows Western showing the ability of a representative, non-limiting synthetic oligonucleotide of the invention to specifically bind to a nucleic acid encoding HDAC-2 to specifically inhibit the expression of HDAC-2 protein. Graph showing blot analysis.

4 is specifically directed to either nucleic acids encoding HDAC-1 or nucleic acids encoding HDAC-1 and HDAC-2 to inhibit expression of HDAV-1 protein or HDAC-1 protein and HDAC-2 protein, respectively. It is a graph showing Western blot analysis showing the ability of the representative, unrestricted synthetic oligonucleotides of the present invention to bind. Mismatched synthetic oligonucleotides were used as negative controls. The same loading in each lane is evidenced by the same expression of actin.

Detailed description of the invention

The present invention provides materials and methods for inhibiting histone deacetylases at the nucleic acid level, and also provides materials and methods for inhibiting specific histone deacetylases at the nucleic acid level. The substances described herein that inhibit histone deacetylase at the nucleic acid level (ie, inhibit transcription and translation) can be used to identify specific histone deacetylases involved in tumorigenesis. In addition, therapeutic compositions that treat and / or alleviate the symptoms of a tumor may be developed using materials of the present invention that specifically inhibit specific histone deacetylases involved in tumor formation.

The materials of the present invention are usefully used as analytical and therapeutic tools, including gene therapy tools. In addition, the present invention provides methods and compositions that can control and fine tune treatment conditions with fewer side effects. Patient and scientific literature referred to herein are well known to those skilled in the art. References, including patients, applications, and GenBank database sequences cited herein are to be embodied by reference to the same extent that each is specifically and individually indicated to be embodied by reference. .

In a first aspect, the present invention provides antisense oligonucleotides that inhibit the expression of histone deacetylases. In some embodiments of the invention, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E. In another embodiment, the oligonucleotides inhibit one or more histone deacetylases or all histone deacetylases.

The antisense oligonucleotides of the invention are complementary to RNA or double chain DNA sites encoding histone deacetylases. In the present invention, "oligonucleotide" includes polymers of two or more deoxyribonucleotides, ribonucleotides or 2'-0-substituted ribonucleotide residues or all combinations thereof. Preferably, the oligonucleotides have about 8 to about 50 nucleoside residues, and most preferably have about 12 to about 30 nucleoside residues. Nucleoside residues may be linked to each other by any of a variety of known internucleoside binding agents. Such internucleoside binders include phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphorus esters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamidates Carbamate, thioether, branched phosphoramidate, branched methylene phosphonate, branched phosphorothioate and sulfone nucleotide linkages. In certain preferred embodiments, such internucleoside binders may be phosphodiester, phosphoroester, phosphorothioate or phosphoramidate binders or combinations thereof. The term oligonucleotide also refers to chemically modified bases or sugars and / or lipophilic groups, intercalating substances, diamines and adamantanes. It includes polymers having additional substituents including without limitation. In the present invention, the term "2'-O-substituted" refers to an -O-low alkyl group or 2 in which the 2 'position of the pentose moiety comprises 1-6 saturated or unsaturated carbon molecules. Substituted by -O-aryl or allyl group having -6 carbon atoms. Here, the alkyl, aryl or allyl group is unsubstituted, for example, halo, hydroxy, trifluoromethyl, cyano, nitro, acyl (acyl), acyloxy, alkoxy, carboxyl, carbalkoxyl or amino group; Alternatively, the 2 ′ position may be substituted with a hydroxy group (to produce ribonucleosides), an amino or a halo group, but not with a 2′-H group.

In the present invention, the term "complementary" means having the ability to hybridize with genomic regions, genes or RNA transcripts thereof under physiological conditions. The hybridization is generally a base between complementary chains to form a base pair of Watson-Crick or Hoogsteen, although other forms of hydrogen bonding or stacking of bases can form hybridization. Occurs as a result of specific hydrogen bonding. In practice, the hybridization can be seen from the observation of inhibition of expression of a particular gene, which occurs at the level of transcription or translation (or both).

Particularly preferred antisense oligonucleotides used in the present invention include chimeric oligonucleotides and hybridized oligonucleotides.

In the present invention, "chimeric oligonucleotide" means an oligonucleotide having one or more forms of internucleoside linking agent. One preferred embodiment of the chimeric oligonucleotide is a chimeric oligonucleotide comprising a phosphorothioate, a phosphodiester or a phosphorodithioate moiety, preferably from about 2 to about 12 nucleotides and alkyl Chimeric oligonucleotides comprising a phosphonate or alkylphosphonothioate moiety (see, eg, Pederson et al., US Pat. Nos. 5,635,377 and 5,366,878). Preferably, the chimeric oligonucleotide comprises at least three consecutive internucleoside linkages selected from phosphodiester and phosphorothioate linkages or combinations thereof.

In the present invention, "crosslinked oligonucleotide" means an oligonucleotide having one or more forms of nucleosides. One preferred embodiment of the hybridizing oligonucleotides comprises ribonucleotides or 2'-0-substituted ribonucleotides (preferably between about 2 and about 12 2'-0-substituted nucleotides) and deoxyribonucleotide sites. . Preferably, the hybridizing oligonucleotides comprise at least three consecutive deoxyribonucleosides and also include ribonucleotides, 2′-O-substituted ribonucleosides or combinations thereof (eg See, for example, Metelev and Agrawal, US Pat. No. 5,652,355).

As long as the oligonucleotide maintains the ability to inhibit the expression of histone deacetylase, the exact nucleotide sequence and chemical structure of the antisense oligonucleotides used in the present invention can be varied. This can be achieved by quantification of the amount of mRNA encoding histone deacetylase, quantification of the amount of histone deacetylase protein, quantification of histone deacetylase activity or in vitro or in vivo . Cell growth analysis of the cells is immediately determined by examining whether the specific antisense oligonucleotide is active by the method of quantitative analysis of the histone deacetylase activity of the cell growth inhibition, all the assays are described in detail herein. It is.

The antisense oligonucleotides used in the present invention are either H-phosphonate chemistry, phosphoramidite chemistry or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., for several cycles). Can be conveniently synthesized on a suitable solid support using well known chemical synthesis methods including applying phosphonate chemistry and phosphoramidite chemistry for other cycles). Suitable solid supports include any standard solid support, such as controlled-pore glass (CPG) used for solid phase oligonucleotide synthesis (e.g., Pon, RT, Methods in Molec. Biol. , 1993 , 20, 465-). 496).

The antisense oligonucleotides of the present invention can be used for various purposes. For example, they can be used to inhibit the activity of histone deacetylase in cell culture or animal systems, or to measure the effect of inhibiting the histone deacetylase activity, thereby reducing the physiological activity of histone deacetylase. It can be used as a "probe". This can be accomplished by applying the antisense oligonucleotides of the invention to cells or animals that inhibit the expression of histone deacetylases and then observing all the phenotypic effects. In this use, the antisense oligonucleotides of the present invention can be preferably used in typical "gene knockout" methods, because they are easier to use and inhibit the activity of histone deacetylases in selected stages of cleavage or differentiation. Because it can be used to suppress. Thus, the method of the present invention can be used as a probe to test the role of histone deacetylase in various stages of the cleavage process.

Preferred antisense oligonucleotides of the invention inhibit transcription of nucleic acid molecules encoding histone deacetylases or inhibit translation of nucleic acid molecules encoding histone deacetylases. Nucleic acids encoding histone deacetylases can be RNA or double-stranded DNA sites, and include intron sequences, untranslated as well as gene-derived coding sequences of histone deacetylase family members. 'And 3' sites, including the border of the intron-exon (exon) without limitation. For human derived sequences, see, eg, Yang et al., Proc. Natl., Acad. Sci. USA , 1996 , 93 (23), 12845-12850; Jurukawa et al., Cytogenet. Cell Genet. , 1996 , 73 (1-2), 130-133; Yang et al., J. Biol. Chem. , 1997 , 272 (44), 28001-28007; Betz et al., Genomics , 1998 , 52 (2), 245-246; Taunton et al., Science , 1996 , 272 (5260), 408-411; And Dangond et al., Biochem. Biophys. Res. Commun. , 1998 , 242 (3), 648-652.

Examples of particularly preferred non-limiting antisense oligonucleotides of the invention include histone deacetylases (eg, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C). , HDAC-D or HDAC-E) is complementary to RNA or double chain DNA sites encoding. Antisense oligonucleotides of the present invention are directed to RNA or double-stranded DNA sites encoding HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D and / or HDAC-E. It is complementary. The sequence of human HDAC-1 can be found in GenBank accession number U50079 (amino acid sequence is set forth in SEQ ID NO: 24 , nucleic acid sequence is set forth in SEQ ID NO: 25 ). The sequence of human HDAC-2 can be found in GenBank Accession Number U31814 (amino acid sequence is set forth in SEQ ID NO: 26 and nucleic acid sequence is set forth in SEQ ID NO: 27 ). The sequence of human HDAC-3 can be found in GenBank Accession Number U75697 (amino acid sequence is set forth in SEQ ID NO: 28 and nucleic acid sequence is set forth in SEQ ID NO: 29 ). The sequence of human HDAC-4 (formerly known as HDAC-A) can be found in GenBank Accession Number AB006626 (amino acid sequence is set forth in SEQ ID NO: 30 , nucleic acid sequence is set forth in SEQ ID NO: 31 ). The sequence of human HDAC-5 (formerly known as HDAC-B) can be found in GenBank Accession Number AB011172 (amino acid sequence is set forth in SEQ ID NO: 32 and nucleic acid sequence is set forth in SEQ ID NO: 33 ). The sequence of human HDAC-C can be found in GenBank Accession Number AC004994 (amino acid sequence is set forth in SEQ ID NO: 34 and nucleic acid sequence is set forth in SEQ ID NO: 35 ). The sequence of human HDAC-D can be found in GenBank Accession Number AC004466 (nucleic acid sequence is set forth in SEQ ID NO: 36 ).

In addition, sequences encoding histone deacetylases from many non-human species are known (see, eg, GenBank Accession Nos. AF006603, AF006602 and AF074882 for rodent histone deacetylases). Thus, the antisense oligonucleotides of the invention may also be complementary to RNA or double chain DNA sites encoding histone deacetylases from non-human animals. In particular, preferred oligonucleotides have about 13 to about 35 nucleotide sequences comprising the nucleotide sequences set forth below in SEQ ID NOs: 1 to 18 . Another particularly preferred oligonucleotide has about 15 to about 26 nucleotide sequences described below. Most preferably, the oligonucleotides described below have a phosphorothioate backbone, 20-26 nucleotides in length and four nucleotides at the 5 'end of the oligonucleotide and four nucleotides at the 3' end, respectively Modified to have a 2'-0-methyl group attached to a sugar moiety.

Antisense Oligonucleotides Specific for Human HDAC-1 (MG2608):

5'-GAA ACG TGA GGG ACT CAG CA-3 '( SEQ ID NO: 1 ).

Antisense oligonucleotides (MG2610) specific for both human HDAC-1 and human HDAC-2 are mixtures of the following four oligonucleotides:

5'-CAG CAA ATT ATG GGT CAT GCG GAT TC-3 '( SEQ ID NO: 2 );

5'-CAG CAA GTT ATG AGT CAT GCG GAT TC-3 '( SEQ ID NO: 3 );

5'-CAG CAA ATT ATG AGT CAT GCG GAT TC-3 '( SEQ ID NO: 4 ); And

5'-CAG CAA GTT ATG GGT CAT GCG GAT TC-3 '( SEQ ID NO: 5 ).

Antisense Oligonucleotides Specific for Human HDAC-2:

5'-TGC TGC TGC TGC TGC TGC CG-3 '( SEQ ID NO: 6 );

5'-CCT CCT GCT GCT GCT GCT GC-3 '( SEQ ID NO: 7 );

5'-GGT TCC TTT GGT ATC TGT TT-3 '( SEQ ID NO: 8 ); And

5'-CTC CTT GAC TGT ACG CCA TG-3 '( SEQ ID NO: 9 );

Antisense oligonucleotides of the invention may optionally be formulated with any pharmaceutically acceptable carrier or diluent (eg, Remington's Pharmaceutical Sciences, 18th Edition, ed.A. Gennaro, Mack Publishing Co., Ltd.). See "preparation of pharmaceutically acceptable formulations" described in Easton, PA, 1990).

In a second aspect, the present invention provides a method of inhibiting histone deacetylase in a cell comprising contacting the cell with an antisense oligonucleotide that inhibits expression of histone deacetylase. Preferably, cell division in contacted cells is inhibited. Accordingly, the antisense oligonucleotides of the present invention are usefully used for the treatment of diseases of humans including benign and malignant tumors by inhibiting the division of cells in contact with the antisense oligonucleotides. The phrase “inhibiting cell division” refers to the histone deacetylase antisense oligonucleotide or histone deacetylase when compared to the growth of non-contact cells with the growth of cells in contact with the oligonucleotide or protein inhibitor. Inhibitors (or combinations thereof) exhibit the ability to delay the growth of cells. Such cell division can be measured by counting contacted and non-contacted cells using a Coulter Cell Counter (Coulter, Miami, FL) or a hemacytometer. If the cells are in solid growth (e.g., solid tumors or organs), these cell division measurements may be performed using a caliper to measure growth or in contact with cells not in contact. It can be obtained by comparing the size of. Preferably, the term includes delaying cell growth by at least 50% of uncontacted cells. More preferably, the term includes delaying cell growth by 100% of uncontacted cells (ie, contacted cells do not increase in number and size). Most preferably, the term includes a reduction in the number or size of cells in contact as compared to cells that are not in contact. Thus, histone deacetylase antisense oligonucleotides or histone deacetylase inhibitors that inhibit cell division in contacted cells may be delayed, stopped, or programmed cell death (ie, programmed cell death, ie, , Apoptosis or necrotic cell death.

In contrast, the phrase “inducing cell division” is used to indicate the presence of histone deacetylase or enzyme activity for cell division in normal cells (ie, not tumor cells). Thus, over-expression of histone deacetylase, which induces cell division, may or may not lead to an increase in cell division; However, inhibition of histone deacetylase, which induces cell division, will lead to inhibition of cell division.

The phrase "inducing cell differentiation" refers to the ability of histone deacetylase antisense oligonucleotides or histone deacetylase inhibitors (or combinations thereof) to induce differentiation of contacted cells as compared to cells not contacted. When used. Thus, when histone deacetylase antisense oligonucleotides or histone deacetylase inhibitors (or both) of the present invention come into contact with tumor cells, they induce differentiation so that they are phylogenetically more differentiated daughter cells. causes the production of cells).

The anti-dividing ability of the antisense oligonucleotides of the present invention induces synchronization of a cell population that grows a-synchronously. For example, the antisense oligonucleotides of the present invention can be used to arrest in vitro G1 or G2 groups of non-neoplastic cell populations that grow in vitro. As a result of such homogenization, for example, genes and / or gene products expressed during the G1 or G2 phase of the cell cycle can be identified. In addition, the homogenization of such cultured cells can be used to test the efficacy of the novel transfection protocol, where the transformation efficiency varies and specific cells of the transformed cells. It depends on the cycle. The use of the antisense oligonucleotides of the present invention can be used to homogenize cell populations and thus help to find improved transformation efficiency.

The use of the antisense oligonucleotides of the invention to inhibit tumor cells is described in detail herein.

In another preferred embodiment, the cells in contact with histone deacetylase antisense oligonucleotides are also in contact with histone deacetylase inhibitors.

In the present invention, "histone deacetylase inhibitor" means an active moiety capable of interacting with histone deacetylase and at the protein level to reduce the activity of histone deacetylase. Histone deacetylase inhibitors include, without limitation, trichostatin A, trichostatin B, thyristostatin C, depudecine, trapoxin, butyrate, SAHA, FR901228 and acetyldinaline (el-Beltagi et al. , Cancer Res. , 1993 , 53 (13), 3008-3014). Histone deacetylase inhibitors are molecules that reduce the activity of histone deacetylase more than reducing the activity of other proteins that are not involved. In a preferred embodiment, the decrease in histone deacetylase activity is at least 5 times, more preferably at least 10 times, and most preferably at least 50 times. In another embodiment, the activity of histone deacetylase is reduced by 100 fold. Preferably, histone deacetylase inhibitors interact with fewer enzymes than all histone deacetylases to reduce activity. "All histone deacetylases" mean all members of both histone deacetylase family proteins from a particular animal species, including HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, Includes without limitation HDAC-C, HDAC-D or HDAC-E, all of which are used herein as “related proteins”. For example, preferred histone deacetylase inhibitors interact with and inhibit HDAC-1 and HDAC-2 but do not inhibit by interacting with HDAC-3. Most preferably, histone deacetylase inhibitors interact with one histone deacetylase (e.g., HDAC-2) to reduce activity but the other histone deacetylase (e.g., HDAC-1). And HDAC-3) does not reduce activity. As described below, preferred histone deacetylase inhibitors are those that interact with histone deacetylase to participate in tumorigenesis to reduce the activity of the enzyme.

Preferably, histone deacetylase inhibitors are functionally linked with antisense oligonucleotides. As described above, the antisense oligonucleotides of the present invention may optionally be formulated with well known pharmaceutically acceptable carriers or diluents. The formulation further comprises one or one or more or additional histone deacetylase antisense oligonucleotide (s) and / or one or more histone deacetylase inhibitor (s) or any other pharmaceutically active substance. It may further include.

In a particularly preferred embodiment of the invention, the antisense oligonucleotides are functionally linked with histone deacetylase inhibitors. The term "operable association" allows antisense oligonucleotides to inhibit the expression of nucleic acids encoding histone deacetylases, and histone deacetylase inhibitors inhibit the activity of histone deacetylases. All linkages of antisense oligonucleotides to histone deacetylase inhibitors that allow it. One or more antisense oligonucleotides of the invention may be functionally linked with one or more histone deacetylase inhibitors. Preferably, the antisense oligonucleotides of the invention that target one particular histone deacetylase (eg, HDAC-2) are functional with histone deacetylase inhibitors that target the same histone deacetylase. Can be connected. Preferred functional linkages are hydrolyzable. Preferably, the hydrolyzable linkage is a covalent linkage between the antisense oligonucleotide and the histone deacetylase inhibitor.

Preferably, the covalent bonds are hydrolyzed by esterases and / or amidases. Examples of such hydrolyzable linkages are well known in the art. Particular preference is given to phosphate esters.

In certain preferred embodiments, the covalent bond is directly bound between the antisense oligonucleotide and the histone deacetylase inhibitor such that the histone deacetylase inhibitor is included in the backbone. In contrast, the covalent bond may be through an extended structure and may combine both antisense oligonucleotides and histone deacetylase inhibitors, for example carbohydrates, peptides or lipids or glycolipids ( It may be formed by binding the antisense oligonucleotide and histone deacetylase inhibitor to a carrier molecule such as glycolipid). Other preferred functional linkages include lipophilic linkages such as the formation of liposomes comprising antisense oligonucleotides covalently linked to lipophilic molecules and histone deacetylase inhibitors. The lipophilic molecule includes without limitation synthetic neoglycolipids such as phosphotidylcholine, cholesterol, phosphatidylethanolamine and sialyllacNAc-HDPE. In certain preferred embodiments, the functional linkage is not a physical linkage, but may simply be present simultaneously in the body, such as when antisense oligonucleotides are present in one liposome and protein inhibitors are present in another liposome.

In a third aspect, the invention provides a therapeutically effective amount of an antisense oligonucleotide of the first aspect of the invention contained in a pharmaceutically acceptable carrier in an animal having at least one tumor cell in the body in a therapeutically effective amount. A method of inhibiting growth of neoplastic cells in an animal comprising administering for a period of time is provided. Preferably, the animal is a mammal and is a particularly domesticated mammal. More preferably, the animal is a human.

The term "neoplastic cell" is used to refer to a cell that shows the growth of an aberrant cell. Preferably, the abnormal growth of neoplastic cells is increased cell growth. Neoplastic cells are hyperplastic cells, cells that are not inhibited by contact growth in vitro, benign tumor cells that cannot metastasize in vivo, or cancer cells that can metastasize in vivo and recur after removal. It may be. The term "tumorigenesis" is used to denote the induction of cell division showing neoplastic growth.

The "therapeutically effective amount" and "therapeutically effective period of time" are used to refer to known dosages and durations to reduce the growth of neoplastic cells. do. Preferably, the administration may be via parenteral, oral, sublingual, transdermal, topical, intranasal or rectal routes. When administered systemically, the therapeutic composition is preferably administered so that the level of antisense oligonucleotides in the blood is from about 0.1 μM to about 10 μM. When administered topically, much lower concentrations are effective than above and tolerated to much higher concentrations. In one of the prior art such therapeutic effects resulting in lower effective concentrations of histone deacetylase inhibitors can vary considerably depending on the tissue, organ or particular animal or patient.

In a preferred embodiment, the therapeutic composition of the present invention is administered systemically enough so that the level of antisense oligonucleotides in the blood is from about 0.01 μM to about 20 μM. In a particularly preferred embodiment, the therapeutic composition is sufficiently administered so that the level of antisense oligonucleotides in the blood is from about 0.05 μM to about 15 μM. In a more preferred embodiment, the level of antisense oligonucleotide in the blood is from about 0.1 μM to about 10 μM.

When administered topically, much lower concentrations than above are effective in treatment. Preferably, the amount of total antisense oligonucleotide ranges from about 0.1 mg to about 200 mg oligonucleotide per kg body weight per day. In the most preferred embodiment, the total amount of antisense oligonucleotide ranges from about 2 mg to about 10 mg oligonucleotide per kg body weight per day. In a particularly preferred embodiment, the amount of therapeutically effective histone deacetylase antisense oligonucleotide is in the range of about 0.5 mg oligonucleotide per kg body weight per day.

In certain preferred embodiments of the third aspect of the invention, the method of the invention comprises administering to the animal an effective amount of a histone deacetylase inhibitor with a pharmaceutically acceptable carrier for a therapeutically effective period of time. Additionally included. Preferably, histone deacetylase inhibitors are functionally linked with antisense oligonucleotides. Methods for functionally linking histone deacetylase inhibitors with histone deacetylase antisense oligonucleotides are described above.

Therapeutic compositions of the present invention comprising a histone deacetylase inhibitor are sufficiently administered systemically so that the level of histone deacetylase inhibitor in the blood is from about 0.01 μM to about 10 μM. In a particularly preferred embodiment, the therapeutic composition is sufficiently administered so that the level of histone deacetylase inhibitor in the blood is from about 0.05 μM to about 10 μM. In a more preferred embodiment, the level of histone deacetylase inhibitor in the blood is about 0.1 μM to about 7 μM. When administered topically, much lower concentrations than above are effective in treatment. Preferably, the amount of total histone deacetylase inhibitor is in the range of about 0.01 mg to about 5 mg protein per kg body weight per day. In a more preferred embodiment, the total amount of histone deacetylase inhibitor is in the range of about 0.1 mg to about 4 mg protein per kg body weight per day. In the most preferred embodiment, the total amount of histone deacetylase inhibitor is in the range of about 0.1 mg to about 1 mg protein per kg body weight per day. In a particularly preferred embodiment, the amount of therapeutically effective synergistic histone deacetylase inhibitor (when administered with antisense oligonucleotide) is 0.1 mg / kg body weight per day.

This aspect of the invention exhibits an enhanced inhibitory effect such that the inhibitors of nucleic acid levels (ie, antisense oligonucleotides) and protein levels (ie histones) are required to exhibit a given inhibitory effect when compared to the amount required when used separately from one another. Reducing the therapeutically effective concentration in one or both deacetylase inhibitors).

Furthermore, one of the antisense oligonucleotides or histone deacetylase inhibitors can have a reduced or increased amount of therapeutically effective cavities by finely adjusting and altering the amount of other elements. Accordingly, the present invention provides a method for defining administration / treatment in an exigency that is particularly urgent for an animal or a particular patient. The therapeutically effective range can be easily determined, for example, by starting with a relatively low amount empirically and sequentially increasing the inhibitory levels.

In a fourth aspect, the present invention provides a method for investigating the role of specific histone deacetylases in the division of cells, including the division of tumor cells. In this method, the type of cell of interest is contacted with an antisense oligonucleotide that inhibits the expression of histone deacetylase, as described in the first aspect of the invention, thereby inhibiting the expression of histone deacetylase in the cell. do. If the contacted cells whose expression of histone deacetylase is inhibited also show inhibition of cell division, the histone deacetylase is involved in the induction of cell division. In this case, if the contacted cells are tumor cells, the contacted tumor cells show inhibition of cell division, and the histone deacetylases whose expression is suppressed become histone deacetylases involved in tumor formation. Preferably, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

Thus, identifying specific histone deacetylases involved in cell division requires that only specific histone deacetylases need to be targeted using antisense oligonucleotides to inhibit cell division or induce differentiation. do. As a result, it is possible to effectively inhibit cell division while using therapeutically effective lower amounts of antisense oligonucleotides. In addition, unwanted side effects resulting from inhibiting all histone deacetylases can be prevented by specifically inhibiting one (or more) histone deacetylase (s) involved in the induction of cell division.

If such histone deacetylases that are involved in the induction of cell division are identified by the antisense oligonucleotides of the first aspect of the present invention, other histone deacetylases not involved in the induction of cell division are inhibited without inhibiting cell division. A histone deacetylase inhibitor that specifically inhibits only histone deacetylase that is involved in the induction of can be prepared. Thus, in a fifth aspect, the present invention provides a method for identifying a histone deacetylase inhibitor that inhibits histone deacetylase, which is involved in the induction of cell division. The method comprises contacting histone deacetylase, identified as involved in the induction of cell division, with a candidate compound and measuring the activity of the contact histone deacetylase. By inhibiting the activity of contacted histone deacetylases, candidate compounds are identified as histone deacetylase inhibitors that inhibit histone deacetylases involved in the induction of cell division.

Determination of the activity of histone deacetylase can be accomplished using known methods. For example, Yoshida et al. Proposed a method for measuring the activity of histone deacetylases by detecting acetylated histones in cells treated with trichostatin A ( J. Biol. Chem. , 1990 , 265, 17174). -17179). Taunton et al. Similarly describe methods for measuring histone deacetylase activity using endogenous and recombinant HDAC-1 ( Science , 1996 , 272, 408-411). Yoshida et al. ( J. Biol. Chem. , 1990 , 265, 17174-17179) and Taunton et al. ( Science , 1996 , 272, 408-411) are all incorporated herein by reference.

Preferably, histone deacetylase inhibitors that inhibit histone deacetylase, which are involved in the induction of cell division, interact with fewer enzymes than all histone deacetylases to decrease activity. Inhibitors.

In a sixth aspect, the present invention provides a method for identifying histone deacetylases involved in the induction of cell differentiation, comprising contacting cells with antisense oligonucleotides that inhibit expression of histone acetylases. Herein, histone deacetylase is identified as a histone deacetylase that is involved in inducing cell differentiation by inducing differentiation in contacted cells. Preferably, the cell is a tumor cell. More preferably, the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D or HDAC-E.

In a seventh aspect, the invention comprises contacting a histone deacetylase identified by the method of the sixth aspect of the invention with a candidate compound and measuring the activity of the histone deacetylase that has been contacted. Provided are methods for identifying histone deacetylase inhibitors that inhibit histone deacetylases involved in induction. Here, candidate compounds are identified as histone deacetylase inhibitors that inhibit histone deacetylase, which is involved in the induction of cell differentiation, due to reduced activity of contacted histone deacetylase. In certain preferred embodiments, histone deacetylase inhibitors interact with fewer enzymes than all histone deacetylases to reduce activity.

In an eighth aspect, the present invention provides histone deacetylase inhibitors identified by the method of the fifth or seventh aspect of the present invention. Preferably, histone deacetylase inhibitors are sufficiently purified.

Fully purified proteins include recombinant protein expression, affinity chromatography, antibody-based affinity chromatography and HPLC (High performance liquid chromatography; see, eg, Fisher (1980), Laboratory Techniques in Biochemistry and Molecular Biology , Work). and Burdon (eds.), see Elsevier). Preferably, fully purified protein refers to at least 80% purified by weight to be purely isolated from other proteins or naturally-occurring organic molecules. More preferably, fully purified protein is at least 90% pure by weight. Most preferably, fully purified protein is at least 95% pure by weight.

In a ninth aspect, the invention provides a cell comprising at least two cells selected from the group consisting of antisense oligonucleotides that inhibit histone deacetylases, histone deacetylase inhibitors, and antisense oligonucleotides that inhibit DNA methyltransferases. Provided are methods for inhibiting division of cells, including contacting with a substance. In one embodiment, the inhibition of contacted cell growth is greater than the inhibition of cell growth in contact with only one substance. In some preferred embodiments, each of the materials selected from the group is sufficiently purified. In a preferred embodiment, the cell is a tumor cell. In another preferred embodiment, the material selected from the group is functionally linked.

Antisense oligonucleotides that inhibit DNA methyltransferases are described in US Pat. No. 5,578,716 to Szyf and Hofe, the entire contents of which are incorporated by reference. DNA methyltransferase inhibitors include 5-aza-2'-deoxycytidine (5-aza-dC), 5-fluoro-2'-deoxycytidine, 5-aza- Cytidine (5-aza0C) or 5,6-dihydro-5-aza-cytidine without limitation.

The following examples are intended to further illustrate certain preferred embodiments of the present invention and are not intended to limit the present invention. Based on the description and examples, other embodiments will be possible to those skilled in the art, which are also included in the present invention.

Example 1 Screening of Antisense Oligonucleotides

To identify antisense oligonucleotides that most effectively inhibit a particular histone deacetylase, several oligonucleotides were selected based on the sequences provided in GenBank Accession Number U50079 for GDAC-1 and GenBank Accession Number U31814 for HDAC-2. Prepared. Some of the screened oligonucleotides are described in Tables 2 and 3 of U.S. Patent No. 60 / 104,804 to Bestereman et al. Filed Oct. 19, 1998, the entirety of which is incorporated herein by reference.

In addition, oligonucleotides that were complementary to both HDAC-1 and HDAC-2 were prepared.

In order to screen the ability of the oligonucleotides to inhibit targeted histone deacetylases, Northern blot analysis was first performed. Specifically, T24 human bladder carcinoma cells (purchased from ATCC, Manassas, VA,) were cultured under the indicated conditions. Cells were washed with phosphate buffered saline (PBS) before adding oligonucleotides. Next, a lipofectin transformation reagent (Gibco-BRL, Mississauga, Ontario) at a concentration of 6.25 μg / ml was added to the serum-free OPTIMEM medium (Gibco / BRL), and then the medium was added to the cells. . Oligonucleotides to be screened were added to other wells of the cells (ie, one oligonucleotide was added per well). The same concentration of oligonucleotides (eg 50 nM) was used for each well. Cells were incubated for 4 hours at 37 ° C. in a cell culture in the presence of lipofectin and oligonucleotides. The cells were washed with PBS and then cultured in medium containing serum. After 24 hours, cells were harvested and the levels of HDAC mRNA determined by Northern blot analysis.

To determine mRNA levels with Northern blots, guanidinium isothiocyanate, except for the additional precipitation of overnight at 4 ° C. using 2 M LiCl to purify RNA from DNA contaminants. It was isolated using a guanidinium isothiocyanate standard procedure (see, for example, Ausubel et al., Current Protocols in Molecular Biology , John Wiley & Sons, New York, NY, 1994). Northern blot analysis was performed according to standard methods. Probes for HDAC-1 and HDAC-2 used full-length cDNA clones amplified by PCR from known sequences for each (eg GenBank Accession Number U50079 and U31814, respectively). The probes were radiolabeled using ³² P-ATP. Northern blots were scanned and quantified using Alpha Imager (Alpha Innovetech).

It is also possible to inhibit the expression of histone deacetylase protein in oligonucleotides that demonstrate the ability to reduce mRNA expression of targeted histone deacetylase (ie, inhibit transcription of histone deacetylase mRNA). Screened for presence. To this end, T24 cells were transformed with oligonucleotides using lipofectin in the manner described above. After 24 hours, cells were lysed according to standard methods. Whole cell extracts (50 μg) were dissolved in SDS / PAGE with a gradient of 7-15% and delivered to PVDF membranes (Amersham, Arlington Heights, IL) and then specific to HDAC-1 and HDAC-2. Western blots were performed using a rabbit polyclonal antibody. Detection was performed using secondary anti-rabbit IgG-HR peroxidase antibody and an enhanced chemiluminescence detection kit (Amersham) according to the manufacturer's instructions.

Based on the results of the present inventors, the antisense oligonucleotides described below, determined by mRNA and protein expression blot analysis, were identified as most effectively inhibiting the expression of targeted histone deacetylases. The oligonucleotides are as described below:

For inhibition of HDAC-1, oligonucleotide number MG2608 has the following sequence:

5′- GAA A CG TGA GGG ACT C AG CA- 3 ′ ( SEQ ID NO: 10 ).

For inhibition of both HDAC-1 and HDAC-2, oligonucleotide number MG2610 mixes four oligonucleotides having the following sequence in a ratio of 25/25/25/25:

5′- CAG C AA ATT ATG GGT CAT GCG G AU UC- 3 ′ ( SEQ ID NO: 11 );

5′- CAG C AA GTT ATG AGT CAT GCG G AU UC- 3 ′ ( SEQ ID NO: 12 );

5′- CAG C AA ATT ATG AGT CAT GCG G AU UC- 3 ′ ( SEQ ID NO: 13 );

5′- CAG C AA GTT ATG GGT CAT GCG G AU UC- 3 ′ ( SEQ ID NO: 14 );

In the inhibition of HDAC-2, the antisense oligonucleotides found to be most effective are shown in Table 1 .

Number of oligonucleotides order SEQ ID NO: Target site MG2628 5'- UGC U GC TGC TGC TGC T GC CG -3 ' 15 121-141 MG2633 5'- CCU C CT GCT GCT GCT G CU GC -3 ' 16 132-152 MG2635 5'- GGU U CC TTT GGT ATC T GU UU -3 ' 17 1605-1625 MG2636 5'- CUC C TT GAC TGT ACG C CA UG -3 ' 18 1-20

(***) The reference number of the target site is consistent with GenBank Accession Number U31814 for HDAC-2.

To investigate the specificity of second-generation histone deacetylase antisense oligonucleotides, mismatch control oligonucleotides were prepared for HDAC-1 (MG2608) and HDAC-1 / 2 (MG2610). The mismatch control oligonucleotides were prepared by substituting the bases of the four nucleotides present at 5 'and 3', where the affinity with the nucleic acid encoding the primarily targeted histone deacetylase is most intense. .

HDAC-1 MISMATCH CONTROL (MG2609) has the following sequence:

5'- CAA U CG TCA GAG ACT C CG AA- 3 '( SEQ ID NO: 19 )

HDAC-1 / 2 MISMATCH CONTROL (MG2637) mixes four oligonucleotides having the following sequence in a ratio of 225/25/25/25.

5'- AAG G AA GTC ATG AAT GAT GCC C AU UG -3 '( SEQ ID NO: 20 );

5'- AAG G AA ATC ATG GAT GAT GCC C AU UG -3 '( SEQ ID NO: 21 );

5'- AAG G AA GTC ATG GAT GAT GCC C AU UG- 3 '( SEQ ID NO: 22 );

5'- AAG G AA ATC ATG AAT GAT GCC C AU UG- 3 '( SEQ ID NO: 23 );

The oligonucleotides (ie, oligonucleotides of SEQ ID NOs: 10-23) are second generation oligonucleotides (ie 4 × 4 hybridization). That is, the oligonucleotide described in SEQ ID NO: 10 to SEQ ID NO: 23 and nucleotides to have been chemically modified, such as: A is 2'-deoxy-ribo Adenosine; C is 2'-deoxyribocytidine; G is 2'-deoxyriboguanosine; T is 2'-deoxyribothymidine; A is riboadenosine; U is uridine; C is ribocytidine; And G represents riboguanosine.

Underlined bases are nucleotides substituted with 2'-methoxyribose. Bases not underlined are deoxyribose nucleosides. The backbone of each oligonucleotide consists of phosphorothioate bonds between adjacent nucleotides.

Next, various oligonucleotides complementary to HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D and HDAC-E were prepared. Such oligonucleotides are based on known nucleic acid sequences of the histone deacetylases (eg, GenBank Accession Number U75697 for HDAC-3). Specific to one of the histone deacetylases by investigating the effect of inhibiting the expression of mRNA and protein in the same manner as described above for HDAC-1, HDAC-1 / 2 and HDAC-2. Antisense oligonucleotides were screened. In addition, oligonucleotides that inhibit one or more histone deacetylases (eg, specific to HDAC-1 / 3 / C) were prepared by mixing antisense oligonucleotides specific for each histone deacetylase. The effect was screened.

Example 2 Inhibition of Histone Deacetylase mRNA Expression Using Antisense Oligonucleotides

To determine the specificity of the antisense oligonucleotides specific for the nucleic acid encoding histone deacetylase and the required dose, the dose dependent inhibition of the oligonucleotide on histone deacetylase mRNA expression was investigated.

To this end, T24 cells were transformed with lipofectin (as described in Example 1) containing 10, 25, 50 or 100 nM oligonucleotides. After 24 hours of incubation, cells were collected and RNA was prepared, followed by Northern blot analysis by the method of Example 1 using radiolabeled HDAC-1 and HDAC-2 cDNA as probes.

Figure 1 shows that the expression of HDAC-1 mRNA is dose-dependently inhibited by both HDAC-1 and HDAC-1 / 2 antisense oligonucleotides at a concentration of 50-100 nM. In contrast, the expression of HDAC-2 mRNA is 50 It was only inhibited by HDAC-1 / 2 antisense oligonucleotide (MG2610) at -100 nM concentration and HDAC-1 antisense oligonucleotide (MG2608) had no effect. The oligonucleotides used in the experiment were first generation oligonucleotides (ie, not chemically modified) and the results are shown in FIG. 1 . 1 shows the results of using oligonucleotides set forth in SEQ ID NO: 1 to SEQ ID NO: 5 .

2 shows that HDAC-2 mRNA is dose-dependently inhibited by HDAC-2 antisense oligonucleotides. All four HDAC-2 antisense oligonucleotides (MG2628, MG2633, MG2635 and MG2636) were able to reduce the expression of HDAC-2 mRNA at 50-100 nM concentration. MG2628 appears to be particularly effective in reducing the expression of HDAC-2 mRNA in this experiment.

The data show that antisense oligonucleotides can be used to target histone deacetylases at the nucleic acid level, thereby reducing the expression of mRNA only 24 hours after exposure to oligonucleotides.

Example 3 Inhibition of Histone Deacetylase Protein Expression Using Second Generation Antisense Oligonucleotides

To investigate the ability of histone deacetylase antisense oligonucleotides to inhibit the expression of proteins, second generation antisense oligonucleotides for HDAC-1, HDAC-1 / 2 and HDAC-2 were prepared. Each second generation antisense oligonucleotide has a backbone that is linked by phosphorothioate linkages between adjacent nucleotides. In addition, the four nucleotide residues present at the 5 'and 3' ends of the oligonucleotide have a sugar residue including the 2'-O-methyl group. This modification of the nucleotide termini acts to increase the binding affinity of the oligonucleotide to the targeted nucleic acid and to increase the stability of the oligonucleotide by inhibiting the susceptibility of the nuclease.

3 shows the ability of second generation HDAC-2 antisense oligonucleotides to inhibit expression of HDAC-2 protein. T24 cells were transformed with lipofectin containing 0, 25 and 50 nM of MG2628 or MG2636 in the same manner as in Example 1. After 24 hours, cells were transformed secondary with the same amount of the same oligonucleotide. After 24 hours again (i.e. 48 hours after the first transformation), the proteins of the cells were isolated and dissolved in SDS-PAGE at a 7-15% gradient, followed by rabbit polyclonal antibodies specific for HDAC-2 ( 1: 500, Santa Cruz Biotech) Western blot was carried out in the same manner as in Example 1. Blot with anti-rabbit IgG-HR peroxidase secondary antibody and visualize using an enhanced chemifluorescence detection kit (Amersham), then blot off and actin to verify that they are equally loaded for each well Re-probe with specific antibody (data not shown).

As can be seen in FIG . 3 , 50 μM of second generation MG2628 or MG2836 was able to inhibit the expression of HDAC-2 protein.

4 shows that HDAC-1 / 2 and HDAC-1 antisense oligonucleotides have a particular ability to inhibit the expression of HDAC-1 and HDAC-2 or HDAC-1 proteins, respectively, when compared to mismatch controls. T24 cells were transformed twice using the method described above using 50 nM oligonucleotides. Cell lysates were prepared 24 hours after the second transformation and dissolved in SDS-PAGE at 7-15% gradient and then transferred to the PVDF membrane. PVDF membrane blots were blotted using anti-HDAC-1 antibodies. After detection with secondary antibody labeled horseradish peroxidase and enhanced chemifluorescence, the blots were removed and re- probed with anti-HDAC-2 antibody. After detection, the blots were removed once again to verify that the same amount of protein was loaded in each lane and reprobed with an antibody specific for actin.

As can be seen in FIG . 4 , the HDAC-1 and HDAC-1 / 2 mismatch control oligonucleotides did not inhibit the expression of HDAC-1 or HDAC-1 and HDAC-2 proteins, respectively. In contrast, HDAC-1 antisense oligonucleotides effectively reduced the expression of HDAC-1 protein and HDAC-1 / 2 antisense oligonucleotides reduced the expression of both HDAC-1 and HDAC-2 proteins.

Example 4 Identification of Histone Deacetylases Involved in Induction of Cell Division

Antisense oligonucleotides of the invention that inhibit the expression of other histone deacetylases were used to screen for histone deacetylases that induce cell division in cultured cells.

To identify histone deacetylases that induce division of normal (ie, non-neoplastic) cells, cultured normal human fibroblasts were transfected with antisense oligonucleotides that inhibit expression of histone deacetylases. Switched. Although standard transformation methods including CaPO ₂ precipitation, electroporation, DEAE-dextran can be used without limitation, transformation with lipofectin transformation reagent (Gibco-BRL) is preferred. After transformation with lipofectin and histone deacetylase antisense oligonucleotides, cells were collected using trypsin at various time points and cells were counted using a hemasitometer or Coulter Cell Counter. In addition, mock transformed control cells (ie, control, lipofectin with nonspecific oligonucleotides) were collected and counted. Microscopically observed what apparent changes (eg, induction of apoptosis) of both antisense oligonucleotides and falsely transformed cells. Antisense oligonucleotides that inhibit the expression of histone deacetylases that inhibit cell division when transformed into normal cells are used to identify histone deacetylases involved in the induction of cell division in normal cells.

To identify histone deacetylases that induce the growth of neoplastic cells, T24 bladder epithelial cancer cells were transformed with histone deacetylase antisense oligonucleotides of the present invention and observed after their growth type and not transformed. Compared to control cells. To this end, one day before transformation, T24 cells (ATCC No. HTB-4) were plated in 10 cm plates at a concentration of 4 × 10 ⁵ cells / dish. At the time of transformation, cells were washed with PBS and then Opti-MEM medium (Gibco-BRL, Mississauga, Ontario) containing 6.25 μg / ml lipofectin transformation reagent was added. The antisense oligonucleotides tested were diluted in the transformation medium to the desired concentration using a 0.1 mM stock solution. After 4 hours of incubation in a 37 ° C., 5% CO ₂ incubator, the plates were washed with PBS and 10 ml fresh cell culture medium was added. T24 cells were transformed for a total of 3 days and split every day to provide optimal transformation conditions. Cells were collected by trypsin treatment at various time periods, and then pellets were separated by centrifugation at 4 ° C. for 5 minutes at 1,100 rpm. Cells were resuspended in PBS and counted with Coulter Particle Counter to determine the total number of cells. Falsely transformed T24 cells (ie, transformed with lipofectin and control oligonucleotides) were similarly grown, collected and counted. Antisense oligonucleotides that inhibit the expression of histone deacetylases that inhibit cell division when transformed into neoplastic cells are used to identify histone deacetylases involved in the induction of cell division in neoplastic cells.

By screening a variety of different histone deacetylase antisense oligonucleotides in normal and neoplastic cells, one can immediately identify histone deacetylases involved in the induction of cell division. Most preferably, the histone deacetylase antisense oligonucleotide of the present invention inhibits the growth of neoplastic cells but does not inhibit the growth of normal cells.

Example 5 Histone Deacetylase Inhibitors Interact with Histone Deacetylase and Reduce Enzyme Activity Involved in Induction of Cell Division

Histone deacetylases that have been identified as involved in the induction of cell division (eg, by the method of Example 4) are used as targets of candidate compounds that interact with enzymes to reduce activity. FR901228 (Fujisawa Pharmaceutical Co., Ltd.) was used as a positive control.

Candidate compounds may be obtained from any source and may be present in nature or synthesized, or may have portions present or synthesized in nature. Candidate compounds may also be designed without limitation to be chemically similar to known histone deacetylase inhibitors, including Trichostatin A, Trichostatin C, Trapoxin, Depudecine, suberoylanilide hydroxamic acid (SAHA), FR901228, and Butyric Acid. have.

Once candidate compounds are identified, such compound pools can be added to histone deacetylases. It is preferable that the histone deacetylase is identified as a histone deacetylase which is involved in the induction of cell division using the antisense oligonucleotide of the present invention. Histone deacetylases can be used, for example, using antibodies specific for particular histone deacetylases (eg, anti-HDAC-1 antibodies available from Santa Cruz Biotech) or prokaryotic or eukaryotic It can also be purified by the method using the histone deacetylase produced recombinantly in the cell. In addition, histone deacetylase may be present in cells normally expressing histone deacetylase.

A group of candidate compounds is added to histone deacetylase and the activity of histone deacetylase is measured. The group of candidate compounds showing histone deacetylase inhibitory activity is sub-divided and the separated ones are selected until only one candidate compound showing histone deacetylase inhibitory activity remains. If a group of candidate compounds is identified as having the ability to inhibit the activity of histone deacetylase, the group may be present in the group using a variety of methods or in compounds that inhibit one or more histone deacetylase proteins. Can be screened to confirm. For example, if a pool is initially screened in cells containing histone deacetylase, the group can be searched sequentially for purified histone deacetylase.

Preferably, the candidate compound (s) found to be histone deacetylase inhibitors inhibit less enzymatic activity than all histone deacetylases. More preferably, the candidate compounds only inhibit histone deacetylases involved in the induction of cell division. Even more preferably, candidate compounds identified as histone deacetylase inhibitors inhibit only one histone deacetylase that is involved in the induction of neoplastic cell growth but not in the induction of normal cell growth.

As another method of identifying candidate compounds that inhibit histone deacetylase, purified histone deacetylase is allowed to adhere to the bottom of the wells in 96-well microplates. Next, candidate compounds (or pools thereof) are added to the plate, with each candidate compound covalently bound to a detectable marker (eg, labeled with biotin). The binding of the candidate compound to the histone deacetylase bound to the plate can be accomplished by adding a secondary reagent (e.g., fluoropores labeled with streptavidin) that binds to the detectable marker and analyzing the plate in a reader It can detect by making it. The ability to inhibit the activity of histone deacetylase is screened for candidate compounds that interact with the purified histone deacetylase identified in the same way.

Example 6 Anti-neoplastic effect of histone deacetylase antisense oligonucleotides on tumor cells in vivo

The purpose of this example is to demonstrate the ability of the histone deacetylase antisense oligonucleotides of the present invention to treat diseases caused by inhibition of histone deacetylase in animals, particularly mammals. In addition, this example provides evidence for the ability of the methods and compositions of the present invention to inhibit the growth of tumors present in domesticated mammals. Subcutaneous injection of 2 × 10 ⁶ preconditioned A549 human pulmonary epithelial cell lines into the flank of female BALB / c nude mice (nude mouse, Taconic Labs, Great Barrington, NY), 8-10 weeks of age. It was. Preconditioning of the cells is achieved by transplanting tumors at least three times in succession in nude mice of the same species. Sequentially, approximately 30 mg of tumor fragments were cut and subcutaneously implanted into the left flank of mice under Forene anesthesia (Abbott Labs., Geneva, Switzerland). When the average volume of tumors reached 100 mm 3, mice were injected daily by injecting oligonucleotide saline containing 0.1 to 6 mg / kg of antisense oligonucleotide (saline preparation, Sigma, St. Louis, Mo.) through the tail vein. Intravenously. The final optimal concentration of oligonucleotides was determined by DOS reaction experiments by standard methods. Tumor volume was calculated according to standard methods every two days after injection (eg, Meyer et al., Int. J. Cancer , 1989 , 43, 851-856). Treatment of the oligonucleotides of the present invention results in tumor weight and volume when compared to a control treated with only saline (i.e., without oligonucleotides) or a control treated with physiological saline and nonspecific oligonucleotides. Decreased significantly. In addition, the activity of histone deacetylase is expected to be significantly reduced compared to the control group treated with saline.

Example 7 Co-anti-neoplastic effect of histone deacetylase antisense oligonucleotides and histone deacetylase inhibitors on tumor cells in vivo

The purpose of this example is to illustrate the ability of the histone deacetylase antisense oligonucleotides and histone deacetylase inhibitors of the invention to inhibit tumor cell growth in mammals. As described in Example 6, mice implanted with A549 tumor cells (average volume 100 mm 3) were treated daily with physiological saline solution containing about 0.1-30 mg / kg of histone deacetylase antisense oligonucleotides. The second group of mice was treated daily with pharmaceutically acceptable compositions comprising about 0.01-5 mg / kg histone deacetylase inhibitor. Some mice received both antisense oligonucleotides and histone deacetylase inhibitors. Among the mice, some groups received antisense oligonucleotides and histone deacetylase inhibitors simultaneously through the tail vein. In the other group, antisense oligonucleotides were administered via the tail vein and histone deacetylase inhibitors were administered subcutaneously. Another group received both antisense oligonucleotides and histone deacetylase inhibitors simultaneously in a subcutaneous injection. Similarly, control mouse groups receive no treatment (eg, only saline), mismatch antisense oligonucleotides, or control compounds that do not inhibit the activity of histone deacetylases, or Mismatch antisense oligonucleotides were administered with the control compound.

Tumor volume was measured using a caliper. When the antisense oligonucleotide and histone deacetylase inhibitor of the present invention were administered, the weight and volume of tumors were significantly reduced compared to the control group. Preferably, the antisense oligonucleotides and histone deacetylase inhibitors inhibit the expression and activity of the same histone deacetylase.

Claims (39)

Antisense oligonucleotides that inhibit the expression of histone deacetylases. The method of claim 1, wherein the histone deacetylase is selected from the group consisting of HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D and HDAC-E. Antisense oligonucleotides characterized by. The antisense oligonucleotide of claim 1, wherein the oligonucleotide inhibits one or more histone deacetylases. 4. The antisense oligonucleotide of claim 3, wherein the oligonucleotide inhibits all histone deacetylases. The antisense oligonucleotide of claim 1, wherein the oligonucleotide inhibits transcription of a nucleic acid molecule encoding histone deacetylase. The oligonucleotide of claim 5, wherein the nucleic acid molecule is selected from a function consisting of genomic DNA, cDNA, and RNA. The antisense oligonucleotide of claim 1, wherein the oligonucleotide inhibits translation of histone deacetylase. The oligonucleotide of claim 1 wherein the oligonucleotide is phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphorus ester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetami Having at least one internucleotide linkage selected from the group consisting of date, carbamate, thioether, branched phosphoramidate, branched methylene phosphonate, branched phosphorothioate, and sulfone internucleotide linkage Antisense oligonucleotide, characterized in that. The antisense oligonucleotide of claim 1, wherein the oligonucleotide is a chimeric oligonucleotide or a hybrid oligonucleotide. The antisense oligonucleotide of claim 1, wherein the oligonucleotide comprises a ribonucleotide or 2′-O-substituted ribonucleotide moiety and a deoxyribonucleotide moiety. A method of inhibiting histone deacetylase in a cell comprising contacting the cell with the antisense oligonucleotide of claim 1. 12. The method of claim 11, wherein the division of cells is inhibited in contacted cells. The method of claim 11, wherein the cell is a neoplastic cell. The method of claim 13, wherein the neoplasm vesicles are present in the animal. 15. The method of claim 14, wherein the neoplastic cell is neoplastic. The method of claim 11, further comprising contacting the cell with a histone deacetylase inhibitor that interacts with the histone deacetylase to reduce the activity of the enzyme. The method of claim 16, wherein the histone deacetylase inhibitor is functionally linked with the antisense oligonucleotide. Neoplastic cells in an animal comprising administering to the animal having at least one tumor cell in the body a therapeutically effective amount of the antisense oligonucleotide of claim 1 in a pharmaceutically acceptable carrier for a therapeutically useful period of time. To inhibit growth. 19. The method of claim 18, wherein the animal is a mammal. 20. The method of claim 19, wherein the mammal is a human. Interacting with histone deacetylase to reduce enzymatic activity, further comprising administering a histone deacetylase inhibitor contained in a pharmaceutically acceptable carrier in a therapeutically effective amount for a therapeutically useful period of time. The method of claim 18. The method of claim 21, wherein the histone deacetylase inhibitor is functionally linked with the antisense oligonucleotide. Contacting cells with antisense oligonucleotides that inhibit the expression of histone deacetylase, and inhibiting cell division in contacted cells with histone deacetylase, which is involved in the induction of cell division. A method of identifying a histone deacetylase that is involved in inducing cell division. The method of claim 23, wherein the cell is a neoplastic cell and the induction of cell division is tumorigenic. The method of claim 23, wherein the histone deacetylase is selected from the group consisting of HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D and HDAC-E. How to feature. Contacting the histone deacetylase identified by the method of claim 23 with the candidate compound and measuring the activity of the contacted histone deacetylase, and inhibiting the activity of the contacted histone deacetylase. A method of identifying a histone deacetylase inhibitor that inhibits histone deacetylase inhibitors involved in the induction of cell division, which is identified as a histone deacetylase inhibitor that inhibits histone deacetylase enzymes involved in the induction of division. 27. The method of claim 26, wherein the histone deacetylase inhibitors interact with fewer enzymes than all histone deacetylases to reduce activity. Contacting the cell with an antisense oligonucleotide that inhibits the expression of histone deacetylase, and identifying histone deacetylase as a histone deacetylase involved in inducing cell differentiation by inducing differentiation in contacted cells. A method for identifying histone deacetylases involved in the induction of cell differentiation. The method of claim 28, wherein the cell is a neoplastic cell. 29. The method of claim 28, wherein the histone deacetylase is selected from the group consisting of HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D and HDAC-E. How to feature. Subjecting the candidate compound to cells by inhibiting the activity of the contacted histone deacetylase, comprising contacting the histone deacetylase identified by the method of claim 28 with the candidate compound and measuring the activity of the contacted histone deacetylase. A method of identifying a histone deacetylase inhibitor that inhibits histone deacetylase inhibitors involved in inducing cell differentiation, which is identified as a histone deacetylase inhibitor that inhibits histone deacetylase enzymes involved in inducing differentiation. 32. The method of claim 31, wherein the histone deacetylase inhibitors interact with fewer enzymes than all histone deacetylases to reduce activity. A histone deacetylase inhibitor as identified by the method of claim 26 or 31. Histone deacetylase inhibitors are sufficiently purified. Division of cells comprising contacting the cell with at least two substances selected from the group consisting of antisense oligonucleotides that inhibit histone deacetylase, histone deacetylase inhibitors, DNA methyltransferases and DNA methyltransferase inhibitors How to suppress it. 36. The method of claim 35, wherein the inhibition of growth of cells in contact is greater than the inhibition of growth of cells in contact with only one substance. 36. The method of claim 35, wherein each substance selected from the group is sufficiently purified. 36. The method of claim 35, wherein the cell is a neoplastic cell. 36. The method of claim 35, wherein the materials selected from the group are functionally linked.