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WO2002018593A2 - Modulation of fas and fasl expression - Google Patents

Modulation of fas and fasl expression Download PDF

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Publication number
WO2002018593A2
WO2002018593A2 PCT/CA2001/000845 CA0100845W WO0218593A2 WO 2002018593 A2 WO2002018593 A2 WO 2002018593A2 CA 0100845 W CA0100845 W CA 0100845W WO 0218593 A2 WO0218593 A2 WO 0218593A2
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WO
WIPO (PCT)
Prior art keywords
fas
human
composition
animal
fasl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CA2001/000845
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French (fr)
Other versions
WO2002018593A3 (en
Inventor
Nigel C. Phillips
Mario C. Filion
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Telesta Therapeutics Inc
Original Assignee
Bioniche Life Sciences Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/CA2000/001467 external-priority patent/WO2001044465A2/en
Priority to JP2002522500A priority Critical patent/JP2004507469A/en
Priority to AU6886301A priority patent/AU6886301A/en
Priority to ES01947067T priority patent/ES2383671T3/en
Priority to EP01947067A priority patent/EP1313853B1/en
Priority to AU2001268863A priority patent/AU2001268863B2/en
Application filed by Bioniche Life Sciences Inc filed Critical Bioniche Life Sciences Inc
Priority to CA002420103A priority patent/CA2420103A1/en
Priority to AT01947067T priority patent/ATE550428T1/en
Priority to MXPA03001812A priority patent/MXPA03001812A/en
Priority to DK01947067.3T priority patent/DK1313853T3/en
Publication of WO2002018593A2 publication Critical patent/WO2002018593A2/en
Publication of WO2002018593A3 publication Critical patent/WO2002018593A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/1138—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 receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates compositions useful for modulating Fas and Fas ligand expression on cells and for modulating efficacy of therapeutic agents.
  • Fas is a type-I membrane protein abundantly expressed by cells in various tissues and particularly on activated T cells, heart cells, kidney cells and hepatocytes.
  • FasL is a type-II transmembrane protein expressed particularly on activated T cells and natural killer cells (Nagata, S. Ann. Rev. Genet. 33:29, 1999), and is expressed constitutively in immune- privileged sites as, for example, the eye and testis (Griffith et al. Science 270:630, 1995).
  • Fas and FasL interactions play an essential role in the regulation of immune cells and in the elimination of autoreactive cells (Sabelko- Downes et al. Curr. Opin. Immunol. 12:330, 2000).
  • Fas/FasL mediates the killing of cancer cells and of virus-infected cells (Famularo et al. Med. Hypoth. 53:50, 1999; Owen-Schaub et al. Int. J. Oncol. 17:5, 2000).
  • FasL expressed on cancer cells, may attack cells of the immune system (O'Connell, J. Exp. Med.
  • FasL expressed on activated T cells, may also participate in tissue damage in fulminant hepatitis and in graft- versus-host disease (Kondo et al. Nature Med. 3:409, 1997; Braun et al. J. Exp. Med. 183:657, 1996).
  • Apoptosis is an active cellular death process characterized by distinctive morphological changes that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al. Int. Rev. Cytol. 68:251, 1980).
  • a molecular hallmark of apoptosis is degradation of cellular nuclear DNA into oligonucleosomal-length fragments as the result of activation of endogenous endonucleases (Wyllie A. Nature 284:555, 1980).
  • Caspases cyste-aspartyl- specific proteases
  • the binding of FasL to Fas activates a cascade of caspases via a FADD adaptor (Fas-associated protein with death domain), which leads to the cleavage of various cellular substrates and to DNA fragmentation (Nagata, S. Ann. Rev. Genet. 33:29, 1999).
  • Synthetic oligonucleotides are polyanionic sequences that can be internalized by cells (Nlassov et al. Biochim. Biophys. Acta 1197:95, 1994) and bind selectively to nucleic acids (Wagner, R. Nature: 372:333, 1994), to specific cellular proteins (Bates et al. J. Biol. Chem. 274:26369, 1999) and to specific nuclear proteins (Scaggiante et al. Eur. J. Biochem. 252:207, 1998), and inhibit cell proliferation. Proliferation is the culmination of the progression of a cell through the cell cycle, resulting in the division of one cell into two cells. Alterations in cell cycle progression occur in all cancers and may result from over-expression of genes, mutation of regulatory genes, or abrogation of DNA damage checkpoints (Hochhauser D. Anti-Cancer Chemotherapeutic Agents 8:903, 1997).
  • Synthetic phosphorothioate oligonucleotides containing unmethylated CpG dinucleotides flanked by two 5' purine and two 3' pyrimidine are reported to induce the synthesis of cytokines by macrophages and B cells, to increase the activity of NK cells and cytotoxic T lymphocytes, and to enhance T- Natl. Acad. Sci. USA93:2879, 1996; Lipford et al. Eur. J. Immunol. 27:2340, 1997).
  • a 20 base synthetic CpG motif is reported to block Fas expression on activated B cells and to block apoptosis induced by anti-Fas monoclonal antibodies (Wang et al. Cell.
  • Fas/FasL The ability to modulate the Fas/FasL system has many clinical applications for use in diseases including, but not limited to, neoplastic autoimmune, degenerative and cardiovascular diseases.
  • Fas/FasL modulating agents have proven to be less than adequate in clinical applications.
  • many of these agents are inefficient, toxic or have significant adverse effects.
  • the present invention fulfills these needs by providing a novel method, employing new compositions comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) n b, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is between 2 and 10, preferably 4 to 8, more preferably 5 to 7, and most preferably 6.
  • the present invention provides new uses for these compositions.
  • This composition is combined with a pharmaceutically acceptable carrier, and is FasL expression in the animal or the human.
  • This composition is also combined with a pharmaceutically acceptable carrier, and is administered to an animal or a human having a disease in an amount effective to modulate Fas and FasL expression in order to treat the disease in the animal or the human.
  • This composition is also administered to an animal or human to modulate the efficacy of Fas and FasL modulatory agents, comprising administration of the composition in an amount effective to modulate efficacy of Fas and FasL modulatory agents to modulate Fas and FasL, or to treat an animal or a human having a disease.
  • compositions of the present invention are to modulate, and preferentially potentiate, the efficacy of a therapeutic agent to treat a disease, comprising administration of the composition in a pharmaceutically acceptable carrier, optionally in combination with a therapeutic agent, in an amount effective to modulate efficacy of the therapeutic agent administered to an animal or a human.
  • the compositions of the present invention may be administered at any time before, during, or after administration of the therapeutic agent, in order to modulate the efficacy of the therapeutic agent.
  • the compositions of the present invention may also be administered in vitro, for example to animal or human cells or tissues.
  • the composition of the present invention comprises a sequence selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) n b, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof provided the total number of bases in the composition is 6.
  • the composition of the present invention comprises this sequence of 6 bases in combination with a pharmaceutically acceptable carrier.
  • the composition of the present invention comprises this sequence of 6 bases in combination with a therapeutic agent and a pharmaceutically acceptable carrier.
  • an object of the present invention is to provide a new composition
  • a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) n b, and a(GC) utilizatb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 2 to 10.
  • sequence synthetic phosphodiester oligodeoxynucleotide
  • Another object of the present invention is to provide a new composition
  • a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) n b, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 4 to 8.
  • sequence synthetic phosphodiester oligodeoxynucleotide
  • Yet another object of the present invention is to provide a new composition
  • a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) n b, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 5 to 7.
  • sequence synthetic phosphodiester oligodeoxynucleotide
  • Another object of the present invention is to provide a new composition
  • a new composition comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) repeatb, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 6.
  • sequence synthetic phosphodiester oligodeoxynucleotide
  • Yet another object of the present invention is to provide for the use of any of the novel compositions described herein for the manufacture of a medicament.
  • Still another object of the present invention is to provide new uses for the compositions of the present invention.
  • Still another object of the present invention is to provide a method to modulate the Fas expression on immune cells.
  • Another object of the present invention is to provide a method to modulate FasL expression on immune cells.
  • Yet another object of the present invention is to provide a method to modulate Fas expression on cancer cells.
  • Yet another object of the present invention is to provide a method to treat a neoplastic disease.
  • Still another object of the present invention is to provide a method to treat an autoimmune disease.
  • Another object of the present invention is to provide a method to treat an inflammatory disease.
  • Yet another object of the present invention is to provide a method to treat a proliferative disease
  • Another object of the present invention is to provide a method to treat a lymphoproliferative disease.
  • Yet another object of the present invention is to provide a method to treat a degenerative disease.
  • Yet another object of the present invention is to provide a method to treat a graft rejection.
  • Another object of the present invention is to provide a method effective to treat an infection.
  • Yet another object of the present invention is to provide a method that modulates the effect of a therapeutic agent to treat disease.
  • Another object of the present invention is to provide a method that potentiates the effect of a FasL modulating agent.
  • Yet another object of the present invention is to provide a method that potentiates the effect of an anti-neoplastic agent.
  • Another object of the present invention is to provide a method that potentiates the effect of an immunostimulatory agent.
  • Still another object of the present invention is to provide a method that potentiates the effect of an immunosuppressive agent.
  • Another object of the present invention is to provide a method that potentiates the effect of an anti-inflammatory agent.
  • Another object of the present invention is to provide a composition that is minimally toxic to the recipient.
  • the present invention fulfills these needs by providing new compositions and new methods of using these compositions.
  • the present compositions comprise a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG) n , (GT) n , a(GT) complicatb, a(GA) n b, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is between 2 and 10, preferably 4 to 8, more preferably 5 to 7, and most preferably 6.
  • the compositions of the present invention further comprise any of the sequences described herein in combination with a pharmaceutically acceptable carrier.
  • the compositions of the present invention further comprise any of the sequences described herein in combination with a therapeutic agent and a pharmaceutically acceptable carrier.
  • the present invention provides new uses for these novel compositions.
  • This composition is combined with a pharmaceutically acceptable carrier, and is administered to an animal or a human an amount effective to modulate Fas and FasL expression in the animal or the human.
  • This composition is also combined with a pharmaceutically acceptable carrier, and is administered to an animal or a human having a disease in an amount effective to modulate Fas and FasL expression in order to treat the disease in the animal or the human.
  • This composition is also administered to an animal or human to modulate the efficacy of Fas and FasL modulatory agents, comprising administration of the composition in an amount effective to modulate efficacy of Fas and FasL modulatory agents to modulate Fas and FasL, or to treat an animal or a human having a disease.
  • composition of the present invention is to modulate, and preferentially potentiate, the efficacy of a therapeutic agent to treat a disease, carrier, optionally in combination with a therapeutic agent, in an amount effective to modulate efficacy of the therapeutic agent administered to an animal or a human.
  • the composition of the present invention may be administered at any time before, during, or after administration of the therapeutic agent, in order to modulate the efficacy of the therapeutic agent.
  • the composition of the present invention comprises a sequence selected from the group consisting of (GG) n , (GT) n , a(GT) n b, a(GA) n b, and a(GC) n b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof provided the total number of bases in the composition is 6.
  • the composition of the present invention comprises this sequence of 6 bases in combination with a pharmaceutically acceptable carrier.
  • the composition of the present invention comprises this sequence of 6 bases in combination with a therapeutic agent and a pharmaceutically acceptable carrier.
  • sequence refers to a synthetic phosphodiester oligodeoxynucleotide comprised of adenine (A), cytosine (C), guanine (G) and thymine (T), with a total number of bases of 2 to 10, preferably 4 to 8, more preferably 5 to 7 and most preferably 6.
  • the word "expression” refers to the cell surface concentration of Fas or of FasL.
  • the words “response” refers to upregulation (increase) or downregulation (decrease) of Fas or of Fas L expression.
  • the word “modulates” refers to changes in the expression of Fas or of FasL. Such changes include upregulation and downregulation of Fas or of FasL expression.
  • the word modulate is also employed to describe the ability of the novel sequences of the present invention to modulate the efficacy of therapeutic agents, including Fas and FasL modulating agents, to treat disease or to modulate Fas or FasL expression.
  • the phrases “therapeutically effective”, “effective amount” and “amount effective to” refer to an amount of a sequence effective to modulate the expression of Fas or of FasL.
  • disease refers to a condition wherein bodily health is impaired.
  • the phrase "therapeutic agent” is any agent approved by a regulatory agency of a country or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use to treat a disease in an animal, including a human.
  • the word “antineoplastic” refers to preventing the development, maturation, proliferation or spread of cancer cells
  • the word “potentiates” refers to a degree of synergism that is greater than additive.
  • the word “synergism” refers to the coordinated action of two or more agents.
  • Administration of an effective amount of a sequence of the present invention to an animal, including a human, is a therapeutic treatment that prevents, treats or eliminates a disease including, but not limited to, neoplastic, autoimmune, proliferative, lymphoproliferative, degenerative, and cardiovascular disease; infection; inflammation; and, graft, tissue and cell rejection.
  • a disease including, but not limited to, neoplastic, autoimmune, proliferative, lymphoproliferative, degenerative, and cardiovascular disease; infection; inflammation; and, graft, tissue and cell rejection.
  • compositions comprising one or more sequences and a pharmaceutically acceptable carrier are prepared by uniformly and intimately bringing into association the sequence and the pharmaceutically acceptable carrier.
  • compositions comprising one or more sequences, a therapeutic agent and a pharmaceutically acceptable carrier are prepared by uniformly and intimately bringing into association the sequence, the therapeutic agent and the pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include liquid carriers, solid carriers or both. Liquid carriers are aqueous carriers, non- aqueous carriers or both and include, but are not limited to, solutions, suspensions and emulsions.
  • Emulsions include, but are not limited to, oil emulsions, water in oil emulsions, water-in-oil-in-water emulsions, site-specific emulsions, long- carriers are biological carriers, chemical carriers or both and include, but are not limited to, viral vector systems, plasmids, particles, microparticles, nanoparticles, microspheres, nanospheres, bacterial cell walls, minipumps, and biodegradable or non-biodegradable natural or synthetic polymers that allow for sustained release of the sequences.
  • Preferred aqueous carriers include, but are not limited to, water, saline and pharmaceutically acceptable buffers.
  • Preferred non-aqueous carriers include, but are not limited to, a mineral oil or a neutral oil including, but not limited to, a diglyceride, a triglyceride, a phospholipid, a lipid, an oil and mixtures thereof, wherein the oil contains an appropriate mix of polyunsaturated and saturated fatty acids.
  • stabilizing agents and excipients may be included regardless of the pharmaceutically acceptable carrier used to present the sequence to the cells.
  • the therapeutic effectiveness of a sequence may be increased by methods including, but not limited to, chemically modifying the base, sugar or phosphate backbone, chemically supplementing or biotechnologically amplifying the sequences using bacterial plasmids containing the appropriate sequences, complexing the sequences to biological or chemical carriers or coupling the sequences to tissue-type or cell-type directed ligands or antibodies.
  • composition of the present invention further comprises a composition comprising a sequence and a therapeutic agent, wherein, when the sequence and the therapeutic agent are combined with a pharmaceutically acceptable carrier and administered to an animal or human having a disease.
  • the sequence modulates and preferentially potentiates the effect of the therapeutic agent on the disease.
  • Therapeutic agents include, but not limited to, anti-neoplastic, anti-inflammatory, anti-autoimmune, anti-degenerative, Fas modulating and FasL modulating agents, or any combination thereof, and radiation therapy, or a combination of radiation therapy with therapeutic agents.
  • These therapeutic agents include, but are not limited to, biologicals, drugs, chemotherapeutic drugs, immunostimulants, immunomodulators, immunotherapeutics, anti-virals, anti- infectives, antibiotics, cytokines, antigens, antibodies, nucleic acids, vaccines, aptabases, nucleic acids, antisense nucleic acids, telomerase inhibitors, caspase biologically engineered and chemically synthesized agents, and agents that target cell death molecules for activation or inactivation.
  • Chemotherapeutic drugs include, but are not limited to, DNA damaging, DNA-alkylating, DNA-cross-linking, anti-tumor antibiotic, topoisomerase inhibiting, purine inhibiting, pyrimidine inhibiting, microtubule stabilizing, microtubule destabilizing, anti-metabolic, hormone antagonist, protein kinase inhibiting, HMG-CoA inhibiting, metaloproteinase inhibiting, CDK inhibiting, cyclin inhibiting, angiogenesis inhibiting, differentiation enhancing and molecular biologically modified viral, bacterial and extotoxic agents.
  • Routes of administration include, but are not limited to, oral, topical, subcutaneous, transdermal, subdermal, intra-muscular, intra-peritoneal, intra- vesical, intra-articular, intra-prostatic, intra-arterial, intra-venous, intra-dermal, intra-cranial, intra-lesional, intra-tumoral, intra-ocular, intra-pulmonary, intra- spinal, placement within cavities of the body, nasal inhalation and impression into skin. It is to be understood that administration of the compositions of the present invention may occur in vivo, ex vivo or in vitro. For example, the compositions of the present invention may be administered to animal or human cells or tissues in vitro.
  • Appropriate doses for in vitro administration are about 1 nM to 1 mM, preferably about 10 nM to 100 ⁇ M, and more preferably about 100 nM to 10 ⁇ M.
  • the volume per dose is preferably about 0.001 to 100 ml per dose, more preferably about 0.01 to 50 ml per dose and most preferably about 0.1 to 30 ml per dose.
  • a sequence in a pharmaceutically acceptable carrier, or sequence plus therapeutic agent in a pharmaceutically acceptable carrier can be administered in a single dose treatment, in multiple dose treatments or continuously infused on a schedule and over a period of time appropriate to the disease being treated, the condition of the recipient and the route of admimstration.
  • the therapeutic agent can be administered before, concurrently with, or after administration of the sequence.
  • the amount of sequence administered per dose is from about 0.001 to 100 mg/kg, more preferably from about 0.01 to 10 mg/ml and most preferably from about 0.1 to 5 mg/kg.
  • the amount of therapeutic agent administered per dose is from about 0.001 to 1000 mg/m 2 or from about 0.01 to 1000 mg/kg, more preferably from about 0.01 to 500 mg/m 2 or about 0.01 to 500 mg/kg and most preferably from about 0.1 to 100 mg/m 2 or about 0.1 to 100 mg/kg.
  • anti- Fas antibodies are employed and are administered in a dose of from about 0.003 to about 0.3 mg/kg, preferably 0.01 to about 0.1 mg/kg.
  • sequence and the particular therapeutic agent administered should be decided by the practitioner using methods known to those skilled in the art and will depend on the type of disease, the severity of the disease, the location of the disease and other clinical factors such as the size, weight and physical condition of the recipient.
  • in vitro assays may optionally be employed to help identify optimal ranges for sequence and for sequence plus therapeutic agent administration.
  • PBMCs Peripheral blood mononuclear cells
  • Ficoll-Hypaque Amersham Pharmacia Biotech, Baie d'Urfee, Quebec, Canada
  • Cancer cells and PBMCs were seeded in 6 well flat-bottom microplates and were maintained at 37°C in a 5% CO 2 atmosphere. Unless stated otherwise, 2 X 10 5 cells/ml were incubated for 48 h with 0 ⁇ g/ml (control) or 100 ⁇ g/ml (5.5 ⁇ M) (treated) of the sequences.
  • Fas and FasL expression were measured by flow cytometry using anti-Fas FITC-conjugated monoclonal antibodies and anti-FasL PE-conjugated monoclonal antibodies in a FACScalibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA) using the CELLQuest program (Becton Dickinson).
  • Results are expressed as percentage (%) increase in the expression of Fas Example 4 Modulation of Fas and FasL on Jurkat leukemia T cells
  • Jurkat human leukemia T cells are an atypical multi-drug resistant human suspension tumor cell model.
  • Jurkat T cells were incubated with the 6 base sequences shown in Table 2.
  • PBMCs were incubated with the 6 base sequences shown in Table 3.
  • T-24 bladder cancer cells are a p53 mutated human cell line T-24 cells
  • UMUC-3 bladder cancer cells are a P-glycoprotein overexpressing human cell line.
  • UMUC-3 cells 1.0 X 10 ⁇ cells/ml were incubated with the 6 base sequences shown in Table 5.
  • Table 5 Percentage increase of Fas and of FasL on UMUC-3 bladder cancer cells
  • OVCAR-3 ovarian cancer cells are a p53 mutated, p21/waf-l/Cip deleted, metastatic human solid tumor model. OVCAR-3 cells were incubated with the 6 base sequences shown in Table 6. Table 6 Percentage increase of Fas and of FasL on OVCAR-3 ovarian cancer cells
  • SK-OV-3 ovarian cancer cells are a p53 mutated, p21/waf-l/Cip deleted, pl5 ink4B ,pl6 ink4 deleted metastatic human solid tumor model.
  • SK-OV 3 cells (1.0
  • LNCaP prostate cancer cells are a TGF-beta 1 receptor negative, androgen-independent, metastatic human solid tumor model. LNCaP cells were incubated with the 6 base sequences shown in Table 8. Table 8 Percentage increase of Fas and of FasL on LNCaP prostate cancer cells
  • MCF-7 human breast cancer cells are a caspase-3 negative, estrogen-dependent human solid tumor model.
  • MCF-7 cells (1 x 10 5 cells/ml) were incubated with the 6 base sequences shown in Table 9.
  • Jurkat human leukemia T cells were pre-incubated with 0.0 ⁇ g/ml (- CHX) or with 0.1 ⁇ g/ml of cycloheximide (+ CHX) for 1 h.
  • Ten ⁇ g/ml or 100 ⁇ g/ml of 6 base SEQ ID NO:5 (GGGTGG) was added to both the - CHX and + CHX cells and the incubation was continued for 24 hours and for 48 hours (Table 10).
  • SEQ ID NO:5 upregulated Fas expression on Jurkat T cells.
  • cycloheximide decreased Fas expression 92% after 24 h and 44% after 48 h.
  • cycloheximide decreased Fas expression 65% after 24 h and 23% after 48 h.
  • Example 13 Synergistic effect of SEQ ID No: 5 (GGGTGG) and agonistic anti-Fas antibodies on inhibition of UMUC-3 bladder cancer cell proliferation
  • UMUC-3 bladder cancer cells were incubated for 48 hours with 0.00, 0.02 and 0.20 ⁇ g/ml of agonistic anti-Fas monoclonal antibodies (clone CH-11 : Coulter-Immunotech, Marseille, France) + 0 or 10 ⁇ g/ml of SEQ ID NO:5.
  • Cell proliferation was measured using dimethylthiazol-diphenyltetrazolium (MTT) reduction (Mosman et al. J. Immunol. Methods 65:55, 1983). MTT was measured at a wavelength of 570 nm using a multiplate spectrophotometer reader (ELX800, Bio-TEK Instruments Inc., Winooski, VT). Table 11 Inhibition of UMUC-3 bladder cancer cell proliferation
  • 6 base SEQ ID NO: 5 potentiated the inhibitory activity of 0.02 and 0.2 ⁇ g/ml of agonistic anti-Fas antibodies on UMUC-3 cell proliferation.
  • Example 14 Additive effect of SEQ ID NO: 5 (GGGTGG) and agonistic anti-Fas antibodies on inhibition of Jurkat leukemia T cell proliferation.
  • Jurkat T leukemia cells were incubated for 48 hours with 0.00, 0.02 and 0.20 ⁇ g/ml of agonistic anti-Fas monoclonal antibodies (clone CH-11) + 0 and 10 ⁇ g/ml of SEQ ID NO:5.
  • Cell proliferation was measured as in Example 13.
  • mice Effect of 6 base sequences and agonistic anti-Fas antibodies on EL-4 murine T EL-4 murine T lymphoma cells are implanted into C57/BL6 lpr/lpr (Fas negative mice). The mice are divided into 30 groups of 10 mice. On day 0, group 1 mice receive saline, group 2 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:5, group 3 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:16, group 4 receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17, group 5 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 18, group 6 mice receive anti-Fas antibodies, group 7 mice receive 1, 10 or 100 mg/kg of SEQ.
  • anti-Fas antibodies are administered at any dose in the range of about 0.003 to about 0.3 mg/kg.
  • Group 1 mice have the most tumor mass
  • groups 2, 3, 4, 5 and 6 mice have less tumor mass than group 1 mice
  • group 7, 8, 9 and 10 mice have the least tumor mass.
  • the efficacy of the SEQ. ID NO:5, the SEQ. ID NO:16 and the SEQ. ID NO:17 is dose-dependent.
  • Example 16 Female (SJL/J x PL/J) FI mice are injected subcutaneously in both femoral regions with an emulsion containing 0.5 mg of myelin basic protein (MBP) mixed with complete Freund's adjuvant. After 24 h, 400 ng of Bordetella pertussis toxin is administered intraperitoneally. On the day of onset (day 0), group 1 mice receive saline, group 2 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:5, group 3 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:16, group 4 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17, group 5 mice receive 1, 10 or 100 mg/kg of SEQ.
  • MBP myelin basic protein
  • group 6 mice receive anti-Fas antibodies
  • group 7 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:5 + anti-Fas antibodies
  • group 8 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:16 + anti-Fas antibodies
  • group 9 receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17 + anti-Fas antibodies
  • group 10 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 18 + anti-Fas antibodies for 3 days by intracisternal administration (20 ⁇ g/day).
  • anti- Fas antibodies are administered at any dose in the range of about 0.003 to about group 1, 2, 3, 4 and 5 mice.
  • Group 7, 8, 9 and 10 mice show the least progression of EAE.
  • the efficacy of the SEQ. ID NO:5, the SEQ. ID NO:16 and the SEQ. ID NO: 17 is dose-dependent

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Abstract

A method employing a compositon comprising a 2 to 10 base synthetic oligonucleotide sequence selected from the group consisting of (GG)n, (GT)n, at (GT)nb, a (GA)nb, and a (GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, for modulation of Fas and FasL expression or for modulation of the efficacy of therapeutic agents. The composition is administered to an animal or human with a pharmaceutically acceptable carrier, and optionally with a therapeutic agent, in an amount effective to modulate Fas and FasL expression, to treat the disease, or to modulate efficacy of the therapeutic agent.

Description

MODULATION OF FAS AND FASL EXPRESSION
FIELD OF THE INVENTION
The present invention relates compositions useful for modulating Fas and Fas ligand expression on cells and for modulating efficacy of therapeutic agents.
BACKGROUND OF THE INVENTION The Fas (Apo-1, CD95) and Fas ligand (FasL, CD95L) system is one of the best-studied cell death systems. Fas is a type-I membrane protein abundantly expressed by cells in various tissues and particularly on activated T cells, heart cells, kidney cells and hepatocytes. FasL is a type-II transmembrane protein expressed particularly on activated T cells and natural killer cells (Nagata, S. Ann. Rev. Genet. 33:29, 1999), and is expressed constitutively in immune- privileged sites as, for example, the eye and testis (Griffith et al. Science 270:630, 1995).
Fas and FasL interactions (Fas/FasL) play an essential role in the regulation of immune cells and in the elimination of autoreactive cells (Sabelko- Downes et al. Curr. Opin. Immunol. 12:330, 2000). In addition, Fas/FasL mediates the killing of cancer cells and of virus-infected cells (Famularo et al. Med. Hypoth. 53:50, 1999; Owen-Schaub et al. Int. J. Oncol. 17:5, 2000). In contrast, FasL, expressed on cancer cells, may attack cells of the immune system (O'Connell, J. Exp. Med. 184:1075, 1996) or facilitate local tumor invasion by killing surrounding tissue (Yoong et al. Am. J. Pathol. 154:693, 1999). FasL, expressed on activated T cells, may also participate in tissue damage in fulminant hepatitis and in graft- versus-host disease (Kondo et al. Nature Med. 3:409, 1997; Braun et al. J. Exp. Med. 183:657, 1996).
Binding of FasL to Fas, or cross-linking of Fas with agonistic antibodies, induces apoptosis (Nagata, S. Ann. Rev. Genet. 33:29, 1999) that results in cell death. Apoptosis is an active cellular death process characterized by distinctive morphological changes that include condensation of nuclear chromatin, cell shrinkage, nuclear disintegration, plasma membrane blebbing, and the formation of membrane-bound apoptotic bodies (Wyllie et al. Int. Rev. Cytol. 68:251, 1980). A molecular hallmark of apoptosis is degradation of cellular nuclear DNA into oligonucleosomal-length fragments as the result of activation of endogenous endonucleases (Wyllie A. Nature 284:555, 1980). Caspases (cysteine-aspartyl- specific proteases) have been implicated as key enzymes in the execution of the late stage of apoptosis. The binding of FasL to Fas activates a cascade of caspases via a FADD adaptor (Fas-associated protein with death domain), which leads to the cleavage of various cellular substrates and to DNA fragmentation (Nagata, S. Ann. Rev. Genet. 33:29, 1999).
Synthetic oligonucleotides are polyanionic sequences that can be internalized by cells (Nlassov et al. Biochim. Biophys. Acta 1197:95, 1994) and bind selectively to nucleic acids (Wagner, R. Nature: 372:333, 1994), to specific cellular proteins (Bates et al. J. Biol. Chem. 274:26369, 1999) and to specific nuclear proteins (Scaggiante et al. Eur. J. Biochem. 252:207, 1998), and inhibit cell proliferation. Proliferation is the culmination of the progression of a cell through the cell cycle, resulting in the division of one cell into two cells. Alterations in cell cycle progression occur in all cancers and may result from over-expression of genes, mutation of regulatory genes, or abrogation of DNA damage checkpoints (Hochhauser D. Anti-Cancer Chemotherapeutic Agents 8:903, 1997).
Synthetic phosphorothioate oligonucleotides containing unmethylated CpG dinucleotides flanked by two 5' purine and two 3' pyrimidine (CpG motifs) are reported to induce the synthesis of cytokines by macrophages and B cells, to increase the activity of NK cells and cytotoxic T lymphocytes, and to enhance T- Natl. Acad. Sci. USA93:2879, 1996; Lipford et al. Eur. J. Immunol. 27:2340, 1997). A 20 base synthetic CpG motif is reported to block Fas expression on activated B cells and to block apoptosis induced by anti-Fas monoclonal antibodies (Wang et al. Cell. Immunol. 180:162, 1997). Irradiation is reported to upregulate the expression of Fas on cancer cells (Sheard et al. Int. J. Cancer 73:757, 1997; Nishioka et al. Int. J. Mol. Med. 3:275, 1999).
The ability to modulate the Fas/FasL system has many clinical applications for use in diseases including, but not limited to, neoplastic autoimmune, degenerative and cardiovascular diseases. However, most prior Fas/FasL modulating agents have proven to be less than adequate in clinical applications. Moreover, many of these agents are inefficient, toxic or have significant adverse effects.
Therefore, there is a continuing need for novel compositions and methods that modulate the expression of Fas and FasL on cells. There is also a need for novel compositions and methods that modulate the efficacy of Fas and FasL modulatory agents on disease. There is also a need for novel compositions and methods that modulate the expression of Fas and FasL on cells in order to treat diseases in animals or humans associated with altered expression of Fas or FasL on cells.
SUMMARY OF THE INVENTION
The present invention fulfills these needs by providing a novel method, employing new compositions comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is between 2 and 10, preferably 4 to 8, more preferably 5 to 7, and most preferably 6. The present invention provides new uses for these compositions. This composition is combined with a pharmaceutically acceptable carrier, and is FasL expression in the animal or the human. This composition is also combined with a pharmaceutically acceptable carrier, and is administered to an animal or a human having a disease in an amount effective to modulate Fas and FasL expression in order to treat the disease in the animal or the human. This composition is also administered to an animal or human to modulate the efficacy of Fas and FasL modulatory agents, comprising administration of the composition in an amount effective to modulate efficacy of Fas and FasL modulatory agents to modulate Fas and FasL, or to treat an animal or a human having a disease. Another new use for this composition of the present invention is to modulate, and preferentially potentiate, the efficacy of a therapeutic agent to treat a disease, comprising administration of the composition in a pharmaceutically acceptable carrier, optionally in combination with a therapeutic agent, in an amount effective to modulate efficacy of the therapeutic agent administered to an animal or a human. The compositions of the present invention may be administered at any time before, during, or after administration of the therapeutic agent, in order to modulate the efficacy of the therapeutic agent. The compositions of the present invention may also be administered in vitro, for example to animal or human cells or tissues.
In a preferred embodiment, the composition of the present invention comprises a sequence selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof provided the total number of bases in the composition is 6. In another preferred embodiment, the composition of the present invention comprises this sequence of 6 bases in combination with a pharmaceutically acceptable carrier. In yet another preferred embodiment, the composition of the present invention comprises this sequence of 6 bases in combination with a therapeutic agent and a pharmaceutically acceptable carrier. These compositions may be used in any of the methods described in the preceding paragraphs and throughout the specification.
The unexpected and surprising ability of the sequences of the present unfulfilled need in the medical arts and provides an important benefit for animals, including humans.
Accordingly, an object of the present invention is to provide a new composition comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)„b, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 2 to 10. Another object of the present invention is to provide a new composition comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 4 to 8.
Yet another object of the present invention is to provide a new composition comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 5 to 7.
Another object of the present invention is to provide a new composition comprising a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)„b, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is 6.
Yet another object of the present invention is to provide novel compositions comprising any of the sequences described herein, in combination with a pharmaceutically acceptable carrier. Another object of the present invention is to provide novel compositions comprising any of the sequences described herein, in combination with a therapeutic agent and a pharmaceutically acceptable carrier.
Yet another object of the present invention is to provide for the use of any of the novel compositions described herein for the manufacture of a medicament.
Still another object of the present invention is to provide new uses for the compositions of the present invention.
Yet another object of the present invention is to provide a method to modulate the Fas expression on cells. Another object of the present invention is to provide a method to modulate
FasL expression on cells.
Still another object of the present invention is to provide a method to modulate the Fas expression on immune cells.
Another object of the present invention is to provide a method to modulate FasL expression on immune cells.
Yet another object of the present invention is to provide a method to modulate Fas expression on cancer cells.
Another object of the present invention is to provide a method to modulate FasL expression on cancer cells. Another object of the present invention is to provide a method to treat a disease in an animal, including a human.
Yet another object of the present invention is to provide a method to treat a neoplastic disease.
Still another object of the present invention is to provide a method to treat an autoimmune disease.
Another object of the present invention is to provide a method to treat an inflammatory disease.
Yet another object of the present invention is to provide a method to treat a proliferative disease Another object of the present invention is to provide a method to treat a lymphoproliferative disease. Yet another object of the present invention is to provide a method to treat a degenerative disease.
Still another object of the present invention is to provide a method to treat a neurodegenerative disease. Another object of the present invention is to provide a method to treat a cardiovascular disease
Yet another object of the present invention is to provide a method to treat a graft rejection.
Another object of the present invention is to provide a method effective to treat an infection.
Yet another object of the present invention is to provide a method that modulates the effect of a therapeutic agent to treat disease.
Yet another object of the present invention is to provide a method that potentiates the effect of a therapeutic agent to treat disease. Still another object of the present invention is to provide a method that potentiates the effect of a Fas modulating agent.
Another object of the present invention is to provide a method that potentiates the effect of a FasL modulating agent.
Yet another object of the present invention is to provide a method that potentiates the effect of an anti-neoplastic agent.
Another object of the present invention is to provide a method that potentiates the effect of an immunostimulatory agent.
Still another object of the present invention is to provide a method that potentiates the effect of an immunosuppressive agent. Another object of the present invention is to provide a method that potentiates the effect of an anti-inflammatory agent.
It is another object of the present invention to provide a composition that is simple to prepare.
Another object of the present invention is to provide a composition that is minimally toxic to the recipient. These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiment and the appended claims.
DETAILED DESCRIPTION
The present invention fulfills these needs by providing new compositions and new methods of using these compositions. The present compositions comprise a synthetic phosphodiester oligodeoxynucleotide (hereinafter, "sequence") selected from the group consisting of (GG)n, (GT)n, a(GT)„b, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof, wherein the total number of bases in the composition is between 2 and 10, preferably 4 to 8, more preferably 5 to 7, and most preferably 6. The compositions of the present invention further comprise any of the sequences described herein in combination with a pharmaceutically acceptable carrier. The compositions of the present invention further comprise any of the sequences described herein in combination with a therapeutic agent and a pharmaceutically acceptable carrier.
The present invention provides new uses for these novel compositions. This composition is combined with a pharmaceutically acceptable carrier, and is administered to an animal or a human an amount effective to modulate Fas and FasL expression in the animal or the human. This composition is also combined with a pharmaceutically acceptable carrier, and is administered to an animal or a human having a disease in an amount effective to modulate Fas and FasL expression in order to treat the disease in the animal or the human. This composition is also administered to an animal or human to modulate the efficacy of Fas and FasL modulatory agents, comprising administration of the composition in an amount effective to modulate efficacy of Fas and FasL modulatory agents to modulate Fas and FasL, or to treat an animal or a human having a disease. Another new use for this composition of the present invention is to modulate, and preferentially potentiate, the efficacy of a therapeutic agent to treat a disease, carrier, optionally in combination with a therapeutic agent, in an amount effective to modulate efficacy of the therapeutic agent administered to an animal or a human. The composition of the present invention may be administered at any time before, during, or after administration of the therapeutic agent, in order to modulate the efficacy of the therapeutic agent.
In a preferred embodiment the composition of the present invention comprises a sequence selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof provided the total number of bases in the composition is 6. In another preferred embodiment, the composition of the present invention comprises this sequence of 6 bases in combination with a pharmaceutically acceptable carrier. In yet another preferred embodiment, the composition of the present invention comprises this sequence of 6 bases in combination with a therapeutic agent and a pharmaceutically acceptable carrier. These compositions may be used in any of the methods described in this patent application.
As used herein the word "sequence" refers to a synthetic phosphodiester oligodeoxynucleotide comprised of adenine (A), cytosine (C), guanine (G) and thymine (T), with a total number of bases of 2 to 10, preferably 4 to 8, more preferably 5 to 7 and most preferably 6.
As used herein, the word "expression" refers to the cell surface concentration of Fas or of FasL.
As used herein, the words "response" refers to upregulation (increase) or downregulation (decrease) of Fas or of Fas L expression. As used herein, the word "modulates" refers to changes in the expression of Fas or of FasL. Such changes include upregulation and downregulation of Fas or of FasL expression. The word modulate is also employed to describe the ability of the novel sequences of the present invention to modulate the efficacy of therapeutic agents, including Fas and FasL modulating agents, to treat disease or to modulate Fas or FasL expression. As used herein, the phrases "therapeutically effective", "effective amount" and "amount effective to" refer to an amount of a sequence effective to modulate the expression of Fas or of FasL.
As used herein, the word "disease" refers to a condition wherein bodily health is impaired. >
As used herein, the phrase "therapeutic agent" is any agent approved by a regulatory agency of a country or a state government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use to treat a disease in an animal, including a human. As used herein, the word "antineoplastic" refers to preventing the development, maturation, proliferation or spread of cancer cells
As used herein, the word "potentiates" refers to a degree of synergism that is greater than additive.
As used herein, the word "synergism" refers to the coordinated action of two or more agents.
Administration of an effective amount of a sequence of the present invention to an animal, including a human, is a therapeutic treatment that prevents, treats or eliminates a disease including, but not limited to, neoplastic, autoimmune, proliferative, lymphoproliferative, degenerative, and cardiovascular disease; infection; inflammation; and, graft, tissue and cell rejection.
Compositions comprising one or more sequences and a pharmaceutically acceptable carrier are prepared by uniformly and intimately bringing into association the sequence and the pharmaceutically acceptable carrier. Compositions comprising one or more sequences, a therapeutic agent and a pharmaceutically acceptable carrier are prepared by uniformly and intimately bringing into association the sequence, the therapeutic agent and the pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include liquid carriers, solid carriers or both. Liquid carriers are aqueous carriers, non- aqueous carriers or both and include, but are not limited to, solutions, suspensions and emulsions. Emulsions include, but are not limited to, oil emulsions, water in oil emulsions, water-in-oil-in-water emulsions, site-specific emulsions, long- carriers are biological carriers, chemical carriers or both and include, but are not limited to, viral vector systems, plasmids, particles, microparticles, nanoparticles, microspheres, nanospheres, bacterial cell walls, minipumps, and biodegradable or non-biodegradable natural or synthetic polymers that allow for sustained release of the sequences.
Preferred aqueous carriers include, but are not limited to, water, saline and pharmaceutically acceptable buffers. Preferred non-aqueous carriers include, but are not limited to, a mineral oil or a neutral oil including, but not limited to, a diglyceride, a triglyceride, a phospholipid, a lipid, an oil and mixtures thereof, wherein the oil contains an appropriate mix of polyunsaturated and saturated fatty acids. Optionally, stabilizing agents and excipients may be included regardless of the pharmaceutically acceptable carrier used to present the sequence to the cells.
The therapeutic effectiveness of a sequence may be increased by methods including, but not limited to, chemically modifying the base, sugar or phosphate backbone, chemically supplementing or biotechnologically amplifying the sequences using bacterial plasmids containing the appropriate sequences, complexing the sequences to biological or chemical carriers or coupling the sequences to tissue-type or cell-type directed ligands or antibodies.
The composition of the present invention further comprises a composition comprising a sequence and a therapeutic agent, wherein, when the sequence and the therapeutic agent are combined with a pharmaceutically acceptable carrier and administered to an animal or human having a disease. The sequence modulates and preferentially potentiates the effect of the therapeutic agent on the disease.
Therapeutic agents include, but not limited to, anti-neoplastic, anti- inflammatory, anti-autoimmune, anti-degenerative, Fas modulating and FasL modulating agents, or any combination thereof, and radiation therapy, or a combination of radiation therapy with therapeutic agents. These therapeutic agents include, but are not limited to, biologicals, drugs, chemotherapeutic drugs, immunostimulants, immunomodulators, immunotherapeutics, anti-virals, anti- infectives, antibiotics, cytokines, antigens, antibodies, nucleic acids, vaccines, aptabases, nucleic acids, antisense nucleic acids, telomerase inhibitors, caspase biologically engineered and chemically synthesized agents, and agents that target cell death molecules for activation or inactivation.
Chemotherapeutic drugs include, but are not limited to, DNA damaging, DNA-alkylating, DNA-cross-linking, anti-tumor antibiotic, topoisomerase inhibiting, purine inhibiting, pyrimidine inhibiting, microtubule stabilizing, microtubule destabilizing, anti-metabolic, hormone antagonist, protein kinase inhibiting, HMG-CoA inhibiting, metaloproteinase inhibiting, CDK inhibiting, cyclin inhibiting, angiogenesis inhibiting, differentiation enhancing and molecular biologically modified viral, bacterial and extotoxic agents. Routes of administration include, but are not limited to, oral, topical, subcutaneous, transdermal, subdermal, intra-muscular, intra-peritoneal, intra- vesical, intra-articular, intra-prostatic, intra-arterial, intra-venous, intra-dermal, intra-cranial, intra-lesional, intra-tumoral, intra-ocular, intra-pulmonary, intra- spinal, placement within cavities of the body, nasal inhalation and impression into skin. It is to be understood that administration of the compositions of the present invention may occur in vivo, ex vivo or in vitro. For example, the compositions of the present invention may be administered to animal or human cells or tissues in vitro. Appropriate doses for in vitro administration are about 1 nM to 1 mM, preferably about 10 nM to 100 μM, and more preferably about 100 nM to 10 μM. Depending on the route of administration, the volume per dose is preferably about 0.001 to 100 ml per dose, more preferably about 0.01 to 50 ml per dose and most preferably about 0.1 to 30 ml per dose. A sequence in a pharmaceutically acceptable carrier, or sequence plus therapeutic agent in a pharmaceutically acceptable carrier, can be administered in a single dose treatment, in multiple dose treatments or continuously infused on a schedule and over a period of time appropriate to the disease being treated, the condition of the recipient and the route of admimstration. Moreover, the therapeutic agent can be administered before, concurrently with, or after administration of the sequence.
Preferably, the amount of sequence administered per dose is from about 0.001 to 100 mg/kg, more preferably from about 0.01 to 10 mg/ml and most preferably from about 0.1 to 5 mg/kg. A sequence plus a chemotherapeutic agent and preferentially potentiate, the effect of the therapeutic agent. Preferably, the amount of therapeutic agent administered per dose is from about 0.001 to 1000 mg/m2 or from about 0.01 to 1000 mg/kg, more preferably from about 0.01 to 500 mg/m2 or about 0.01 to 500 mg/kg and most preferably from about 0.1 to 100 mg/m2 or about 0.1 to 100 mg/kg. In one embodiment of a therapeutic agent, anti- Fas antibodies are employed and are administered in a dose of from about 0.003 to about 0.3 mg/kg, preferably 0.01 to about 0.1 mg/kg.
The particular sequence and the particular therapeutic agent administered, the amount per dose, the dose schedule and the route of administration should be decided by the practitioner using methods known to those skilled in the art and will depend on the type of disease, the severity of the disease, the location of the disease and other clinical factors such as the size, weight and physical condition of the recipient. In addition, in vitro assays may optionally be employed to help identify optimal ranges for sequence and for sequence plus therapeutic agent administration.
The following examples will serve to further illustrate the present invention without, at the same time, however, constituting any limitation thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the invention.
Example 1 Preparation of sequences Phosphodiester sequences were prepared by Sigma-Genosys (Woodlands,
TX) using Abacus Segmented Synthesis Technology. Unless stated otherwise, the sequences were dispersed in autoclaved deionized water or in a pharmaceutically acceptable buffer such as, but not limited to, saline immediately prior to use.
Example 2
Cells All cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in the medium recommended by the ATCC. Table 1 shows the cell lines, their origins and their properties.
Table 1 Cell lines
Figure imgf000015_0001
Peripheral blood mononuclear cells (hereinafter, "PBMCs") were isolated from human blood by Ficoll-Hypaque (Amersham Pharmacia Biotech, Baie d'Urfee, Quebec, Canada) density gradient centrifugation. Cancer cells and PBMCs were seeded in 6 well flat-bottom microplates and were maintained at 37°C in a 5% CO2 atmosphere. Unless stated otherwise, 2 X 105 cells/ml were incubated for 48 h with 0 μg/ml (control) or 100 μg/ml (5.5 μM) (treated) of the sequences.
Example 3
Measurement of Fas and of FasL at the cell surface
Fas and FasL expression were measured by flow cytometry using anti-Fas FITC-conjugated monoclonal antibodies and anti-FasL PE-conjugated monoclonal antibodies in a FACScalibur Flow Cytometer (Becton Dickinson, San Jose, CA, USA) using the CELLQuest program (Becton Dickinson).
Results are expressed as percentage (%) increase in the expression of Fas Example 4 Modulation of Fas and FasL on Jurkat leukemia T cells
Jurkat human leukemia T cells are an atypical multi-drug resistant human suspension tumor cell model. Jurkat T cells were incubated with the 6 base sequences shown in Table 2.
Table 2 Percentage increase of Fas and of FasL on Jurkat human leukemia T cells
Figure imgf000016_0001
Example 5 Modulation of Fas and of FasL on PBMCs
PBMCs were incubated with the 6 base sequences shown in Table 3.
Table 3 Percentage increase of Fas and of FasL on PBMCs
Figure imgf000017_0001
Example 6 Modulation of Fas and of FasL on T-24 bladder cancer cells T-24 bladder cancer cells are a p53 mutated human cell line T-24 cells
(1.0 X 1Q5 cells/ml) were incubated with the 6 base sequences shown in Table 4. Table 4 Percentage increase of Fas and of FasL on T-24 bladder cancer cells
Figure imgf000018_0001
Example 7 Modulation of Fas and of FasL on UMUC-3 bladder cancer cells
UMUC-3 bladder cancer cells are a P-glycoprotein overexpressing human cell line. UMUC-3 cells (1.0 X 10^ cells/ml) were incubated with the 6 base sequences shown in Table 5. Table 5 Percentage increase of Fas and of FasL on UMUC-3 bladder cancer cells
Figure imgf000019_0001
Example 8 Modulation of Fas and of FasL on OVCAR-3 ovarian cancer cells
OVCAR-3 ovarian cancer cells are a p53 mutated, p21/waf-l/Cip deleted, metastatic human solid tumor model. OVCAR-3 cells were incubated with the 6 base sequences shown in Table 6. Table 6 Percentage increase of Fas and of FasL on OVCAR-3 ovarian cancer cells
Figure imgf000020_0001
Example 9 Modulation of Fas and of FasL on SK-OV 3 ovarian cancer cells
SK-OV-3 ovarian cancer cells are a p53 mutated, p21/waf-l/Cip deleted, pl5ink4B,pl6ink4 deleted metastatic human solid tumor model. SK-OV 3 cells (1.0
X 10^ cells/ml) were incubated with the 6 base sequences shown in Table 7. Table 7 Percentage increase of Fas and FasL on SK-OV-3 ovarian cancer cells
Figure imgf000021_0001
Example 10 Modulation of Fas and of FasL on LNCaP prostate cancer cells
LNCaP prostate cancer cells are a TGF-beta 1 receptor negative, androgen-independent, metastatic human solid tumor model. LNCaP cells were incubated with the 6 base sequences shown in Table 8. Table 8 Percentage increase of Fas and of FasL on LNCaP prostate cancer cells
Figure imgf000022_0001
Example 11 Modulation of Fas and of FasL on MCF-7 breast cancer cells
MCF-7 human breast cancer cells are a caspase-3 negative, estrogen- dependent human solid tumor model. MCF-7 cells (1 x 105 cells/ml) were incubated with the 6 base sequences shown in Table 9. Table 9 Percentage increase of Fas and of FasL on MCF-7 breast cancer cells
Figure imgf000023_0001
Example 12 Inhibition of Fas expression on Jurkat human leukemia T cells by cycloheximide
Jurkat human leukemia T cells were pre-incubated with 0.0 μg/ml (- CHX) or with 0.1 μg/ml of cycloheximide (+ CHX) for 1 h. Ten μg/ml or 100 μg/ml of 6 base SEQ ID NO:5 (GGGTGG) was added to both the - CHX and + CHX cells and the incubation was continued for 24 hours and for 48 hours (Table 10). Table 10 Percentage increase of Fas on Jurkat human leukemia T cells
Figure imgf000024_0001
As shown in Table 10, SEQ ID NO:5 upregulated Fas expression on Jurkat T cells. With 10 μg/ml of SEQ ID NO:5, cycloheximide decreased Fas expression 92% after 24 h and 44% after 48 h. With 100 μg/ml of SEQ ID NO: 5 cycloheximide decreased Fas expression 65% after 24 h and 23% after 48 h. These data suggest that 6 base SEQ ID NO:5 stimulates de novo synthesis of Fas.
Example 13 Synergistic effect of SEQ ID No: 5 (GGGTGG) and agonistic anti-Fas antibodies on inhibition of UMUC-3 bladder cancer cell proliferation
UMUC-3 bladder cancer cells were incubated for 48 hours with 0.00, 0.02 and 0.20 μg/ml of agonistic anti-Fas monoclonal antibodies (clone CH-11 : Coulter-Immunotech, Marseille, France) + 0 or 10 μg/ml of SEQ ID NO:5. Cell proliferation was measured using dimethylthiazol-diphenyltetrazolium (MTT) reduction (Mosman et al. J. Immunol. Methods 65:55, 1983). MTT was measured at a wavelength of 570 nm using a multiplate spectrophotometer reader (ELX800, Bio-TEK Instruments Inc., Winooski, VT). Table 11 Inhibition of UMUC-3 bladder cancer cell proliferation
Figure imgf000025_0001
As shown in Table 11, 6 base SEQ ID NO: 5 potentiated the inhibitory activity of 0.02 and 0.2 μg/ml of agonistic anti-Fas antibodies on UMUC-3 cell proliferation.
Example 14 Additive effect of SEQ ID NO: 5 (GGGTGG) and agonistic anti-Fas antibodies on inhibition of Jurkat leukemia T cell proliferation.
Jurkat T leukemia cells were incubated for 48 hours with 0.00, 0.02 and 0.20 μg/ml of agonistic anti-Fas monoclonal antibodies (clone CH-11) + 0 and 10 μg/ml of SEQ ID NO:5. Cell proliferation was measured as in Example 13.
Table 12 Inhibition of Jurkat human leukemia T cell proliferation
Figure imgf000025_0002
As shown in Table 12, the effects of 6 base SEQ ID NO:5 and of 0.02 and 0.2 μg/ml of agonistic anti-Fas antibodies on Jurkat T cell proliferation were additive.
Example 15
Effect of 6 base sequences and agonistic anti-Fas antibodies on EL-4 murine T EL-4 murine T lymphoma cells are implanted into C57/BL6 lpr/lpr (Fas negative mice). The mice are divided into 30 groups of 10 mice. On day 0, group 1 mice receive saline, group 2 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:5, group 3 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:16, group 4 receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17, group 5 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 18, group 6 mice receive anti-Fas antibodies, group 7 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 5 + anti-Fas antibodies, group 8 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 16 + anti-Fas antibodies, group 9 receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17 + anti-Fas antibodies, group 10 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 18 + anti-Fas antibodies. In these different groups, anti-Fas antibodies are administered at any dose in the range of about 0.003 to about 0.3 mg/kg. Group 1 mice have the most tumor mass, groups 2, 3, 4, 5 and 6 mice have less tumor mass than group 1 mice, and group 7, 8, 9 and 10 mice have the least tumor mass. The efficacy of the SEQ. ID NO:5, the SEQ. ID NO:16 and the SEQ. ID NO:17 is dose-dependent.
Example 16 Female (SJL/J x PL/J) FI mice are injected subcutaneously in both femoral regions with an emulsion containing 0.5 mg of myelin basic protein (MBP) mixed with complete Freund's adjuvant. After 24 h, 400 ng of Bordetella pertussis toxin is administered intraperitoneally. On the day of onset (day 0), group 1 mice receive saline, group 2 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:5, group 3 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:16, group 4 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17, group 5 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 18, group 6 mice receive anti-Fas antibodies, group 7 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:5 + anti-Fas antibodies, group 8 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO:16 + anti-Fas antibodies, group 9 receive 1, 10 or 100 mg/kg of SEQ. ID NO: 17 + anti-Fas antibodies, group 10 mice receive 1, 10 or 100 mg/kg of SEQ. ID NO: 18 + anti-Fas antibodies for 3 days by intracisternal administration (20 μg/day). In these different groups, anti- Fas antibodies are administered at any dose in the range of about 0.003 to about group 1, 2, 3, 4 and 5 mice. Group 7, 8, 9 and 10 mice show the least progression of EAE. The efficacy of the SEQ. ID NO:5, the SEQ. ID NO:16 and the SEQ. ID NO: 17 is dose-dependent

Claims

CLAIMSWe claim:
1. A composition comprising a synthetic oligonucleotide sequence of 2 to 10 bases, selected from the group consisting of (GG)n, (GT)n, a(GT)nb, a(GA)nb, and a(GC)nb, wherein n is an integer between 1 and 3, and a and b are independently either none or one or more As, Cs, Gs, or Ts, or combinations thereof.
2. The composition of Claim 1, wherein the sequence is 4 to 8 bases.
3. The composition of Claim 1, wherein the sequence is 5 to 7 bases.
4. The composition of Claim 1, wherein the sequence is 6 bases.
5. The composition of Claim 1, wherein the sequence is any one of SEQ ID NO: l to SEQ ID NO.Tδ.
6. The composition of any one of Claims 1 to 5, further comprising a pharmaceutically acceptable carrier.
7. The composition of any one of Claims 1 to 5, further comprising a therapeutic agent.
8. A method of modulating Fas or FasL expression in an animal or a human comprising administration of any one of Claims 1 to 5 and a pharmaceutically acceptable carrier to the animal or the human in an amount effective to modulate Fas or FasL expression in the animal or the human.
9. A method of modulating efficacy of a therapeutic agent in an animal or human comprising administration of an effective amount of the composition of any one of Claims 1 to 7, before, concurrently with, or after administration of the therapeutic agent, wherein the amount is effective to modulate efficacy of the therapeutic agent.
10. The method of Claims 8 or 9, wherein the animal or human has a disease.
11. The method of Claim 10, wherein the disease is cancer.
12. The method of Claim 11, wherein the cancer is selected from the group consisting of ovarian, prostate, breast, bladder, and leukemia.
1 3 . The method of Claim 10, wherein the disease is autoimmune
14. The method of Claim 8 or 9, wherein the animal or human has inflammation, infection, graft rejection, tissue rejection or cell rejection.
15. Use of any one of the compositions of Claims 1 to 5 in the manufacture of a medicament.
16. Use of any one of the compositions of Claims 1 to 7 for modulation of Fas or FasL expression in cells of an animal or a human, comprising administration to the animal or the human of any one of the compositions of Claims 1 to 7 in an amount effective to modulate Fas or FasL expression in the cells of the animal or the human.
17. Use of any one of the compositions of Claims 1 to 6 for modulation of efficacy of a therapeutic agent comprising administration to the animal or the human of any one of the compositions of Claims 1 to 6 in an amount effective to modulate efficacy of the therapeutic agent.
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AT01947067T ATE550428T1 (en) 2000-08-29 2001-06-12 MODULATION OF FAS AND FASL EXPRESSION BY A SYNTHETIC OLIGONUCLEOTIDE AND AN ANTI-FAS ANTIBODY
ES01947067T ES2383671T3 (en) 2000-08-29 2001-06-12 Modulation of Fas and FasL expression by a synthetic oligonucleotide phosphodiester and an anti-Fas antibody
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