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WO2000071703A2 - Inhibition d'histone deacetylase - Google Patents

Inhibition d'histone deacetylase Download PDF

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Publication number
WO2000071703A2
WO2000071703A2 PCT/IB2000/001252 IB0001252W WO0071703A2 WO 2000071703 A2 WO2000071703 A2 WO 2000071703A2 IB 0001252 W IB0001252 W IB 0001252W WO 0071703 A2 WO0071703 A2 WO 0071703A2
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Prior art keywords
histone deacetylase
hdac
cell
antisense
ohgonucleotide
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PCT/IB2000/001252
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English (en)
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WO2000071703A3 (fr
Inventor
Alan R. Macleod
Zuomei Li
Jeffrey M. Besterman
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Methylgene Inc.
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Priority to EP00954830A priority Critical patent/EP1173562A2/fr
Priority to JP2000620080A priority patent/JP2003500052A/ja
Priority to AU67182/00A priority patent/AU6718200A/en
Priority to CA002366408A priority patent/CA2366408A1/fr
Priority to KR1020017014048A priority patent/KR20020007398A/ko
Publication of WO2000071703A2 publication Critical patent/WO2000071703A2/fr
Publication of WO2000071703A3 publication Critical patent/WO2000071703A3/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/345—Spatial arrangement of the modifications having at least two different backbone modifications
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • This invention relates to the inhibition of histone deacetylase expression and enzymatic activity.
  • histone deacetylases Deacetylation of the core histones H1-H4 is mediated by a two related families of enzymes called the histone deacetylases.
  • One family of histone deacetylases includes
  • HDAC-1, HDAC-2, and HDAC-3 A second family of histone deacetylases includes HDAC- 4 (formerly HDAC-A), HDAC-5 (formerly HDAC-B), HDAC-C, HDAC-D, and HDAC-E.
  • Histone deacetylase activity is thought to modulate the accessibility of transcription factors to enhancer and promoter elements. Indeed, an enrichment of underacetylated histone H4 has been found in transcriptionally silent regions of the genome (Taunton et al., Science 272: 408-411, 1996).
  • Trichostatin A an antibiotic isolated from Streptomyces, has been shown to inhibit histone deacetylase activity and arrest cell cycle progression in cells in the Gl and G2 phases (Yoshida et aL, J. Biol. Chem. 265:
  • the known inhibitors of histone deacetylase are all natural product and are all small molecules that inhibit histone deacetylase activity at the protein level. Moreover, all of the known histone deacetylase inhibitors are non-specific for a particular histone deacetylase enzyme, and more or less inhibit all members of both the histone deacetylase families equally.
  • the invention provides methods and reagents for inhibiting histone deacetylases at a nucleic acid level, as well as for inhibiting expression of a specific histone by inhibiting expression at the nucleic acid level.
  • the invention allows the identification of and specific inhibition of a specific histone deacetylase involved in tumorigenesis.
  • the invention provides an antisense oligonucleotide that inhibits the expression of a histone deacetylase.
  • the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E.
  • the oligonucleotide inhibits more than one histone deacetylase, or the oligonucleotide inhibits all histone deacetylases.
  • the oligonucleotide is a chimeric oligonucleotide or a hybrid ohgonucleotide.
  • the oligonucleotide inhibits transcription of a nucleic acid molecule encoding the histone deacetylase.
  • the nucleic acid molecule may be genomic DNA (e.g., a gene), cDNA, or RNA.
  • the oligonucleotide inhibits translation of the histone deacetylase.
  • the antisense oligonucleotide has at least one internucleotide linkage selected from the group consisting of phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate and sulfone internucleotide linkages.
  • the oligonucleotide comprises a ribonucleotide or 2'-O-substituted ribonucleotide region and a deoxyribonucleotide region.
  • the invention provides a method for inhibiting a histone deacetylase in a cell comprising contacting the cell with the antisense oligonucleotide of the first aspect of the invention.
  • cell proliferation is inhibited in the contacted cell.
  • the cell is a neoplastic cell which may be in an animal, including a human, and which may be in a neoplastic growth.
  • the method of the second aspect of the invention further comprises contacting the cell with a histone deacetylase protein inhibitor that interacts with and reduces the enzymatic activity of the histone deacetylase.
  • the histone deacetylase protein inhibitor is operably associated with the antisense oligonucleotide.
  • the invention provides a method for inhibiting neoplastic cell growth in an animal comprising administering to an animal having at least one neoplastic cell present in its body a therapeutically effective amount of the antisense ohgonucleotide of the first aspect of the invention with a pharmaceutically acceptable carrier for a therapeutically effective period of time.
  • the method further comprises administering to the animal a therapeutically effective amount of a histone deacetylase protein inhibitor that interacts with and reduces the enzymatic activity of the histone deacetylase with a pharmaceutically acceptable carrier for a therapeutically effective period of time.
  • a histone deacetylase protein inhibitor is operably associated with the antisense oligonucleotide.
  • the invention provides a method for identifying a histone deacetylase that is involved in induction of cell proliferation comprising contacting a cell with an antisense oligonucleotide that inhibits the expression of a histone deacetylase, wherein inhibition of cell proliferation in the contacted cell identifies the histone deacetylase as a histone deacetylase that is involved in induction of cell proliferation.
  • the cell is a neoplastic cell, and the induction of cell proliferation is tumorigenesis.
  • the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E.
  • the invention provides a method for identifying a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell proliferation comprising contacting a histone deacetylase identified by the method of the fourth aspect of the invention with a candidate compound and measuring the enzymatic activity of the contacted histone deacetylase, wherein a reduction in the enzymatic activity of the contacted histone deacetylase identifies the candidate compound as a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell proliferation.
  • the histone deacetylase protein inhibitor interacts with and reduces the enzymatic activity of fewer than all histone deacetylases.
  • the invention provides a method for identifying a histone deacetylase that is involved in induction of cell differentiation comprising contacting a cell with an antisense ohgonucleotide that inhibits the expression of a histone deacetylase, wherein induction of differentiation in the contacted cell identifies the histone deacetylase as a histone deacetylase that is involved in induction of cell differentiation.
  • the cell is a neoplastic cell.
  • the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E.
  • the invention provides a method for identifying a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell differentiation comprising contacting a histone deacetylase identified by the method of the sixth aspect of the invention with a candidate compound and measuring the enzymatic activity of the contacted histone deacetylase, wherein a reduction in the enzymatic activity of the contacted histone deacetylase identifies the candidate compound as a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell differentiation.
  • the histone deacetylase protein inhibitor interacts with and reduces the enzymatic activity of fewer than all histone deacetylases.
  • the invention provides a histone deacetylase protein inhibitor identified by the method of the fifth or the seventh aspects of the invention.
  • the histone deacetylase protein inhibitor is substantially pure.
  • the invention provides a method for inhibiting cell proliferation in a cell comprising contacting a cell with at least two of the reagents selected from the group consisting of an antisense oligonucleotide that inhibits a histone deacetylase, a histone deacetylase protein inhibitor, an antisense ohgonucleotide that inhibits a DNA methyltransferase, and a DNA methyltransferase protein inhibitor.
  • the inhibition of cell growth of the contacted cell is greater than the inhibition of cell growth of a cell contacted with only one of the reagents.
  • each of the reagents selected from the group is substantiaUy pure.
  • the cell is a neoplastic cell.
  • the reagents selected from the group are operably associated. According to the invention, reagents found to specifically inhibit a histone deacetylase involved in neoplasia may be used as therapeutic agents to inhibit neoplastic cell growth in patients suffering from neoplasia.
  • an antisense oligonucleotide that inhibits the expression of a histone deacetylase may be administered with a pharmaceutically-acceptable carrier (e.g., physiological sterile saline solution) via any route of administration to a patient suffering from neoplasia or hyperplasia in an attempt to alleviate any resulting disease symptom (e.g., death).
  • a pharmaceutically-acceptable carrier e.g., physiological sterile saline solution
  • an antisense oligonucleotide that inhibits the expression of a histone deacetylase may be incorporated into a gene therapy expression vector (e.g., a replication-deficient adenoviral vector), and phage particles carrying such vectors may be delivered with a pharmaceutically-acceptable carrier directly to the cells of the neoplastic or hyperplastic growth.
  • a gene therapy expression vector e.g., a replication-deficient adenoviral vector
  • phage particles carrying such vectors may be delivered with a pharmaceutically-acceptable carrier directly to the cells of the neoplastic or hyperplastic growth.
  • Pharmaceutically-acceptable carriers and their formulations are well- known and generally described in, for example, Remington's Pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990).
  • Figure 1 is a graphic representation of a Northern blotting analysis showing the dose- dependent abilities of representative, nonlimitmg, synthetic ohgonucleotides according to the invention that specifically bind to either HDAC-1-encoding nucleic acid or both HDAC-1- and HDAC-2-encoding nucleic acids to inhibit expression of HDAC-1 mRNA or both HDAC-1 mRNA and HDAC-2 mRNA, respectively.
  • Figure 2 is a graphic representation of a Northern blotting analysis showing the dose- dependent abilities of representative, nonlimiting, synthetic oligonucleotides according to the invention that specifically bind to HDAC-2-encoding nucleic acid to inhibit expression of HDAC-2 mRNA.
  • Figure 3 is a graphic representation of a Western blotting analysis showing the abilities of representative, nonlimiting, synthetic ohgonucleotides according to the invention that specifically bind to HDAC-2-encoding nucleic acid to specifically inhibit expression of HDAC-2 protein.
  • Figure 4 is a graphic representation of a Western blotting analysis showing the abilities of representative, nonlimiting, synthetic ohgonucleotides according to the invention that specifically bind to either HDAC-1 -encoding nucleic acid or both HDAC-1- and HDAC- 2-encoding nucleic acid to inhibit expression of HDAC-1 protein or both HDAC-1 protein and HDAC-2 protein, respectively. Mismatched synthetic oligonucleotides were used as negative controls. Equal loading of all lanes is evidenced by the equivalent expression of actin.
  • the invention provides methods and reagents for inhibiting a histone deacetylase at a nucleic acid level, as well as for inhibiting a specific histone deacetylase at the nucleic acid level.
  • the reagents described herein that inhibit histone deacetylase at the nucleic acid level i.e., inhibiting transcription and translation
  • therapeutical compositions for treating and/or alleviating the symptoms of neoplasia may be developed using the reagents of the invention that specifically inhibit a particular histone deacetylase involved in neoplasia.
  • the reagents according to the invention are useful as analytical tools and as therapeutic tools, including as gene therapy tools.
  • the invention also provides methods and compositions which may be manipulated and fine-tuned to fit the condition(s) to be treated while producing fewer side effects.
  • GenBank database sequences that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be inco ⁇ orated by reference.
  • the invention provides an antisense ohgonucleotide that inhibits the expression of a histone deacetylase.
  • the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E.
  • the ohgonucleotide inhibits more than one histone deacetylase, or the ohgonucleotide inhibits all histone deacetylases.
  • the antisense ohgonucleotides according to the invention are complementary to a region of RNA or double-stranded DNA that encodes a histone deacetylase.
  • the term "ohgonucleotide” includes polymers of two or more deoxyribonucleosides, ribonucleosides, or 2'-O-substituted ribonucleoside residues, or any combination thereof.
  • such oligonucleotides have from about 8 to about 50 nucleoside residues, and most preferably from about 12 to about 30 nucleoside residues.
  • the nucleoside residues may be coupled to each other by any of the numerous known intemucleoside linkages.
  • Such intemucleoside linkages include without limitation phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleotide linkages.
  • these intemucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof.
  • ohgonucleotide also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines, and adamantane.
  • the term "2'-O-substituted” means substitution of the 2' position of the pentose moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl, or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or such 2' substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or
  • the term "complementary" means having the abihty to hybridize to a genomic region, a gene, or an RNA transcript thereof under physiological conditions.
  • Such hybridization is ordinarily the result of base-specific hydrogen bonding between complementary strands, preferably to form Watson-Crick or Hoogsteen base pairs, although other modes of hydrogen bonding, as well as base stacking can lead to hybridization.
  • such hybridization can be inferred from the observation of specific gene expression inhibition, which may be at the level of transcription or translation (or both).
  • antisense ohgonucleotides utilized in this aspect of the invention include chimeric ohgonucleotides and hybrid ohgonucleotides.
  • a "chimeric ohgonucleotide” refers to an ohgonucleotide having more than one type of intemucleoside linkage.
  • One preferred embodiment of such a chimeric ohgonucleotide is a chimeric ohgonucleotide comprising a phosphorothioate, phosphodiester or phosphorodithioate region, preferably comprising from about 2 to about 12 nucleotides, and an alkylphosphonate or alkylphosphonothioate region (see e.g., Pederson et al. U.S. Patent Nos. 5,635,377 and 5,366,878).
  • such chimeric oligonucleotides contain at least three consecutive intemucleoside linkages selected from phosphodiester and phosphorothioate linkages, or combinations thereof.
  • a "hybrid oligonucleotide” refers to an ohgonucleotide having more than one type of nucleoside.
  • One preferred embodiment of such a hybrid oligonucleotide comprises a ribonucleotide or 2'-O-substituted ribonucleotide region, preferably comprising from about 2 to about 12 2'-O-substituted nucleotides, and a deoxyribonucleotide region.
  • such a hybrid oligonucleotide will contain at least three consecutive deoxyribonucleosides and will also contain ribonucleosides, 2'-O- substituted ribonucleosides, or combinations thereof (see e.g., Metelev and Agrawal, U.S. Patent No. 5,652,355).
  • nucleotide sequence and chemical structure of an antisense ohgonucleotide utilized in the invention can be varied, so long as the ohgonucleotide retains its ability to inhibit expression of a histone deacetylase. This is readily determined by testing whether the particular antisense oligonucleotide is active by quantitating the amount of mRNA encoding a histone deacetylase, quantitating the amount of histone deacetylase protein, quantitating the histone deacetylase enzymatic activity, or quantitating the abihty of histone deacetylase to inhibit cell growth in a an in vitro or in vivo cell growth assay, all of which are described in detail in this specification.
  • Antisense oligonucleotides utilized in the invention may conveniently be synthesized on a suitable solid support using well-known chemical approaches, including H-phosphonate chemistry, phosphoramidite chemistry, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (i.e., H-phosphonate chemistry for some cycles and phosphoramidite chemistry for other cycles).
  • Suitable sohd supports include any of the standard solid supports used for solid phase ohgonucleotide synthesis, such as controlled- pore glass (CPG) (see, e.g., Pon, R. T., Methods in Molec. Biol. 20: 465-496, 1993).
  • CPG controlled- pore glass
  • Antisense oligonucleotides according to the invention are useful for a variety of purposes. For example, they can be used as "probes" of the physiological function of histone deacetylase by being used to inhibit the activity of histone deacetylase in an experimental cell culture or animal system and to evaluate the effect of inhibiting such histone deacetylase activity. This is accomplished by administering to a cell or an animal an antisense oligonucleotide that inhibits histone deacetylase expression according to the invention and observing any phenotypic effects.
  • the antisense oligonucleotides according to the invention is preferable to traditional "gene knockout" approaches because it is easier to use, and can be used to inhibit histone deacetylase activity at selected stages of development or differentiation.
  • the method according to the invention can serve as a probe to test the role of histone deacetylation in various stages of development.
  • Preferred antisense ohgonucleotides of the invention inhibit either the transcription of a nucleic acid molecule encoding the histone deacetylase, or the translation of a nucleic acid molecule encoding the histone deacetylase.
  • Histone deacetylase-encoding nucleic acids may be RNA or double stranded DNA regions and include, without limitation, intronic sequences, untranslated 5' and 3' regions, intron-exon boundaries as well as coding sequences from a histone deacetylase family member gene. For human sequences, see e.g., Yang et al., Proc. Natl. Acad. Sci.
  • antisense oligonucleotides of the invention are complementary to regions of RNA or double- stranded DNA encoding a histone deacetylase (e.g., HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E).
  • the antisense ohgnouncleotides according to the invention are complementary to regions of RNA or double-stranded DNA that encode HDAC-1, HDAC-2, HDAC-3, HDAC- 4, HDAC-5, HDAC-C, HDAC-D, and/or HDAC-E.
  • the sequence of human HDAC-1 can be found in GenBank Accession No.
  • U50079 (amino acid sequence in SEQ ID NO:24; nucleic acid sequence in SEQ ID NO:25.
  • the sequence of human HDAC-2 can be found in GenBank Accession No. U31814 (amino acid sequence in SEQ ID NO: 26; nucleic acid sequence in SEQ ID NO: 27).
  • the sequence of human HDAC-3 can be found in GenBank Accession No. U75697 (amino acid sequence in SEQ ID NO: 28; nucleic acid sequence in SEQ ID NO: 29).
  • the sequence of human HDAC-4 (formerly human HDAC-A) in GenBank Accession No. AB006626 (amino acid sequence in SEQ ID NO: 30; nucleic acid sequence in SEQ ID NO: 31).
  • human HDAC-5 (formerly human HDAC-B) can be found in GenBank Accession No. ABOl 1172 (amino acid sequence in SEQ ID NO: 32; nucleic acid sequence in SEQ ID NO: 33).
  • the sequence of human HDAC-C can be found in GenBank Accession No. AC004994 (amino acid sequence in SEQ ID NO: 34; nucleic acid sequence in SEQ ID NO: 35).
  • the sequence of human HDAC-D can be found in GenBank Accession No. AC004466 (nucleic acid sequence in SEQ ID NO: 36).
  • the antisense oligonucleotides of the invention may also be complementary to regions of RNA or double-stranded DNA that encode histone deacetylases from non-human animals.
  • preferred ohgonucleotides have nucleotide sequences of from about 13 to about 35 nucleotides which include the nucleotide sequences shown below as SEQ ID NOs: 1-18.
  • ohgonucleotides have nucleotide sequences of from about 15 to about 26 nucleotides of the nucleotide sequences shown below.
  • the ohgonucleotides shown below have phosphorothioate backbones, are 20-26 nucleotides in length, and are modified such that the terminal four nucleotides at the 5' end of the ohgonucleotide and the terminal four nucleotides at the 3' end of the ohgonucleotide each have 2' -O- methyl groups attached to their sugar residues.
  • Antisense ohgonucleotide specific for human HDAC-1 (MG2608): 5'-GAA ACG TGA GGG ACT CAG CA-3' (SEQ ID NO: 1).
  • Antisense ohgonucleotide specific for both human HDAC-1 and human HDAC-2 is a 25/25/25/25 mixture of four ohgonucleotides: 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 oligonucleotide specific for human HDAC-2 5'-TGC TGC TGC TGC TGC TGC TGC CG-3' (MG2628; SEQ ID NO: 6); 5'-CCT CCT GCT GCT GCT GCT GC-3' (MG2633; SEQ ID NO: 7); 5'-GGT TCC TTT GGT ATC TGT TT-3' (MG2635; SEQ ID NO: 8); and 5'-CTC CTT GAC TGT ACG CCA TG-3' (MG2636; SEQ ID NO: 9).
  • the antisense oligonucleotides according to the invention may optionally be formulated with any of the well known pharmaceutically acceptable carriers or diluents (see preparation of pharmaceutically acceptable formulations in, e.g.. Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990).
  • the invention provides a method for inhibiting a histone deacetylase in a cell comprising contacting the cell with the antisense ohgonucleotide that inhibits the expression of a histone deacetylase.
  • cell prohferation is inhibited in the contacted cell.
  • the antisense oligonucleotides according to the invention are useful in therapeutic approaches to human diseases including benign and malignant neoplasms by inhibiting cell prohferation in cells contacted with the antisense ohgonucleotides.
  • inhibitorting cell prohferation is used to denote an ability of a histone deacetylase antisense ohgonucleotide or a histone deacetylase protein inhibitor (or combination thereof) to retard the growth of cells contacted with the ohgonucleotide or protein inhibitor, as compared to cells not contacted.
  • Such an assessment of cell proliferation can be made by counting contacted and non-contacted cells using a Coulter Cell Counter (Coulter, Miami, FL) or a hemacyto meter.
  • the term includes a retardation of cell prohferation that is at least 50% of non-contacted cells. More preferably, the term includes a retardation of cell prohferation that is 100% of non- contacted cells (i.e., the contacted cells do not increase in number or size). Most preferably, the term includes a reduction in the number or size of contacted cells, as compared to non- contacted cells.
  • a histone deacetylase antisense oligonucleotide or a histone deacetylase protein inhibitor that inhibits cell proliferation in a contacted cell may induce the contacted cell to undergo growth retardation, to undergo growth arrest, to undergo programmed cell death (i.e., to apoptose), or to undergo necrotic cell death.
  • the phrase "inducing cell prohferation” is used to denote the requirement of the presence or enzymatic activity of a histone deacetylase for cell proliferation in a normal (i.e., non-neoplastic) cell.
  • a histone deacetylase that induces cell proliferation may or may not lead to increased cell proliferation; however, inhibition of a histone deacetylase that induces cell proliferation will lead to inhibition of cell proliferation.
  • inducing cell differentiation is used to denote the ability of a histone deacetylase antisense oligonucleotide or histone deacetylase protein inhibitor (or combination thereof) to induce differentiation in a contacted cell as compared to a cell that is not contacted.
  • a neoplastic cell when contacted with a histone deacetylase antisense ohgonucleotide or histone deacetylase protein inhibitor (or both) of the invention, may be induced to differentiate, resulting in the production of a daughter cell that is phylogenetically more advanced than the contacted cell.
  • the cell prohferation inhibiting ability of the antisense ohgonucleotides according to the invention allows the synchronization of a population of a-synchronously growing cells.
  • the antisense oligonucleotides of the invention may be used to arrest a population of non- neoplastic cells grown in vitro in the Gl or G2 phase of the cell cycle.
  • Such synchronization allows, for example, the identification of gene and/or gene products expressed during the Gl or G2 phase of the cell cycle.
  • Such a synchronization of cultured cells may also be useful for testing the efficacy of a new transfection protocol, where transfection efficiency varies and is dependent upon the particular cell cycle phase of the cell to be transfected.
  • Use of the antisense ohgonucleotides of the invention allows the synchronization of a population of cells, thereby aiding detection of enhanced transfection efficiency.
  • anti-neoplstic utility of the antisense ohgonucleotides according to the invention is described in detail elsewhere in this specification.
  • the cell contacted with a histone deacetylase antisense ohgonucleotide is also contacted with a histone deacetylase protein inhibitor.
  • histone deacetylase protein inhibitor denotes an active moiety capable of interacting with a histone deacetylase at the protein level and reducing the activity of that histone deacetylase.
  • Histone deacetylase protein inhibitors include, without limitation, trichostatin A, trichostatin B, trichostatin C, depudecin, trapoxin, butyrate, suberoylanilide hydroxamic acid (SAHA), FR901228 (Fujisawa Pharmaceuticals), and acetyldinaline (el-Beltagi et al., Cancer Res. 53(13):3008-3014, 1993).
  • a histone deacetylase protein inhibitor is a molecule that reduces the activity of a histone deacetylase to a greater extent than it reduces the activity of any unrelated protein. In a preferred embodiment, such reduction of the activity of a histone deacetylase is at least 5-fold, more preferably at least 10-fold, most preferably at least 50-fold. In another embodiment, the activity of a histone deacetylase is reduced 100-fold.
  • a histone deacetylase protein inhibitor interacts with and reduces the activity of fewer than all histone deacetylases.
  • all histone deacetylases is meant all of the members of both of the histone deacetylase families of proteins from a particular species of animal and includes, without limitation, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E, all of which are considered “related proteins," as used herein.
  • a preferred histone deacetylase protein inhibitor interacts with and inhibits HDAC- 1 and HDAC-2, but does not interact with and inhibit HDAC-3.
  • a histone deacetylase protein inhibitor interacts with and reduces the activity of one histone deacetylase (e.g., HDAC-2), but does not interact with or reduce the activities of the other histone deacetylases (e.g., HDAC-1 and HDAC-3).
  • a preferred histone deacetylase protein inhibitor is one that interacts with and reduces the enzymatic activity of a histone deacetylase that is involved in tumorigenesis.
  • the histone deacetylase protein inhibitor is operably associated with the antisense oligonucleotide.
  • the antisense oligonucleotides according to the invention may optionally be formulated well known pharmaceutically acceptable carriers or diluents.
  • This formulation may further contain one or more one or more additional histone deacetylase antisense oligonucleotide(s), and/or one or more histone deacetylase protein inhibitor(s), or it may contain any other pharmacologically active agent.
  • the antisense oligonucleotide is in operable association with a histone deacetylase protein inhibitor.
  • operable association includes any association between the antisense ohgonucleotide and the histone deacetylase protein inhibitor which allows an antisense oligonucleotide to inhibit histone deacetylase-encoding nucleic acid expression and allows the histone deacetylase protein inhibitor to inhibit histone deacetylase enzymic activity.
  • One or more antisense ohgonucleotide of the invention may be operably associated with one or more histone deacetylase protein inhibitor.
  • an antisense oligonucleotide of the invention that targets one particular histone deacetylase is operably associated with a histone deacetylase protein inhibitor which targets the same histone deacetylase.
  • a preferred operable association is a hydrolyzable.
  • the hydrolyzable association is a covalent linkage between the antisense oligonucleotide and the histone deacetylase protein inhibitor.
  • such covalent linkage is hydrolyzable by esterases and/or amidases. Examples of such hydrolyzable associations are well known in the art. Phosphate esters are particularly preferred.
  • the covalent linkage may be directly between the antisense oligonucleotide and the histone deacetylase protein inhibitor so as to integrate the histone deacetylase protein inhibitor into the backbone.
  • the covalent linkage may be through an extended structure and may be formed by covalently linking the antisense ohgonucleotide to the histone deacetylase protein inhibitor through coupling of both the antisense oligonucleotide and the histone deacetylase protein inhibitor to a carrier molecule such as a carbohydrate, a peptide or a lipid or a glycohpid.
  • Other preferred operable associations include lipophilic association, such as formation of a hposome containing an antisense ohgonucleotide and the histone deacetylase protein inhibitor covalently linked to a lipophilic molecule and thus associated with the liposome.
  • hpophilic molecules include without hmitation phosphotidylcholine, cholesterol, phosphatidylethanolamine, and synthetic neoglycohpids, such as syalyllacNAc-HDPE.
  • the operable association may not be a physical association, but simply a simultaneous existence in the body, for example, when the antisense ohgonucleotide is associated with one liposome and the protein effector is associated with another hposome.
  • the invention provides a method for inhibiting neoplastic cell proliferation in an animal comprising administering to an animal having at least one neoplastic cell present in its body a therapeutically effective amount of the antisense oligonucleotide of the first aspect of the invention with a pharmaceutically acceptable carrier for a therapeutically effective period of time.
  • the animal is a mammal, particularly a domesticated mammal. Most preferably, the animal is a human.
  • the term "neoplastic cell" is used to denote a cell that shows aberrant cell growth.
  • the aberrant cell growth of a neoplastic cell is increased cell growth.
  • a neoplastic cell may be a hyperplastic cell, a cell that shows a lack of contact inhibition of growth in vitro, a benign tumor cell that is incapable of metastasis in vivo, or a cancer cell that is capable of metastases in vivo and that may recur after attempted removal.
  • tumorgenesis is used to denote the induction of cell proliferation that leads to the development of a neoplastic growth.
  • therapeutically effective amount and “therapeutically effective period of time” are used to denote known treatments at dosages and for periods of time effective to reduce neoplastic cell growth.
  • such administration should be parenteral, oral, sublingual, transdermal, topical, intranasal, or intrarectal.
  • the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of antisense oligonucleotide from about 0.1 ⁇ M to about 10 ⁇ M.
  • concentrations typically be effective, and much higher concentrations may be tolerated.
  • concentrations may vary considerably depending on the tissue, organ, or the particular animal or patient to be treated according to the invention.
  • the therapeutic composition of the invention is administered systemically at a sufficient dosage to attain a blood level of antisense ohgonucleotide from about 0.01 ⁇ M to about 20 ⁇ M. In a particularly preferred embodiment, the therapeutic composition is administered at a sufficient dosage to attain a blood level of antisense ohgonucleotide from about 0.05 ⁇ M to about 15 ⁇ M. In a more preferred embodiment, the blood level of antisense ohgonucleotide is from about 0.1 ⁇ M to about 10 ⁇ M.
  • a total dosage of antisense oligonucleotide will range from about 0.1 mg to about 200 mg ohgonucleotide per kg body weight per day. In a more preferred embodiment, a total dosage of antisense ohgonucleotide will range from about 1 mg to about 20 mg oligonucleotide per kg body weight per day. In a most preferred embodiment, a total dosage of antisense oligonucleotide will range from about 2 mg to about 10 mg ohgonucleotide per kg body weight per day. In a particularly preferred embodiment, the therapeutically effective amount of a histone deacetylase antisense oligonucleotide is about 0.5 mg ohgonucleotide per kg body weight per day.
  • the method further comprises administering to the animal a therapeutically effective amount of a histone deacetylase protein inhibitor with a pharmaceutically acceptable carrier for a therapeutically effective period of time.
  • a histone deacetylase protein inhibitor is operably associated with the antisense oligonucleotide. Methods for the operable association of a histone deacetylase protein inhibitor with a histone deacetylase antisense oligonucleotide are described above.
  • the histone deacetylase protein inhibitor-containing therapeutic composition of the invention is administered systemically at a sufficient dosage to attain a blood level histone deacetylase protein inhibitor from about O.Ol ⁇ M to about lO ⁇ M.
  • the therapeutic composition is administered at a sufficient dosage to attain a blood level of histone deacetylase protein inhibitor from about 0.05 ⁇ M to about lO ⁇ M.
  • the blood level of histone deacetylase protein inhibitor is from about 0. l ⁇ M to about 7 ⁇ M.
  • concentrations than this may be effective.
  • a total dosage of histone deacetylase protein inhibitor will range from about 0.01 mg to about 5 mg protein effector per kg body weight per day.
  • a total dosage of histone deacetylase protein inhibitor will range from about 0.1 mg to about 4 mg protein effector per kg body weight per day. In a most preferred embodiment, a total dosage of histone deacetylase protein inhibitor will range from about 0.1 mg to about 1 mg protein effector per kg body weight per day. In a particularly preferred embodiment, the therapeutically effective synergistic amount of histone deacetylase protein inhibitor (when administered with an antisense ohgonucleotide) is 0.1 mg per kg body weight per day.
  • This aspect of the invention results in an improved inhibitory effect, thereby reducing the therapeutically effective concentrations of either or both of the nucleic acid level inhibitor (i.e., antisense oligonucleotide) and the protein level inhibitor (i.e., histone deacetylase protein inhibitor) required to obtain a given inhibitory effect as compared to those necessary when either is used individually.
  • the therapeutically effective synergistic amount of either the antisense ohgonucleotide or the histone deacetylase inhibitor may be lowered or increased by fine tuning and altering the amount of the other component.
  • the invention therefore provides a method to tailor the administration/treatment to the particular exigencies specific to a given animal species or particular patient.
  • the invention provides a method for investigating the role of a particular histone deacetylase in cellular prohferation, including the proliferation of neoplastic cells.
  • the cell type of interest is contacted with an amount of an antisense ohgonucleotide that inhibits the expression of a histone deacetylase, as described for the first aspect according to the invention, resulting in inhibition of expression of the histone deacetylase in the cell.
  • the histone deacetylase is involved in the induction of cell prohferation.
  • the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E.
  • histone deacetylase protein inhibitors may be generated that specifically inhibit the histone deacetylase involved in inducing cell proliferation, while not inhibiting other histone deacetylases not involved in inducing cell proliferation. Accordingly, in a fifth aspect, the invention provides a method for identifying a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in the induction of cell prohferation.
  • This method comprises contacting a histone deacetylase identified as being involved in inducing cell proliferation with a candidate compound and measuring the enzymatic activity of the contacted histone deacetylase.
  • a reduction in the enzymatic activity of the contacted histone deacetylase identifies the candidate compound as a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell prohferation.
  • Measurement of the enzymatic activity of a histone deacetylase can be achieved using known methodologies. For example, Yoshida et al. (J. Biol. Chem.
  • the histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell proliferation is a histone deacetylase protein inhibitor that interacts with and reduces the enzymatic activity of fewer than all histone deacetylases.
  • the invention provides a method for identifying a histone deacetylase that is involved in induction of cell differentiation comprising contacting a cell with an antisense oligonucleotide that inhibits the expression of a histone deacetylase, wherein induction of differentiation in the contacted cell identifies the histone deacetylase as a histone deacetylase that is involved in induction of cell differentiation.
  • the cell is a neoplastic cell.
  • the histone deacetylase is HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, or HDAC-E.
  • the invention provides a method for identifying a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell differentiation comprising contacting a histone deacetylase identified by the method of the sixth aspect of the invention with a candidate compound and measuring the enzymatic activity of the contacted histone deacetylase, wherein a reduction in the enzymatic activity of the contacted histone deacetylase identifies the candidate compound as a histone deacetylase protein inhibitor that inhibits a histone deacetylase that is involved in induction of cell differentiation.
  • the histone deacetylase protein inhibitor interacts with and reduces the enzymatic activity of fewer than all histone deacetylases.
  • the invention provides a histone deacetylase protein inhibitor identified by the method of the fifth or the seventh aspects of the invention.
  • the histone deacetylase protein inhibitor is substantially pure.
  • Substantially purified proteins can be achieve by any standard method including, without limitation, expression of recombinant protein, affinity chromatography, antibody- based affinity purification, and high performance hquid chromatography (HPLC; see, e.g., Fisher (1980) Laboratory Techniques in Biochemistry and Molecular Biology, Work and Burdon (eds.), Elsevier).
  • a substantially purified protein is at least 80%, by weight, pure in that it is free from other proteins or naturally-occurring organic molecules. More preferably, a substantially purified protein is at least 90% pure, by weight. Most preferably, a substantially purified protein is at least 95% pure, by weight.
  • the invention provides a method for inhibiting cell proliferation in a cell comprising contacting a cell with at least two of the reagents selected from the group consisting of an antisense ohgonucleotide that inhibits a histone deacetylase, a histone deacetylase protein inhibitor, an antisense ohgonucleotide that inhibits a DNA methyltransferase, and a DNA methyltransferase protein inhibitor.
  • the inhibition of cell growth of the contacted cell is greater than the inhibition of cell growth of a cell contacted with only one of the reagents.
  • each of the reagents selected from the group is substantially pure.
  • the cell is a neoplastic cell.
  • the reagents selected from the group are operably associated.
  • DNA methyltransferase protein inhibitors include, without hmitation, 5-aza-2'- deoxycytidine (5-aza-dC), 5-fluoro-2'-deoxycytidine, 5-aza-cytidine (5-aza-C), or 5,6- dihydro-5-aza-cytidine.
  • Example 1 Screening of Antisense Oligonucleotides To identify which antisense ohgonucleotides were most effective at inhibiting a specific histone deacetylase, a number of oligonucleotides were generated based on the sequences provided in GenBank Accession Number U50079 for HDAC-1 and GenBank Accession Number U31814 for HDAC-2.
  • HDAC-1 and HDAC-2 are HDAC-1 and HDAC-2.
  • T24 human bladder carcinoma cells (commercially available from the American Type Culture Collection (ATCC), Manassas, VA) were grown under suggested conditions. Before addition of ohgonucleotides, cells were washed with PBS (phosphate buffered saline). Next, hpofectin transfection reagent (Gibco-BRL Mississauga, Ontario), at a concentration of 6.25 ⁇ g/ml, was added to serum free OPTIMEM medium (GIBCO/BRL), which was then added to the cells.
  • PBS phosphate buffered saline
  • Ohgonucleotides to be screened were then added to different wells of cells (i.e., one oligonucleotide per well of cells). The same concentration of oligonucleotide (e.g., 50 nM) was used per well of cells. The cells were allowed to incubate with lipofectin and ohgonucleotide for 4 hours at 37 C in a cell culture incubator. The cells were then washed with PBS and returned to full serum-containing medium. Twenty- four hours later, the cells were harvested for determination of HD AC mRNA levels by Northern blotting analysis.
  • oligonucleotide e.g., 50 nM
  • RNA was prepared from cells by the guanidinium isothiocyanate standard procedure (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1994), with the exception of an additional precipitation step in 2 M LiCl overnight at 4°C to purify RNA from cellular DNA contamination.
  • Northern blotting analysis was performed according to standard protocols.
  • Probes for HDAC-1 and HDAC-2 were full length cDNA clones generated by PCR amplification from the known sequences for each (e.g., GenBank Accession Nos. U50079 and U31814, respectively). These probes were radio labelled with
  • the oligonucleotides which showed an ability to reduce the mRNA expression of a targeted histone deacetylase i.e., were able to inhibit transcription of the histone deacetylase mRNA
  • T24 cells were transfected with ohgonucleotide using lipofectin as described above. Twenty-four hours later, the cells were lysed according to standard procedures.
  • the whole cell extracts (50 ⁇ g) were resolved on 7-15% gradient SDS PAGE, transferred to PVDF membrane (Amersham, Arlington Heights, IL), and subjected to Western blotting analysis with rabbit polyclonal HDACl- and HDAC-2 specific antibodies (1:500, Santa Cmz Biotech., Santa Cruz, CA) were used. Detection was accomplished with a secondary anti- rabbit IgG-HR peroxidase antibody and an enhanced chemiluminescence detection kit (Amersham) accordingly to manufacturer's instructions. Based on our results, the following antisense ohgonucleotides were identified as being most effective at inhibiting the expression of targeted histone deacetylase as determined by both mRNA and protein expression blotting analysis.
  • oliognucleotides are as follows: For inhibition of HDAC-1, Ohgonucleotide No. MG2608 having the sequence: 5'-GAA ACG TGA GGG ACT CAG CA-3' (SEQ ID NO: 10). For inhibition of both HDAC- 1 and HDAC-2, Oligonucleotide No. MG2610 is a
  • Table I shows the antisense ohgonucleotides found to be most effective:
  • HDAC-1 HDAC-1
  • HDAC-1 / 2 HDAC-1 / 2
  • HDAC- 1 MISMATCH CONTROL has the sequence: 5'-CAA UCG TCA GAG ACT CCG AA-3' (SEQ ID NO: 19).
  • HDAC-1 / 2 MISMATCH CONTROL (MG2637), has a 225/25/25/25 mixture of four ohgonucleotides having the sequences:
  • oligonucleotides having SEQ ID NOs: 10-23 were second generation oligonucleotides (i.e., 4x4 hybrids). That is, oligonucleotides having SEQ ID NOs: 10-23 were chemically modified as follows: A equals 2'-deoxyriboadenosine; C equals 2'- deoxyribocytidine; G equals 2'-deoxyriboguanosine; T equals 2'-deoxyribothymidine; A equals riboadenosine; U equals uridine; C equals ribocytidine; and G equals riboguanosine.
  • underlined bases were 2'-methoxyribose substituted nucleotides.
  • Non-underlined bases indicate deoxyribose nucleosides.
  • the backbone of each oligonucleotide consisted of a phosphorothioate linkage between adjoining nucleotides.
  • a number of oligonucleotides are next generated which are complementary to HDAC-3, HDAC-4, HDAC-5, HDAC-C, HDAC-D, and HDAC-E. These ohgonucleotides are based on the known nucleic acid sequences of these histone deacetylases (see, e.g., GenBank Accession No. U75697 for HDAC-3).
  • Antisense oligonucleotides specific for one of these histone deacetylases are screened for efficacy at inhibiting expression of mRNA and protein as described above for HDAC-1, HDAC-1 / 2, and HDAC-2.
  • antisense ohgonucleotides that inhibit more than one histone deacetylase e.g., HDAC-1 / 3 / C- specific
  • HDAC-1 / 3 / C- specific are also generated by mixing antisense ohgonucleotides specific for each histone deacetylase and screened for efficacy.
  • Oligonucleotides To determine the specificity and dose requirements of the antisense ohgonucleotides specific for histone deacetylase-encoding nucleic acid, the dose dependent inhibition of these ohgonucleotides on histone deacetylase mRNA expression was examined.
  • T24 ceUs were transfected using lipofectin (as described in Example 1) using 10, 25, 50, or 100 nM oligonucleotide.
  • the ceUs were harvested twenty-four hours foUowing transfection, RNA prepared, and Northern blotting analysis performed as described in Example 1 using radio labelled HDAC-1 and HDAC-2 cDNA as probe.
  • Fig. 1 shows the dose dependent inhibition of HDAC-1 mRNA expression by both HDAC-1 and HDAC-1 / 2 antisense ohgonucleotides at 50-100 nM.
  • HDAC-2 mRNA expression was inhibited by only the HDAC-1 / 2 antisense ohgonucleotide (MG2610) at 50-100 nM, while the HDAC-1 antisense oligonucleotide (MG2608) had no effect.
  • the oligonucleotides used in the experiment, the results of which are shown in Fig. 1, were first generation oligonucleotides (i.e., were not chemically modified).
  • the oligonucleotides used to obtain the results shown in Fig. 1 had sequences of SEQ ID NOs: 1- 5.
  • Fig. 2 shows the dose-dependent inhibition of HDAC-2 mRNA by HDAC-2 antisense oligonucleotide.
  • AU four HDAC-2 antisense oligonucleotide were able to reduce the level of HDAC-2 mRNA expression at 50-100 nM.
  • MG2628 appeared particularly efficacious at reducing HDAC-2 mP T expression in this experiment.
  • Second Generation Antisense Oligonucleotides To determine the ability of histone deacetylase antisense ohgonucleotides to inhibit protein expression, second generation versions of the HDAC-1, HDAC-1 / 2, and HDAC-2 antisense ohgonucleotides were generated. Each of these second generation antisense ohgonucleotides had a backbone consisting of a phosphorothioate linkage between each adjoining nucleotide. Moreover, the four terminal nucleotide residues at both the 5' and 3' ends of the olgonucleotide had sugar residues comprising a 2'-O-methyl group.
  • This modification to the terminal nucleotide residues served to increase binding affinity of the ohgonucleotide to the targeted nucleic acid, and to increase the stability of the ohgonucleotide by inhibiting nuclease susceptibility.
  • Fig. 3 shows the abUity of second generation HDAC-2 antisense ohgonucleotides to inhibit HDAC-2 protein expression.
  • T24 ceUs were transfected with 0, 25, or 50 nM
  • ceUs were transfected a second time with the same amount of the same oligonucleotide. Twenty- four hours after this (i.e., 48 hours after the first transfection), ceUular proteins were prepared, resolved on 7-15% gradient SDS-PAGE, and subjected to Western blotting analysis as described in Example 1 with rabbit polyclonal HDAC2 specific antibody (1:500, Santa Cruz Biotech).
  • Fig. 3 shows the specific ability of the HDAC-1 / 2 and HDAC- , antisense ohgonucleotides to inhibit protein expression of both HDAC-1 and HDAC-2 or HDAC-1, respectively, when compared to the mismatch controls.
  • T24 cells were transfected twice as described above with 50 nM oligonucleotide.
  • Cell lysates were prepared twenty-four hours following the second transfection, resolved on 7-15% gradient SDS-PAGE, and transferred to PNDF membrane.
  • the PVDF membrane blot was first blotted with anti-HDAC-1 antibody. FoUowing detection with horseradish peroxidase-labeUed secondary antibody and enhanced chemiluminescence, the blot was stripped, and re-probed with anti-HDAC-2 antibody. FoUowing detection, the blot was stripped for a second time and re-probed with an actin- specific antibody to verify equal protein loading in the lanes.
  • HDAC-1 and HDAC-1 / 2 mismatch control oligonucleotides failed to inhibit HDAC-1 or HDAC-1 and HDAC-2 protein expression, respectively.
  • HDAC-1 antisense ohgonucleotide effectively reduced expression of HDAC-1 protein
  • HDAC-1 / 2 antisense oligonucleotide reduced protein expression of both HDAC- 1 and HDAC-2.
  • Example 4 Identification of A Histone Deacetylase Involved in Induction of Cell Prohferation
  • Antisense ohgonucleotides that inhibit expression of different histone deacetylases are screened to identify a histone deacetylase that induces cell prohferation in cultured ceUs.
  • cultured normal human fibroblast ceUs are transfected with an antisense ohgonucleotide that inhibits the expression of a histone deacetylase.
  • an antisense ohgonucleotide that inhibits the expression of a histone deacetylase. While any standard transfection protocol may be employed, including, without limitation, CaPO 4 precipitation, electroporation, DEAE-dextran), transfection using the lipofectin transfection reagent (Gibco- BRL) is preferred.
  • An antisense oligonucleotide that inhibits the expression of a histone deacetylase that is found to inhibit cell proliferation when transfected into a normal cell identifies a histone deacetylase that is involved in induction of ceU prohferation in normal cells.
  • T24 bladder carcinoma ceUs are transfected with histone deacetylase antisense oligonucleotides according to the invention and their growth pattern is observed and compared to that of untransfected control cells.
  • T24 ceUs ATCC No. HTB-4
  • T24 ceUs ATCC No. HTB-4
  • cells are washed with phosphate buffered saline (PBS) and 5 ml of Opti-MEM media
  • the cells are resuspended in PBS and counted on a Coulter Particle Counter to determine the total cell number.
  • Mock-transfected T24 ceUs transfected with lipofectin and a control ohgonucleotide
  • An antisense ohgonucleotide that inhibits the expression of a histone deacetylase that is found to inhibit ceU proliferation when transfected into a neoplastic cell identifies a histone deacetylase that is involved in induction of cell proliferation in neoplastic ceUs.
  • a histone deacetylase antisense oligonucleotide of the invention is one that inhibits cell proliferation of neoplastic cells, but does not inhibit cell proliferation in normal cells.
  • Example 5 A Histone Deacetylase Protein Inhibitor that Interacts With and Reduces the Enzymatic Activity of A Histone Deacetylase Involved in the Induction of Cell Proliferation
  • a histone deacetylase that is identified as being involved in the induction of cell proliferation is used as a target for candidate compounds designed to interact with and inhibit its enzymatic activity.
  • FR901228 available from Fujisawa Pharmaceuticals
  • Candidate compounds can be derived from any source and may be naturally- occurring or synthetic, or may have naturaUy-occurring and synthetic components.
  • Candidate compounds may also be designed to chemically resemble any of the known histone deacetylase protein inhibitors, including, without hmitation, trichostatin A, trichostatin C, trapoxin, depudecin, suberoylanilide hydro xamic acid (SAHA), FR901228, and butyrate.
  • a pool of such compounds may be added to a histone deacetylase.
  • a histone deacetylase is preferably one that is identified using the antisense ohgonucleotides of the invention as a histone deacetylase involved in induction of ceU prohferation.
  • the histone deacetylase may be purified, for example, by using antibodies specific to that particular histone deacetylase (e.g., anti-HDAC-1 antibody commercially available from Santa Cruz Biotech.) or by recombinant production of the histone deacetylase in prokaryotic or eukaryotic cells.
  • the histone deacetylase may also be present in a ceU which normally expresses the histone deacetylase.
  • Pools of candidate compounds are added to the histone deacetylase, and the enzymatic activity of the histone deacetylase is measured.
  • a pool of candidate compounds showing such a histone deacetylase inhibiting activity is sub-divided, and the subdivisions tested until one candidate compound is isolated having a histone deacetylase inhibiting activity. It will be understood that once a pool of candidate compounds is identified as having an ability to inhibit histone deacetylase enzymatic activity, the pool may be screened via various methods to ascertain the presence within the pool or one or more histone deacetylase protein inhibitor compounds.
  • the pool may be subsequently screened on purified histone deacetylase.
  • the candidate compound(s) found to be a histone & acetylase protein inhibitor inhibits the activity of fewer than aU histone deacetylases. More preferably, such a candidate compound inhibits only those histone deacetylases that are involved in the induction of cell proliferation. Even more preferably, the candidate compound that is identified as a histone deacetylase protein inhibitor is one that inhibits only one histone deacetylase, where that one histone deacetylase is involved in the induction of cell prohferation.
  • the candidate compound that is identified as a histone deacetylase protein inhibitor is one that inhibits only one histone deacetylase, where that one histone deacetylase is involved in the induction of cell prohferation in neoplastic ceUs, but is not involved in the induction of cell prohferation in normal cells.
  • a candidate compound that is a histone deacetylase protein inhibitor purified histone deacetylase is allowed to adhere to the bottom of wells in a 96-weU microtiter plate.
  • Candidate compounds (or pools thereof) are then added to the plate, where each candidate compound has been modified with the covalent attachment of a detectable marker (e.g., a biotin label). Binding of the candidate compound to the plate- bound histone deacetylase is detected via addition of a secondary reagent that binds to the detectable marker (e.g., a streptavidin- labelled fluorophore), and subsequent analysis of the plate on a micro-titer plate reader.
  • a secondary reagent that binds to the detectable marker
  • Candidate compounds thus identified which interact with purified histone deacetylase are then screened for an abUity to inhibit the enzymatic activity of the histone deacetylase.
  • Example 6 Anti-Neoplastic Effect of Histone Deacetylase Antisense Oligonucleotide on Tumor Cells in Vivo
  • the purpose of this example is to illustrate the abihty of the histone deacetylase antisense ohgonucleotide of the invention to treat diseases responsive to histone deacetylase inhibition in animals, particularly mammals.
  • This example further provides evidence of the ability of the methods and compositions of the invention to inhibit tumor growth in domesticated mammal.
  • Eight to ten week old female BALB/c nude mice (Taconic Labs, Great Barrington, NY) are injected subcutaneously in the flank area with 2 x 10 6 preconditioned A549 human lung carcinoma cells.
  • Preconditioning of these cells is done by a minimum of three consecutive tumor transplantations in the same strain of nude mice. Subsequently, tumor fragments of approximately 30 mgs are excised and implanted subcutaneously in mice, in the left flank area under Forene anesthesia (Abbott Labs., Geneva, Switzerland). When the tumors reaches a mean volume of 100 mm 3 , the mice are treated intravenously, by daily bolous infusion into the taU vein, with ohgonucleotide saline preparations containing 0.1-6 mg/kg of antisense oligonucleotide (Sigma, St. Louis, MO). The optimal final concentration of the oligonucleotide is established by dose response experiments according to standard protocols.
  • Tumor volume is calculated according to standard methods every second day post infusion (e.g., Meyer et al., Int. J. Cancer 43:851- 856 (1989)).
  • Treatment with the ohgonucleotides according to the invention causes a significant reduction in tumor weight and volume relative to controls treated with saline only (i.e., no ohgonucleotide) or controls treated with sahne plus a control, non-specific ohgonucleotide.
  • the activity of histone deacetylase when measured is expected to be significantly reduced relative to saline treated controls.
  • Example 7 Synergistic Anti-Neoplastic Effect of Histone Deacetylase Antisense Oligonucleotide and Histone Deacetylase Protein Inhibitor on Tumor Cells in Vivo
  • the purpose of this example is to Ulustrate the abihty of the histone deacetylase antisense ohgonucleotide and the histone deacetylase protein inhibitor of the invention to inhibit tumor growth in a mammal.
  • mice bearing implanted A549 tumors (mean volume 100 mm 3 ) are treated daUy with saline preparations containing from about 0.1 mg to about 30 mg per kg body weight of histone deacetylase antisense ohgonucleotide.
  • mice A second group of mice is treated daily with pharmaceutically acceptable preparations containing from about 0.01 mg to about 5 rag per kg body weight of histone deacetylase protein inhibitor.
  • Some mice receive both the antisense oligonucleotide and the histone deacetylase protein inhibitor.
  • one group may receive the antisense oligonucleotide and the histone deacetylase protein inhibitor simultaneously intravenously via the tail vein.
  • Another group may receive the antisense ohgonucleotide via the tail vein, and the histone deacetylase protein inhibitor subcutaneously.
  • Yet another group may receive both the antisense oligonucleotide and the histone deacetylase protein inhibitor simultaneously via a subcutaneous injection.
  • mice ⁇ e similarly estabhshed which receive no treatment e.g., saline only
  • a mismatch antisense oligonucleotide only e.g., a control compound that does not inhibit histone deacetylase activity
  • a control compound that does not inhibit histone deacetylase activity e.g., a control compound that does not inhibit histone deacetylase activity
  • a control compound that does not inhibit histone deacetylase activity e.g., a control compound that does not inhibit histone deacetylase activity
  • mismatch antisense ohgonucleotide with control compound e.g., a control compound that does not inhibit histone deacetylase activity
  • Tumor volume is measured with calipers.
  • Treatment with the antisense oligonucleotide plus the histone deacetylase protein inhibitor according to the invention causes a significant reduction in tumor weight and volume relative to controls.
  • the antisense oligonucleotide and the histone deacetylase protein inhibitor inhibit the expression and activity of the same histone deacetylase.

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  • Investigating Or Analysing Biological Materials (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne l'inhibition de l'expression et de l'activité enzymatique d'histone déacétylase et, en particulier, l'inhibition d'une histone déacétylase spécifique. L'invention concerne également des compositions comprenant des oligonucléotides antisens, ainsi que des procédés d'utilisation de celles-ci pour inhiber une histone déacétylase. Enfin, l'invention concerne également des procédés permettant d'identifier une histone déacétylase intervenant dans l'induction de la prolifération cellulaire, ainsi que des procédés permettant d'identifier des composés qui réagissent mutuellement avec l'activité enzymatique d'une telle histone déacétylase et réduisent cette activité.
PCT/IB2000/001252 1999-05-03 2000-05-03 Inhibition d'histone deacetylase WO2000071703A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP00954830A EP1173562A2 (fr) 1999-05-03 2000-05-03 Inhibition d'histone deacetylase
JP2000620080A JP2003500052A (ja) 1999-05-03 2000-05-03 ヒストン脱アセチル酵素の抑制
AU67182/00A AU6718200A (en) 1999-05-03 2000-05-03 Inhibition of histone deacetylase
CA002366408A CA2366408A1 (fr) 1999-05-03 2000-05-03 Inhibition d'histone deacetylase
KR1020017014048A KR20020007398A (ko) 1999-05-03 2000-05-03 히스톤 탈아세틸화효소의 억제

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13228799P 1999-05-03 1999-05-03
US60/132,287 1999-05-03

Publications (2)

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WO2000071703A2 true WO2000071703A2 (fr) 2000-11-30
WO2000071703A3 WO2000071703A3 (fr) 2001-07-19

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EP (1) EP1173562A2 (fr)
JP (1) JP2003500052A (fr)
KR (1) KR20020007398A (fr)
AU (1) AU6718200A (fr)
CA (1) CA2366408A1 (fr)
WO (1) WO2000071703A2 (fr)

Cited By (37)

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WO2001067107A1 (fr) * 2000-03-04 2001-09-13 Imperial College Innovations Limited Modulation de l'histone deacetylase
WO2002036783A2 (fr) * 2000-10-31 2002-05-10 Bayer Aktiengesellschaft Regulation de l'histone deacetylase chez l'homme
WO2002069947A2 (fr) * 2001-01-12 2002-09-12 Methylgene, Inc. Procede permettant d'inhiber specifiquement l'histone deacetylase-4
WO2003006652A2 (fr) * 2000-03-24 2003-01-23 Methylgene, Inc. Inhibition d'isoformes specifiques d'histone deacetylase
WO2002055688A3 (fr) * 2001-01-10 2003-04-10 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Inhibiteurs de l'histone deacetylase dans le diagnostique et le traitement de neoplasmes de la thyroide
WO2003075839A2 (fr) 2002-03-04 2003-09-18 Aton Pharma, Inc. Procedes d'induction de differenciation terminale
WO2003080864A1 (fr) * 2002-03-26 2003-10-02 Exhonit Therapeutics Sa Histone deacetylase: nouvelle cible moleculaire de la neurotoxicite
FR2837838A1 (fr) * 2002-03-26 2003-10-03 Exonhit Therapeutics Sa Nouvelle cible moleculaire de la neurotoxicite
WO2004005513A2 (fr) * 2002-07-03 2004-01-15 Methylgene, Inc. Procede d'inhibition specifique de l'histone deacetylase-7 et 8
US6706686B2 (en) 2001-09-27 2004-03-16 The Regents Of The University Of Colorado Inhibition of histone deacetylase as a treatment for cardiac hypertrophy
EP1400806A1 (fr) * 2002-09-18 2004-03-24 G2M Cancer Drugs AG Criblage pour inhibiteurs de l'histone déacétylase
JP2006509010A (ja) * 2002-12-05 2006-03-16 インペリアル・カレッジ・イノベイションズ・リミテッド アポトーシスの制御
WO2006093053A1 (fr) 2005-03-02 2006-09-08 Astellas Pharma Inc. Nouveau marqueur pd pour inhibiteur d’histone deacetylase
US7148257B2 (en) 2002-03-04 2006-12-12 Merck Hdac Research, Llc Methods of treating mesothelioma with suberoylanilide hydroxamic acid
US7154002B1 (en) 2002-10-08 2006-12-26 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7169801B2 (en) 2003-03-17 2007-01-30 Takeda San Diego, Inc. Histone deacetylase inhibitors
WO2008041053A2 (fr) 2005-05-20 2008-04-10 Methylgene, Inc. Inhibiteurs de la signalisation du récepteur vegf et du récepteur hgf
US7456219B2 (en) 2002-03-04 2008-11-25 Merck Hdac Research, Llc Polymorphs of suberoylanilide hydroxamic acid
US7566535B2 (en) 2002-03-07 2009-07-28 University Of Delaware Enhanced oligonucleotide-mediated nucleic acid sequence alteration
US7642253B2 (en) 2005-05-11 2010-01-05 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7642275B2 (en) 2004-12-16 2010-01-05 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7732475B2 (en) 2005-07-14 2010-06-08 Takeda San Diego, Inc. Histone deacetylase inhibitors
EP2201947A2 (fr) 2003-08-26 2010-06-30 Merck HDAC Research, LLC Utilisation de SAHA pour traiter le mésotheliome
EP2226072A1 (fr) 2003-08-29 2010-09-08 Aton Pharma, Inc. Combinaisons à base d'acide suberoylanilide hydroxamique et d'agents antimétabolites pour le traitement du cancer
US7838520B2 (en) 2001-09-14 2010-11-23 Methylgene, Inc. Inhibitors of histone deacetylase
EP2269609A2 (fr) 2001-10-16 2011-01-05 Sloan-Kettering Institute for Cancer Research Traitement des maladiees neurodégénératives et le cancer du cerveau avec l'acide hydroxamique subéroylanilide (le SAHA)
US8030344B2 (en) 2007-03-13 2011-10-04 Methylgene Inc. Inhibitors of histone deacetylase
US8093295B2 (en) 2005-05-20 2012-01-10 Merck Sharp & Dohme Corp. Formulations of suberoylanilide hydroxamic acid and methods for producing the same
WO2012118632A1 (fr) 2011-02-28 2012-09-07 Ning Xi Quinoléines substituées et leurs méthodes d'utilisation
EP2532657A2 (fr) 2008-10-14 2012-12-12 Sunshine Lake Pharma Co., Ltd Composés et procédés d'utilisation
WO2014022116A2 (fr) 2012-07-28 2014-02-06 Calitor Sciences, Llc Composés de pyrazolone substituée et leurs procédés d'utilisation
WO2014130375A1 (fr) 2013-02-21 2014-08-28 Calitor Sciences, Llc Composés hétéro-aromatiques en tant que modulateurs de pi3 kinase
US8957027B2 (en) 2006-06-08 2015-02-17 Celgene Corporation Deacetylase inhibitor therapy
US9539303B2 (en) 2006-04-24 2017-01-10 Celgene Corporation Treatment of Ras-expressing tumors
WO2017067447A1 (fr) 2015-10-19 2017-04-27 Sunshine Lake Pharma Co., Ltd. Sel d'un inhibiteur d'egfr, forme cristalline correspondante et utilisations correspondantes
US9636298B2 (en) 2014-01-17 2017-05-02 Methylgene Inc. Prodrugs of compounds that enhance antifungal activity and compositions of said prodrugs
EP3299019A1 (fr) 2012-11-14 2018-03-28 Calitor Sciences, LLC Composés hétéroaromatiques comme modulateurs de la kinase pi3 et procédés d'utilisation

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US7250514B1 (en) 2002-10-21 2007-07-31 Takeda San Diego, Inc. Histone deacetylase inhibitors
WO2015051035A1 (fr) 2013-10-01 2015-04-09 The J. David Gladstone Institutes Compositions, systèmes et procédés pour le criblage de médicament de bruit d'expression génétique et leurs utilisations

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YOSHIDA M ET AL: "POTENT AND SPECIFIC INHIBITION OF MAMMALIAN HISTONE DEACETYLASE BOTH IN VIVO AND IN VITRO BY TRICHOSTATIN A" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 265, no. 28, 5 October 1990 (1990-10-05), pages 17174-17179, XP000616087 ISSN: 0021-9258 cited in the application *

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WO2001067107A1 (fr) * 2000-03-04 2001-09-13 Imperial College Innovations Limited Modulation de l'histone deacetylase
WO2003006652A3 (fr) * 2000-03-24 2004-05-13 Methylgene Inc Inhibition d'isoformes specifiques d'histone deacetylase
WO2003006652A2 (fr) * 2000-03-24 2003-01-23 Methylgene, Inc. Inhibition d'isoformes specifiques d'histone deacetylase
WO2002036783A2 (fr) * 2000-10-31 2002-05-10 Bayer Aktiengesellschaft Regulation de l'histone deacetylase chez l'homme
WO2002036783A3 (fr) * 2000-10-31 2003-05-01 Bayer Ag Regulation de l'histone deacetylase chez l'homme
WO2002055688A3 (fr) * 2001-01-10 2003-04-10 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Inhibiteurs de l'histone deacetylase dans le diagnostique et le traitement de neoplasmes de la thyroide
AU2002249938B2 (en) * 2001-01-10 2006-12-21 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Histone deacetylase inhibitors in diagnosis and treatment of thyroid neoplasms
WO2002069947A2 (fr) * 2001-01-12 2002-09-12 Methylgene, Inc. Procede permettant d'inhiber specifiquement l'histone deacetylase-4
WO2002069947A3 (fr) * 2001-01-12 2003-10-09 Methylgene Inc Procede permettant d'inhiber specifiquement l'histone deacetylase-4
US7838520B2 (en) 2001-09-14 2010-11-23 Methylgene, Inc. Inhibitors of histone deacetylase
US6706686B2 (en) 2001-09-27 2004-03-16 The Regents Of The University Of Colorado Inhibition of histone deacetylase as a treatment for cardiac hypertrophy
US6946441B2 (en) 2001-09-27 2005-09-20 Regents of the University of Colorado, A Body Corporation Inhibition of histone deacetylase as a treatment for cardiac hypertrophy
US7879865B2 (en) 2001-10-16 2011-02-01 Sloan-Kettering Institute For Cancer Research Treatment of cancer of the brain using histone deacetylase inhibitors
EP2269609A2 (fr) 2001-10-16 2011-01-05 Sloan-Kettering Institute for Cancer Research Traitement des maladiees neurodégénératives et le cancer du cerveau avec l'acide hydroxamique subéroylanilide (le SAHA)
US7375137B2 (en) 2002-03-04 2008-05-20 Merck Hdac Research, Llc Methods of treating cancer with HDAC inhibitors
US7851509B2 (en) 2002-03-04 2010-12-14 Merck Hdac Research, Llc Polymorphs of suberoylanilide hydroxamic acid
US7456219B2 (en) 2002-03-04 2008-11-25 Merck Hdac Research, Llc Polymorphs of suberoylanilide hydroxamic acid
EP2322160A1 (fr) 2002-03-04 2011-05-18 Merck HDAC Research, LLC Procédés d'induction de différenciation terminale
US7847122B2 (en) 2002-03-04 2010-12-07 Merck Hdac Research, Llc Polymorphs of suberoylanilide hydroxamic acid
EP2266552A2 (fr) 2002-03-04 2010-12-29 Merck HDAC Research, LLC Procédés d'induction de différentiation de terminal
US8101663B2 (en) 2002-03-04 2012-01-24 Merck Hdac Research, Llc Polymorphs of suberoylanilide hydroxamic acid
US7148257B2 (en) 2002-03-04 2006-12-12 Merck Hdac Research, Llc Methods of treating mesothelioma with suberoylanilide hydroxamic acid
US7399787B2 (en) 2002-03-04 2008-07-15 Merck Hdac Research, Llc Methods of treating cancer with HDAC inhibitors
WO2003075839A2 (fr) 2002-03-04 2003-09-18 Aton Pharma, Inc. Procedes d'induction de differenciation terminale
US7732490B2 (en) 2002-03-04 2010-06-08 Merck Hdac Research, Llc Methods of treating cancer
US8067472B2 (en) 2002-03-04 2011-11-29 Merck Hdac Research, Llc Methods of treating Hodgkin's and non-Hodgkin's lymphoma
EP2082737A2 (fr) 2002-03-04 2009-07-29 Merck HDAC Research, LLC Procédés d'induction de différentiation de terminal
US7652069B2 (en) 2002-03-04 2010-01-26 Merck Hdac Research, Llc Polymorphs of suberoylanilide hydroxamic acid
US7566535B2 (en) 2002-03-07 2009-07-28 University Of Delaware Enhanced oligonucleotide-mediated nucleic acid sequence alteration
WO2003080864A1 (fr) * 2002-03-26 2003-10-02 Exhonit Therapeutics Sa Histone deacetylase: nouvelle cible moleculaire de la neurotoxicite
FR2837838A1 (fr) * 2002-03-26 2003-10-03 Exonhit Therapeutics Sa Nouvelle cible moleculaire de la neurotoxicite
WO2004005513A2 (fr) * 2002-07-03 2004-01-15 Methylgene, Inc. Procede d'inhibition specifique de l'histone deacetylase-7 et 8
WO2004005513A3 (fr) * 2002-07-03 2004-07-01 Methylgene Inc Procede d'inhibition specifique de l'histone deacetylase-7 et 8
US20060110735A1 (en) * 2002-09-18 2006-05-25 Thorsten Heinzel Use of molecular markers for the preclinical and clinical profiling of inhibitors of enzymes having histone deacetylase activity
AU2003267386B2 (en) * 2002-09-18 2010-07-01 Karlsruher Institut Fur Technologie The use of molecular markers for the preclinical and clinical profiling of inhibitors of enzymes having histone deacetylase activity
WO2004027418A3 (fr) * 2002-09-18 2004-05-13 G2M Cancer Drugs Ag Utilisation de marqueurs moleculaires pour le profilage preclinique et clinique d'inhibiteurs d'enzymes possedant une activite d'histone-desacetylase
WO2004027418A2 (fr) * 2002-09-18 2004-04-01 G2M Cancer Drugs Ag Utilisation de marqueurs moleculaires pour le profilage preclinique et clinique d'inhibiteurs d'enzymes possedant une activite d'histone-desacetylase
EP1400806A1 (fr) * 2002-09-18 2004-03-24 G2M Cancer Drugs AG Criblage pour inhibiteurs de l'histone déacétylase
US7399884B2 (en) 2002-10-08 2008-07-15 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7154002B1 (en) 2002-10-08 2006-12-26 Takeda San Diego, Inc. Histone deacetylase inhibitors
JP2006509010A (ja) * 2002-12-05 2006-03-16 インペリアル・カレッジ・イノベイションズ・リミテッド アポトーシスの制御
US7381825B2 (en) 2003-03-17 2008-06-03 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7375228B2 (en) 2003-03-17 2008-05-20 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7169801B2 (en) 2003-03-17 2007-01-30 Takeda San Diego, Inc. Histone deacetylase inhibitors
EP2201947A2 (fr) 2003-08-26 2010-06-30 Merck HDAC Research, LLC Utilisation de SAHA pour traiter le mésotheliome
EP2226072A1 (fr) 2003-08-29 2010-09-08 Aton Pharma, Inc. Combinaisons à base d'acide suberoylanilide hydroxamique et d'agents antimétabolites pour le traitement du cancer
US7642275B2 (en) 2004-12-16 2010-01-05 Takeda San Diego, Inc. Histone deacetylase inhibitors
WO2006093053A1 (fr) 2005-03-02 2006-09-08 Astellas Pharma Inc. Nouveau marqueur pd pour inhibiteur d’histone deacetylase
US7642253B2 (en) 2005-05-11 2010-01-05 Takeda San Diego, Inc. Histone deacetylase inhibitors
US8329726B2 (en) 2005-05-20 2012-12-11 Methylgene Inc. Inhibitors of VEGF receptor and HGF receptor signaling
US8450372B2 (en) 2005-05-20 2013-05-28 Merck Sharp & Dohme Corp. Formulations of suberoylanilide hydroxamic acid and methods for producing same
WO2008041053A2 (fr) 2005-05-20 2008-04-10 Methylgene, Inc. Inhibiteurs de la signalisation du récepteur vegf et du récepteur hgf
US8093264B2 (en) 2005-05-20 2012-01-10 Methylgene Inc. Fused heterocycles as inhibitors of VEGF receptor and HGF receptor signaling
US8093295B2 (en) 2005-05-20 2012-01-10 Merck Sharp & Dohme Corp. Formulations of suberoylanilide hydroxamic acid and methods for producing the same
US8288440B2 (en) 2005-05-20 2012-10-16 Merck Sharp & Dohme Corp. Formulations of suberoylanilide hydroxamic acid and methods for producing same
US7732475B2 (en) 2005-07-14 2010-06-08 Takeda San Diego, Inc. Histone deacetylase inhibitors
US7741494B2 (en) 2005-07-14 2010-06-22 Takeda San Diego, Inc. Histone deacetylase inhibitors
US9539303B2 (en) 2006-04-24 2017-01-10 Celgene Corporation Treatment of Ras-expressing tumors
US9259452B2 (en) 2006-06-08 2016-02-16 Gelgene Corporation Deacetylase inhibitor therapy
US8957027B2 (en) 2006-06-08 2015-02-17 Celgene Corporation Deacetylase inhibitor therapy
US8354445B2 (en) 2007-03-13 2013-01-15 Methylgene Inc. Inhibitors of histone deacetylase
US8030344B2 (en) 2007-03-13 2011-10-04 Methylgene Inc. Inhibitors of histone deacetylase
EP2532657A2 (fr) 2008-10-14 2012-12-12 Sunshine Lake Pharma Co., Ltd Composés et procédés d'utilisation
WO2012118632A1 (fr) 2011-02-28 2012-09-07 Ning Xi Quinoléines substituées et leurs méthodes d'utilisation
WO2014022116A2 (fr) 2012-07-28 2014-02-06 Calitor Sciences, Llc Composés de pyrazolone substituée et leurs procédés d'utilisation
WO2014022117A1 (fr) 2012-07-28 2014-02-06 Calitor Sciences, Llc Composés de pyrazolone substituée et leurs procédés d'utilisation
EP3299019A1 (fr) 2012-11-14 2018-03-28 Calitor Sciences, LLC Composés hétéroaromatiques comme modulateurs de la kinase pi3 et procédés d'utilisation
WO2014130375A1 (fr) 2013-02-21 2014-08-28 Calitor Sciences, Llc Composés hétéro-aromatiques en tant que modulateurs de pi3 kinase
US9636298B2 (en) 2014-01-17 2017-05-02 Methylgene Inc. Prodrugs of compounds that enhance antifungal activity and compositions of said prodrugs
WO2017067447A1 (fr) 2015-10-19 2017-04-27 Sunshine Lake Pharma Co., Ltd. Sel d'un inhibiteur d'egfr, forme cristalline correspondante et utilisations correspondantes

Also Published As

Publication number Publication date
JP2003500052A (ja) 2003-01-07
KR20020007398A (ko) 2002-01-26
CA2366408A1 (fr) 2000-11-30
EP1173562A2 (fr) 2002-01-23
AU6718200A (en) 2000-12-12
WO2000071703A3 (fr) 2001-07-19

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