WO2006122409A1 - Antimicrobial molecules and their uses - Google Patents

Antimicrobial molecules and their uses Download PDF

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Publication number
WO2006122409A1
WO2006122409A1 PCT/CA2006/000798 CA2006000798W WO2006122409A1 WO 2006122409 A1 WO2006122409 A1 WO 2006122409A1 CA 2006000798 W CA2006000798 W CA 2006000798W WO 2006122409 A1 WO2006122409 A1 WO 2006122409A1
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oligonucleotide
formulation
nucleotides
sequence
length
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PCT/CA2006/000798
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French (fr)
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Andrew Vaillant
Jean-Marc Juteau
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Replicor Inc.
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Publication of WO2006122409A1 publication Critical patent/WO2006122409A1/en

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    • 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
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • 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
    • C12N2310/17—Immunomodulatory nucleic acids
    • 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
    • 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
    • C12N2310/32—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
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • the present invention relates with the identification and use of antimicrobial oligonucleotides that act by a sequence independent mechanism, and includes the discovery that the antimicrobial activity is greater for larger oligonucleotides.
  • Microbial infections affect humans and animals and are an important cause of morbidity around the world.
  • Microbial infectious agents include protozoan parasites, bacteria and fungi for which antimicrobial agents are often available.
  • antimicrobial agents are associated with undesirable side effects and the problem of microbial resistance to such agents is a growing problem.
  • Malaria is a serious and sometimes fatal disease caused by a protozoan parasite. Patients with malaria typically are very sick with high fevers, shaking chills, and flu-like illness.
  • malaria parasites can infect humans: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. Malaria is a leading cause of death and disease worldwide, especially in developing countries. There is currently no malaria vaccine approved for human use.
  • Treatment options include: chloroquine, sulfadoxine-pyrimethamine, mefloquine, atovaquone-proguanil, quinine, doxycycline, artemisin derivatives and primaquine.
  • Pathogenic microorganisms such as protozoan parasites, bacteria and fungi, use different pathogenesis or virulence factors to colonize hosts. Adherence to the host tissue or cell is an important factor in the initiation of pathogenesis. Also, the adherence to the cell surface can initiate the entry of microbial pathogens into the host cell.
  • oligonucleotides mainly antisense ONs
  • a specific antisense effect could not be demonstrated over three cycles of the parasite.
  • Both the 18mer antisense and sense oligonucleotides inhibited invasion of red blood cells at similar concentrations to dextran sulfate as determined by microscopy and [ HJhypoxanthine incorporation into DNA (Kanagaratnam et al, 1998, Int. J. Biochem. Cell Biol. 30: 979-985).
  • PS-ONs phosphosphorothioate-oligonucleotides
  • the invention relates to oligonucleotides (ONs) acting predominantly by a sequence independent mode of action for the treatment of microbial infections.
  • the invention also relates to ONs and their use as therapeutic agents, and more particularly for their use in methods of treatment and formulations for the treatment of diseases involving infection by microorganisms.
  • an antimicrobial oligonucleotide formulation comprising at least one oligonucleotide having an antimicrobial activity against a target microorganism, said activity occurring principally by a sequence independent mode of action.
  • the oligonucleotide formulation further comprises at least one delivery system.
  • the oligonucleotide formulation of the present invention comprises an oligonucleotide of at least 15 nucleotides in length; 20 nucleotides in length; 25 nucleotides in length; 30 nucleotides in length; 35 nucleotides in length; preferably 40 nucleotides in length; 45 nucleotides in length; 50 nucleotides in length; more preferably 60 nucleotides in length; or 80 nucleotides in length.
  • the oligonucleotide formulation comprises an oligonucleotide of 20-30 nucleotides in length; 30-40 nucleotides in length; preferably 40-50 nucleotides in length; 50-60 nucleotides in length; more preferably 60-70 nucleotides in length; or 70-80 nucleotides in length.
  • the oligonucleotide formulation of the present invention comprises an oligonucleotide which is not complementary to any equal length portion of a microbial genomic sequence. More preferably, the genomic sequence is of a human or of a non human animal.
  • an oligonucleotide formulation comprising an oligonucleotide containing at least 10 contiguous nucleotides of randomer sequence; more preferably 20 nucleotides of randomer sequence; 30 nucleotides of randomer sequence; or 40 nucleotides of randomer sequence.
  • the oligonucleotide formulation of the present invention comprises a randomer oligonucleotide.
  • the oligonucleotide formulation comprises an oligonucleotide having a homopolymer sequence of at least 10 contiguous A nucleotides; 10 contiguous T nucleotides; 10 contiguous U nucleotides; 10 contiguous G nucleotides; 10 contiguous I nucleotide analogs; or 10 contiguous C nucleotides.
  • the oligonucleotide formulation comprises an oligonucleotide which is a homopolymer sequence of C nucleotides.
  • the oligonucleotide formulation comprises an oligonucleotide having a poly AT sequence at least 10 nucleotides in length; a poly AC sequence at least 10 nucleotides in length; a poly AG sequence at least 10 nucleotides in length; a polyAU sequence at least 10 nucleotides in length; a poly AI sequence at least 10 nucleotides in length; a polyGC sequence at least 10 nucleotides in length; a polyGT sequence at least 10 nucleotides in length; a polyGU sequence at least 10 nucleotides in length; a polyGI sequence at least 10 nucleotides in length; a polyCT sequence at least 10 nucleotides in length; a polyCU sequence at least 10 nucleotides in length; a polyCI sequence at least 10 nucleotides in length; a polyTI sequence at least 10 nucleotides in length; a polyTU sequence at least 10 nucleotides in
  • the oligonucleotide formulation comprises an oligonucleotide having at least one phosphodiester linkage. [0022] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one ribonucleotide.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one modification to its chemical structure, more preferably at least two different modifications to its chemical structure.
  • an oligonucleotide formulation comprising an oligonucleotide having at least one sulfur modification.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorothioated linkage; at least one phosphorodithioated linkage; or at least one boranophosphate linkage.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one sulfur modified nucleobase moiety such as one sulfur modified ribose moiety, one 2' modification to the ribose moiety, one 2'-0 alkyl modified ribose moiety, one 2'-0 methyl modified ribose, one 2'-methoxyethyl modified ribose, or one 2'-FANA modified ribose.
  • one sulfur modified nucleobase moiety such as one sulfur modified ribose moiety, one 2' modification to the ribose moiety, one 2'-0 alkyl modified ribose moiety, one 2'-0 methyl modified ribose, one 2'-methoxyethyl modified ribose, or one 2'-FANA modified ribose.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one methylphosphonate linkage.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphorothioate backbone.
  • GAA glycol nucleic acid
  • the oligonucleotide formulation comprises an oligonucleotide having at least one locked nucleic acid portion.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorodiamidate morpholino portion.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one abasic nucleic acid.
  • the oligonucleotide formulation comprises an oligonucleotide having a linker to form a concatemer of two or more oligonucleotide sequences.
  • the oligonucleotide formulation of the present invention comprises an oligonucleotide linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, an improved ability to be formulated, a detectable signal, higher antimicrobial activity, better pharmacokinetic properties, specific tissue distribution and lower toxicity.
  • the oligonucleotide formulation comprises an oligonucleotide linked or conjugated to a PEG molecule; or linked or conjugated to a cholesterol molecule.
  • the oligonucleotide formulation comprises an oligonucleotide linked or conjugated to a cholesterol molecule.
  • the oligonucleotide formulation comprises a double stranded oligonucleotide.
  • the oligonucleotide formulation comprises an oligonucleotide having at least one base which is capable of hybridizing via non- Watson-Crick interactions.
  • the oligonucleotide formulation comprises an oligonucleotide having a portion complementary to a genome.
  • the oligonucleotide formulation comprises an oligonucleotide that binds to one or more cellular components.
  • the oligonucleotide formulation comprises an oligonucleotide that interacts with one or more cellular components, wherein said interaction resulting in inhibition of a protein activity or expression.
  • the oligonucleotide formulation comprises an oligonucleotide wherein at least a portion of the sequence of said oligonucleotide is derived from a genome.
  • the oligonucleotide of the present invention has at most 90%, preferably 80%, more preferably 75% identity with the genomic sequence.
  • an oligonucleotide formulation comprising an oligonucleotide that targets a protozoan parasite; Plasmodium sp; Plasmodium falciparum; or a bacterium. More preferably, the oligonucleotide targets Escherichia sp; Streptococcus sp; a fungus.
  • the oligonucleotide mixture comprises a mixture of at least two different oligonucleotides. More preferably, the oligonucleotide formulation of the present invention comprises a mixture of at least ten different oligonucleotides or at least 100 different oligonucleotides; or at least 1000 different oligonucleotides; or at least 10 6 different oligonucleotides.
  • an antimicrobial pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to the present invention; and a pharmaceutically acceptable carrier. More preferably, the antimicrobial pharmaceutical composition is adapted for the treatment, control, or prevention of a microbial infection disease.
  • the microbial infection disease is a protozoan parasite infection; is malaria; a bacterial infection.
  • an antimicrobial pharmaceutical composition according to the present invention, adapted for delivery by a mode selected from the group consisting of ocular administration, eye drop administration, oral ingestion, subcutaneous injection, intramuscular injection, and intravenous injection.
  • the antimicrobial pharmaceutical composition further comprises a delivery system; at least one other antimicrobial drug in combination; a non-nucleotidic antimicrobial in combination; an antimicrobial antisense oligonucleotide in combination; more preferably an antimicrobial RNAi- inducing oligonucleotide.
  • said microbial infection disease is a protozoan parasite infection; is malaria; is a bacterial infection; is a fungal infection.
  • said subject is a human or a non-human animal.
  • a method for the prophylaxis or treatment of a microbial infection disease in a subject comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to the present invention, or antimicrobial pharmaceutical composition according to any the present invention.
  • said microbial infection disease is a protozoan parasite infection; is malaria; is a bacterial infection; is a fungal infection.
  • said subject is a human or a non-human animal.
  • the present invention is concerned with the identification and use of antimicrobial ONs that act by a sequence independent mechanism, and includes the discovery that the antimicrobial activity is greater for larger ONs.
  • antisense or aptameric ONs have been tested for antimicrobial activity.
  • antisense or aptameric ONs are typically sequence-specific and target either intracellular mRNA or a protein, and typically are about 16-25 nucleotides in length.
  • the antimicrobial effect of randomer ONs is sequence independent. Considering the volumes and concentrations of ONs used in these tests, it is theoretically impossible that a particular sequence is present at more than 1 copy in the mixture. This means than there can be no antisense or sequence-specific aptameric effect in these ONs randomers. In all examples, should the microbial inhibition effect be caused by the sequence-specificity of the ONs, such effect would thus have to be caused by only one molecule, a result that does not appear possible. For example, for an ON randomer 40 bases in length, any particular sequence in the population would theoretically represent only 1/4 40 or 1/8.27X10 "25 of the total fraction.
  • ONs appears to have potentially broad-spectrum activity against many types of diseases involving microbial infection. Therefore to test this hypothesis, several ON randomers of different sizes and chemical type were selected to be tested in various microorganisms.
  • the present invention discloses that ONs have an antimicrobial activity that is sequence independent, dependent on size, and chemical modification. These results suggest that ONs have an antimicrobial activity by inhibiting adherence of microorganisms to the cell and/or inhibiting entry of microorganisms into cells.
  • a modification of the ON such as, but not limited to, a phosphorothioate modification or other sulfur modifications, appears to be beneficial for antimicrobial activity.
  • Such sulfur modifications may include without restriction mono and diphosphorothioation of the phosphodiester linkage, 4' or 5' thiolation of the uracil moiety, 5' thiolation of the cytidine moiety, 2' or 4' thiolation of the thymine moiety, 6' thiolation of the guanine moiety, sulfur modifications to any other nucleobase moiety and sulfur modifications to the ribose moiety of any nucleotide.
  • ONs may have more than one sulfur substitution on each nucleotide, which can potentially increase the activity.
  • any single or multiple sulfur substitution may be combined with other modifications known to improve properties of ONs.
  • ONs of this invention may also have chemical modifications including without restriction: any 2' ribose modification including 2'-0 methyl, 2'-fluorine, 2'-FANA, 2'-methoxyethyl, locked nucleic acids, methylphosphonates, boraophosphates and phosphorodiamidate morpholino oligomers.
  • ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone capable of forming stable antiparallel duplexes following the Watson-Crick base pairing rules (Zhang , et al, 2005, J. Am. Chem. Soc. 127(12): 4174-4175).
  • GNA glycol nucleic acid
  • Such GNA may comprise phosphorothioate linkages or other appropriate modifications.
  • One aspect of the invention provides an antimicrobial ON targeting microorganisms.
  • Such an ON comprises at least one active ON and is adapted for use as an antimicrobial agent.
  • ONs of this invention may be in the form of a formulation targeting microbial agents involved in microbial infection diseases.
  • a formulation comprises at least one active ON and is adapted for use as an antimicrobial agent.
  • the ONs of this invention may be in the form of a pharmaceutical composition useful for treating (or prophylaxis of) microbial infection diseases, which may be approved by a regulatory agency for use in humans or in non- human animals, and/or against a particular disease.
  • a pharmaceutical composition comprises at least one therapeutically active ON and is adapted for use as an antimicrobial agent.
  • This pharmaceutical composition may include physiologically and/or pharmaceutically acceptable carriers. The characteristics of the carrier may depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain other active factors and/or agents which enhance activity.
  • the invention provides a method for the prophylaxis or treatment of a microbial infections in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable ON as described herein, e.g., a sequence independent ON at least 6 nucleotides in length, more preferably 15 nucleotides in length, or a pharmaceutical composition or formulation containing such ON.
  • a pharmacologically acceptable ON as described herein, e.g., a sequence independent ON at least 6 nucleotides in length, more preferably 15 nucleotides in length, or a pharmaceutical composition or formulation containing such ON.
  • the infection is related to a disease or condition indicated herein as related to a microbial infection
  • the subject is a type of subject as indicated herein, e.g., human, non-human animal, non-human mammal, bird and the like
  • the treatment is for a microbial disease or disease with a microbial etiology.
  • the microbial infections targeted by ONs, formulation, pharmaceutical composition, use for or method of treatment described herein are caused by a microbe, which is defined in the present invention as a microorganism such as a protozoan parasite, a bacteria or a fungus such as, without restriction, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Acanthamoeba sp., Trypanosoma brucei rhodesiense, Trypanosoma brucei gambiense, Trypanosoma cruzi, Entamoeba histolytica, Babesia microti, Babesia divergens, Balantidium coli, Blastocystis hominis, Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora sp., Giardia intestinalis ('also known as Giardia lamblia)
  • the present invention involves the discovery that oligonucleotides (ONs), e.g., oligodeoxynucleotides (ODNs), including modified oligonucleotides, can have a therapeutic application through a sequence independent mode of action. It is not necessary for the oligonucleotide to be complementary to any sequence or to have a particular distribution of nucleotides in order to have activity. Such an oligonucleotide can even be prepared as a randomer. [0062] In addition, the inventors have discovered that different length oligonucleotides have different activity.
  • the present invention discloses that the length of oligonucleotide that produces maximal effect when modified with sulfur linkages is typically in the range of 30-120 nucleotides but not restricted to these length.
  • this invention provides oligonucleotide agents that can have activity against diseases and conditions described herein. Such agents are particularly advantageous in view of the limited therapeutic options currently available.
  • the ONs, e.g., ODNs, of the present invention are useful in therapy for treating or preventing diseases and conditions described herein.
  • Such treatments are applicable to many types of patients and treatments, including, for example, the prophylaxis or treatment of diseases and conditions described herein.
  • a first aspect of the invention concerns oligonucleotides, e.g., purified oligonucleotides, where the activity occurs principally by a sequence independent (e.g., non-sequence complementary or non-sequence dependant aptameric activity) mode of action, and formulations containing such oligonucleotides.
  • sequence independent e.g., non-sequence complementary or non-sequence dependant aptameric activity
  • Oligonucleotides useful in the present invention can be of various lengths, e.g., at least 6, 10, 14, more preferably 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length.
  • the oligonucleotide can be in a range, e.g., a range defined by taking any two of the preceding listed values as inclusive end points of the range, for example 10-20, more preferably 15-50, 20-30, 20-40, 30-40, 30-50, 40-50, 40-60, 40-80, 50-60, 50-70, 60-70, 70-80, 60-120, and 80-120 nucleotides.
  • a minimum length or length range is combined with any other of the oligonucleotide specifications listed herein for the present oligonucleotides.
  • the nucleotide can include various modifications, e.g., stabilizing modifications, and thus can include at least one modification in the phosphodiester linkage and/or on the sugar, and/or on the base.
  • the oligonucleotide can include one or more phosphorothioate linkages, phosphorodithioate linkages, and/or methylphosphonate linkages.
  • Different chemically compatible modified linkages can be combined, e.g., modifications where the synthesis conditions are chemically compatible.
  • the oligonucleotides can include phosphodiester linkages, e.g., include at least one phosphodiester linkage, or at least 5, 10, 20, 30% or more phosphodiester linkages.
  • oligonucleotide has modified linkages throughout, e.g., phosphorothioate; has a 3'- and/or 5 '-cap; includes a terminal 3 '-5' linkage; the oligonucleotide is or includes a concatemer consisting of two or more oligonucleotide sequences joined by a linker(s).
  • the present invention further provides an oligonucleotide, wherein said oligonucleotide is linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, higher protein interaction, an improved ability to be formulated for delivery, a detectable signal, higher activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity.
  • the oligonucleotide includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% modified linkages, e.g., phosphorothioate, phosphorodithioate, and/or methylphosphonate.
  • At least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or all of the nucleotides are modified at the 2'-position of the ribose, e.g., 2'-OMe, T- F, 2'-amino.
  • modified linkages are combined with T- modifications in oligonucleotides, for example, at least 30% modified linkages and at least 30% 2 '-modifications; or respectively at least 40% and 40%, at least 50% and 50%, at least 60% and 60%, at least 70% and 70%, at least 80% and 80%, at least 90% and 90%, 100% and 100%.
  • the oligonucleotide includes at least 30, 40, 50, 60, 70, 80, 90, or 100% modified linkages and at least 30, 40, 50, 60, 70, 80, 90, or 100% 2 '-modifications where embodiments include each combination of listed modified linkage percentage and 2 '-modification percentage (e.g., at least 50% modified linkage and at least 80% 2 '-modifications, and at least 80% modified linkages and 100% 2 '-modifications).
  • the modified linkages are phosphorothioate linkages; the modified linkages are phosphorodithioate linkages; the 2 '-modifications are 2'-OMe; the 2 '-modifications are 2'-fluoro; the 2 '-modifications are a combination of 2'-0Me and 2'-fluoro; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-0Me; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'- OMe; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2'- modifications are a combination of 2'--
  • the oligonucleotide is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides in length, or is in a length range defined by taking any two of the specified lengths as inclusive endpoints of the range.
  • all but 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the internucleotidic linkages and/or 2'-positions of the ribose moiety are modified, e.g., with linkages modified with phosphorothioate, phosphorodithioate, or methylphosphonate linkages and/or 2'-0Me, 2'-F, and/or 2'-amino modifications of the ribose moiety.
  • the oligonucleotide includes at least 1, 2, 3, or 4 ribonucleotides, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90%, or even 100% ribonucleotides.
  • the oligonucleotide includes non-nucleotide groups in the chain (i.e., form part of the chain backbone) and/or as side chain moieties, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more, or up to 5, 10, 20% or more of the chain moieties and/or side chain moieties.
  • the oligonucleotide is free of self-complementary sequences longer than 5, 8, 10, 15, 20, 25, 30 nucleotides; the oligonucleotide is free of catalytic activity, e.g., cleavage activity against RNA; the oligonucleotide does not induce an RNAi mechanism.
  • the oligonucleotide binds protein involved in a disease or condition described in the present invention ; the sequence of the oligonucleotide (or the oligonucleotide has at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence) is derived from a genome; the activity of an oligonucleotide with a sequence derived from a genome is not superior to a randomer oligonucleotide or a random oligonucleotide of the same length; the oligonucleotide includes a portion complementary to a genome sequence and a portion not complementary to a genome sequence; unless otherwise indicated, the sequence of the oligonucleotide includes A(x), C(x), G(x), T(x), U(x), I(x), AC(x), AG(x), AT(x), AU(x), CG(x), CT(x), CU(x), GT(x), GU(x), C(x), C(x),
  • the oligonucleotide is at least 15, 20, 25, 29, 30, 32, 34, 35, 36, 38, 40, 45, 46, 50, 60, 70, 80, 90, 100, 110, 120, 140, or 160 nucleotides in length or is in a range defined by taking any two of the listed values as inclusive endpoints, or the length of the specified repeat sequence is at least a length or in a length range just specified);
  • the oligonucleotide includes a combination of repeat sequences (e.g., repeat sequences as specified above), including, for example, each combination of the above monomer and/or dimer repeats taken 2, 3, or 4 at a time;
  • the oligonucleotide is single stranded (RNA or DNA);
  • the oligonucleotide is double stranded (RNA or DNA);
  • the oligonucleotide includes at least one Gquartet or CpG portion;
  • the oligonucleotide includes a portion complementary to a
  • phosphorothioated ONs containing only (or at least primarily) pyrimidine nucleotides, including cytosine and/or thymidine and/or other pyrrolidines are resistant to low pH and polycytosine oligonucleotides showed increased resistance to a number of nucleases, thereby providing two important characteristics for oral administration of an ON.
  • the oligonucleotide has at least 80, 90, or 95, or 100% modified internucleotidic linkages (e.g., phosphorothioate or phosphorodithoiate) and the pyrimidine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100%, i.e.; is a pyrimidine oligonucleotide or the cytosine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or 100% i.e. is a polycytosine oligonucleotide.
  • modified internucleotidic linkages e.g., phosphorothioate or phosphorodithoiate
  • the length is at least 29, 30, 32, 34, 36, 38, 40, 45, 50, 60, 70, or 80 nucleotides, or is in a range of 20-28, 25-35, 29- 40, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, or 70-80, or is in a range defined by taking any two of the listed values as inclusive endpoints of the range.
  • the oligonucleotide is at least 50, 60, 70, 80, or 90% cytosine; at least 50, 60, 70, 80, or 90% thymidine (and may have a total pyrimidine content as listed above).
  • the oligonucleotide contains a listed percentage of either cytosine or thymidine, and the remainders of the pyrimidine nucleotides are the other of cytosine and thymidine. Also in certain embodiments, the oligonucleotide includes at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, or more contiguous pyrimidine nucleotides, e.g., as C nucleotides, T nucleotides, or CT dinucleotide pairs.
  • the pyrimidine oligonucleotide consists only of pyrimidine nucleotides; includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non- pyrimidine moieities; includes 1-5, 6-10, 11-15, or at least 16 non-pyrimidine backbone moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20 non- nucleotide moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20 purine nucleotides.
  • the linkages between such moieties or between such moieties and nucleotides are at least 25, 35, 50, 70, 90, or 100 % as resistant to acidic conditions as PS linkages in a 40-mer polyC oligonucleotide as evaluated by gel electrophoresis under conditions appropriate for the size and chemistry of the oligonucleotide.
  • Oligonucleotides can also be used in combinations, e.g., as a mixture.
  • Such combinations or mixtures can include, for example, at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10000, 100,000, 1,000,000, or more different oligonucleotides, e.g., any combination of oligonucleotides are described herein.
  • Such combinations or mixtures can, for example, be different sequences and/or different lengths and/or different modifications and/or different linked or conjugated molecules.
  • pluralities of oligonucleotides have a minimum length or are in a length range as specified above for oligonucleotides.
  • at least one, a plurality, or each of the oligonucleotides can have any of the other properties specified herein for individual oligonucleoties (which can also be in any consistent combination).
  • the sequence of the oligonucleotide is not perfectly complementary to any equal length portion of the a genome sequence, or the oligonucleotide has at most 60%, preferably 50%, more preferably 40% complementarity to any equal length portion of the genomic sequence, the oligonucleotide sequence does not consist essentially of polyA, polyC, polyG, polyT, Gquartet, or a TG-rich sequence.
  • TG-rich indicates that the sequence of the oligonucleotide consists of at least 50 percent T and G nucleotides, or if so specified, at least 60, 70, 80, 90, or 95% T and G, or even 100%.
  • the invention provides a mixture of oligonucleotides that includes at least two different oligonucleotides as described herein, e.g., at least 2, 3, 4, 5, 7, 10, 50, 100, 1000, 10,000, 100,000, 1,000,000, or even more.
  • oligonucleotide includes at least 20 linked nucleotides.
  • the oligonucleotide may also include additional, non-nucleotide moieties, which may form part of the backbone of the oligonucleotide chain. Unless otherwise indicated, when non-nucleotide moieities are present in the backbone, at least 10 of the linked nucleotides are contiguous.
  • sequence independent mode of action indicates that the particular biological activity is not dependent on a particular oligonucleotide sequence in the oligonucleotide.
  • the activity does not depend on sequence dependent hybridization such as with antisense activity, or a particular sequence resulting in a sequence dependent aptameric interaction.
  • non-sequence complementary mode of action indicates that the mechanism by which the material exhibits an effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect.
  • sequence complementary mode of action means that the effect of a material involves hybridization of complementary nucleic acid sequences or sequence specific aptameric interaction.
  • sequence complementary mode of action means that the activity of the oligonucleotide satisfies at least one of the 5 tests provided herein (see Examples).
  • the oligonucleotide satisfies test 1, test 2, test 3, test 4 or test 5; the oligonucleotide satisfies a combination of two of the tests, i.e., tests 1 & 2; tests 1 & 3; tests 1 & 4, tests 2 & 3, tests 2 & 4, tests 3 & 4, tests 1 & 5, tests 2 & 5, tests 3 & 5, test 4 & 5; the oligonucleotide satisfies a combination of 3 of the 5 tests; the oligonucleotide satisfies a combination of 4 of the 5 tests, i.e., the oligonucleotide satisfies all of tests 1, 2, 3, 4 & 5.
  • a related aspect concerns an oligonucleotide randomer or randomer formulation that contains at least one randomer, where the activity of the randomer occurs principally by a sequence independent, e.g., non-sequence complementary mode of action.
  • a randomer formulation can, for example, include a mixture of randomers of different lengths, e.g., at least 2, 3, 5, 10, or more different lengths, or other mixtures as described herein.
  • the phrase "derived from a genome” indicates that a particular sequence has a nucleotide base sequence that has at least 70% identity to a genomic nucleotide sequence or its complement (e.g., is the same as or complementary to a genomic sequence), or is a corresponding RNA sequence.
  • the term indicates that the sequence is at most 60% identical to a genomic sequence of a particular gene involved in a disease or condition against which the oligonucleotide is directed, or to its complementary sequence.
  • the identity is at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence.
  • Genome can be from an animal, e.g. a human, from a microorganism, e.g.
  • the invention also provides a pharmaceutical composition that includes a therapeutically effective amount of a pharmacologically acceptable, oligonucleotide or mixture of oligonucleotides as described herein, e.g., at least 6 nucleotides in length or other length as listed herein, where the activity of the oligonucleotide occurs principally by a sequence independent, e.g., non-sequence complementary or non- sequence dependent aptamer, mode of action, and a pharmaceutically acceptable carrier.
  • a sequence independent e.g., non-sequence complementary or non- sequence dependent aptamer, mode of action
  • the oligonucleotide or a combination or mixture of oligonucleotides is as specified hereinabove for individual oligonucleotides or combinations or mixtures of oligonucleotides.
  • the pharmaceutical compositions are approved for administration to a human, or a non- human animal such as a non-human primate.
  • the pharmaceutical composition can be formulated for delivery by a mode selected from the group consisting of but not restricted to oral ingestion, oral mucosal delivery, intranasal drops or spray, intraocular injection, subconjonctival injection, eye drops, ear drops, by inhalation, intratracheal injection or spray, intrabronchial injection or spray, intrapleural injection, intraperitoneal injection perfusion or irrigation, intrathecal injection or perfusion, intracranial injection or perfusion, intramuscular injection, intravenous injection or perfusion, intraarterial injection or perfusion, intralymphatic injection or perfusion, subcutaneous injection or perfusion, intradermal injection, topical skin application, by organ perfusion, by topical application during surgery, intratumoral injection, topical application, gastric injection perfusion or irrigation, enteral injection or perfusion, colonic injection perfusion or irrigation, rectal injection perfusion or irrigation, by rectal suppository or enema, by urethral suppository or injection, intravesical injection
  • the composition includes a delivery system, e.g., targeted to specific cells or tissues; a liposomal formulation, another drug, e.g., a non-nucleotide polymer, an antisense molecule, a siRNA, or a small molecule drug.
  • a delivery system e.g., targeted to specific cells or tissues
  • a liposomal formulation e.g., another drug, e.g., a non-nucleotide polymer, an antisense molecule, a siRNA, or a small molecule drug.
  • the oligonucleotide, oligonucleotide preparation, oligonucleotide formulation, or pharmaceutical composition has an in vitro IC 50 or EC 50 Of 10, 5, 2, 1, 0.50, 0.20, 0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 jtiM or less.
  • the pharmaceutical composition contains at least one polypyrimidine oligonucleotide as described herein. In view of the resistance to low pH discovered for polypyrimidine oligonucleoides; in certain embodiments such a composition is adapted for delivery to an acidic in vivo site, e.g., oral delivery or vaginal delivery.
  • the term "acidic site” means a site that has a pH of less than 7. Examples include the stomach (pH generally 1-2), the vagina (pH generally 4-5 but may be lower), and to a lesser degree, the skin (pH generally 4-6).
  • the phrase "adapted for oral delivery" and like terms indicate that the composition is sufficiently resistant to acidic pH to allow oral administration without a clinically excessive loss of activity, e.g., an excessive first pass loss due to stomach acidity of less than 50% (or is indicated, less than 40%, 30%, 20%, 10%, or 5%).
  • small molecule means that the molecular weight of the molecule is 1500 daltons or less. In some cases, the molecular weight is 1000, 800, 600, 500, or 400 daltons or less.
  • the invention provides a kit that includes at least one oligonucleotide, oligonucleotide mixture, oligonucleotide formulation, or pharmaceutical composition that includes such oligonucleotide, oligonucleotide mixture, or oligonucleotide formulation in a labeled package, where the activity of the oligonucleotide occurs principally by a sequence independent e.g., non-sequence complementary or non-sequence dependent aptameric, mode of action and the label on the package indicates that the oligonucleotide can be used against at least one disease or condition.
  • a sequence independent e.g., non-sequence complementary or non-sequence dependent aptameric, mode of action indicates that the oligonucleotide can be used against at least one disease or condition.
  • the kit includes a pharmaceutical composition that includes at least one oligonucletide as described herein. In one embodiment, the kit contains a mixture of at least two different oligonucleotides.
  • the oligonucleotide is adapted for in vivo use in an animal and/or the label indicates that the oligonucleotide or composition is acceptable and/or approved for use in an animal;
  • the animal is a mammal, such as human, or a non-human mammal such as bovine, porcine, a ruminant, ovine, or equine;
  • the animal is a non-human animal;
  • the animal is a bird, the kit is approved by a regulatory agency such as the U.S. Food and Drug Administration or equivalent agency for use in an animal, e.g., a human.
  • the different random oligonucleotides comprises randomers of different lengths; the random oligonucleotides can have different sequences or can have sequence in common, such as the sequence of the shortest oligos of the plurality; and/or the different random oligonucleotides comprise a plurality of oligonucleotides comprising a randomer segment at least 5 nucleotides in length or the different random oligonucleotides include a plurality of randomers of different lengths.
  • Other oligonucleotides e.g., as described herein oligonucleotides, can be tested in a particular system.
  • the invention provides a method for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide at least 6 nucleotides in length, more preferably 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • a sequence independent oligonucleotide at least 6 nucleotides in length, more preferably 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • the invention provides a use for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide at least 6 nucleotides in length, more preferably 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • a sequence independent oligonucleotide at least 6 nucleotides in length, more preferably 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s).
  • the disease or condition targeted be any of those listed herein as suitable for inhibition using the present invention
  • the subject is a type of subject as indicated herein, e.g., human, non-human animal, non- human mammal, bird, plant, and the like
  • the treatment is for a condition or a disease as indicated hereinabove.
  • the invention provides a method for the prophylaxis or treatment of a in an acidic environment in a subject, comprising administering to a subject in need of such a treatment a therapeutically effective amount of at least one pharmacologically acceptable pharmaceutical composition of the invention, said composition being adapted for administration to an acidic in vivo site.
  • the invention provides a use for the prophylaxis or treatment in an acidic environment in a subject, comprising administering to a subject in need of such a treatment a therapeutically effective amount of at least one pharmacologically acceptable pharmaceutical composition of the invention, said composition being adapted for administration to an acidic in vivo site.
  • the oligonucleotide is a polypyrimidine oligonucleotide (or a formulation or pharmaceutical composition containing such polypyrimidine oligonucleotide), which may be adapted for oral or vaginal administration, e.g., as described herein.
  • terapéuticaally effective amount refers to an amount that is sufficient to effect a therapeutically or prophylactically significant reduction of a disease or condition when administered to a typical subject of the intended type.
  • oligonucleotide, formulation, or composition should be administered in a therapeutically effective amount.
  • the oligonucleotide(s) having a sequence independent mode of action is not associated with a transfection agent; the oligonucleotide(s) having a sequence independent mode of action is not encapsulated in liposomes and/or non-liposomal lipid particles.
  • the oligonucleotide(s) having a sequence independent mode of action is in a pharmaceutical composition or is administered in conjunction with (concurrently or sequentially) an oligonucleotide that acts principally by a sequence dependent mode of action, e.g., antisense oligonucleotide or siRNA, where the oligonucleotide(s) having a sequence dependent mode of action can be associated with a transfection agent and/or encapsulated in liposomes and/or non-liposomal lipid particles.
  • a sequence dependent mode of action e.g., antisense oligonucleotide or siRNA
  • the invention provides a polymer mix that includes at least one oligonucleotide and at least one non-nucleotide polymer.
  • a polymer mix that includes at least one oligonucleotide and at least one non-nucleotide polymer.
  • the invention provides an oligonucleotide randomer, where the randomer is at least 6 nucleotides in length, more preferably 15 nucleotides in length.
  • the randomer has a length as specified above for oligonucleotides; the randomer includes at least one phosphorothioate linkage, the randomer includes at least one phosphorodithioate linkage or other modification as listed herein; the randomer oligonucleotides include at least one non-randomer segment (such as a segment complementary to a selected nucleic acid sequence), which can have a length as specified above for oligonucleotides; the randomer is in a preparation or pool of preparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or 700 ⁇ mol, 1 , 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 mole of randomer, or a range defined by taking any two different values from the preceding as inclusive end points, or is synthesized at one of the listed scales or scale ranges.
  • the randomer includes at least one phosphorothioate linkage, the randomer includes at least one phosphorodithioate linkage
  • oligonucleotide means oligodeoxynucleotide or oligodeoxyribonucleotide or oligoribonucleotide.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs thereof. This term includes oligonucleotides composed of naturally- occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly.
  • Oligonucleotides that include backbone and/or other modifications can also be referred to as oligonucleosides. Except otherwise specified, oligonucleotide definition includes homopolymers, heteropolymers, randomers (see below), random sequence oligonucleotides, genomic -derived sequence oligonucleotides and oligonucleotides purified from natural sources.
  • sequence independent activity indicates that the mechanism by which the material exhibits an antimicrobial effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect nor it is due to a sequence-specific aptameric activity.
  • a “sequence dependant mode of action or activity” means that the antimicrobial effect of a material involves hybridization of complementary nucleic acid sequences, or involves a sequence-specific aptameric interaction.
  • antimicrobial means inhibiting, stopping or preventing the growth or colonization of microorganisms.
  • antimicrobial also means inhibiting, stopping of preventing the adherence to tissues, the adherence to cells, the invasion of tissues, the invasion of cells, or the entry into cells.
  • An antimicrobial compound can be used to treat a disease whose etiology is based on a microbial infection.
  • microbe As used herein the terms microbe, microbial (also included in the word antimicrobial) and microorganism refer, if not specified, to protozoan parasites, bacteria and fungi.
  • microbial infection when referring to the term disease means a disease involving unwanted colonization of or growth in a subject by one or more microorganisms.
  • antimicrobial refers to an effect due to the presence of ONs or other material that inhibiting, stopping of preventing the growth of a microorganism compared to untreated microorganisms, in a system or organism.
  • antimicrobial ONs will have antimicrobial activity against multiple microorganisms.
  • antimicrobial oligonucleotide formulation refers to a preparation that includes at least one antimicrobial oligonucleotide that is adapted for use as an antimicrobial agent.
  • the formulation includes the ON or ONs, and can contain other materials that do not interfere with their use as an antimicrobial agents in vivo. Such other materials can include without restriction diluents, excipients, carrier materials, delivery systems and/or other antimicrobial materials.
  • composition refers to an antimicrobial ON formulation that includes a physiologically or pharmaceutically acceptable carrier or excipient. Such compositions can also include other components that do not make the composition unsuitable for administration to a desired subject, e.g., a human.
  • the phrase "adapted for use as an antimicrobial agent" indicates that the material exhibits an antimicrobial effect and does not include any component or material that makes it unsuitable for use in inhibiting microbial growth in an in vivo system, e.g., for administering to a subject such as a human subject.
  • the term "subject" refers to a living higher organism, including, for example, animals such as mammals, e.g., humans, non-human primates, non-human animals and plants, e.g., fruit trees.
  • the term "randomer” is intended to mean a single stranded nucleic acid polymer, modified or not, having degenerate sequences at every position, such as NNNNNNNNNN.
  • Each degenerate nucleotide position actually exists as a random population of the five naturally occurring bases on the nucleotide (adenine, guanine, cytosine, thymine, uracil) at this particular position, resulting in a completely degenerate pool of ONs of the same size but having no sequence identity as a population.
  • Randomers can also include nucleobases which do not occur naturally including without restriction hypoxanthine, xanthosine, imidazole, 2- aninopurines or 5-nitroindole.
  • the term randomer can apply to a sequence or a portion of a sequence.
  • degenerate means that a sequence is made of a mix of nucleotides.
  • a completely degenerate sequence means that A, C, G, and T (or other nucleobases) are randomly used at each position of the sequence and nucleotide position are identified by N (see randomer definition).
  • a degenerate sequence means also that at least two nucleobases are randomly used at each position of the sequence. Degenerate can apply to a sequence, a portion of a sequence or one nucleotide position in a sequence.
  • the term "delivery system” refers to a component or components that, when combined with an ON as described herein, facilitates the transfer of ONs inside cells, increases the amount of ONs that contact the intended location in vivo, and/or extends the duration of its presence at the target or increases its circulating lifetime in vivo, e.g., by at least 10, 20, 50, or 100%, or even more as compared to the amount and/or duration in the absence of the delivery system.
  • the term delivery system also means encapsulation system or encapsulation reagent.
  • To encapsulate ONs means to put in contact an ON with a delivery system or an encapsulation reagent.
  • An ON in contact with a delivery system can be referred to as an "encapsulated ON".
  • terapéuticaally effective amount refers to an amount that is sufficient to effect a therapeutically or prophylactically significant reduction in microbial growth when administered to a typical subject of the intended type.
  • the ON, formulation, or composition should be administered in a therapeutically effective amount.
  • oligonucleotides having each linkage phosphorothioated and each ribonucleotide modified at the 2'-position of the ribose may have antimicrobial activity but do not trigger RNase H activity , a property desirable for traditional antisense ONs but completely dispensable for the activity described in this present invention.
  • Results demonstrate that modifications at the 2 '-position of each ribose of PS-ONs renders the ON more resistant to nucleases in comparison with a PS-ON comprising the same modifications but only at both ends (gapmer). Gapmers are preferentially used in the antisense technology. Nuclease resistance of PS-ONs including modifications at the 2'-position of each ribose could display beneficial properties, such as improved pharmakokinetics and/or oral availability.
  • PS-ONs that include modifications at the 2'-position of each ribose show desirable characteristics
  • PS-ONs with substantial numbers of modifications at the 2 '-position of ribose could also display desirable characteristics, e.g., modification at least 50 % of the riboses and more preferably 80% or even more.
  • modified ONs are useful in this invention.
  • Such modified ONs include, for example, ONs containing modified backbones or non- natural internucleoside linkages.
  • ONs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Such modified ON backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate and borano- phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5 ' to 5' or 2' to 2' linkage.
  • Oligonucleotides having inverted polarity typically include a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • Some exemplary modified ON backbones that do not include a phosphodiester linkage have backbones that are formed by short chain alkyl or cycloalkyl interaucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH 2 component parts.
  • Modified ONs may also contain one or more substituted sugar moieties.
  • such oligonucleotides can include one of the following 2 '-modifications: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C 2 to Cio alkenyl and alkynyl, or 2'-O-(O-carboran-l-yl)methyl.
  • Particular examples are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) ⁇ OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON [(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to 10.
  • exemplary ONs include one of the following 2'-modifications: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 .
  • OCF 3 SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an ON, or a group for improving the pharmacodynamic properties of an ON.
  • Examples include 2'-methoxyethoxy (2'-0-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., HeIv. Chim.
  • LNAs Locked Nucleic Acids
  • the linkage can be a methelyne (-CH 2 -)- group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in International patent application publication Nos WO 98/39352 and WO 99/14226, which are incorporated herein by reference in their entireties.
  • modifications include sulfur-nitrogen bridge modifications, such as locked nucleic acid as described in Orum et al. (2001, Curr. Opin. MoI. Ther. 3: 239- 243).
  • the 2 '-modification may be in the arabino (up) position or ribo (down) position. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Exemplary U.S. patents describing the preparation of such modified sugar structures include, for example, U.S. patents Nos.
  • the linker may, for example, consist of modified nucleotides or non-nucleotide units.
  • the linker provides flexibility to the ON concatemer. Use of such ON concatemers can provide a facile method to synthesize a final molecule, by joining smaller ON building blocks to obtain the desired length.
  • a 12 carbon linker (C 12 phosphoramidite) can be used to join two or more ON concatemers and provide length, stability, and flexibility.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). ONs may also include base modifications or substitutions.
  • Modified bases include other synthetic and naturally-occurring bases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl(-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-
  • Additional modified bases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those described in U.S.
  • Another modification includes phosphorodithioate linkages. Knowing that phosphorodithioate ONs (PS2-ONs) and PS-ONs have a similar binding affinity to proteins (Tonkinson et al, 1994, Antisense Res. Dev. 4: 269-278; Cheng et al, 1997, J. MoI. Recogn. 10: 101-107) and knowing that a possible mechanism of action of ONs is binding to protein involved in microbial infection, it could be desirable to include phosphorodithioate linkages on the antimicrobial ONs described in the present invention.
  • ONs prepared by conventional methods consist of a mixture of diastereomers by virtue of the asymmetry around the phosphorus atom involved in the internucleotide linkage. This may affect the stability of the binding between ONs and targets such as proteins involved in microbial infection. Previous data showed that protein binding is significantly stereo-dependent (Yu et al, 2000, Bioorg. Med. Chem. 8: 275-284). Thus, using stereodefined or stereo-enriched ONs could improve their protein binding properties and improve their antimicrobial efficacy.
  • modified oligonucleotides containing phosphorothioate or dithioate linkages may also contain one or more substituted sugar moieties particularly modifications at the sugar moieties including, without restriction, 2 '-ethyl, 2'-ethoxy, 2'-methoxy, 2'-aminopropoxy, T- allyl, 2'-fluoro, 2'-pentyl, 2 '-propyl, 2'-dimethylaminooxyethoxy, and T- dimethylaminoethoxyethoxy.
  • the 2'-modification may be in the arabino (up) position or ribo (down) position.
  • a preferred 2'-arabino modification is 2'-fluoro. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Moreover ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone (Zhang L, et al (2005) J. Am. Chem. Soc. 127(12):4174-5). Such GNA may comprise phosphorothioate linkages and may comprise only pyrimidine bases.
  • GNA glycol nucleic acid
  • Such GNA may comprise phosphorothioate linkages and may comprise only pyrimidine bases.
  • the present oligonucleotides can be prepared in an ON formulation or pharmaceutical composition.
  • the present ONs may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Exemplary United States patents that describe the preparation of such uptake, distribution and/or absorption assisting formulations include, for example, U.S.
  • the ONs, formulations, and compositions of the invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • prodrug versions of the present oligonucleotides are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in Gosselin et al. (International patent application publication No WO 93/24510) and in Imbach et al., (International patent application publication No WO 94/26764 and U.S. patent No. 5,770,713), which are hereby incorporated by reference in their entireties.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the present compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Many such pharmaceutically acceptable salts are known and can be used in the present invention.
  • useful examples of pharmaceutically acceptable salts include but are not limited to salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and salts formed from element
  • the present invention also includes pharmaceutical compositions and formulations which contain the antimicrobial ONs of the invention.
  • Such pharmaceutical compositions may be administered in a number of ways such as intraocular, subconjunctival, by eye drop or topically to the eye.
  • administrations include topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery); pulmonary, e.g., by inhalation or insufflations of powders or aerosols, including by nebulizer; intratracheal; intranasal; epidermal and transdermal; oral; or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion.
  • compositions and formulations for administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Other formulations include those in which the ONs of the invention are in mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g.
  • ONs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, ONs may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, laurie acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, 1 - dodecylazacycloheptan-2-one, an acylcarnitine, an acyl choline, or a C MO alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • a C MO alkyl ester e.g. isopropylmyristate IPM
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Exemplary surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Exemplary bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24, 25-dihydro-fusidate, sodium glycodihydrofusidate.
  • DCA chenodeoxycholic acid
  • UDCA ursodeoxychenedeoxycholic acid
  • cholic acid dehydrocholic acid
  • deoxycholic acid deoxycholic acid
  • glucholic acid glycholic acid
  • glycodeoxycholic acid taurocholic acid
  • taurodeoxycholic acid sodium tauro-24, 25-dihydro-fusidate, sodium glycodihydrofusidate.
  • Exemplary fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium).
  • arachidonic acid arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • a particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further exemplary penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • ON complexing agents include poly-amino acids; polyimines; polyacrytates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches.
  • Particularly advantageous complexing agents include chitosan, N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino- methylethylene P(TDAE), polyaminostyrene (e.g.
  • PLGA poly(DL-lactic-co- glycolic acid
  • PEG polyethyleneglycol
  • compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • the pharmaceutical formulations of the present invention which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaking the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • the formulations and compositions of the present invention may be prepared and formulated as emulsions.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ m in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.
  • Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be either water-in-oil (w/o) or of the oil- in-water (o/w) variety.
  • Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion.
  • Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199).
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • HLB hydrophile/lipophile balance
  • Surfactants may be classified into different classes based on the nature of the hydrophilic group: non-ionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • Naturally occurring emulsif ⁇ ers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsif ⁇ ers especially in combination with surfactants and in viscous preparations.
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • a large variety of non-emulsifying materials is also included in emulsion formulations and contributes to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong inter-facial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers,
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p- hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • the compositions of ONs are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • micro-emulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system.
  • microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).
  • Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
  • microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML31O), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants.
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML31
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1 -propanol, and 1 -butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al, Pharmaceutical Research, 1994, 11 : 1385- 1390; Ritschet, Met/i. Find. Exp. Clin. PharmacoL, 1993, 13: 205).
  • Micro-emulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11: 1385; Ho et al., J. Pharm. Set, 1996, 85: 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of ONs and nucleic acids from the gastrointestinal tract.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories - surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et ah, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).
  • liposome refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers, i.e., liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior.
  • the aqueous portion typically contains the composition to be delivered.
  • lipid vesicles In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. Additional factors for liposomes include the lipid surface charge, and the aqueous volume of the liposomes.
  • liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
  • liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target.
  • liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine.
  • Neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • DOPE dioleoyl phosphatidylethanolamine
  • Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC.
  • PC phosphatidylcholine
  • Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome include one or more glycolipids, such as monosialoganglioside G MI , or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • Liposomes that include lipids derivatized with one or more hydrophilic polymers, and methods of preparation are described, for example, in Sunamoto et al, Bull. Chem. Soc. Jpn., 1980, 53: 2778 (a nonionic detergent, 2Ci 2 15G, that contains a PEG moiety); Ilium et al, FEBS Lett., 1984, 167: 79 (hydrophilic coating of polystyrene particles with polymeric glycols); Sears, U.S.
  • Liposomes that include nucleic acids have been described, for example, in Thierry et al, International patent application publication No WO 96/40062 (methods for encapsulating high molecular weight nucleic acids in liposomes); Tagawa et al, U.S. patent No 5,264,221 (protein-bonded liposomes containing RNA); Rahman et al, U.S. patent No 5,665,710 (methods of encapsulating oligodeoxynucleotides in liposomes); Love et al, International patent application publication No WO 97/04787 (liposomes that include antisense oligonucleotides).
  • Transfersomes are highly deformable lipid aggregates which are attractive for drug delivery vehicles.
  • Transfersomes may be described as lipid droplets which are so highly deformable that they can penetrate through pores which are smaller than the droplet.
  • Transfersomes are adaptable to the environment in which they are used, for example, they are shape adaptive, self-repairing, frequently reach their targets without fragmenting, and often self-loading.
  • Transfersomes can be made, for example, by adding surface edge-activators, usually surfactants, to a standard liposomal composition.
  • surfactants surfactants
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are usable over a wide range of pH values, and with typical HLB values from 2 to about 18 depending on structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters; and nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isothionates, acyl laurates and sulfosuccinates, and phosphates.
  • the alkyl sulfates and soaps are the most commonly used anionic surfactants.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines, with the quaternary ammonium salts used most often.
  • Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • surfactants The use of surfactants in drug products, formulations and in emulsions has been reviewed in Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).
  • penetration enhancers are used in or with a composition to increase the delivery of nucleic acids, particularly ONs across membranes of animals.
  • Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating nonsurfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these classes of penetration enhancers is described below in greater detail.
  • surfactants are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of ONs through the mucosa is enhanced.
  • penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et at., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252), each of which is incorporated herein by reference in its entirety.
  • Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 -monocaprate, 1 -dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci-io alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and diglycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Le
  • bile salts include any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro- fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington 's Pharmaceutical Sciences,
  • chelating agents can be regarded as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of ONs through the mucosa is enhanced.
  • chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618: 315-339).
  • chelating agents include disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1-33; Buur e? ⁇ /., J. Control ReL, 1990, 14: 43-51).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen laureth-9
  • N-amino acyl derivatives of beta-diketones enamine
  • non-chelating non-surfactant penetration enhancing compounds are compounds that do not demonstrate significant chelating agent or surfactant activity, but still enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1-33).
  • penetration enhancers examples include unsaturated cyclic ureas, 1 -alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal antiinflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al,, J. Pharm. Pharmacol, 1987, 39: 621-626).
  • nucleic acids may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • carrier compound can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • the coadministration of a nucleic acid and a carrier compound often with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney
  • the recovery of a partially phosphorothioated ON in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2-disulfonic acid (Miyao et al.,AntisenseRes. Dev., 1995, 5: 1 15-121; Takakura et al., Antisense & NucL Acid Drug Dev., 1996, 6: 177-183), each of which is incorporated herein by reference in its entirety.
  • a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal, and is typically liquid or solid.
  • a pharmaceutical carrier is generally selected to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition, in view of the intended administration mode.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
  • binding agents e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxyprop
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
  • compositions may additionally contain other components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacif ⁇ ers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacif ⁇ ers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran, and/or stabilizers.
  • a pharmaceutical composition containing (a) one or more antimicrobial ONs and (b) one or more other chemo therapeutic agents which function by a similar or a different mechanism.
  • chemotherapeutic agents include but are not limited to maltose tetrapalmitate, maltose tripalmitate, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethytmetamine,
  • chemotherapeutic agents may be used individually (e.g., 5-FU and an ON), sequentially (e.g., 5-FU and an ON for a period of time followed by MTX and ON), or in combination with one or more other such chemotherapeutic agents (e.g., 5-EU, MTX and an ON, or 5-FU, radiotherapy and an ON).
  • Chemotherapeutic agents can be anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids.
  • ONs of this invention used in the pharmaceutical composition or formulation or to practice a method of treating a human or an animal can be carried out in a variety of conventional ways for example using ocular, by eye drop, oral, subcutaneous, intravenous, intraperitoneal, intramuscular, intrathecal, intranasal, by inhalation, by enema, transdermal, sublingual and dermal routes.
  • the pharmaceutical composition or ON formulation of the invention may further contain other drugs for the treatment of microbial infection diseases.
  • additional factors and/or agents may be included in the pharmaceutical composition, for example, to produce a synergistic effect with the ONs of the invention.
  • an antimicrobial ONs demonstrating low, preferably the lowest possible, homology with the human (or other subject organism's) genome is designed.
  • One goal is to obtain an ON that will show the lowest toxicity due to interactions with human or animal genome sequence(s) and/or mRNAs.
  • the first step is to produce the desired length sequence of the ON, e.g., by aligning nucleotides A, C, G, T/U in a random fashion, manually or, more commonly, using a computer program.
  • the second step is to compare the ON sequence with a library of human sequences such as GenBank and/or the Ensemble Human Genome Database.
  • the sequence generation and comparison can be performed repetitively, if desired, to identify a sequence or sequences having a desired low homology level with the subject genome. It is desirable for the ON sequence to have the lowest homology possible with the entire genome, while also minimizing self interaction.
  • the last step is to test the ON in an assay to measure antimicrobial activity.
  • sequence independent ON sequence portion(s) is/are coupled with antisense sequence portion(s) to increase the activity of the final ON.
  • the non-specific portion of the ON is described in the present invention.
  • the antisense portion can be complementary to a microbial or host pathogenenesis-related gene mRNA or to other genes important for the progression of microbial infection diseases.
  • sequence independent sequence portion(s) is/are coupled with a G-rich motif ON portion(s) to improve the activity of the final ON.
  • the non-specific portion of the ON is described in the present invention.
  • the G-rich motif portion can, as non-limiting examples, include, CpG, Gquartet, and/or CG that are described in the literature as stimulators of the immune system.
  • Another approach is to use an ON composed of one or more types of non- Watson-Crick nucleotides/nucleosides.
  • Such ONs can mimic PS-ONs and other modifications with some of the following characteristics similar to PS-ONs: a) the total charge; b) the space between the units; c) the length of the chain; d) a net dipole with accumulation of negative charge on one side; e) the ability to bind to proteins f) the ability to be with delivery systems, h) an acceptable therapeutic index, i) an antimicrobial activity.
  • the ON can have a phosphorothioate backbone but is not limited to it.
  • Another approach is to use a polymer mimicking the activity of ONs described in the present invention to obtain inhibition of microbial activity.
  • anionic polymers were shown to bind to proteins. These polymers belong to several classes: (1) sulfate esters of polysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2) polymers containing sulfonated benzene or naphthalene rings and naphthalene sulfonate polymers; (3) polycarboxylates (acrylic acid polymers); and acetyl phthaloyl cellulose (Neurath et al, 2002, BMC Infect Dis 2: 27); and (4) abasic ONs (Takeshita et al, 1987, J.
  • Non-nucleotide protein binding polymers are described in the literature.
  • the polymers described herein can mimic ONs described in this invention and may have the following characteristics similar to ONs: a) the length of the chain; b) a net dipole with accumulation of negative charge on one side; c) the ability to bind to proteins; d) the ability to be encapsulated by a delivery system, e) an acceptable therapeutic index, f) an antimicrobial activity.
  • the antimicrobial polymer may preferably be a polyanion displaying similar space between its units as compared to a PS-ON.
  • the antimicrobial polymer may display a similar hydrophobicity than PS-ON.
  • Example 1 Sulfur modified ONs have sequence-independent anti-malarial activity.
  • Plasmodium falciparum laboratory line 3D7 was synchronized by sorbitol treatment, late stage schizonts was purified by floatation on 65% Percoll and used for erythrocyte invasion assays.
  • the parasitaemia of purified-schizonts was determined by Giemsa staining. Uninfected human erythrocytes and media were added to the schizonts to achieve a final hematocrit of 2% (2x10 8 erythrocytes ml "1 ) and a final parasitemia of 2 % (4x 10 erythrocytes ml " ).
  • Inhibition efficiency (%) (1- Inv (treatment) cone / Inv (control) cone) x lOO
  • Example 2 The anti-malarial activity of sulfur modified ONs is size dependent.
  • the anti-malarial activity of sulfur modified ONs is dependent on sulphur modification.
  • REP 2086 (SEQ ID NO: 83), which is a sulphur-less version of REP 2107 (SEQ ID NO: 103), had negligible anti-malarial activity.
  • REP 2086 (SEQ ID NO: 83)
  • REP 2107 (SEQ ID NO: 103)
  • Example 4 Sulfur modified ONs have sequence-independent anti-bacterial activity.
  • Escherichia coli strain 2787 a strain isolated from a case of infantile diarrhea, with previously demonstrated adhesion to human cells mediated by a bacterial glycoprotein (Benz and Schmidt, MoI. Microbiol., 40(6): 1403-13, 2001) was used for these experiments.
  • strain 2787 was grown overnight at 37° with agitation in I-medium (15g peptone, 3g yeast extract, 6g NaCl and Ig glucose, per L). The bacterial culture was diluted in I-medium to an absorbance of 0.2 at 600 run, approximately corresponding to a concentration of 10 8 cfu/mL.
  • HEp2 cells ATCC # CCL-23, human cells
  • DMEM Dubecco's Modified Eagle Medium
  • Hyclone#SH3054103 10% Bovine Growth Serum
  • the cells were grown overnight in a cell culture incubator with a controlled atmosphere at 37° and 5% CO 2 and were visually inspected for uniform adhesion in the wells.
  • Serial dilutions of the compound were made in sterile microfuge tubes in 1 mL cell culture medium. Two controls of 1 mL of medium with no compound were added.
  • 10 ⁇ L of the resuspended bacteria was added (i.e. 10 6 cfu for 2.5xlO 5 cells, an m.o.i. of 4), and the mix was incubated at 37° for 15 min.
  • the media of the cells was then discarded and the cells washed once with warmed DMEM.
  • the test compounds were then transferred into the cell containing wells.
  • the plate was incubated for 3 hours in a cell culture incubator.
  • the cells were then washed three times with PBS (Gibco- Invitrogen#14190-144) and then 100 uL of 0.1 % Triton XlOO was added to the cells, mixed by pipetting and incubated at room temperature for 10 min.
  • the bacteria recovered from the well without cells corresponds to the total number of bacteria that were present in the test (inoculum) and the bacteria recovered from the well without compound is the total number of bacteria that can adhere to the cells (negative control).
  • PBMEC cells (porcine brain microvascular endothelial cells) were seeded at 8X10 4 cells/wells in 8 wells of 24-wells plate (Falcon) and incubated at 37°C, 5%CO 2 in humid atmosphere in PBMEC complete media (1 : 1 mixture of Iscove's modified Dulbecco's medium and Ham's F-12 medium (Gibco); supplemented with 7,5% heat-inactivated fetal bovine serum, sodium bicarbonate, L-glutamine, human transferrin, N-acetyl-cysteine, hypoxanthine, porcine heparin, human fibroblast growth factor-basic and ⁇ - mercaptoethanol with antibiotics for 24h. Then, complete media with antibiotics was removed and replaced by PBMEC complete media without antibiotic for an additional 24h. On the day of the adhesion assay, the cells were visually inspected to confirm the confluency of the layer,
  • Streptococcus suis serotype 2 virulent strain 31533 was grown overnight at 37°C with agitation (120 RPM) in Todd Hewith broth (Difco), washed twice with sterile phosphate buffer saline (PBS) and diluted to 10 8 CFU/ml in PBMEC complete media. Bacterial suspension (11 ⁇ l of 10 8 CFU/ml) was added to eight microtubes. PBMEC complete media and test compounds were added in order to obtain the final concentrations 100 ⁇ M, 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M and 0 ⁇ M for the compound and 10 6 CFU/ml for the bacteria.
  • Cell lysates were diluted 1/10 in PBS. Fifty ⁇ l of each undiluted and 10-fold diluted cell lysate were plated on a Todd Hewitt agar using the Autoplate 4000 (Spiral Biotech). The next day, bacteria were counted and the numbers of colony forming units were calculated. Bacteria recovered from the well 0 ⁇ M is the total number that can adhere to the cells (negative control). The experiment was repeated three times on three different days.
  • Antimicrobial ONs with increased pH resistance, lower serum protein binding and superior nuclease resistance are provided.
  • a phosphorothioate randomer labeled at the 3' end with FITC (the bait) is diluted to 2nM in assay buffer (1OmM Tris, pH7.2, 8OmM NaCl, 1OmM EDTA, 10OmM b-mercaptoethanol and 1% tween 20). This oligo is then mixed with the appropriate amount of non heat-inactivated FBS. Following randomer-FBS interaction, the complexes are challenged with various unlabelled randomers to assess their ability to displace the bait from its complex. Displaced bait is measured by fluorescence polarization. The displacement curve was used to determine Kd.
  • Antimicrobial phosphorothioated polypyrimidine ONs exhibit acid and nuclease resistance.
  • the phosphorothioated 40mer ONs containing only the pyrimidine nucleotides cytosine (polyC, REP 2031, SEQ ID NO: 31) or thymidine (polyT, REP 2030, SEQ ID NO: 30) or the polyTC heteropolymer (REP 2056, SEQ ID NO: 52) had equivalent acid resistance compared to the fully 2'-O-methylated randomers whether phosphorothioated (REP 2107, SEQ ID NO: 103) or not (REP 2086, SEQ ID NO: 83).
  • phosphorothioated oligonucleotides containing only the purine nucleotide adenosine (poly A, REP 2029, SEQ ID NO: 29) or any adenosine or guanosine nucleotides (REP 2033, SEQ ID NO: 33; REP 2055, SEQ ID NO: 51; REP 2057, SEQ ID NO: 53) showed no greater acid resistance compared to unmodified DNA.
  • PII phosphodiesterase 11
  • Sl Sl nuclease
  • Exol Exonuclease 1
  • PS all linkages phosphorothioated
  • PII phosphodiesterase 11
  • Sl Sl nuclease
  • Exol Exonuclease 1
  • PS all linkages phosphorothioated
  • 2'0Me all riboses are 2'0 methylated.
  • - complete degradation
  • ++++ no degredation
  • PS phosphorothioate
  • 2'0Me 2'-O-methyl modification of the ribose.
  • the pyrimidine content of such an oligonucleotide is more than 50%, more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%.
  • these results show the potential of a method of treatment using oral administration of a therapeutically effective amount of at least one pharmacologically acceptable ON composed of pyrimidine nucleotides.
  • These results also show the potential of ONs containing high levels of pyrimidine nucleotides as a component of an ON formulation.
  • Example 7 Tests for Determining if an Oligonucleotide has sequence-independent activity.
  • TEST#1 Effect of partial degeneracy of a candidate ON on its antimicrobial efficacy.
  • L the number of bases in the candidate ON
  • X the number of bases on each end of the oligo to be made degenerate (but having the same chemistry as the candidate ON)
  • the IC50 generation will be performed using the antibacterial assays described herein. If the candidate ON is claimed to have an activity against another microbiological agent then the IC50 values shall be generated by a test of antimicrobial efficacy accepted by the pharmaceutical industry. IC50 values shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using these tests, the IC 50 of the candidate ON shall be compared to its degenerate counterpart. If the IC50 of the partially degenerate ON is less than 5 -fold greater than the original candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the ON shall be deemed to have sequence independent activity.
  • This test serves to compare the antimicrobial efficacy of a candidate ON with the antimicrobial efficacy of a randomer ON of equivalent size and chemistry in the same microbe.
  • the IC50 values shall be generated using the assays described herein. If the candidate ON is claimed to have an antimicrobial activity against microbial agents other than bacteria or malarial parasites, then the IC50 values shall be generated by a test of antimicribial efficacy accepted by the pharmaceutical industry. IC50 values shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using this test, the IC50 of the candidate ON shall be compared to an ON randomer of equivalent size and chemistry. If the IC50 of the ON randomer is less than 5-fold greater than the candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the candidate ON shall be deemed to have sequence-independent activity.
  • This test serves to compare the efficacy of a candidate ON against a target microbe whose genome is homologous to the candidate ON with the efficacy of the candidate ON against a second microbe whose genome has no homology to that candidate ON but is in the same genus. For example, if a candidate ON is reported to have activity against E. coli, its activity against E.coli will be compared to its activity against Escherichia sp.(not coli) . The comparison of the relative activities of the candidate ON in the target microbe and the second microbe is accomplished by using the activities of an ON randomer of the same length and chemistry in both microbes to normalize the IC50 values for the candidate ON obtained in the two microbes.
  • the IC50 generation will be determined in this microbe using one of the assays described herein, or other assays known in the art. Similarly, IC50 generation will be performed for the candidate ON against a second microbe using one of the assays as described herein or an assay accepted by the industry for a microorganisme whose genome has no homology to the sequence of the candidate ON but is from the same genus. IC50 generation is also performed for a randomer of equivalent size and chemistry against each of the microbes. The IC50 of the ON randomer against the two microbes are used to normalize the IC50 values for the candidate ON against the two microbes as follows.
  • the candidate ON shall be deemed to have sequence independent activity if the fold difference in IC50 between the two microbes is less than 5.
  • This test serves to determine if a candidate ON has a drug-like activity in a microbe where the sequence of the candidate ON is not homologous to any portion of the microbial genome and the microbial is from a different genus or family.
  • the candidate ON shall be tested using one of the assays described herein for the acceptable formulation to the pharmaceutical industry such that the sequence of the candidate ON tested is not homologous to any portion of the genome of the microbe to be used.
  • An IC50 value shall be generated using a minimum of seven concentrations of the candidate ON, with three or more points in the linear range.
  • the candidate ON shall be deemed to have a drug-like activity. If the candidate ON is deemed to have a drug-like activity in a microbe from a different family for which the candidate ON is not complementary and thus can have no sequence dependent antisense activity, it shall be considered to have sequence- independent activity.
  • sequence-independent antimicrobial activity of ONs occurs outside the cell.
  • ONs are not readily cell permeable, they must be delivered across the cell membrane by an appropriate carrier to have antisense activity in an in vitro context.
  • anti-microbial activity of antisense ONs by definition is dependent on delivery inside cells for activity. If a particular sequence-specific candidate ON has in vitro antimicrobial activity when used naked, it must benefit from the sequence-independent properties of ONs described in this invention.
  • the activity if the candidate ON shall be assessed using the assays decribed herein where these specific microbes are the target of the candidate ON. For other microbes, the activity shall be assessed using an assay acceptable to the pharmaceutical industry.
  • the antimicrobial activity of the naked candidate ON shall be compared to that of the encapsulated (for transfection) candidate ON (using identical candidate ON concentrations in both naked and encapsulated conditions).
  • the activity shall be measured by a dose response curve with not less than 7 concentrations, at least 3 of which fall in the linear range which includes the 50% inhibition of microbial activity.
  • the IC50 (the concentration which reduces microbial activity 50%) shall be calculated by linear regression of the linear range of the dose response curve as defined above. If the IC50 of the naked candidate ON is less than 5 fold greater than that of the encapsulated candidate ON, then the activity of the candidate ON shall be deemed to have sequence-independent activity.
  • a threshold of at least a 5-fold difference between the IC50s of said compounds have been set. This threshold ensures the reliability of the assessment of the above mentioned tests.
  • the thresholds described in tests 1 to 3 and 5 above are the default thresholds. If specifically indicated, other thresholds can be used in the comparison tests 1 to 3 and 5 described above. Thus for example, if specifically indicated, the threshold for determining whether an ON is acting with sequence-independent activity can be any of 10-fold, 8-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.5-fold, or equal.
  • the threshold described in test 4 above is also a default threshold.
  • the threshold for determining whether an ON has sequence-independent activity in test 4 can be an IC50 of less than lOuM, 5uM, 1 uM, 0.8 uM, 0.6uM, 0.5uM, 0.4 uM, 0.3 uM, 0.2 uM or 0.1 uM.
  • the ON can be required to satisfy any two (e.g., tests 1 & 2, 1 & 3, 1 & 4, 1 & 5, 2 & 3, 2& 4, 2 & 5, 3 & 4, and 3 & 5), any three (e.g., tests 1 & 2 & 3, 1 & 2 & 4, 1,& 2 & 5, 1 & 3 & 4, 1 & 3 & 5, 2 & 3 & 4, and 2 & 4 & 5), any 4 of the tests (e.g., 1 & 2 & 3 & 4, 1 & 2 & 3 & 5, and 2 & 3 & 4 & 5) at a default threshold, or if specifically indicated, at another threshold(s) as indicated above.
  • Example 8 Sulfur modified ONs have a therapeutic effect against malaria infection.
  • REP 2006 SEQ ID NO: 6
  • REP 2031 SEQ ID NO: 31
  • a 40mer fully phosphorothioated poly deoxycytosine nucleotide was assessed in mice infected with Plasmodium chabaudi adami DS parasitized red blood cells. Parasitemia was followed each day by monotring the percentage of infected red blood cells (Table 10)

Abstract

The present invention relates with the identification and use of antimicrobial oligonucleotides that act by a sequence independent mechanism, and includes the discovery that the antimicrobial activity is greater for larger oligonucleotides.

Description

ANTIMICROBIAL MOLECULES AND THEIR USES
FIELD OF THE INVENTION
[0001] The present invention relates with the identification and use of antimicrobial oligonucleotides that act by a sequence independent mechanism, and includes the discovery that the antimicrobial activity is greater for larger oligonucleotides.
BACKGROUND OF THE INVENTION
[0002] Microbial infections affect humans and animals and are an important cause of morbidity around the world. Microbial infectious agents include protozoan parasites, bacteria and fungi for which antimicrobial agents are often available. However, some antimicrobial agents are associated with undesirable side effects and the problem of microbial resistance to such agents is a growing problem.
[0003] Malaria is a serious and sometimes fatal disease caused by a protozoan parasite. Patients with malaria typically are very sick with high fevers, shaking chills, and flu-like illness. Four kinds of malaria parasites can infect humans: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. Malaria is a leading cause of death and disease worldwide, especially in developing countries. There is currently no malaria vaccine approved for human use. Treatment options include: chloroquine, sulfadoxine-pyrimethamine, mefloquine, atovaquone-proguanil, quinine, doxycycline, artemisin derivatives and primaquine. However, the type of treatment and success depend on the type (species) of the infecting parasite, the area where the infection was acquired, the drug-resistance status and the clinical status of the patient. .Resistance to these drugs and serious side effects from their use has been reported.
[0004] Pathogenic microorganisms, such as protozoan parasites, bacteria and fungi, use different pathogenesis or virulence factors to colonize hosts. Adherence to the host tissue or cell is an important factor in the initiation of pathogenesis. Also, the adherence to the cell surface can initiate the entry of microbial pathogens into the host cell.
[0005] The use of oligonucleotides (ONs), mainly antisense ONs, in the treatment of microbial infectious disease has been described in the scientific literature. [0006] In a study on malaria, a specific antisense effect could not be demonstrated over three cycles of the parasite. Both the 18mer antisense and sense oligonucleotides inhibited invasion of red blood cells at similar concentrations to dextran sulfate as determined by microscopy and [ HJhypoxanthine incorporation into DNA (Kanagaratnam et al, 1998, Int. J. Biochem. Cell Biol. 30: 979-985). Another group reported the exogenous delivery of phosphosphorothioate-oligonucleotides (PS-ONs) between 0.01 and 0.5 microM significantly inhibited Plasmodium falciparum growth compared with sense sequence controls suggesting sequence specific inhibition. This inhibition was shown to occur during maturation stages (Noonpakdee et al., 2003, Biochem. Biophys. Res. Commun. 302: 659-664). A PS-ON targeted to the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, was able to kill amastigotes-the intracellular stage of the parasite-in murine macrophages in culture (Ramazeilles et al, 1994, Proc. Natl. Acad. Sci. USA 91 : 7859-7863). Mishra et al. showed a better anti-parasitic antivity of PS ONs against Leishmania at lower pH (4.5), at an elevated temperature (350C) and in presence of cationic poly-1-lysine (Mishra et al., 2001 , Biochem. Pharmacol 62: 569- 580).
[0007] For bacterial infections, Good et al. (2001, Nat Biotechnol., 19(4): 360- 364)showed that a 9- to 12-mer peptide-nucleic-acid (PNA) ON, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli while retaining target specificity. It was also shown that morpholino antisense ONs inhibit gene expression in E. coli but have limited cellular due to the outer membrane. Uptake across the outer membrane was achieved by coupling phosphorodiamidate morpholino oligomers (PMOs) to a peptide (Geller et al, 2003, Antimicrob. Agents Chemother., 2003, 47(10): 3233-3239). Another study demonstrated that antisense PS-ONs, introduced by heat shock or electroporation in E. coli, reduced LacZ expression or in another case increased the bactericidal activity of norfloxacin (White et al. 1997, Antimicrobial Agents and Chemotherapy 41(12): 2699-2704).
[0008] A study on fungi showed the possibility to modestly impair growth of Cryptococcus neoformans at 370C by using a 30bp antisense ON targeting the gene CNAl (Gorlach et al. 2002, Microbiology 148: 213-219). Antisense ONs have been successfully introduced by electroporation or direct uptake in order to downregulate the prohibitin negative function on cell cycle of Pneumocystis carinii (de Monbrison, 2002, J. Microbiol. Methods 50: 211-213).
[0009] There are methods for treating or preventing microbial infection caused by protozoan parasites, bacteria and fungi. However, there are problems in the treatment of such infections including chronic and recurrent infections, toxicity, and drug resistance. Thus, there is a need for novel compounds, methods of treatment and formulations to cure, prevent or control microbial infections.
SUMMARY OF THE INVENTION
[0010] The invention relates to oligonucleotides (ONs) acting predominantly by a sequence independent mode of action for the treatment of microbial infections. The invention also relates to ONs and their use as therapeutic agents, and more particularly for their use in methods of treatment and formulations for the treatment of diseases involving infection by microorganisms.
[0011] In accordance with the present invention, there is provided an antimicrobial oligonucleotide formulation comprising at least one oligonucleotide having an antimicrobial activity against a target microorganism, said activity occurring principally by a sequence independent mode of action.
[0012] In a further embodiment of the present invention, the oligonucleotide formulation further comprises at least one delivery system.
[0013] In an additional embodiment, the oligonucleotide formulation of the present invention comprises an oligonucleotide of at least 15 nucleotides in length; 20 nucleotides in length; 25 nucleotides in length; 30 nucleotides in length; 35 nucleotides in length; preferably 40 nucleotides in length; 45 nucleotides in length; 50 nucleotides in length; more preferably 60 nucleotides in length; or 80 nucleotides in length.
[0014] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide of 20-30 nucleotides in length; 30-40 nucleotides in length; preferably 40-50 nucleotides in length; 50-60 nucleotides in length; more preferably 60-70 nucleotides in length; or 70-80 nucleotides in length. [0015] In a further embodiment, the oligonucleotide formulation of the present invention comprises an oligonucleotide which is not complementary to any equal length portion of a microbial genomic sequence. More preferably, the genomic sequence is of a human or of a non human animal.
[0016] In accordance with the present invention, there is provided an oligonucleotide formulation comprising an oligonucleotide containing at least 10 contiguous nucleotides of randomer sequence; more preferably 20 nucleotides of randomer sequence; 30 nucleotides of randomer sequence; or 40 nucleotides of randomer sequence.
[0017] In a further embodiment, the oligonucleotide formulation of the present invention comprises a randomer oligonucleotide.
[0018] In another embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide having a homopolymer sequence of at least 10 contiguous A nucleotides; 10 contiguous T nucleotides; 10 contiguous U nucleotides; 10 contiguous G nucleotides; 10 contiguous I nucleotide analogs; or 10 contiguous C nucleotides.
[0019] In another embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide which is a homopolymer sequence of C nucleotides.
[0020] In another embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide having a poly AT sequence at least 10 nucleotides in length; a poly AC sequence at least 10 nucleotides in length; a poly AG sequence at least 10 nucleotides in length; a polyAU sequence at least 10 nucleotides in length; a poly AI sequence at least 10 nucleotides in length; a polyGC sequence at least 10 nucleotides in length; a polyGT sequence at least 10 nucleotides in length; a polyGU sequence at least 10 nucleotides in length; a polyGI sequence at least 10 nucleotides in length; a polyCT sequence at least 10 nucleotides in length; a polyCU sequence at least 10 nucleotides in length; a polyCI sequence at least 10 nucleotides in length; a polyTI sequence at least 10 nucleotides in length; a polyTU sequence at least 10 nucleotides in length; or a polyUI sequence at least 10 nucleotides in length.
[0021] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one phosphodiester linkage. [0022] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one ribonucleotide.
[0023] Preferably, the oligonucleotide formulation comprises an oligonucleotide having at least one modification to its chemical structure, more preferably at least two different modifications to its chemical structure.
[0024] In accordance to the present invention, there is also provided an oligonucleotide formulation comprising an oligonucleotide having at least one sulfur modification.
[0025] Preferably, the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorothioated linkage; at least one phosphorodithioated linkage; or at least one boranophosphate linkage.
[0026] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one sulfur modified nucleobase moiety such as one sulfur modified ribose moiety, one 2' modification to the ribose moiety, one 2'-0 alkyl modified ribose moiety, one 2'-0 methyl modified ribose, one 2'-methoxyethyl modified ribose, or one 2'-FANA modified ribose.
[0027] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one methylphosphonate linkage.
[0028] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphorothioate backbone.
[0029] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one locked nucleic acid portion.
[0030] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one phosphorodiamidate morpholino portion.
[0031] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one abasic nucleic acid.
[0032] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide having a linker to form a concatemer of two or more oligonucleotide sequences. [0033] According to the present invention, the oligonucleotide formulation of the present invention comprises an oligonucleotide linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, an improved ability to be formulated, a detectable signal, higher antimicrobial activity, better pharmacokinetic properties, specific tissue distribution and lower toxicity.
[0034] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide linked or conjugated to a PEG molecule; or linked or conjugated to a cholesterol molecule.
[0035] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide linked or conjugated to a cholesterol molecule.
[0036] In a further embodiment, the oligonucleotide formulation comprises a double stranded oligonucleotide.
[0037] In another embodiment, the oligonucleotide formulation comprises an oligonucleotide having at least one base which is capable of hybridizing via non- Watson-Crick interactions.
[0038] According to an embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide having a portion complementary to a genome.
[0039] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide that binds to one or more cellular components.
[0040] In a further embodiment, the oligonucleotide formulation comprises an oligonucleotide that interacts with one or more cellular components, wherein said interaction resulting in inhibition of a protein activity or expression.
[0041] In one embodiment of the present invention, the oligonucleotide formulation comprises an oligonucleotide wherein at least a portion of the sequence of said oligonucleotide is derived from a genome. The oligonucleotide of the present invention has at most 90%, preferably 80%, more preferably 75% identity with the genomic sequence. [0042] In accordance with the present invention, there is also provided an oligonucleotide formulation comprising an oligonucleotide that targets a protozoan parasite; Plasmodium sp; Plasmodium falciparum; or a bacterium. More preferably, the oligonucleotide targets Escherichia sp; Streptococcus sp; a fungus.
[0043] In a further embodiment, the oligonucleotide mixture comprises a mixture of at least two different oligonucleotides. More preferably, the oligonucleotide formulation of the present invention comprises a mixture of at least ten different oligonucleotides or at least 100 different oligonucleotides; or at least 1000 different oligonucleotides; or at least 106 different oligonucleotides.
[0044] In accordance with the present invention, there is also provided an antimicrobial pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to the present invention; and a pharmaceutically acceptable carrier. More preferably, the antimicrobial pharmaceutical composition is adapted for the treatment, control, or prevention of a microbial infection disease.
[0045] In a further embodiment, the microbial infection disease is a protozoan parasite infection; is malaria; a bacterial infection.
[0046] In an additional embodiment, there is provided an antimicrobial pharmaceutical composition according to the present invention, adapted for delivery by a mode selected from the group consisting of ocular administration, eye drop administration, oral ingestion, subcutaneous injection, intramuscular injection, and intravenous injection.
[0047] In a further embodiment, the antimicrobial pharmaceutical composition further comprises a delivery system; at least one other antimicrobial drug in combination; a non-nucleotidic antimicrobial in combination; an antimicrobial antisense oligonucleotide in combination; more preferably an antimicrobial RNAi- inducing oligonucleotide.
[0048] In accordance with the present invention, there is provided a use of a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to the present invention, or antimicrobial pharmaceutical composition according to the present invention for the prophylaxis or treatment of a microbial infection disease in a subject. More preferably, said microbial infection disease is a protozoan parasite infection; is malaria; is a bacterial infection; is a fungal infection.
[0049] In addition, said subject is a human or a non-human animal.
[005Oj In accordance to the present invention, there is also provided a method for the prophylaxis or treatment of a microbial infection disease in a subject, comprising administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to the present invention, or antimicrobial pharmaceutical composition according to any the present invention. More preferably, said microbial infection disease is a protozoan parasite infection; is malaria; is a bacterial infection; is a fungal infection. In addition, said subject is a human or a non-human animal.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0051] The present invention is concerned with the identification and use of antimicrobial ONs that act by a sequence independent mechanism, and includes the discovery that the antimicrobial activity is greater for larger ONs.
[0052] As described previously, antisense or aptameric ONs have been tested for antimicrobial activity. However, such antisense or aptameric ONs are typically sequence-specific and target either intracellular mRNA or a protein, and typically are about 16-25 nucleotides in length.
[0053] As demonstrated in the present invention, the antimicrobial effect of randomer ONs is sequence independent. Considering the volumes and concentrations of ONs used in these tests, it is theoretically impossible that a particular sequence is present at more than 1 copy in the mixture. This means than there can be no antisense or sequence-specific aptameric effect in these ONs randomers. In all examples, should the microbial inhibition effect be caused by the sequence-specificity of the ONs, such effect would thus have to be caused by only one molecule, a result that does not appear possible. For example, for an ON randomer 40 bases in length, any particular sequence in the population would theoretically represent only 1/440 or 1/8.27X10"25 of the total fraction. Given that 1 mole = 6.022X1023 molecules, and the fact that our largest synthesis is currently done at the 15 micromole scale, all possible sequences will not be present and also, each sequence is present most probably as only one copy. Of course, one skilled in the art applying the teaching of the present invention could also use sequence specific ONs, but utilize the sequence independent activity discovered in the present invention. Accordingly, the present invention is not to be restricted to non-sequence complementary ONs, but disclaims what has been disclosed in the prior art regarding sequence-specific antisense ONs for treating microbial infection diseases.
[0054] According to the discussion above and the data reported herein, ONs appears to have potentially broad-spectrum activity against many types of diseases involving microbial infection. Therefore to test this hypothesis, several ON randomers of different sizes and chemical type were selected to be tested in various microorganisms. The present invention discloses that ONs have an antimicrobial activity that is sequence independent, dependent on size, and chemical modification. These results suggest that ONs have an antimicrobial activity by inhibiting adherence of microorganisms to the cell and/or inhibiting entry of microorganisms into cells.
[0055] One skilled in the art applying the teaching of the present invention could also use ONs with different chemical modifications. A modification of the ON, such as, but not limited to, a phosphorothioate modification or other sulfur modifications, appears to be beneficial for antimicrobial activity. Such sulfur modifications may include without restriction mono and diphosphorothioation of the phosphodiester linkage, 4' or 5' thiolation of the uracil moiety, 5' thiolation of the cytidine moiety, 2' or 4' thiolation of the thymine moiety, 6' thiolation of the guanine moiety, sulfur modifications to any other nucleobase moiety and sulfur modifications to the ribose moiety of any nucleotide. Moreover, ONs may have more than one sulfur substitution on each nucleotide, which can potentially increase the activity. Finally, any single or multiple sulfur substitution may be combined with other modifications known to improve properties of ONs. ONs of this invention may also have chemical modifications including without restriction: any 2' ribose modification including 2'-0 methyl, 2'-fluorine, 2'-FANA, 2'-methoxyethyl, locked nucleic acids, methylphosphonates, boraophosphates and phosphorodiamidate morpholino oligomers. Moreover ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone capable of forming stable antiparallel duplexes following the Watson-Crick base pairing rules (Zhang , et al, 2005, J. Am. Chem. Soc. 127(12): 4174-4175). Such GNA may comprise phosphorothioate linkages or other appropriate modifications.
[0056] One aspect of the invention provides an antimicrobial ON targeting microorganisms. Such an ON comprises at least one active ON and is adapted for use as an antimicrobial agent.
[0057] In another aspect, ONs of this invention may be in the form of a formulation targeting microbial agents involved in microbial infection diseases. Such a formulation comprises at least one active ON and is adapted for use as an antimicrobial agent.
[0058] In another aspect, the ONs of this invention may be in the form of a pharmaceutical composition useful for treating (or prophylaxis of) microbial infection diseases, which may be approved by a regulatory agency for use in humans or in non- human animals, and/or against a particular disease. Such a pharmaceutical composition comprises at least one therapeutically active ON and is adapted for use as an antimicrobial agent. This pharmaceutical composition may include physiologically and/or pharmaceutically acceptable carriers. The characteristics of the carrier may depend on the route of administration. The pharmaceutical composition of the invention may also contain other active factors and/or agents which enhance activity.
[0059] In yet another aspect, the invention provides a method for the prophylaxis or treatment of a microbial infections in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable ON as described herein, e.g., a sequence independent ON at least 6 nucleotides in length, more preferably 15 nucleotides in length, or a pharmaceutical composition or formulation containing such ON. In particular embodiments the infection is related to a disease or condition indicated herein as related to a microbial infection; the subject is a type of subject as indicated herein, e.g., human, non-human animal, non-human mammal, bird and the like; the treatment is for a microbial disease or disease with a microbial etiology.
[0060] In particular embodiments, the microbial infections targeted by ONs, formulation, pharmaceutical composition, use for or method of treatment described herein are caused by a microbe, which is defined in the present invention as a microorganism such as a protozoan parasite, a bacteria or a fungus such as, without restriction, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Acanthamoeba sp., Trypanosoma brucei rhodesiense, Trypanosoma brucei gambiense, Trypanosoma cruzi, Entamoeba histolytica, Babesia microti, Babesia divergens, Balantidium coli, Blastocystis hominis, Cryptosporidium parvum, Cryptosporidium hominis, Cyclospora sp., Giardia intestinalis ('also known as Giardia lamblia) Leishmania sp., Pneumocystis jiroveci (previously classified as Pneumocystis carini), Toxoplasma gondii, Trichomonas vaginalis, Bacillus anthracis, Clostridium botulinum, Brucella sp., Mycobacterium ulcerans, Campylobacter jejuni, Chlamydia pneumoniae, Vibrio cholerae, Escherichia coli (of many different serotypes, categorized into four major groups according to virulence mechanisms: enterotoxigenic; enter opathogenic; enteroinvasive; and enteroaggregative^, Escherichia coli serotype O157:H7, Corynebacterium diphtheriae, Streptococcus pneumoniae, Campylobacte rsp., Salmonella sp., Burkholderia (formerly Pseudomonas) mallei, Streptococcus pyogenes, group A streptococcus, Streptococcus agalactiae, group B streptococcus, Haemophilus influenzae, Mycobacterium leprae, Helicobacter pylori, Legionella pneumophila, Leptospires Listeria monocytogenes, Neisseria meningitides, Neisseria gonorrhoeae Treponema pallidum Mycobacterium avium complex, Mycoplasma pneumoniae, Nocardia asteroids, Bordetella pertussis, Streptococcus pneumoniae, Staphylococcus aureus, Chlamydia psittaci, Streptobacillus moniliformis, Salmonella enteritidis, Shigella sp., Chlamydia trachomatis, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestisis, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Coccidioides immitis, Histoplasma capsulatum and Sporothrix schenckii. In the above list the species include also the unspecified genus, e.g. Escherichia coli includes also Escherichia sp.
[0061] The present invention involves the discovery that oligonucleotides (ONs), e.g., oligodeoxynucleotides (ODNs), including modified oligonucleotides, can have a therapeutic application through a sequence independent mode of action. It is not necessary for the oligonucleotide to be complementary to any sequence or to have a particular distribution of nucleotides in order to have activity. Such an oligonucleotide can even be prepared as a randomer. [0062] In addition, the inventors have discovered that different length oligonucleotides have different activity. For example, the present invention discloses that the length of oligonucleotide that produces maximal effect when modified with sulfur linkages is typically in the range of 30-120 nucleotides but not restricted to these length. In view of the present discoveries concerning properties of oligonucleotides, this invention provides oligonucleotide agents that can have activity against diseases and conditions described herein. Such agents are particularly advantageous in view of the limited therapeutic options currently available.
[0063] Therefore, the ONs, e.g., ODNs, of the present invention are useful in therapy for treating or preventing diseases and conditions described herein. Such treatments are applicable to many types of patients and treatments, including, for example, the prophylaxis or treatment of diseases and conditions described herein.
[0064] A first aspect of the invention concerns oligonucleotides, e.g., purified oligonucleotides, where the activity occurs principally by a sequence independent (e.g., non-sequence complementary or non-sequence dependant aptameric activity) mode of action, and formulations containing such oligonucleotides.
[0065] Oligonucleotides useful in the present invention can be of various lengths, e.g., at least 6, 10, 14, more preferably 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70, 80, 90, 100, 110, 120, 140, 160, or more nucleotides in length. Likewise, the oligonucleotide can be in a range, e.g., a range defined by taking any two of the preceding listed values as inclusive end points of the range, for example 10-20, more preferably 15-50, 20-30, 20-40, 30-40, 30-50, 40-50, 40-60, 40-80, 50-60, 50-70, 60-70, 70-80, 60-120, and 80-120 nucleotides. In a particular embodiment, a minimum length or length range is combined with any other of the oligonucleotide specifications listed herein for the present oligonucleotides.
[0066] The nucleotide can include various modifications, e.g., stabilizing modifications, and thus can include at least one modification in the phosphodiester linkage and/or on the sugar, and/or on the base. For example, the oligonucleotide can include one or more phosphorothioate linkages, phosphorodithioate linkages, and/or methylphosphonate linkages. Different chemically compatible modified linkages can be combined, e.g., modifications where the synthesis conditions are chemically compatible. While modified linkages are useful, the oligonucleotides can include phosphodiester linkages, e.g., include at least one phosphodiester linkage, or at least 5, 10, 20, 30% or more phosphodiester linkages. Additional useful modifications include, without restriction, modifications at the 2 '-position of the sugar, such as 2'- O-alkyl modifications such as 2'-O-methyl modifications, 2'-amino modifications, T- halo modifications such as 2'-fluoro; acyclic nucleotide analogs. Other modifications are also known in the art and can be used. In particular embodiments, the oligonucleotide has modified linkages throughout, e.g., phosphorothioate; has a 3'- and/or 5 '-cap; includes a terminal 3 '-5' linkage; the oligonucleotide is or includes a concatemer consisting of two or more oligonucleotide sequences joined by a linker(s).
[0067] The present invention further provides an oligonucleotide, wherein said oligonucleotide is linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, higher protein interaction, an improved ability to be formulated for delivery, a detectable signal, higher activity, better pharmacokinetic properties, specific tissue distribution, lower toxicity.
[0068] In a certain embodiment, the oligonucleotide includes at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 100% modified linkages, e.g., phosphorothioate, phosphorodithioate, and/or methylphosphonate.
[0069] In a certain embodiment, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%, or all of the nucleotides are modified at the 2'-position of the ribose, e.g., 2'-OMe, T- F, 2'-amino.
[0070] In a further embodiment, modified linkages are combined with T- modifications in oligonucleotides, for example, at least 30% modified linkages and at least 30% 2 '-modifications; or respectively at least 40% and 40%, at least 50% and 50%, at least 60% and 60%, at least 70% and 70%, at least 80% and 80%, at least 90% and 90%, 100% and 100%. In certain embodiments, the oligonucleotide includes at least 30, 40, 50, 60, 70, 80, 90, or 100% modified linkages and at least 30, 40, 50, 60, 70, 80, 90, or 100% 2 '-modifications where embodiments include each combination of listed modified linkage percentage and 2 '-modification percentage (e.g., at least 50% modified linkage and at least 80% 2 '-modifications, and at least 80% modified linkages and 100% 2 '-modifications). In a particular embodiment, in each of the combinations percentages described, the modified linkages are phosphorothioate linkages; the modified linkages are phosphorodithioate linkages; the 2 '-modifications are 2'-OMe; the 2 '-modifications are 2'-fluoro; the 2 '-modifications are a combination of 2'-0Me and 2'-fluoro; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-0Me; the modified linkages are phosphorothioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'- OMe; the modified linkages are phosphorodithioate linkages and the 2 '-modifications are 2'-fluoro; the modified linkages are phosphorodithioate linkages and the 2'- modifications are a combination of 2'-0Me and 2'-fluoro. In a certain embodiment of oligonucleotides as described herein that combine a particular percentage of modified linkages and a particular percentage of 2 '-modifications, the oligonucleotide is at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides in length, or is in a length range defined by taking any two of the specified lengths as inclusive endpoints of the range.
[0071] In a certain embodiment, all but 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the internucleotidic linkages and/or 2'-positions of the ribose moiety are modified, e.g., with linkages modified with phosphorothioate, phosphorodithioate, or methylphosphonate linkages and/or 2'-0Me, 2'-F, and/or 2'-amino modifications of the ribose moiety.
[0072] In another embodiment, the oligonucleotide includes at least 1, 2, 3, or 4 ribonucleotides, or at least 10, 20, 30, 40, 50, 60, 70, 80, 90%, or even 100% ribonucleotides.
[0073] In a particular embodiment, the oligonucleotide includes non-nucleotide groups in the chain (i.e., form part of the chain backbone) and/or as side chain moieties, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more, or up to 5, 10, 20% or more of the chain moieties and/or side chain moieties.
[0074] In certain embodiments, the oligonucleotide is free of self-complementary sequences longer than 5, 8, 10, 15, 20, 25, 30 nucleotides; the oligonucleotide is free of catalytic activity, e.g., cleavage activity against RNA; the oligonucleotide does not induce an RNAi mechanism. [0075] In a particular embodiment, the oligonucleotide binds protein involved in a disease or condition described in the present invention ; the sequence of the oligonucleotide (or the oligonucleotide has at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence) is derived from a genome; the activity of an oligonucleotide with a sequence derived from a genome is not superior to a randomer oligonucleotide or a random oligonucleotide of the same length; the oligonucleotide includes a portion complementary to a genome sequence and a portion not complementary to a genome sequence; unless otherwise indicated, the sequence of the oligonucleotide includes A(x), C(x), G(x), T(x), U(x), I(x), AC(x), AG(x), AT(x), AU(x), CG(x), CT(x), CU(x), GT(x), GU(x), TU(x), AI(x), IC(x), IG(x), IT(x) IU(x) where x is 2, 3, 4, 5, 6, ... 60 ... 120 (in particular embodiments the oligonucleotide is at least 15, 20, 25, 29, 30, 32, 34, 35, 36, 38, 40, 45, 46, 50, 60, 70, 80, 90, 100, 110, 120, 140, or 160 nucleotides in length or is in a range defined by taking any two of the listed values as inclusive endpoints, or the length of the specified repeat sequence is at least a length or in a length range just specified); the oligonucleotide includes a combination of repeat sequences (e.g., repeat sequences as specified above), including, for example, each combination of the above monomer and/or dimer repeats taken 2, 3, or 4 at a time; the oligonucleotide is single stranded (RNA or DNA); the oligonucleotide is double stranded (RNA or DNA); the oligonucleotide includes at least one Gquartet or CpG portion; the oligonucleotide includes a portion complementary to a mRNA and is at least 29, 37, or 38 nucleotides in length (or other length as specified above); the oligonucleotide includes at least one non-Watson-Crick oligonucleotide and/or at least one nucleotide that participates in non-Watson-Crick binding with another nucleotide and/or at least one nucleotide that cannot form base pairs with other nucleotides; the oligonucleotide is a random oligonucleotide, the oligonucleotide is a randomer or includes a randomer portion, e.g., a randomer portion that has a length of at least 5, 10, 15, 20, 25, 30, 35, 40 or more contiguous oligonucleotides or a length as specified above for oligonucleotide length or at least 10, 20, 30, 40, 50, 60, 70, 80, 90% or all the nucleotides are randomer; the oligonucleotide is linked or conjugated at one or more nucleotide residues to a molecule that modifies the characteristics of the oligonucleotide, e.g. to provide higher stability (such as stability in serum or stability in a particular solution), lower serum interaction, higher cellular uptake, higher protein interaction, improved ability to be formulated for delivery, a detectable signal, improved pharmacokinetic properties, specific tissue distribution, and/or lower toxicity.
[0076] It was also discovered that phosphorothioated ONs containing only (or at least primarily) pyrimidine nucleotides, including cytosine and/or thymidine and/or other pyrrolidines are resistant to low pH and polycytosine oligonucleotides showed increased resistance to a number of nucleases, thereby providing two important characteristics for oral administration of an ON. Thus, in certain embodiments, the oligonucleotide has at least 80, 90, or 95, or 100% modified internucleotidic linkages (e.g., phosphorothioate or phosphorodithoiate) and the pyrimidine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or 100%, i.e.; is a pyrimidine oligonucleotide or the cytosine content is more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or 100% i.e. is a polycytosine oligonucleotide. In certain embodiments, the length is at least 29, 30, 32, 34, 36, 38, 40, 45, 50, 60, 70, or 80 nucleotides, or is in a range of 20-28, 25-35, 29- 40, 30-40, 35-45, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, or 70-80, or is in a range defined by taking any two of the listed values as inclusive endpoints of the range. In a particular embodiment, the oligonucleotide is at least 50, 60, 70, 80, or 90% cytosine; at least 50, 60, 70, 80, or 90% thymidine (and may have a total pyrimidine content as listed above). In a particular embodiment, the oligonucleotide contains a listed percentage of either cytosine or thymidine, and the remainders of the pyrimidine nucleotides are the other of cytosine and thymidine. Also in certain embodiments, the oligonucleotide includes at least 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, or more contiguous pyrimidine nucleotides, e.g., as C nucleotides, T nucleotides, or CT dinucleotide pairs. In certain embodiments, the pyrimidine oligonucleotide consists only of pyrimidine nucleotides; includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non- pyrimidine moieities; includes 1-5, 6-10, 11-15, or at least 16 non-pyrimidine backbone moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20 non- nucleotide moieties; includes at least one, 1-20, 1-5, 6-10, 11-15, or 16-20 purine nucleotides. Preferably, in embodiments in which non-nucleotide moieities are present, the linkages between such moieties or between such moieties and nucleotides are at least 25, 35, 50, 70, 90, or 100 % as resistant to acidic conditions as PS linkages in a 40-mer polyC oligonucleotide as evaluated by gel electrophoresis under conditions appropriate for the size and chemistry of the oligonucleotide. [0077] Oligonucleotides can also be used in combinations, e.g., as a mixture. Such combinations or mixtures can include, for example, at least 2, 3, 4, 5, 10, 20, 50, 100, 1000, 10000, 100,000, 1,000,000, or more different oligonucleotides, e.g., any combination of oligonucleotides are described herein. Such combinations or mixtures can, for example, be different sequences and/or different lengths and/or different modifications and/or different linked or conjugated molecules. In a particular embodiment of such combinations or mixtures, pluralities of oligonucleotides have a minimum length or are in a length range as specified above for oligonucleotides. In another embodiment of such combinations or mixtures, at least one, a plurality, or each of the oligonucleotides can have any of the other properties specified herein for individual oligonucleoties (which can also be in any consistent combination).
[0078] In certain embodiments, the sequence of the oligonucleotide is not perfectly complementary to any equal length portion of the a genome sequence, or the oligonucleotide has at most 60%, preferably 50%, more preferably 40% complementarity to any equal length portion of the genomic sequence, the oligonucleotide sequence does not consist essentially of polyA, polyC, polyG, polyT, Gquartet, or a TG-rich sequence.
[0079] As used in connection with the present oligos, the term "TG-rich" indicates that the sequence of the oligonucleotide consists of at least 50 percent T and G nucleotides, or if so specified, at least 60, 70, 80, 90, or 95% T and G, or even 100%.
[0080] In a related aspect, the invention provides a mixture of oligonucleotides that includes at least two different oligonucleotides as described herein, e.g., at least 2, 3, 4, 5, 7, 10, 50, 100, 1000, 10,000, 100,000, 1,000,000, or even more.
[0081] Specification of particular lengths for oligonucleotides, e.g., at least 20 nucleotides in length, means that the oligonucleotide includes at least 20 linked nucleotides. Unless clearly indicated to the contrary, the oligonucleotide may also include additional, non-nucleotide moieties, which may form part of the backbone of the oligonucleotide chain. Unless otherwise indicated, when non-nucleotide moieities are present in the backbone, at least 10 of the linked nucleotides are contiguous.
[0082] As used herein in connection with the action of an oligonucleotide, "sequence independent mode of action" indicates that the particular biological activity is not dependent on a particular oligonucleotide sequence in the oligonucleotide. For example, the activity does not depend on sequence dependent hybridization such as with antisense activity, or a particular sequence resulting in a sequence dependent aptameric interaction. Similarly, the phrase "non-sequence complementary mode of action" indicates that the mechanism by which the material exhibits an effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect. Conversely, a "sequence complementary mode of action" means that the effect of a material involves hybridization of complementary nucleic acid sequences or sequence specific aptameric interaction. Thus, indicating that the activity of a material is due to a sequence independent mode of action" or that the activity is "not primarily due to a sequence complementary mode of action" means that the activity of the oligonucleotide satisfies at least one of the 5 tests provided herein (see Examples). In particular embodiments, the oligonucleotide satisfies test 1, test 2, test 3, test 4 or test 5; the oligonucleotide satisfies a combination of two of the tests, i.e., tests 1 & 2; tests 1 & 3; tests 1 & 4, tests 2 & 3, tests 2 & 4, tests 3 & 4, tests 1 & 5, tests 2 & 5, tests 3 & 5, test 4 & 5; the oligonucleotide satisfies a combination of 3 of the 5 tests; the oligonucleotide satisfies a combination of 4 of the 5 tests, i.e., the oligonucleotide satisfies all of tests 1, 2, 3, 4 & 5.
[0083] A related aspect concerns an oligonucleotide randomer or randomer formulation that contains at least one randomer, where the activity of the randomer occurs principally by a sequence independent, e.g., non-sequence complementary mode of action. Such a randomer formulation can, for example, include a mixture of randomers of different lengths, e.g., at least 2, 3, 5, 10, or more different lengths, or other mixtures as described herein.
[0084] The phrase "derived from a genome" indicates that a particular sequence has a nucleotide base sequence that has at least 70% identity to a genomic nucleotide sequence or its complement (e.g., is the same as or complementary to a genomic sequence), or is a corresponding RNA sequence. In a particular embodiment of the present invention, the term indicates that the sequence is at most 60% identical to a genomic sequence of a particular gene involved in a disease or condition against which the oligonucleotide is directed, or to its complementary sequence. In particular embodiments, the identity is at most 60%, preferably 50%, more preferably 40% identity with a genomic sequence. Genome can be from an animal, e.g. a human, from a microorganism, e.g. a virus, a bacteria, a parasite, or from plant. [0085] The invention also provides a pharmaceutical composition that includes a therapeutically effective amount of a pharmacologically acceptable, oligonucleotide or mixture of oligonucleotides as described herein, e.g., at least 6 nucleotides in length or other length as listed herein, where the activity of the oligonucleotide occurs principally by a sequence independent, e.g., non-sequence complementary or non- sequence dependent aptamer, mode of action, and a pharmaceutically acceptable carrier. In a particular embodiment, the oligonucleotide or a combination or mixture of oligonucleotides is as specified hereinabove for individual oligonucleotides or combinations or mixtures of oligonucleotides. In a particular embodiment, the pharmaceutical compositions are approved for administration to a human, or a non- human animal such as a non-human primate.
[0086] In a particular embodiment, the pharmaceutical composition can be formulated for delivery by a mode selected from the group consisting of but not restricted to oral ingestion, oral mucosal delivery, intranasal drops or spray, intraocular injection, subconjonctival injection, eye drops, ear drops, by inhalation, intratracheal injection or spray, intrabronchial injection or spray, intrapleural injection, intraperitoneal injection perfusion or irrigation, intrathecal injection or perfusion, intracranial injection or perfusion, intramuscular injection, intravenous injection or perfusion, intraarterial injection or perfusion, intralymphatic injection or perfusion, subcutaneous injection or perfusion, intradermal injection, topical skin application, by organ perfusion, by topical application during surgery, intratumoral injection, topical application, gastric injection perfusion or irrigation, enteral injection or perfusion, colonic injection perfusion or irrigation, rectal injection perfusion or irrigation, by rectal suppository or enema, by urethral suppository or injection, intravesical injection perfusion or irrigation, or intraarticular injection.
[0087] In a particular embodiment, the composition includes a delivery system, e.g., targeted to specific cells or tissues; a liposomal formulation, another drug, e.g., a non-nucleotide polymer, an antisense molecule, a siRNA, or a small molecule drug.
[0088] In a particular embodiment, the oligonucleotide, oligonucleotide preparation, oligonucleotide formulation, or pharmaceutical composition has an in vitro IC50 or EC50 Of 10, 5, 2, 1, 0.50, 0.20, 0.10, 0.09. 0.08, 0.07, 0.75, 0.06, 0.05, 0.045, 0.04, 0.035, 0.03, 0.025, 0.02, 0.015, or 0.01 jtiM or less. [0089] In a particular embodiment, the pharmaceutical composition contains at least one polypyrimidine oligonucleotide as described herein. In view of the resistance to low pH discovered for polypyrimidine oligonucleoides; in certain embodiments such a composition is adapted for delivery to an acidic in vivo site, e.g., oral delivery or vaginal delivery.
[0090] As used in relation to in vivo administration of the present oligonucleotides and compositions, the term "acidic site" means a site that has a pH of less than 7. Examples include the stomach (pH generally 1-2), the vagina (pH generally 4-5 but may be lower), and to a lesser degree, the skin (pH generally 4-6).
[0091] As used herein, the phrase "adapted for oral delivery" and like terms indicate that the composition is sufficiently resistant to acidic pH to allow oral administration without a clinically excessive loss of activity, e.g., an excessive first pass loss due to stomach acidity of less than 50% (or is indicated, less than 40%, 30%, 20%, 10%, or 5%).
[0092] As used herein in connection with agents and drugs or test compounds, the term "small molecule" means that the molecular weight of the molecule is 1500 daltons or less. In some cases, the molecular weight is 1000, 800, 600, 500, or 400 daltons or less.
[0093] In another aspect, the invention provides a kit that includes at least one oligonucleotide, oligonucleotide mixture, oligonucleotide formulation, or pharmaceutical composition that includes such oligonucleotide, oligonucleotide mixture, or oligonucleotide formulation in a labeled package, where the activity of the oligonucleotide occurs principally by a sequence independent e.g., non-sequence complementary or non-sequence dependent aptameric, mode of action and the label on the package indicates that the oligonucleotide can be used against at least one disease or condition.
[0094] In another embodiment of the present invention, it is provided that the kit includes a pharmaceutical composition that includes at least one oligonucletide as described herein. In one embodiment, the kit contains a mixture of at least two different oligonucleotides. In one embodiment, the oligonucleotide is adapted for in vivo use in an animal and/or the label indicates that the oligonucleotide or composition is acceptable and/or approved for use in an animal; the animal is a mammal, such as human, or a non-human mammal such as bovine, porcine, a ruminant, ovine, or equine; the animal is a non-human animal; the animal is a bird, the kit is approved by a regulatory agency such as the U.S. Food and Drug Administration or equivalent agency for use in an animal, e.g., a human.
[0095] In a particular embodiment, the different random oligonucleotides comprises randomers of different lengths; the random oligonucleotides can have different sequences or can have sequence in common, such as the sequence of the shortest oligos of the plurality; and/or the different random oligonucleotides comprise a plurality of oligonucleotides comprising a randomer segment at least 5 nucleotides in length or the different random oligonucleotides include a plurality of randomers of different lengths. Other oligonucleotides, e.g., as described herein oligonucleotides, can be tested in a particular system.
[0096] In yet another aspect, the invention provides a method for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide at least 6 nucleotides in length, more preferably 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s). In a further embodiment, the invention provides a use for the prophylaxis or treatment in a subject by administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide as described herein, e.g., a sequence independent oligonucleotide at least 6 nucleotides in length, more preferably 15 nucleotides in length, or an pharmaceutical composition or formulation or mixture containing such oligonucleotide(s). In a particular embodiment, the disease or condition targeted be any of those listed herein as suitable for inhibition using the present invention; the subject is a type of subject as indicated herein, e.g., human, non-human animal, non- human mammal, bird, plant, and the like; the treatment is for a condition or a disease as indicated hereinabove.
[0097] In yet another aspect, the invention provides a method for the prophylaxis or treatment of a in an acidic environment in a subject, comprising administering to a subject in need of such a treatment a therapeutically effective amount of at least one pharmacologically acceptable pharmaceutical composition of the invention, said composition being adapted for administration to an acidic in vivo site.
[0098] In yet another aspect, the invention provides a use for the prophylaxis or treatment in an acidic environment in a subject, comprising administering to a subject in need of such a treatment a therapeutically effective amount of at least one pharmacologically acceptable pharmaceutical composition of the invention, said composition being adapted for administration to an acidic in vivo site.
[0099] In particular embodiments, the oligonucleotide is a polypyrimidine oligonucleotide (or a formulation or pharmaceutical composition containing such polypyrimidine oligonucleotide), which may be adapted for oral or vaginal administration, e.g., as described herein.
[0100] The term "therapeutically effective amount" refers to an amount that is sufficient to effect a therapeutically or prophylactically significant reduction of a disease or condition when administered to a typical subject of the intended type. In aspects involving administration of an oligonucleotide to a subject, typically the oligonucleotide, formulation, or composition should be administered in a therapeutically effective amount.
[0101] In a certain embodiment involving oligonucleotide formulations, pharmaceutical compositions, treatment and prophylactic methods and/or treatment and prophylactic uses described herein, the oligonucleotide(s) having a sequence independent mode of action is not associated with a transfection agent; the oligonucleotide(s) having a sequence independent mode of action is not encapsulated in liposomes and/or non-liposomal lipid particles. In a certain embodiment, the oligonucleotide(s) having a sequence independent mode of action is in a pharmaceutical composition or is administered in conjunction with (concurrently or sequentially) an oligonucleotide that acts principally by a sequence dependent mode of action, e.g., antisense oligonucleotide or siRNA, where the oligonucleotide(s) having a sequence dependent mode of action can be associated with a transfection agent and/or encapsulated in liposomes and/or non-liposomal lipid particles.
[0102] In yet another aspect, the invention provides a polymer mix that includes at least one oligonucleotide and at least one non-nucleotide polymer. JOl 03] In yet another aspect, the invention provides an oligonucleotide randomer, where the randomer is at least 6 nucleotides in length, more preferably 15 nucleotides in length. In particular embodiments the randomer has a length as specified above for oligonucleotides; the randomer includes at least one phosphorothioate linkage, the randomer includes at least one phosphorodithioate linkage or other modification as listed herein; the randomer oligonucleotides include at least one non-randomer segment (such as a segment complementary to a selected nucleic acid sequence), which can have a length as specified above for oligonucleotides; the randomer is in a preparation or pool of preparations containing at least 5, 10, 15, 20, 50, 100, 200, 500, or 700 μmol, 1 , 5, 7, 10, 20, 50, 100, 200, 500, or 700 mmol, or 1 mole of randomer, or a range defined by taking any two different values from the preceding as inclusive end points, or is synthesized at one of the listed scales or scale ranges.
[0104] In connection with modifying characteristics of an oligonucleotide by linking or conjugating with another molecule or moiety, the modifications in the characteristics are evaluated relative to the same oligonucleotide without the linked or conjugated molecule or moiety.
[0105] In the context of the present invention, unless specifically limited or specified the term "oligonucleotide (ON)" means oligodeoxynucleotide or oligodeoxyribonucleotide or oligoribonucleotide. Thus, "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA) and/or analogs thereof. This term includes oligonucleotides composed of naturally- occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Examples of modifications that can be used are described herein. Oligonucleotides that include backbone and/or other modifications can also be referred to as oligonucleosides. Except otherwise specified, oligonucleotide definition includes homopolymers, heteropolymers, randomers (see below), random sequence oligonucleotides, genomic -derived sequence oligonucleotides and oligonucleotides purified from natural sources.
[0106] As used herein in connection with the antimicrobial action of a material, the phrase "sequence independent activity" or "sequence independent mode of action" indicates that the mechanism by which the material exhibits an antimicrobial effect is not due to hybridization of complementary nucleic acid sequences, e.g., an antisense effect nor it is due to a sequence-specific aptameric activity. Conversely, a "sequence dependant mode of action or activity" means that the antimicrobial effect of a material involves hybridization of complementary nucleic acid sequences, or involves a sequence-specific aptameric interaction.
[0107] As used herein the term "antimicrobial" means inhibiting, stopping or preventing the growth or colonization of microorganisms. The term antimicrobial also means inhibiting, stopping of preventing the adherence to tissues, the adherence to cells, the invasion of tissues, the invasion of cells, or the entry into cells. An antimicrobial compound can be used to treat a disease whose etiology is based on a microbial infection.
[0108] As used herein the terms microbe, microbial (also included in the word antimicrobial) and microorganism refer, if not specified, to protozoan parasites, bacteria and fungi.
[0109] As used herein the term "microbial infection" when referring to the term disease means a disease involving unwanted colonization of or growth in a subject by one or more microorganisms.
[0110] As used herein in connection with ONs or other materials, the term "antimicrobial" refers to an effect due to the presence of ONs or other material that inhibiting, stopping of preventing the growth of a microorganism compared to untreated microorganisms, in a system or organism. In a certain embodiment of the present invention, antimicrobial ONs will have antimicrobial activity against multiple microorganisms.
[0111] The term "antimicrobial oligonucleotide formulation" refers to a preparation that includes at least one antimicrobial oligonucleotide that is adapted for use as an antimicrobial agent. The formulation includes the ON or ONs, and can contain other materials that do not interfere with their use as an antimicrobial agents in vivo. Such other materials can include without restriction diluents, excipients, carrier materials, delivery systems and/or other antimicrobial materials.
[0112] As used herein, the term "pharmaceutical composition" refers to an antimicrobial ON formulation that includes a physiologically or pharmaceutically acceptable carrier or excipient. Such compositions can also include other components that do not make the composition unsuitable for administration to a desired subject, e.g., a human.
[0113] As used in connection with an antimicrobial formulation, pharmaceutical composition, or other material, the phrase "adapted for use as an antimicrobial agent" indicates that the material exhibits an antimicrobial effect and does not include any component or material that makes it unsuitable for use in inhibiting microbial growth in an in vivo system, e.g., for administering to a subject such as a human subject.
[0114] As used herein in connection with administration of an antimicrobial material, the term "subject" refers to a living higher organism, including, for example, animals such as mammals, e.g., humans, non-human primates, non-human animals and plants, e.g., fruit trees.
[0115] In the present invention, the term "randomer" is intended to mean a single stranded nucleic acid polymer, modified or not, having degenerate sequences at every position, such as NNNNNNNNNN. Each degenerate nucleotide position actually exists as a random population of the five naturally occurring bases on the nucleotide (adenine, guanine, cytosine, thymine, uracil) at this particular position, resulting in a completely degenerate pool of ONs of the same size but having no sequence identity as a population. Randomers can also include nucleobases which do not occur naturally including without restriction hypoxanthine, xanthosine, imidazole, 2- aninopurines or 5-nitroindole. The term randomer can apply to a sequence or a portion of a sequence.
[0116] In the present invention, the term degenerate means that a sequence is made of a mix of nucleotides. A completely degenerate sequence means that A, C, G, and T (or other nucleobases) are randomly used at each position of the sequence and nucleotide position are identified by N (see randomer definition). A degenerate sequence means also that at least two nucleobases are randomly used at each position of the sequence. Degenerate can apply to a sequence, a portion of a sequence or one nucleotide position in a sequence.
[0117] As used herein, the term "delivery system" refers to a component or components that, when combined with an ON as described herein, facilitates the transfer of ONs inside cells, increases the amount of ONs that contact the intended location in vivo, and/or extends the duration of its presence at the target or increases its circulating lifetime in vivo, e.g., by at least 10, 20, 50, or 100%, or even more as compared to the amount and/or duration in the absence of the delivery system. The term delivery system also means encapsulation system or encapsulation reagent. To encapsulate ONs means to put in contact an ON with a delivery system or an encapsulation reagent. An ON in contact with a delivery system can be referred to as an "encapsulated ON".
[0118] The term "therapeutically effective amount" refers to an amount that is sufficient to effect a therapeutically or prophylactically significant reduction in microbial growth when administered to a typical subject of the intended type. In aspects involving administration of an antimicrobial ON to a subject, typically the ON, formulation, or composition should be administered in a therapeutically effective amount.
Phosphorothioation and 2' sugar modification
[0119] The incorporation of phosphorothioate linkages and ribonucleotide modifications, including 2'-O-methyl and other 2' sugar modifications, into oligonucleotides of this invention, is useful for improving characteristics of sequence- independent antimicrobial oligonucleotides. Results demonstrate that modification at the 2'-position of each ribose reduces the general interaction of the PS-ONs with serum proteins and renders them significantly more resistant to low pH. These properties promise to increase the pharmacokinetic performance and reduce the toxic side effects normally seen with PS-ONs. For example, their pH resistance makes them more suitable for oral delivery. Also their lowered interaction with serum proteins promises to improve their pharmacokinetic behaviour without affecting their antimicrobial activity. Thus, oligonucleotides having each linkage phosphorothioated and each ribonucleotide modified at the 2'-position of the ribose, e.g., 2'-O-methyl modifications, may have antimicrobial activity but do not trigger RNase H activity , a property desirable for traditional antisense ONs but completely dispensable for the activity described in this present invention. Results also demonstrate that modifications at the 2 '-position of each ribose of PS-ONs renders the ON more resistant to nucleases in comparison with a PS-ON comprising the same modifications but only at both ends (gapmer). Gapmers are preferentially used in the antisense technology. Nuclease resistance of PS-ONs including modifications at the 2'-position of each ribose could display beneficial properties, such as improved pharmakokinetics and/or oral availability.
[0120] In addition, while PS-ONs that include modifications at the 2'-position of each ribose show desirable characteristics, PS-ONs with substantial numbers of modifications at the 2 '-position of ribose could also display desirable characteristics, e.g., modification at least 50 % of the riboses and more preferably 80% or even more.
Oligonucleotide Modifications and Synthesis
[0121] As indicated herein, modified ONs are useful in this invention. Such modified ONs include, for example, ONs containing modified backbones or non- natural internucleoside linkages. ONs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
[0122] Such modified ON backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, carboranyl phosphate and borano- phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5 ' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity typically include a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.
[0123] Preparation of oligonucleotides with phosphorus-containing linkages as indicated above are described, for example, in U.S. patent Nos 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821 ; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is incorporated by reference herein in its entirety.
[0124] Some exemplary modified ON backbones that do not include a phosphodiester linkage have backbones that are formed by short chain alkyl or cycloalkyl interaucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, 0, S and CH2 component parts. Particularly advantageous are backbone linkages that include one or more charged moieties. Examples of U.S. patents describing the preparation of the preceding oligonucleotides include U.S. patents Nos 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489, 677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is incorporated by reference herein in its entirety.
[0125] Modified ONs may also contain one or more substituted sugar moieties. For example, such oligonucleotides can include one of the following 2 '-modifications: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to Cio alkenyl and alkynyl, or 2'-O-(O-carboran-l-yl)methyl. Particular examples are O[(CH2)nO]mCH3, O(CH2)~OCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON [(CH2)nCH3)]2, where n and m are from 1 to 10. Other exemplary ONs include one of the following 2'-modifications: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3. OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an ON, or a group for improving the pharmacodynamic properties of an ON. Examples include 2'-methoxyethoxy (2'-0-CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., HeIv. Chim. Acta, 1995, 78: 486-504) i.e., an alkoxyalkoxy group; 2'-dimethy-laminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE; and 2'- dirnethylarninoethoxyethoxy (also known as 2'-O- dimethylaminoethoxyethyl or T- DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2.
[0126] Other modifications include Locked Nucleic Acids (LNAs) in which the T- hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage can be a methelyne (-CH2-)- group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in International patent application publication Nos WO 98/39352 and WO 99/14226, which are incorporated herein by reference in their entireties.
[0127] Other modifications include sulfur-nitrogen bridge modifications, such as locked nucleic acid as described in Orum et al. (2001, Curr. Opin. MoI. Ther. 3: 239- 243).
[0128] Other modifications include 2'-methoxy (2'-0-CH3), 2'-methoxyethyl (2'O- CH2-CH3 ), 2 '-ethyl, 2'-ethoxy, 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'-O-allyl (2'-0-CH2-CH=CH2) and 2'-fluoro (2'-F).
[0129] The 2 '-modification may be in the arabino (up) position or ribo (down) position. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Exemplary U.S. patents describing the preparation of such modified sugar structures include, for example, U.S. patents Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393, 878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567, 811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627, 053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792, 747; and 5,700,920, each of which is incorporated by reference herein in its entirety. [0130] Still other modifications include an ON concatemer consisting of multiple ON sequences joined by a linker(s). The linker may, for example, consist of modified nucleotides or non-nucleotide units. In some embodiments, the linker provides flexibility to the ON concatemer. Use of such ON concatemers can provide a facile method to synthesize a final molecule, by joining smaller ON building blocks to obtain the desired length. For example, a 12 carbon linker (C 12 phosphoramidite) can be used to join two or more ON concatemers and provide length, stability, and flexibility.
[0131] As used herein, "unmodified" or "natural" bases (nucleobases) include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). ONs may also include base modifications or substitutions. Modified bases include other synthetic and naturally-occurring bases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl(-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional modified bases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (IH- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido [5,4-b][l,4]benzoxazin- 2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those described in U.S. patent No 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.
[0132] Another modification includes phosphorodithioate linkages. Knowing that phosphorodithioate ONs (PS2-ONs) and PS-ONs have a similar binding affinity to proteins (Tonkinson et al, 1994, Antisense Res. Dev. 4: 269-278; Cheng et al, 1997, J. MoI. Recogn. 10: 101-107) and knowing that a possible mechanism of action of ONs is binding to protein involved in microbial infection, it could be desirable to include phosphorodithioate linkages on the antimicrobial ONs described in the present invention.
[0133] Another approach to modify ONs is to produce stereodefined or stereo- enriched ONs as described in Yu et al. (2000, Bioorg. Med. Chem. 8: 275-284) and in Inagawa et al. (2002, FEBS Lett. 25: 48-52). ONs prepared by conventional methods consist of a mixture of diastereomers by virtue of the asymmetry around the phosphorus atom involved in the internucleotide linkage. This may affect the stability of the binding between ONs and targets such as proteins involved in microbial infection. Previous data showed that protein binding is significantly stereo-dependent (Yu et al, 2000, Bioorg. Med. Chem. 8: 275-284). Thus, using stereodefined or stereo-enriched ONs could improve their protein binding properties and improve their antimicrobial efficacy.
[0134] The incorporation of modifications such as those described above can be utilized in many different incorporation patterns and levels. That is, a particular modification need not be included at each nucleotide or linkage in an ON, and different modifications can be utilized in combination in a single ON, or even on a single nucleotide.
[0135] As examples and in accordance with the description above, modified oligonucleotides containing phosphorothioate or dithioate linkages may also contain one or more substituted sugar moieties particularly modifications at the sugar moieties including, without restriction, 2 '-ethyl, 2'-ethoxy, 2'-methoxy, 2'-aminopropoxy, T- allyl, 2'-fluoro, 2'-pentyl, 2 '-propyl, 2'-dimethylaminooxyethoxy, and T- dimethylaminoethoxyethoxy. The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-fluoro. Similar modifications may also be made at other positions on the ON, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2 '-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. ONs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Moreover ONs may have a structure of or comprise a portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphodiester backbone (Zhang L, et al (2005) J. Am. Chem. Soc. 127(12):4174-5). Such GNA may comprise phosphorothioate linkages and may comprise only pyrimidine bases.
Oligonucleotide formulations and pharmaceutical compositions
[01361 The present oligonucleotides can be prepared in an ON formulation or pharmaceutical composition. Thus, the present ONs may also be mixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Exemplary United States patents that describe the preparation of such uptake, distribution and/or absorption assisting formulations include, for example, U.S. patents Nos 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is incorporated herein by reference in its entirety.
[0137] The ONs, formulations, and compositions of the invention include any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
[0138] The term "prodrug" indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular embodiments, prodrug versions of the present oligonucleotides are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in Gosselin et al. (International patent application publication No WO 93/24510) and in Imbach et al., (International patent application publication No WO 94/26764 and U.S. patent No. 5,770,713), which are hereby incorporated by reference in their entireties.
[0139] The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the present compounds: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Many such pharmaceutically acceptable salts are known and can be used in the present invention.
[0140] For ONs, useful examples of pharmaceutically acceptable salts include but are not limited to salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and salts formed from elemental anions such as chlorine, bromine, and iodine.
[0141] The present invention also includes pharmaceutical compositions and formulations which contain the antimicrobial ONs of the invention. Such pharmaceutical compositions may be administered in a number of ways such as intraocular, subconjunctival, by eye drop or topically to the eye.
[0142] Other examples of administrations include topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery); pulmonary, e.g., by inhalation or insufflations of powders or aerosols, including by nebulizer; intratracheal; intranasal; epidermal and transdermal; oral; or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion.
[0143] Pharmaceutical compositions and formulations for administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Other formulations include those in which the ONs of the invention are in mixed with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP, dioleoylphosphatidyl ethanolamine DOTMA) and other delivering agents or molecules. ONs may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, ONs may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, laurie acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, 1 - dodecylazacycloheptan-2-one, an acylcarnitine, an acyl choline, or a C MO alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
[0144 j Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Exemplary surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Exemplary bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenedeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24, 25-dihydro-fusidate, sodium glycodihydrofusidate. Exemplary fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further exemplary penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. ON complexing agents include poly-amino acids; polyimines; polyacrytates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches. Particularly advantageous complexing agents include chitosan, N-trimethytchitosan, poly-L-lysine, polyhistidine, polyorithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino- methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylatc), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co- glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG).
[0145] Compositions and formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
[0146] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. [0147] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaking the product.
[0148] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
[0149] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
Emulsions
[0150] The formulations and compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (lids.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 2, p. 335; Higuchi et al., in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil- in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in- oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.
[0151] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199). [0152] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: non-ionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
[0153] Naturally occurring emulsifϊers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifϊers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
[0154] A large variety of non-emulsifying materials is also included in emulsion formulations and contributes to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0155] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong inter-facial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
[0156] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p- hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
[0157] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture has been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailabiity standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
[0158] In one embodiment of the present invention, the compositions of ONs are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically micro-emulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
[0159] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
[0160] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML31O), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1 -propanol, and 1 -butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
[0161] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al, Pharmaceutical Research, 1994, 11 : 1385- 1390; Ritschet, Met/i. Find. Exp. Clin. PharmacoL, 1993, 13: 205). Micro-emulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11: 1385; Ho et al., J. Pharm. Set, 1996, 85: 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of ONs and nucleic acids from the gastrointestinal tract.
[0162] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories - surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et ah, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).
Liposomes
[0163] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles offer specificity and extended duration of action for drug delivery. Thus, as used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers, i.e., liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion typically contains the composition to be delivered. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. Additional factors for liposomes include the lipid surface charge, and the aqueous volume of the liposomes.
[0164] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
[0165] For topical administration, there is evidence that liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target.
[0166] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
[0167] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome include one or more glycolipids, such as monosialoganglioside GMI, or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Without being bound by any particular theory, it is believed that for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the increase in circulation half-life of these sterically stabilized liposomes is due to a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Lett, 1987, 223: 42; Wu et al, Cancer Research, 1993, 53: 3765).
[0168] Various liposomes that include one or more glycolipids have been reported in Papahadjopoulos et ah, Ann. N. Y. Acad. ScL, 1987, 507: 64 (monosiatoganglioside GMI, galactocerebroside sulfate and phosphatidylinositol); Gabizon et al, Proc. Natl. Acad. ScL USA., 1988, 85: 6949;Allen et al, US. patent No 4,837,028 and International patent application publication No WO 88/04924 (sphingomyelin and the ganglioside GMI or a galactocerebroside sulfate ester); Webb et al, U.S. paten No 5,543,152 (sphingomyelin); Lim et al, International patent application publication No WO 97/13499 (1,2-sn-dimyristoylphosphatidylcholine).
[0169] Liposomes that include lipids derivatized with one or more hydrophilic polymers, and methods of preparation are described, for example, in Sunamoto et al, Bull. Chem. Soc. Jpn., 1980, 53: 2778 (a nonionic detergent, 2Ci215G, that contains a PEG moiety); Ilium et al, FEBS Lett., 1984, 167: 79 (hydrophilic coating of polystyrene particles with polymeric glycols); Sears, U.S. patents Nos 4,426,330 and 4,534, 899 (synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)); Klibanov et al, FEBS Lett., 1990, 268: 235 (phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate); Blume et al, Biochimica et Biophysica Acta, 1990, 1029: 91 (PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG); Fisher, European Patent No EP 0 445 131 Bl and International patent application publication No WO 90/04384 (covalently bound PEG moieties on liposome external surface); Woodle et al, U.S. patents Nos 5,013,556 and 5,356,633, and Martin et al, U.S. patent No 5,213,804 and European Patent No EP 0 496 813 Bl (liposome compositions containing 1-20 mole percent of PE derivatized with PEG); Martin et al, International patent application publication No WO 91/05545 and U.S. patent No 5,225,212 and in Zalipsky et al, International patent application publication No WO 94/20073 (liposomes containing a number of other lipid-polymer conjugates); Choi et al., International patent application publication No WO 96/10391 (liposomes that include PEG-modified ceramide lipids); Miyazaki et ah, U.S. patent No 5,540,935, and Tagawa et al, U.S. patent No 5,556,948 (PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces).
[0170] Liposomes that include nucleic acids have been described, for example, in Thierry et al, International patent application publication No WO 96/40062 (methods for encapsulating high molecular weight nucleic acids in liposomes); Tagawa et al, U.S. patent No 5,264,221 (protein-bonded liposomes containing RNA); Rahman et al, U.S. patent No 5,665,710 (methods of encapsulating oligodeoxynucleotides in liposomes); Love et al, International patent application publication No WO 97/04787 (liposomes that include antisense oligonucleotides).
[0171] Another type of liposome, transfersomes are highly deformable lipid aggregates which are attractive for drug delivery vehicles. (Cevc et al, 1998, Biochim Biophys Acta. 1368(2): 201-15.) Transfersomes may be described as lipid droplets which are so highly deformable that they can penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, for example, they are shape adaptive, self-repairing, frequently reach their targets without fragmenting, and often self-loading. Transfersomes can be made, for example, by adding surface edge-activators, usually surfactants, to a standard liposomal composition. Surfactants
[0172] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0173] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and are usable over a wide range of pH values, and with typical HLB values from 2 to about 18 depending on structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters; and nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant class.
[0174] Surfactant molecules that carry a negative charge when dissolved or dispersed in water are classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isothionates, acyl laurates and sulfosuccinates, and phosphates. The alkyl sulfates and soaps are the most commonly used anionic surfactants.
[0175] Surfactant molecules that carry a positive charge when dissolved or dispersed in water are classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines, with the quaternary ammonium salts used most often.
[0176] Surfactant molecules that can carry either a positive or negative charge are classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. [0177] The use of surfactants in drug products, formulations and in emulsions has been reviewed in Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).
Penetration Enhancers
[0178] In some embodiments, penetration enhancers are used in or with a composition to increase the delivery of nucleic acids, particularly ONs across membranes of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
[0179] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating nonsurfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of these classes of penetration enhancers is described below in greater detail.
[0180] In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of ONs through the mucosa is enhanced. These penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et at., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252), each of which is incorporated herein by reference in its entirety.
[0181] Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1 -monocaprate, 1 -dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci-io alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and diglycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1-33; El Hariri et al, J. Pharm. Pharmacol, 1992, 44: 651-654), each of which is incorporated herein by reference in its entirety.
[0182] The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro- fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington 's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto ct al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al, J. Pharm:. ScL, 1990, 79, 579-583).
[0183] In the present context, chelating agents can be regarded as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of ONs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618: 315-339). Without limitation, chelating agents include disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1-33; Buur e? α/., J. Control ReL, 1990, 14: 43-51).
[0184] As used herein, non-chelating non-surfactant penetration enhancing compounds are compounds that do not demonstrate significant chelating agent or surfactant activity, but still enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1-33). Examples of such penetration enhancers include unsaturated cyclic ureas, 1 -alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal antiinflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al,, J. Pharm. Pharmacol, 1987, 39: 621-626).
[0185] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
[0186] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, often with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs. For example, the recovery of a partially phosphorothioated ON in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2-disulfonic acid (Miyao et al.,AntisenseRes. Dev., 1995, 5: 1 15-121; Takakura et al., Antisense & NucL Acid Drug Dev., 1996, 6: 177-183), each of which is incorporated herein by reference in its entirety.
Excipients
[0187] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal, and is typically liquid or solid. A pharmaceutical carrier is generally selected to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition, in view of the intended administration mode. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycotate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
[0188] Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
[0189] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Other Pharmaceutical Composition Components
[0190] The present compositions may additionally contain other components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifϊers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
[0191] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran, and/or stabilizers.
[0192] In a certain embodiment of the invention, it is provided a pharmaceutical composition containing (a) one or more antimicrobial ONs and (b) one or more other chemo therapeutic agents which function by a similar or a different mechanism. Examples of such chemotherapeutic agents include but are not limited to maltose tetrapalmitate, maltose tripalmitate, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethytmetamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, A- hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5- FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP- 16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin, and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al, eds., Rahway, NJ. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and an ON), sequentially (e.g., 5-FU and an ON for a period of time followed by MTX and ON), or in combination with one or more other such chemotherapeutic agents (e.g., 5-EU, MTX and an ON, or 5-FU, radiotherapy and an ON). Chemotherapeutic agents can be anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids.
[0193] Administration of ONs of this invention used in the pharmaceutical composition or formulation or to practice a method of treating a human or an animal can be carried out in a variety of conventional ways for example using ocular, by eye drop, oral, subcutaneous, intravenous, intraperitoneal, intramuscular, intrathecal, intranasal, by inhalation, by enema, transdermal, sublingual and dermal routes.
[0194] The pharmaceutical composition or ON formulation of the invention may further contain other drugs for the treatment of microbial infection diseases. Such additional factors and/or agents may be included in the pharmaceutical composition, for example, to produce a synergistic effect with the ONs of the invention.
[0195] In another approach, an antimicrobial ONs demonstrating low, preferably the lowest possible, homology with the human (or other subject organism's) genome is designed. One goal is to obtain an ON that will show the lowest toxicity due to interactions with human or animal genome sequence(s) and/or mRNAs. The first step is to produce the desired length sequence of the ON, e.g., by aligning nucleotides A, C, G, T/U in a random fashion, manually or, more commonly, using a computer program. The second step is to compare the ON sequence with a library of human sequences such as GenBank and/or the Ensemble Human Genome Database. The sequence generation and comparison can be performed repetitively, if desired, to identify a sequence or sequences having a desired low homology level with the subject genome. It is desirable for the ON sequence to have the lowest homology possible with the entire genome, while also minimizing self interaction. The last step is to test the ON in an assay to measure antimicrobial activity.
[0196] In another approach, sequence independent ON sequence portion(s) is/are coupled with antisense sequence portion(s) to increase the activity of the final ON. The non-specific portion of the ON is described in the present invention. The antisense portion can be complementary to a microbial or host pathogenenesis-related gene mRNA or to other genes important for the progression of microbial infection diseases.
[0197] In another approach, sequence independent sequence portion(s) is/are coupled with a G-rich motif ON portion(s) to improve the activity of the final ON. The non-specific portion of the ON is described in the present invention. The G-rich motif portion can, as non-limiting examples, include, CpG, Gquartet, and/or CG that are described in the literature as stimulators of the immune system.
[0198] Another approach is to use an ON composed of one or more types of non- Watson-Crick nucleotides/nucleosides. Such ONs can mimic PS-ONs and other modifications with some of the following characteristics similar to PS-ONs: a) the total charge; b) the space between the units; c) the length of the chain; d) a net dipole with accumulation of negative charge on one side; e) the ability to bind to proteins f) the ability to be with delivery systems, h) an acceptable therapeutic index, i) an antimicrobial activity. The ON can have a phosphorothioate backbone but is not limited to it. Examples of non- Watson-Crick nucleotides/nucleosides are described in Kool (2002 Ace. Chem. Res. 35:936-943) and Takeshita et al. (1987, J. Biol. Chem. 262: 10171-10179), where ONs containing synthetic abasic sites are described.
[0199] Another approach is to use a polymer mimicking the activity of ONs described in the present invention to obtain inhibition of microbial activity. As described in the literature, several anionic polymers were shown to bind to proteins. These polymers belong to several classes: (1) sulfate esters of polysaccharides (dextrin and dextran sulfates; cellulose sulfate); (2) polymers containing sulfonated benzene or naphthalene rings and naphthalene sulfonate polymers; (3) polycarboxylates (acrylic acid polymers); and acetyl phthaloyl cellulose (Neurath et al, 2002, BMC Infect Dis 2: 27); and (4) abasic ONs (Takeshita et al, 1987, J. Biol. Chem. 262: 10171-10179). Other examples of non-nucleotide protein binding polymers are described in the literature. The polymers described herein can mimic ONs described in this invention and may have the following characteristics similar to ONs: a) the length of the chain; b) a net dipole with accumulation of negative charge on one side; c) the ability to bind to proteins; d) the ability to be encapsulated by a delivery system, e) an acceptable therapeutic index, f) an antimicrobial activity. In order to mimic the effect of an ON, the antimicrobial polymer may preferably be a polyanion displaying similar space between its units as compared to a PS-ON. Also to mimic the effect of an ON, the antimicrobial polymer may display a similar hydrophobicity than PS-ON.
[0200] The present invention would be readily understood by referring to the following examples which are given to illustrate the invention rather than to limits its scope.
ON that can be used in the present invention are listed in the following:
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Example 1 Sulfur modified ONs have sequence-independent anti-malarial activity.
[0201] The anti-malarial activity of a degenerate 40 mer sulfur modified (phosphorothioated) ON (REP 2006; SEQ ID NO: 6) was assessed in vitro using two assays:
In vitro erythrocyte invasion assay
[0202] Plasmodium falciparum laboratory line 3D7 was synchronized by sorbitol treatment, late stage schizonts was purified by floatation on 65% Percoll and used for erythrocyte invasion assays. The parasitaemia of purified-schizonts was determined by Giemsa staining. Uninfected human erythrocytes and media were added to the schizonts to achieve a final hematocrit of 2% (2x108 erythrocytes ml"1) and a final parasitemia of 2 % (4x 10 erythrocytes ml" ). 160 ul of this suspension was added to wells of a 96 well flat bottom microtitre plate containing 40 ul of different concentrations of drug diluted in distilled water. Different concentrations of drug were tested in triplicates. Distilled water/buffer was used as a control. Giemsa staining was used to determine invasion rates. The percentage of erythrocytes that were infected with P. falciparum following incubation for 26-30 hours at 370C under 90 % N2, 5% O2, 5% CO2 were scored to determine the invasion rates. The invasion inhibition efficiencies were determined using invasion rates measured in presence of different concentrations (cone) of drug (Inv (treatment) cone) and control bufferd (Inv (control) cone) as follows:
Inhibition efficiency (%) = (1- Inv (treatment) cone / Inv (control) cone) x lOO
In vitro growth assay using [3 H] -Hypoxanthine
[0203] To assess the effects of various drugs on parasite growth 100 ul of parasite culture (0.8-1.0% parasitaemia) at late ring stage was incubated with different concentrations of drug in triplicate for 24h, after which [3H] -hypoxanthine (1.2 uCi per well) was added and the culture was maintained for an additional 24h. The mature parasite cultures were lysed by freeze and thawing method and the lysed culture was collected on glass-fiber filters and the uptake of [3H] -hypoxanthine was counted on a scintillation counter. Uptake of [3H] -hypoxanthine by drug treated parasite was compared with control cultures.
[0204] Both of these assays demonstrated a dose dependent inhibition of both malarial invasion and growth (Table 1). Table 1 Antimalarial activity of REP 2006 (SEQ ID NO: 6)
Figure imgf000061_0001
[0205] These data demonstrate sequence-independent anti-malarial activity of sulfur modified ONs
Example 2 The anti-malarial activity of sulfur modified ONs is size dependent.
[0206] The activity of various sizes of PS-ON randomers in inhibiting the invasion of P. falciparum into erythrocytes was determined as described in another example. The results of this test demonstrated a size dependent activity of PS-ONs in inhibiting erthythrocyte invasion by P. falciparum (Table 2) with longer PS-ONs having more potent activity.
Table 2 The anti-malarial activity of PS-ON randomers is size dependent.
Figure imgf000061_0002
Example 3
The anti-malarial activity of sulfur modified ONs is dependent on sulphur modification.
[0207] The activity of ON equivalently sized randomers which were fully phosphorothioated (REP 2006, SEQ ID NO: 6), fully 2'-O-methylated (REP 2086, SEQ ID NO: 83) or fully phosphorothioated and fully 2'-O-methylated (REP 2107, SEQ ID NO: 103) were tested for their ability to inhibit the invasion of erythrocytes by P. falciparum as described in the present invention. The results of this test demonstrated that ONs containing a sulphur modification (REP 2006, SEQ ID NO: 6; and REP 2107, SEQ ID NO: 103) were effective in preventing P. falciparum invasion of erythrocytes. REP 2086 (SEQ ID NO: 83), which is a sulphur-less version of REP 2107 (SEQ ID NO: 103), had negligible anti-malarial activity. Thus the presence of a sulphur modification on ONs confers anti-malarial activity to these compounds.
Table 3 Antimalarial activity of sulphur and non-sulfur ON randomers.
Figure imgf000062_0001
Example 4 Sulfur modified ONs have sequence-independent anti-bacterial activity.
[0208] To determine if sulfur modified ONs have anti-bacterial activity, a completely degenerate 40mer sulfur modified (phosphorothioation) ON (REP 2006, SEQ ID NO:6) was tested for anti-bacterial activity in two assays:
Adhesion of E. coli
[0209] Escherichia coli strain 2787, a strain isolated from a case of infantile diarrhea, with previously demonstrated adhesion to human cells mediated by a bacterial glycoprotein (Benz and Schmidt, MoI. Microbiol., 40(6): 1403-13, 2001) was used for these experiments.
[0210] For each experiment, strain 2787 was grown overnight at 37° with agitation in I-medium (15g peptone, 3g yeast extract, 6g NaCl and Ig glucose, per L). The bacterial culture was diluted in I-medium to an absorbance of 0.2 at 600 run, approximately corresponding to a concentration of 108 cfu/mL. HEp2 cells (ATCC # CCL-23, human cells) were seeded at 2.5xlO5 cells per well in 8 wells of a 24 wells plate and grown in Dubecco's Modified Eagle Medium (DMEM, Gibco- Invitrogen# 12430054) supplemented with 10% Bovine Growth Serum (Hyclone#SH3054103), without antibiotics. The cells were grown overnight in a cell culture incubator with a controlled atmosphere at 37° and 5% CO2 and were visually inspected for uniform adhesion in the wells. Serial dilutions of the compound were made in sterile microfuge tubes in 1 mL cell culture medium. Two controls of 1 mL of medium with no compound were added. In each of these tubes, 10 μL of the resuspended bacteria was added (i.e. 106 cfu for 2.5xlO5 cells, an m.o.i. of 4), and the mix was incubated at 37° for 15 min. The media of the cells was then discarded and the cells washed once with warmed DMEM. The test compounds were then transferred into the cell containing wells. The plate was incubated for 3 hours in a cell culture incubator. The cells were then washed three times with PBS (Gibco- Invitrogen#14190-144) and then 100 uL of 0.1 % Triton XlOO was added to the cells, mixed by pipetting and incubated at room temperature for 10 min. Luria-Bertani broth (LB, Lennox modification), 900 uL, was then added and the wells were scraped to recover all the bacteria. All the recovered bacteria were 10-fold serially diluted in LB from 1/100 up to 1/10,000; 100 uL of each dilution were spread on an LB agar plate which were incubated at 37° overnight. The next day the bacteria were counted and the number of colony forming units inferred. The bacteria recovered from the well without cells corresponds to the total number of bacteria that were present in the test (inoculum) and the bacteria recovered from the well without compound is the total number of bacteria that can adhere to the cells (negative control). The experiment was repeated 3 times on three different days.
Adhesion of S. suis
[0211] The adhesion assay was performed as followed. Briefly, PBMEC cells (porcine brain microvascular endothelial cells) were seeded at 8X104 cells/wells in 8 wells of 24-wells plate (Falcon) and incubated at 37°C, 5%CO2 in humid atmosphere in PBMEC complete media (1 : 1 mixture of Iscove's modified Dulbecco's medium and Ham's F-12 medium (Gibco); supplemented with 7,5% heat-inactivated fetal bovine serum, sodium bicarbonate, L-glutamine, human transferrin, N-acetyl-cysteine, hypoxanthine, porcine heparin, human fibroblast growth factor-basic and β- mercaptoethanol with antibiotics for 24h. Then, complete media with antibiotics was removed and replaced by PBMEC complete media without antibiotic for an additional 24h. On the day of the adhesion assay, the cells were visually inspected to confirm the confluency of the layer,
[0212] Streptococcus suis serotype 2 virulent strain 31533 was grown overnight at 37°C with agitation (120 RPM) in Todd Hewith broth (Difco), washed twice with sterile phosphate buffer saline (PBS) and diluted to 108 CFU/ml in PBMEC complete media. Bacterial suspension (11 μl of 108 CFU/ml) was added to eight microtubes. PBMEC complete media and test compounds were added in order to obtain the final concentrations 100 μM, 10 μM, 5μM, 1 μM, 0.5μM, 0.1 μM, 0.05μM and 0 μM for the compound and 106 CFU/ml for the bacteria. Mixtures were incubated for 15 min, at 37°C. After the 15 min-incubation period, the media of the cells was removed and replaced by one ml of the mixtures contained in the microtubes. The plate was centrifuged at 800 X g for 10 min and incubated for 2h in a humid atmosphere, at 370C, 5%CO2. The monolayers were then washed five times with sterile PBS and incubated for 10 min at 37°C in the presence of 200 μl of 0,05% trypsin-0,03% EDTA. Then, 800 μl of ice-cold water was added, and the cells were disrupted by scrapping the bottom of the well and by repeated pipetting. Cell lysates were diluted 1/10 in PBS. Fifty μl of each undiluted and 10-fold diluted cell lysate were plated on a Todd Hewitt agar using the Autoplate 4000 (Spiral Biotech). The next day, bacteria were counted and the numbers of colony forming units were calculated. Bacteria recovered from the well 0 μM is the total number that can adhere to the cells (negative control). The experiment was repeated three times on three different days.
[0213] The results from these experiments (Table 4) showed a dose dependent inhibition of the adhesion of both E. coli and S. suis suggesting a potential antibacterial activity for REP 2006 (SEQ ID NO: 6). These data demonstrate the sequence-independent activity of sulfur modified ONs.
Table 4 Inhibition of bacterial adherence by REP 2006 (SEQ ID NO: 6).
Figure imgf000065_0001
Example 5
Antimicrobial ONs with increased pH resistance, lower serum protein binding and superior nuclease resistance.
[0214] It is shown in this example the effect of combining unmodified linkages, phosphorothiate linkages, 2'-0 methyl modified riboses and unmodified ribonucleotides on the serum protein interaction and chemical stability of a 40 base randomer ON, that can be used as a antimicrobial agent.
[0215] All randomers were prepared using standard solid phase, batch synthesis at the University of Calgary Core DNA Services lab on a 1 or 15 uM synthesis scale, deprotected and desalted on a 50cm Sephadex G-25 column.
[0216] To determine serum protein interaction, a phosphorothioate randomer labeled at the 3' end with FITC (the bait) is diluted to 2nM in assay buffer (1OmM Tris, pH7.2, 8OmM NaCl, 1OmM EDTA, 10OmM b-mercaptoethanol and 1% tween 20). This oligo is then mixed with the appropriate amount of non heat-inactivated FBS. Following randomer-FBS interaction, the complexes are challenged with various unlabelled randomers to assess their ability to displace the bait from its complex. Displaced bait is measured by fluorescence polarization. The displacement curve was used to determine Kd.
[0217] pH resistance was determined by incubation of randomers in PBS adjusted to the appropriate pH with HCl. 24 hours after incubation, samples were neutralized with IM TRJS, pH 7.4 and run on denaturing acryalmide gels and visualized following EtBr staining. [0218] For these experiments, we compared the behaviours of different modified ON randomers: REP 2006 (SEQ ID NO: 6), REP 2024 (SEQ ID NO: 24), REP 2107 (SEQ ID NO: 103), REP 2086 (SEQ ID NO: 83) and REP 2060 (SEQ ID NO: 56).
[0219] The relative affinity of these ON randomers for serum proteins was determined as described above. The results of these experiments showed that REP 2107 (SEQ ID NO: 103) has a lower affinity to serum proteins than REP 2006 (SEQ ID NO: 6) or REP 2024 (SEQ ID NO: 24) (Table 5 in this example) and that there was no interaction detected between REP 2086 (SEQ ID NO: 83) and serum proteins. Moreover, at saturation of competition, REP 2107 (SEQ ID NO: 103) was less effective at displacing bound bait than REP 2006 (SEQ ID NO: 6)or REP 2024 (SEQ ID NO: 24) (Table 6 in this example).
Table 5 Serum protein affinity of various randomers.
Figure imgf000066_0001
Table 6 Displacement of bait randomer at saturation.
Figure imgf000066_0002
[0220] The pH stability of these randomers was tested in the range of pH 1 to pH 7 over 24 hours of incubation at 37 deg C. While REP 2006 (SEQ ID NO: 6) and REP 2024 (SEQ ID NO: 24) showed significant degredation at pH 3 and complete degradation at pH 2.5, REP 2107 (SEQ ID NO: 103), REP 2086 (SEQ ID NO: 83) and 2060 (SEQ ID NO: 56) were completely stable at pH 1 after 24h of incubation.
[0221] It is demonstrated here that the incorporation of 2'-0 methyl modifications in PS-ON randomers lowers serum protein binding and improve low pH resistance. The fully 2'-O-methylated, fully phosphorothioated randomer (REP 2107, SEQ ID NO: 103) has a weaker interaction with serum proteins and shows a significantly increased resistance to low pH induced hydrolysis.
[0222] 40 mer randomers of various chemistries were assessed for their ability to resist degradation by various nucleases for 4 hours at 37 deg C (Table 7 in this example). While most chemistries exhibited resistance to more than one nuclease, only REP 2107 (SEQ ID NO: 103) was resistant to all four nucleases tested. It is important to note that REP 2024 (SEQ ID NO: 24; which has 2'-O methyl modifications at the 4 riboses at each end of the molecule) showed the same resistance profile as its parent molecule REP 2006 (SEQ ID NO: 6), being sensitive to Sl nuclease degradation while 2107 (SEQ ID NO: 103; fully 2'-0 methyl modified) was resistant to this enzyme. These results suggest that fully 2'-0 methyl modified and fully phosphorothioated ON will be the most effective of the tested oligonucleotides in resisting degradation by nucleases in the blood.
Table 7 Resistance to various nucleases by different randomer chemistries.
Figure imgf000067_0001
Example 6
Antimicrobial phosphorothioated polypyrimidine ONs exhibit acid and nuclease resistance.
[0223] To determine the extent of acid resistance of ONs that can be used as antimicrobial agents, various 40 base ONs having different chemistries and/or sequences are incubated in PBS buffered to different pH values for 24 hours at 37 deg C. The degradation of these ONs was assessed by urea-polyacryamide gel electrophoresis (Table 8).
[0224] The results of these studies show that randomer ONs (containing both pyrimidine and purine nucleotides) are resistant to acidic pH only when they were fully 2'-O-methylated. Our data indicated that even partially 2'-O-methylated ONs (gapmers, REP 2024, SEQ ID NO: 24) do not display any significant increase in acid resistance compared to fully phosphorothioated ONs. Even fully phosphorothioated randomers show no increased pH resistance compared to unmodified ONs. In contrast, it was noted that the phosphorothioated 40mer ONs containing only the pyrimidine nucleotides cytosine (polyC, REP 2031, SEQ ID NO: 31) or thymidine (polyT, REP 2030, SEQ ID NO: 30) or the polyTC heteropolymer (REP 2056, SEQ ID NO: 52) had equivalent acid resistance compared to the fully 2'-O-methylated randomers whether phosphorothioated (REP 2107, SEQ ID NO: 103) or not (REP 2086, SEQ ID NO: 83). Contrary to the results for the polypyrimidine oligonucleotides, phosphorothioated oligonucleotides containing only the purine nucleotide adenosine (poly A, REP 2029, SEQ ID NO: 29) or any adenosine or guanosine nucleotides (REP 2033, SEQ ID NO: 33; REP 2055, SEQ ID NO: 51; REP 2057, SEQ ID NO: 53) showed no greater acid resistance compared to unmodified DNA.
[0225] These results are significant because the preferred way described in the prior art to achieve greater acid resistance compared to phosphorothioated ONs was to add 2'-O-methyl modifications (or other 2'-ribose modifications) or other modifications. The present data demonstrates that the 2'-O-methyl ribose modification or other 2'- ribose modifications are not required if the ON is a polypyrimidine (i.e. contains only pyrimidine nucleotides [e.g. homopolymers of cytosine or thymidine or a heteropolymer of cytosines and thymidines]) to achieve pH and nuclease resistance. The presence of purines (A or G) even in the presence of pyrimidines, can render ONs acid labile.
Table 8 Acid stability of various 40 mer ONs
Figure imgf000069_0001
PII = phosphodiesterase 11, Sl = Sl nuclease, Exol = Exonuclease 1, PS = all linkages phosphorothioated, 2'OMe = all riboses are 2'0 methylated. +++ = no degradation, ++ = less than 5-% degradation, -/+ = more than 90% degradation, ~ = completely degraded.
[0226] To determine the extent of ON nucleotide composition and modifications on nuclease resistance, various 40 base ONs having different nucleotide compositions and modifications were incubated in the presence of various endo and exonucleases for 4 hours at 37 deg C. The degradation of these ONs was assessed by urea- polyacryamide gel electrophoresis.
[0227] The results of these studies showed that randomer ONs were resistant to all four enzymes tested (phosphodiesterase II [Sigma], Sl nuclease [Fermentas], Bal31 [New England Biolabs] and exonuclease 1 [New England Biolabs]) only when they were fully phosphorothioated and fully 2'-0 -methylated (Table 9). Omission of any of these modifications in randomers resulted in increased sensitivity to one or more of the nucleases tested. It was noted that the fully phosphorothioated, partially 2'-O - methylated randomer (REP 2024, SEQ ID NO: 24) was equivalent in nuclease resistance to REP 2006 (SEQ ID NO: 6), indicated that 2'-O- methylation may be required on each nucleotide of a phosphorothioated ON to achieve the optimal nuclease resistance. However, it was also noted that the phosphorothioated 40mer polypyrimidine poly cytosine (poly C, REP 2031, SEQ ID NO: 31) had equivalent nuclease resistance compared to the fully phosphorothioated, fully 2'0 methylated randomer (REP 2107, SEQ ID NO: 103).
[0228] These results are significant because the prior art teaches that the preferred way to enhance nuclease resistance of phosphorothioated ONs is to add 2'-0 -methyl modifications, other 2'- ribose modifications, or other modifications. This new data demonstrates that the 2'-O-methyl modification or other 2'-ribose modifications or any other modifications are not required to enhance nuclease resistance if the ON is fully phosphorothioated and consists of a homopolymer of cytosines.
Table 9 Nuclease resistance of various 40 mer ONs.
Figure imgf000071_0001
PII = phosphodiesterase 11, Sl = Sl nuclease, Exol = Exonuclease 1, PS = all linkages phosphorothioated, 2'0Me = all riboses are 2'0 methylated. - = complete degradation, ++++ = no degredation, PS = phosphorothioate, 2'0Me = 2'-O-methyl modification of the ribose.
[0229] These results demonstrate that phosphorothioated ONs containing only pyrimidine nucleotides, including cytosine and/or thymidine and/or other pyrimidines are resistant to low pH and phosphorothioated ONs containing only cytosine nucleotides exhibit superior nuclease resistance, two important characteristics for oral administration of an ON. Thus, high pyrimidine nucleotide content of an ON is advantageous to provide resistance to low pH resistance and high cytosine content is advantagaeous to provide improved nuclease resistance. For example, in certain embodiments, the pyrimidine content of such an oligonucleotide is more than 50%, more than 60%, or more than 70%, or more than 80%, or more than 90%, or 100%. Furthermore, these results show the potential of a method of treatment using oral administration of a therapeutically effective amount of at least one pharmacologically acceptable ON composed of pyrimidine nucleotides. These results also show the potential of ONs containing high levels of pyrimidine nucleotides as a component of an ON formulation.
Example 7 Tests for Determining if an Oligonucleotide has sequence-independent activity.
[0230] It is shown herein that sulfur modified ONs have broad spectrum antimicrobial activity. Moreover, it is shown that the activity of such ONs are sequence-independent. Of course any one skilled in the art could prepare sequence- specific ONs, for example an antisense ON targeting an mRNA of a particular microorganisme and incorporating sulfur or other modifications. However such an ON would have benefited from the ON modifications that are described herein and the fact that it is demonstrated herein that the activity of such a modified ON is sequence independent and size dependent. An ON shall be considered to have sequence-independent activity if it meets the criteria of any one of the 5 tests outlined below. An ON having a reasonable part of its function due to a sequence-independent activity shall be considered to benefit from the inventions described herein.
TEST#1 Effect of partial degeneracy of a candidate ON on its antimicrobial efficacy.
[0231] This test serves to measure the antimicrobial activity of a candidate ON sequence when part of its sequence is made degenerate. If the degenerate version of the candidate ON having the same chemistry retains its activity as described below, is it deemed to have sequence-independent activity. Candidate ONs will be made degenerate according to the following rule:
L = the number of bases in the candidate ON X = the number of bases on each end of the oligo to be made degenerate (but having the same chemistry as the candidate ON)
If L is even, then X=integer (L/4) If L is odd, then X=integer ((L+l)/4) X must be equal to or greater than 4
[0232] If the candidate ON is claimed to have activity against bacteria or malarial parasites, the IC50 generation will be performed using the antibacterial assays described herein. If the candidate ON is claimed to have an activity against another microbiological agent then the IC50 values shall be generated by a test of antimicrobial efficacy accepted by the pharmaceutical industry. IC50 values shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using these tests, the IC 50 of the candidate ON shall be compared to its degenerate counterpart. If the IC50 of the partially degenerate ON is less than 5 -fold greater than the original candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the ON shall be deemed to have sequence independent activity.
TEST #2 Comparison of antimicrobial activity of a candidate ON with an ON randomer.
[0233] This test serves to compare the antimicrobial efficacy of a candidate ON with the antimicrobial efficacy of a randomer ON of equivalent size and chemistry in the same microbe.
[0234] If the candidate ON is claimed to have an antimicrobial activity against bacteria or malarial parasites, then the IC50 values shall be generated using the assays described herein. If the candidate ON is claimed to have an antimicrobial activity against microbial agents other than bacteria or malarial parasites, then the IC50 values shall be generated by a test of antimicribial efficacy accepted by the pharmaceutical industry. IC50 values shall be generated using a minimum of seven concentrations of compound, with three or more points in the linear range of the dose response curve. Using this test, the IC50 of the candidate ON shall be compared to an ON randomer of equivalent size and chemistry. If the IC50 of the ON randomer is less than 5-fold greater than the candidate ON (based on minimum triplicate measurements, standard deviation not to exceed 15% of mean) then the candidate ON shall be deemed to have sequence-independent activity.
TEST #3
Comparison of antimicrobial activity of a candidate ON in two non-homologous microbes from the same genus
[0235] This test serves to compare the efficacy of a candidate ON against a target microbe whose genome is homologous to the candidate ON with the efficacy of the candidate ON against a second microbe whose genome has no homology to that candidate ON but is in the same genus. For example, if a candidate ON is reported to have activity against E. coli, its activity against E.coli will be compared to its activity against Escherichia sp.(not coli) . The comparison of the relative activities of the candidate ON in the target microbe and the second microbe is accomplished by using the activities of an ON randomer of the same length and chemistry in both microbes to normalize the IC50 values for the candidate ON obtained in the two microbes.
[0236] Thus, if the candidate ON is claimed to have an antimicrobial activity against a certain microbe, then the IC50 generation will be determined in this microbe using one of the assays described herein, or other assays known in the art. Similarly, IC50 generation will be performed for the candidate ON against a second microbe using one of the assays as described herein or an assay accepted by the industry for a microorganisme whose genome has no homology to the sequence of the candidate ON but is from the same genus. IC50 generation is also performed for a randomer of equivalent size and chemistry against each of the microbes. The IC50 of the ON randomer against the two microbes are used to normalize the IC50 values for the candidate ON against the two microbes as follows.
[0237] An equivalent algebraic transformation is applied to the IC50 of the candidate ON and the ON randomer in the first (homologous) microbe such that the IC50 of the randomer is now 1.
[0238] An equivalent algebraic transformation is applied to the IC50 of the candidate ON and the ON randomer in the second (non-homologous) microbe such that the IC50 of the randomer in now 1. [0239] The fold difference in the IC50s for the candidate ON in the homologus versus the non-homologous microbe is calculated by dividing the transformed IC50 of the candidate ON in the non-homologous microbe by the transformed IC50 of the candidate ON in the homologous microbe.
[0240] The candidate ON shall be deemed to have sequence independent activity if the fold difference in IC50 between the two microbes is less than 5.
TEST #4 Antimicrobial activity of a candidate ON in a different genus
[0241] This test serves to determine if a candidate ON has a drug-like activity in a microbe where the sequence of the candidate ON is not homologous to any portion of the microbial genome and the microbial is from a different genus or family. Thus the candidate ON shall be tested using one of the assays described herein for the acceptable formulation to the pharmaceutical industry such that the sequence of the candidate ON tested is not homologous to any portion of the genome of the microbe to be used. An IC50 value shall be generated using a minimum of seven concentrations of the candidate ON, with three or more points in the linear range. If the resulting dose response curve indicates a drug-like activity (which can typically be seen as a decay or sigmoidal curve, having reduced anti-microbial efficacy with decreasing concentrations of candidate ON) and the IC50 generated from the curve is less than 10 uM, the candidate ON shall be deemed to have a drug-like activity. If the candidate ON is deemed to have a drug-like activity in a microbe from a different family for which the candidate ON is not complementary and thus can have no sequence dependent antisense activity, it shall be considered to have sequence- independent activity.
Test #5 Extracellular antimicrobial activity of a candidate ON
[0242] The sequence-independent antimicrobial activity of ONs occurs outside the cell. The state of the art in ON technology teaches that, since ONs are not readily cell permeable, they must be delivered across the cell membrane by an appropriate carrier to have antisense activity in an in vitro context. Thus, the anti-microbial activity of antisense ONs by definition is dependent on delivery inside cells for activity. If a particular sequence-specific candidate ON has in vitro antimicrobial activity when used naked, it must benefit from the sequence-independent properties of ONs described in this invention.
[0243] The activity if the candidate ON shall be assessed using the assays decribed herein where these specific microbes are the target of the candidate ON. For other microbes, the activity shall be assessed using an assay acceptable to the pharmaceutical industry.
[0244] Using the appropriate assay, the antimicrobial activity of the naked candidate ON shall be compared to that of the encapsulated (for transfection) candidate ON (using identical candidate ON concentrations in both naked and encapsulated conditions). The activity shall be measured by a dose response curve with not less than 7 concentrations, at least 3 of which fall in the linear range which includes the 50% inhibition of microbial activity. The IC50 (the concentration which reduces microbial activity 50%) shall be calculated by linear regression of the linear range of the dose response curve as defined above. If the IC50 of the naked candidate ON is less than 5 fold greater than that of the encapsulated candidate ON, then the activity of the candidate ON shall be deemed to have sequence-independent activity.
Thresholds used in these tests
[0245] The purpose of these tests are to determine by a reasonable analysis, if ONs benefit from or utilize the sequence-independent antimicrobial properties of ONs which have been described herein and is acting with sequence-independent activity. Of course anyone skilled in the art will realize that, given the inherent variability of all testing methodologies, especially antimicrobial testing methods, a determination of differences in antimicrobial activity between two compounds may not be reliably concluded if the threshold is set at a 2 or 3 fold differences between the activities of said compounds. This is due to the fact that variations from experiment to experiment with the same compound in these assays can yield IC50s which vary in this range. Thus, to be reasonably certain that real differences between the activities of two compounds (e.g. two ONs) exist, a threshold of at least a 5-fold difference between the IC50s of said compounds have been set. This threshold ensures the reliability of the assessment of the above mentioned tests. [0246] The thresholds described in tests 1 to 3 and 5 above are the default thresholds. If specifically indicated, other thresholds can be used in the comparison tests 1 to 3 and 5 described above. Thus for example, if specifically indicated, the threshold for determining whether an ON is acting with sequence-independent activity can be any of 10-fold, 8-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.5-fold, or equal. The threshold described in test 4 above is also a default threshold. If specifically indicated, the threshold for determining whether an ON has sequence-independent activity in test 4 can be an IC50 of less than lOuM, 5uM, 1 uM, 0.8 uM, 0.6uM, 0.5uM, 0.4 uM, 0.3 uM, 0.2 uM or 0.1 uM.
[0247] Similarly, though the default is that satisfying any one of the above 5 tests is sufficient, if specifically indicated, the ON can be required to satisfy any two (e.g., tests 1 & 2, 1 & 3, 1 & 4, 1 & 5, 2 & 3, 2& 4, 2 & 5, 3 & 4, and 3 & 5), any three (e.g., tests 1 & 2 & 3, 1 & 2 & 4, 1,& 2 & 5, 1 & 3 & 4, 1 & 3 & 5, 2 & 3 & 4, and 2 & 4 & 5), any 4 of the tests (e.g., 1 & 2 & 3 & 4, 1 & 2 & 3 & & 5, and 2 & 3 & 4 & 5) at a default threshold, or if specifically indicated, at another threshold(s) as indicated above.
Example 8 Sulfur modified ONs have a therapeutic effect against malaria infection.
[0248] The effect of REP 2006 (SEQ ID NO: 6), a 40mer fully degenerate, fully phosphorothioated oligodeoxynucleotides, and REP 2031 (SEQ ID NO: 31), a 40mer fully phosphorothioated poly deoxycytosine nucleotide, to prevent progression of malaria in vivo was assessed in mice infected with Plasmodium chabaudi adami DS parasitized red blood cells. Parasitemia was followed each day by monotring the percentage of infected red blood cells (Table 10)
Table 10
Percentage of infected red blood cells (n=8) in mice following infection with Plasmodium chabaudi adami DS parasitized red blood cells.
Figure imgf000078_0001
[0249] These data show that both REP 2006 (SEQ ID NO: 6) and REP 2031 (SEQ ID NO: 31) significantly reduced the progression of infection in mice and that sulfur modified oligonucleotides are a potential therapeutic treatment for malarial infections and other microbial infections.
[0250] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. An antimicrobial oligonucleotide formulation comprising at least one oligonucleotide having an antimicrobial activity against a target microorganism, said activity occuring principally by a sequence independent mode of action.
2. The oligonucleotide of claim 1, wherein said oligonucleotide formulation further comprises at least one delivery system.
3. The oligonucleotide formulation of any one of claims 1 -2, wherein said oligonucleotide is at least 15 nucleotides in length.
4. The oligonucleotide formulation of any one of claims 1-3, wherein said oligonucleotide is at least 20 nucleotides in length.
5. The oligonucleotide formulation of any one of claim 1-4, wherein said oligonucleotide is at least 25 nucleotides in length.
6. The oligonucleotide formulation of any one of claim 1-5, wherein said oligonucleotide is at least 30 nucleotides in length.
7. The oligonucleotide formulation of any one of claims 1-6, wherein said oligonucleotide is at least 35 nucleotides in length.
8. The oligonucleotide formulation of any one of claims 1-7, wherein said oligonucleotide is at least 40 nucleotides in length.
9. The oligonucleotide formulation of any one of claims 1-8, wherein said oligonucleotide is at least 45 nucleotides in length.
10. The oligonucleotide formulation of any one of claims 1-9, wherein said oligonucleotide is at least 50 nucleotides in length.
11. The oligonucleotide formulation of any one of claims 1-10, wherein said oligonucleotide is at least 60 nucleotides in length.
12. The oligonucleotide formulation of any one of claims 1-11, wherein said oligonucleotide is at least 80 nucleotides in length.
13. The oligonucleotide formulation of any one of claims 1 -6, wherein said oligonucleotide is 20-30 nucleotides in length.
14. The oligonucleotide formulation of any one of claims 1-8, 13, wherein said oligonucleotide is 30-40 nucleotides in length.
15. The oligonucleotide formulation of any one of claims 1-10, 13-14, wherein said oligonucleotide is 40-50 nucleotides in length.
16. The oligonucleotide formulation of any one of claims 1-11, 13-15, wherein said oligonucleotide is 50-60 nucleotides in length.
17. The oligonucleotide formulation of any one of claims 1-11, 13-16, wherein said oligonucleotide is 60-70 nucleotides in length.
18. The oligonucleotide formulation of any one of claims 1-12, 13-17 wherein said oligonucleotide is 70-80 nucleotides in length.
19. The oligonucleotide formulation of any one of claims 1-18, wherein said oligonucleotide is not complementary to any equal length portion of a microbial genomic sequence.
20. The oligonucleotide formulation of any one of claims 1-19, wherein said genomic sequence is of a human.
21. The oligonucleotide formulation of any one of claims 1-20, wherein said genomic sequence is of a non human animal.
22. The oligonucleotide formulation of any one of claims 1-21, wherein said oligonucleotide comprises at least 10 contiguous nucleotides of randomer sequence.
23. The oligonucleotide formulation of any one of claims 1-22, wherein said oligonucleotide comprises at least 20 nucleotides of randomer sequence.
24. The oligonucleotide formulation of any one of claims 1-23, wherein said oligonucleotide comprises at least 30 nucleotides of randomer sequence.
25. The oligonucleotide formulation of any one of claims 1-24, wherein said oligonucleotide comprises at least 40 nucleotides of randomer sequence.
26. The oligonucleotide formulation of any one of claims 1-18, wherein said oligonucleotide is a randomer oligonucleotide.
27. The oligonucleotide formulation of any one of claims 1 -25, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous A nucleotides.
28. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous T nucleotides.
29. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous U nucleotides.
30. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous G nucleotides.
31. The oligonucleotide formulation of any one of claims 1 -25, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous I nucleotide analogs.
32. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a homopolymer sequence of at least 10 contiguous C nucleotides.
33. The oligonucleotide formulation of any one of claims 1-18, wherein said oligonucleotide is a homopolymer of C nucleotides.
34. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a poly AT sequence at least 10 nucleotides in length.
35. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyAC sequence at least 10 nucleotides in length.
36. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a poly AG sequence at least 10 nucleotides in length.
37. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a poly AU sequence at least 10 nucleotides in length.
38. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a poly AI sequence at least 10 nucleotides in length.
39. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyGC sequence at least 10 nucleotides in length.
40. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyGT sequence at least 10 nucleotides in length.
41. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyGU sequence at least 10 nucleotides in length.
42. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyGI sequence at least 10 nucleotides in length.
43. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyCT sequence at least 10 nucleotides in length.
44. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyCU sequence at least 10 nucleotides in length.
45. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyCI sequence at least 10 nucleotides in length.
46. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyTI sequence at least 10 nucleotides in length.,
47. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyTU sequence at least 10 nucleotides in length.
48. The oligonucleotide formulation of any one of claims 1-25, wherein said oligonucleotide comprises a polyUI sequence at least 10 nucleotides in length.
49. The oligonucleotide formulation of any one of claims 1-48, wherein said oligonucleotide comprises at least one phosphodiester linkage.
50. The oligonucleotide formulation of any one of claims 1-48, wherein said oligonucleotide comprises at least one ribonucleotide.
51. The oligonucleotide formulation of any of claims 1-48, wherein said oligonucleotide comprises at least one modification to its chemical structure.
52. The oligonucleotide formulation of any one of claims 1-48, 51, wherein said oligonucleotide comprises at least two different modifications to its chemical structure
53. The oligonucleotide formulation of claim 52, wherein one of said at least two different modifications is a sulfur modification.
54. The oligonucleotide formulation of any one of claims 52-53, wherein one of said at least two different modifications is a phosphorothioated linkage.
55. The oligonucleotide formulation of any one of claims 52-54, wherein one of said at least two different modifications is a phosphorodithioated linkage.
56. The oligonucleotide formulation of any one of claims 52-55, wherein one of said at least two different modifications is a boranophosphate linkage.
57. The oligonucleotide formulation of any one of claims 52-56, wherein one of said at least two different modifications is a sulfur modified nucleobase moiety.
58. The oligonucleotide formulation of any of claims 52-57 wherein one of said at least two different modifications is a sulfur modified ribose moiety.
59. The oligonucleotide formulation of any one of claims 1-58, wherein said oligonucleotide comprises at least one 2' modification to the ribose moiety.
60. The oligonucleotide formulation of claim 59, wherein said at least one 2' modification to the ribose moiety is a 2'-0 alkyl modified ribose moiety.
61. The oligonucleotide formulation of any one of claims 59-60, wherein said at least one 2' modification to the ribose moiety is a 2'-0 methyl modified ribose.
62. The oligonucleotide formulation of any one of claims 59-61 , wherein said at least one 2' modification to the ribose moiety is a 2'-methoxyethyl modified ribose.
63. The oligonucleotide formulation of any one of claims 59-62, wherein said at least one 2' modification to the ribose moiety is a 2'-FANA modified ribose.
64. The oligonucleotide formulation of any one of claims 1-63, wherein said oligonucleotide comprises at least one methylphosphonate linkage.
65. The oligonucleotide formulation of any one of claims 1-64, wherein said oligonucleotide comprises at least one portion consisting of glycol nucleic acid (GNA) with an acyclic propylene glycol phosphorothioate backbone.
66. The oligonucleotide formulation of any of one claims 1-65, wherein said oligonucleotide comprises at least one locked nucleic acid portion.
67. The oligonucleotide formulation of any one of claims 1-66, wherein said oligonucleotide comprises at least one phosphorodiamidate morpholino portion.
68. The oligonucleotides formulation of any one of claims 1-67, wherein said oligonucleotides comprises at least one abasic nucleic acid.
69. The oligonucleotide formulation of any one of claims 1-68, wherein said oligonucleotide comprises a linker to form a concatemer of two or more oligonucleotide sequences.
70. The oligonucleotide formulation of any one of claims 1-69, wherein said oligonucleotide is linked or conjugated at one or more nucleotide residues, to a molecule modifying the characteristics of the oligonucleotide to obtain one or more characteristics selected from the group consisting of higher stability, lower serum interaction, higher cellular uptake, an improved ability to be formulated, a detectable signal, higher antimicrobial activity, better pharmacokinetic properties, specific tissue distribution and lower toxicity.
71. The oligonucleotide formulation of claim 70 wherein said oligonucleotide is linked or conjugated to a PEG molecule.
72. The oligonucleotide formulation of claim 70 wherein said oligonucleotide is linked or conjugated to a cholesterol molecule.
73. The oligonucleotide formulation of any one of claims 1-21 and 27-72, wherein said oligonucleotide is double stranded.
74. The oligonucleotide formulation of any one of claims 1-73, wherein said oligonucleotide comprises at least one base which is capable of hybridizing via non- Watson-Crick interactions.
75. The oligonucleotide formulation of any one of claims 1-18 and 22-25 and 27- 32 and 34-74, wherein said oligonucleotide comprises a portion complementary to a genome.
76. The oligonucleotide formulation of any one of claims 1-75, wherein said oligonucleotide binds to one or more cellular components.
77. The oligonucleotide formulation of any one of claims 1-76, wherein said oligonucleotide interacts with one or more cellular components, wherein said interaction results in inhibition of a protein activity or expression.
78. The oligonucleotide formulation of any one of claims 1-18 and 22-25 and 27- 32 and 34-77, wherein at least a portion of the sequence of said oligonucleotide is derived from a genome.
79. The oligonucleotide formulation of any one of claims 1-18, 22-25, 27-32 and 34-78 wherein said oligonucleotide has at most 90%, preferably 80%, more preferably 75% identity with a genomic sequence.
80. The oligonucleotide formulation of any one of claims 1-79, wherein said oligonucleotide targets a protozoan parasite.
81. The oligonucleotide formulation of any one of claims 1 -79, wherein said oligonucleotide targets Plasmodium sp.
82. The oligonucleotide formulation of any one of claims 1-79, wherein said oligonucleotide targets Plasmodium falciparum.
83. The oligonucleotide formulation of any one of claims 1-79, wherein said oligonucleotide targets a bacterium.
84. The oligonucleotide formulation of any one of claims 1-79, wherein said oligonucleotide targets Escherichia sp.
85. The oligonucleotide formulation of any one of claims 1-79, wherein said oligonucleotide targets Streptococcus sp.
86. The oligonucleotide formulation of any one of claims 1-79, wherein said oligonucleotide targets a fungus.
87. The oligonucleotide formulation of any one of claims 1-86, comprising a mixture of at least two different oligonucleotides.
88. The oligonucleotide formulation of any one of claims 1-87, comprising a mixture of at least ten different oligonucleotides.
89. The oligonucleotide formulation of any one of claims 1-88, comprising a mixture of at least 100 different oligonucleotides.
90. The oligonucleotide formulation of any one of claims 1-89, comprising a mixture of at least 1000 different oligonucleotides.
91. The oligonucleotide formulation of any one of claims 1 -90, comprising a mixture of at least 106 different oligonucleotides.
92. An antimicrobial pharmaceutical composition comprising
a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to any one of claims 1 to 91 ; and
a pharmaceutically acceptable carrier.
93. The antimicrobial pharmaceutical composition of claim 92, adapted for the treatment, control, or prevention of a microbial infection disease.
94. The antimicrobial pharmaceutical composition of claim 93, wherein said microbial infection disease is a protozoan parasite infection.
95. The antimicrobial pharmaceutical composition of claim 94, wherein said microbial infection disease is malaria.
96. The antimicrobial pharmaceutical composition of claim 95, wherein said microbial infection disease is a bacterial infection.
97. The antimicrobial pharmaceutical composition of any one of claims 92-96, adapted for delivery by a mode selected from the group consisting of ocular administration, eye drop administration, oral ingestion, subcutaneous injection, intramuscular injection, and intravenous injection.
98. The antimicrobial pharmaceutical composition of claim 97, wherein said composition further comprises a delivery system.
99. The antimicrobial pharmaceutical composition of claim 98 wherein said composition further comprises at least one other antimicrobial drug in combination.
100. The antimicrobial pharmaceutical composition of claim 99, wherein said composition further comprises a non-nucleotidic antimicrobial in combination.
101. The antimicrobial pharmaceutical composition of claim 100, wherein said composition further comprises an antimicrobial antisense oligonucleotide in combination.
102. The antimicrobial pharmaceutical composition of claim 101, wherein said composition further comprises an antimicrobial RNAi-inducing oligonucleotide.
103. Use of a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to any of claims 1-91, or antimicrobial pharmaceutical composition according to any of claims 92-102 for the prophylaxis or treatment of a microbial infection disease in a subject.
104. The use of claim 103, wherein said microbial infection disease is a protozoan parasite infection.
105. The use of claim 104, wherein said microbial infection disease is malaria.
106. The use of claim 105, wherein said microbial infection disease is a bacterial infection.
107. The use of claim 105, wherein said microbial infection disease is a fungal infection.
108. The use of claim 105, wherein wherein said subject is a human
109. The use of claim 103, wherein said subject is a non-human animal.
110. A method for the prophylaxis or treatment of a microbial infection disease in a subject, comprising
administering to a subject in need of such treatment a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide formulation according to any of claims 1-91, or antimicrobial pharmaceutical composition according to any one of claims 92-102.
111. The method of claim 110, wherein said microbial infection disease is a protozoan parasite infection.
112. The method of claim 110, wherein said microbial infection disease is malaria.
113. The method of claim 110, wherein said microbial infection disease is a bacterial infection.
114. The method of claim 110, wherein said microbial infection disease is a fungal infection.
115. The method of any of claims 110-114, wherein said subject is a human.
116. The method of any of claims 110-114, wherein said subject is a non-human animal.
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