EP1086116A1 - Compositions destinees a l'administration pulmonaire d'acides nucleiques et procedes - Google Patents

Compositions destinees a l'administration pulmonaire d'acides nucleiques et procedes

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Publication number
EP1086116A1
EP1086116A1 EP99923263A EP99923263A EP1086116A1 EP 1086116 A1 EP1086116 A1 EP 1086116A1 EP 99923263 A EP99923263 A EP 99923263A EP 99923263 A EP99923263 A EP 99923263A EP 1086116 A1 EP1086116 A1 EP 1086116A1
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EP
European Patent Office
Prior art keywords
oligonucleotide
antisense
pharmaceutical composition
isis
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99923263A
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German (de)
English (en)
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EP1086116A4 (fr
Inventor
Clarence Frank Bennett
David J. Ecker
Phillip Dan Cook
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Publication of EP1086116A1 publication Critical patent/EP1086116A1/fr
Publication of EP1086116A4 publication Critical patent/EP1086116A4/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—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
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

  • the present invention relates to compositions and methods for the delivery of nucleic acid therapeutics and diagnostics to the lung of an animal, particularly a human . More particularly, the present invention is directed to compositions and methods for the pulmonary delivery of oligonucleotide therapeutics and diagnostics, including antisense oligonucleotides. In some preferred embodiments, the present invention is directed to methods and compositions for pulmonary delivery of oligonucleotide therapeutic compositions comprising penetration enhancers, carrier compounds and/or transfection agents.
  • Oligonucleotides have been administered by various routes. For example, oligonucleotides administered by parenteral routes have been shown to be effective for the treatment of diseases and/or disorders. See, e.g., U.S.
  • Patent No. 5,595,978, January 21, 1997 to Draper et al . which discloses intravitreal injection as a means for the direct delivery of antisense oligonucleotides to the vitreous humor of the mammalian eye. See also, Robertson, Nature Biotechnology, 1997, 15:209 and Anon., Genetic
  • oligonucleotides via the lung for the treatment of pulmonary disorders are attractive because oligonucleotide is delivered directly to the target organ.
  • ⁇ yce J. ., Exp . Opin . Invest . Drugs (1997) 6 (9) .1149-1156; Schreier, H.,
  • Antisense oligonucleotides have been shown to demonstrate antisense effect upon cells of various diseases or disorders, including cancer. See, for example, Dosaka- Akita et al . , Cancer Res. 55, 1559-1564 (1995) (inhibition of proliferation by L-myc antisense DNA for the transitional initiation site in human small cell lung cancer) .
  • compositions which can effectively provide for the pulmonary delivery of nucleic acids, particularly oligonucleotides, more particularly oligonucleotides having one or more chemical modifications, together with methods for using such compositions to deliver such oligonucleotides and nucleic acids into the lung of an animal.
  • the present invention is directed to these, as well as other, important ends.
  • the present invention is directed to compositions and methods for pulmonary delivery of oligonucleotides .
  • the present invention provides pharmaceutical compositions for pulmonary delivery of an oligonucleotide comprising at least one oligonucleotide or bioequivalent thereof .
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.
  • an nucleic acid therapeutic or diagnostic composition comprising: preparing a nucleic acid therapeutic or diagnostic composition; aerosolizing the nucleic acid composition; and introducing the aerosolized nucleic acid composition into the lung of a mammal; wherein the aerosolized nucleic acid composition comprises at least one oligonucleotide.
  • the present invention provides methods of treating an animal having or suspected of having a disease or disorder that is treatable with one or more nucleic acids comprising administering a therapeutically effective amount of an aerosolized nucleic acid composition to the lung of the animal, wherein the aerosolized nucleic acid composition comprises at least one oligonucleotide or bioequivalent thereof.
  • the present invention provides methods of investigating the role of gene or gene product in an animal other than a human comprising administering a therapeutically effective amount of an aerosolized nucleic acid composition to the lung of the animal, wherein the aerosolized nucleic acid composition comprises at least one oligonucleotide or bioequivalent thereof.
  • the present invention provides methods for delivering an oligonucleotide therapeutic or diagnostic compound to the lung of an animal comprising applying to said lung a pharmaceutical composition in accordance with the present invention.
  • the oligonucleotide is delivered within cells of said lung, preferably to animal that is known or suspected to suffer from a disease or disorder.
  • the disease or disorder is asthma, a cancer of the lung, pulmonary fibrosis, rhinovirus, tuberculosis, bronchitis, or pneumonia.
  • the pharmaceutical composition is in aqueous media.
  • the aqueous media is sterilized, pyrogen free water.
  • the aqueous media is saline solution.
  • the aqueous media is a buffer.
  • the pharmaceutical composition is a powder.
  • the oligonucleotide is an antisense oligonucleotide, which preferably modulates, the expression of a protein or modulates a rate of cellular proliferation.
  • the antisense oligonucleotide modulates expression of a cellular adhesion protein.
  • antisense oligonucleotide is antisense to a genetic sequence implicated in a disease or disorder, preferably, asthma, a cancer of the lung, pulmonary fibrosis, rhinovirus, tuberculosis, bronchitis, or pneumonia.
  • the antisense oligonucleotide is antisense to a portion of a gene coding for a cytokine.
  • the antisense oligonucleotide is antisense to a portion of a gene coding for ICAM-1, ELAM-1, VCAM-1, B7-1, B7-2, CD40, LFA-3, PECAM-1, a ras oncogene, an H-ras oncogene, a K-ras oncogene, or Protein Kinase C.
  • the antisense oligonucleotide contains one or more phosphodiester linkages. In further preferred embodiments, the antisense oligonucleotide contains one or more phosphorothioate linkages. In further preferred embodiments, the pharmaceutical composition comprises more than one antisense oligonucleotide.
  • the nucleic acid therapeutic composition is an aerosolized solution consisting essentially of an antisense oligonucleotide in saline solution.
  • the oligonucleotide is a ribozyme, an external guide sequence, or an antisense peptide nucleic acid.
  • the oligonucleotide is an aptamer or a molecular decoy.
  • the aqueous media of the pharmaceutical composition is sterilized, pyrogen free buffer solution.
  • the nucleic acid therapeutic composition is aerosolized solution consists essentially of an antisense oligonucleotide in buffer solution.
  • methods are provided for modulating the expression of a gene in an animal comprising administering to said animal the pharmaceutical composition of the invention.
  • the present invention also provides medical devices for pulmonary delivery of an aerosol comprising a pharmaceutical composition in accordance with the present invention.
  • the medical device is a nebulizer.
  • the present invention provides novel compounds comprising at least one moiety of Formula:
  • Ri has the formula -0-R 5 -0-R 6 ; R 5 and R 6 are independently alkyl having from 1 to about five carbons; and Q is 5-methylcytosine.
  • R 5 is -CH 2 -CH 2 - and R 6 is -CH 3 .
  • compounds are provided having the formula:
  • R ⁇ has the formula -0-R 5 -0-R 6 ;
  • R 5 and R 6 are independently alkyl having from 1 to about five carbons
  • Q is 5-methylcytosine
  • M is an internucleoside linkage
  • B is a nucleobase; each R 2 is H, OH, F, or a group of formula R 7 -(R 8 ) V ; R 7 is C 3 -C 20 alkyl, C 4 -C 20 alkenyl, C 2 -C 20 alkynyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyloxy, or C 2 -C 20 alkynyloxy;
  • R 8 is hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, 0-alkyl, S-alkyl, NH-alkyl, N-dialkyl, 0- aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, inter- calator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, or a group that enhances the pharmaco-
  • R 3 is H or a hydroxyl protecting group
  • R 4 is H, OH, an internucleoside linkage, a linker connected to a solid support, or a group of formula -O-Pr where Pr is a hydroxyl protecting group
  • m and n are each independently from 0 to about 50.
  • R 5 is -CH 2 -CH 2 - and R 6 is -CH 3 .
  • each R x is -0-CH 2 - CH 2 -0-CH 3 .
  • each R 2 is -0-CH 2 -CH 2 -0-CH 3
  • B is selected from the group consisting of 5-methylcytosine, adenine, guanine, uracil and thymine.
  • oligonucleotides comprising one or more 5- methylcytosine-2 ' -methoxyethoxy nucleosidic moieties.
  • compositions comprising a compound of the invention.
  • Figure 1 is a plot showing that oligonucleotides were uniformly nebulized, and that the size of the resultant particles is not altered over time.
  • Figure 2 shows nebulization of oligonucleotide (ISIS 2503; 40 mg/mL) by a PulmoAide Nebulizer (Apguard Medical, Inc., Woodland Hills, CA) for a period of 20 minutes.
  • the mist coming out of the nebulizer was collected in an impinger and was analyzed for oligonucleotide content by ultraviolet absorption.
  • the straight line of the graph indicates that the nebulization was uniform over the course of the experiment .
  • the present invention provides compositions and methods for the pulmonary delivery of oligonucleotides and other nucleic acids to the lung of an animal .
  • the present invention provides compositions and methods for modulating the in vivo expression of a gene in an animal through the pulmonary administration of an antisense oligonucleotide, thereby bypassing the complications and expense which may be associated with intravenous and other routes of administration.
  • Enhanced delivery of the oligonucleotides and other nucleic acids to the lung of an animal is achieved through the use of the compositions and methods of the invention.
  • oligonucleotides are rapidly eliminated from plasma and accumulate mainly in the liver and kidney after i.v. administration (Miyao et al . ,
  • One means of ameliorating first pass clearance effects is to increase the dose of an administered drug, thereby compensating for proportion of drug lost to first pass clearance. Although this may be readily achieved with i.v. administration by, for example, simply providing more of the drug to an animal, other factors influence the bioavailability of administred drugs .
  • the present invention provides pharmaceutical compositions for pulmonary administration of large molecule therapeutics such as oligonucleotides comprising the oligonucleotide and at least one substance which facilitates the transport of a drug across the mucous membrane (s) of the lung (so called “mucosal penetration enhancers,” also known as “absorption enhancers” or simply as “penetration enhancers”).
  • oligonucleotides also known as “absorption enhancers” or simply as “penetration enhancers”
  • the present invention provides compositions and methods for pulmonary delivery of one or more nucleic acids to an animal.
  • the term "animal” is meant to encompass humans as well as other mammals, as well as reptiles, fish, amphibians, and birds.
  • pulmonary delivery refers to the administration, directly or otherwise, to a portion of the lung of an animal.
  • the term “lung” has its accustomed meaning as the chief organ of respiration (i.e. gas exchange) in an animal.
  • the term “pulmonary delivery” subsumes the absorption of the delivered component from the interior surface of lung, into the lung tissue.
  • the present invention provides compositions and methods for the pulmonary administration of oligonucleotides.
  • the compositions can contain carrier compounds, penetration enhancing agents, and/or transfection agents.
  • carrier compound refers to a nucleic acid, or analog thereof, which is inert
  • nucleic acid (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, typically 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, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.
  • the recovery of a partially phosphorothioated oligonucleotide in hepatic tissue is 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 . , Antisense Res . Dev. , 1995, 5:115; Takakura et al . , Antisense & Nucl . Acid Drug Dev. , 1996, 6 : 177 ) .
  • 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 .
  • the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e . g. , pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers ( e . g.
  • 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.
  • disintegrates e.g., starch, sodium starch glycolate, etc.
  • wetting agents e.g., sodium lauryl sulphate, etc.
  • the present invention employs oligonucleotides for use in antisense modulation of the function of DNA or messenger RNA (mRNA) encoding a protein the modulation of which is desired, and ultimately to regulate the amount of such a protein.
  • mRNA messenger RNA
  • Hybridization of an antisense oligonucleotide with its mRNA target interferes with the normal role of mRNA and causes a modulation of its function in cells .
  • mRNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, turnover or degradation of the mRNA and possibly even independent catalytic activity which may be engaged in by the RNA.
  • the overall effect of such interference with mRNA function is modulation of the expression of a protein, wherein “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of the protein. In the context of the present invention, inhibition is the preferred form of modulation of gene expression.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases .
  • An oligonucleotide is a polymer of repeating units generically known as a nucleotides .
  • An unmodified (naturally occurring) nucleotide has three components: (1) a nitrogenous base linked by one of its nitrogen atoms to (2) a 5-carbon cyclic sugar and (3) a phosphate, esterified to carbon 5 of the sugar.
  • the phosphate of a first nucleotide is also esterified to carbon 3 of the sugar of a second, adjacent nucleotide.
  • the "backbone” of an unmodified oligonucleotide consists of (2) and (3) , that is, sugars linked together by phosphodiester linkages between the carbon 5 (5') position of the sugar of a first nucleotide and the carbon 3 (3') position of a second, adjacent nucleotide.
  • a “nucleoside” is the combination of (1) a nucleobase and (2) a sugar in the absence of (3) a phosphate moiety (Kornberg, A., DNA Replication, W.H.
  • Oligonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid. Such oligonucleotides which specifically hybridize to a portion of the sense strand of a gene are commonly described as "antisense.” In the context of the invention,
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds .
  • “Complementary, as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • oligonucleotide and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable.
  • An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a decrease or loss of function, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vi tro assays, under conditions in which the assays are performed.
  • Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutic agents.
  • antisense oligonucleotides which are able to inhibit gene expression with extraordinar specificity, are often used by those of ordinary skill to elucidate the function of particular genes, for example to distinguish between the functions of various members of a biological pathway.
  • This specific inhibitory effect has, therefore, been harnessed by those skilled in the art for research uses .
  • the specificity and sensitivity of oligonucleotides is also harnessed by those of skill in the art for therapeutic uses.
  • the following U.S. patents demonstrate palliative, therapeutic and other methods utilizing antisense oligonucleotides.
  • U. S. Patent No. 5,135,917 provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression.
  • Patent No. 5,098,890 is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions.
  • U.S. Patent No. 5,087,617 provides methods for treating cancer patients with antisense oligonucleotides.
  • U.S. Patent No. 5,166,195 provides oligonucleotide inhibitors of Human Immunodeficiency Virus (HIV) .
  • U.S. Patent No. 5,004,810 provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication.
  • U.S. Patent No. 4,806,463 provides antisense oligonucleotides and methods using them to inhibit HTLV- III replication.
  • U.S. Patent No. 5,286,717 provides oligonucleotides having a complementary base sequence to a portion of an oncogene.
  • U.S. Patent No. 5,276,019 and U.S. Patent No. 5,264,423 are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells.
  • 4,689,320 is directed to antisense oligonucleotides as antiviral agents specific to cytomegalovirus (CMV) .
  • CMV cytomegalovirus
  • U.S. Patent No. 5,098,890 provides oligonucleotides complementary to at least a portion of the mRNA transcript of the human c-myb gene.
  • U.S. Patent No. 5,242,906 provides antisense oligonucleotides useful in the treatment of latent Epstein-Barr virus (EBV) infections.
  • EBV Epstein-Barr virus
  • Other examples of antisense oligonucleotides are provided herein.
  • the oligonucleotides in accordance with this invention preferably comprise from about 8 to about 30 nucleotides.
  • oligonucleotides comprise from about 15 to 25 nucleotides.
  • a nucleotide is a base-sugar combination suitably bound to an adjacent nucleotide through a phosphodiester, phosphorothioate or other covalent linkage.
  • the term "oligonucleotide” includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions which function similarly. Such modified or substituted oligonucleotides may be preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases .
  • Oligonucleotides are also useful in determining the nature, function and potential relationship to body or disease states in animals of various genetic components of the body.
  • the function of a gene has been chiefly examined by the construction of loss-of-function mutations in the gene (i.e., "knock-out” mutations) in an animal (e.g., a transgenic mouse) .
  • Such tasks are difficult, time-consuming and cannot be accomplished for genes essential to animal development since the "knock-out" mutation would produce a lethal phenotype.
  • the loss-of-function phenotype cannot be transiently introduced during a particular part of the animal ' s life cycle or disease state; the "knock-out” mutation is always present.
  • Antisense knockouts that is, the selective modulation of expression of a gene by antisense oligonucleotides, rather than by direct genetic manipulation, overcomes these limitations (see, for example, Albert et al . , Trends in
  • the present invention further encompasses compositions employing ribozymes.
  • Synthetic RNA molecules and derivatives thereof that catalyze highly specific endoribonuclease activities are known as ribozymes.
  • the cleavage reactions are catalyzed by the RNA molecules themselves .
  • the sites of self- catalyzed cleavage are located within highly conserved regions of RNA secondary structure (Buzayan et al . , Proc. Natl . Acad. Sci . U. S .A . , 1986, 83 , 8859; Forster et al . ,
  • RNA molecules Naturally occurring autocatalytic RNA molecules have been modified to generate ribozymes which can be targeted to a particular cellular or pathogenic RNA molecule with a high degree of specificity.
  • ribozymes serve the same general purpose as antisense oligonucleotides (i.e., modulation of expression of a specific gene) and, like oligonucleotides, are nucleic acids possessing significant portions of single- strandedness . That is, ribozymes have substantial chemical and functional identity with oligonucleotides and are thus considered to be equivalents for purposes of the present invention.
  • oligonucleotides may be formulated in the compositions of the invention and used for therapeutic, palliative or prophylactic purposes according to the methods of the invention.
  • Such other biologically active oligonucleotides include, but are not limited to, antisense compounds including, inter alia, antisense oligonucleotides, antisense PNAs and ribozymes (described supra) and EGSs, as well as aptamers and molecular decoys (described infra) .
  • EGSs are antisense compounds that direct of an endogenous nuclease (RNase P) to a targeted nucleic acid (Forster et al . , Science, 1990, 249, 783; Guerrier-Takada et al . , Proc.
  • Antisense compounds may alternatively or additionally comprise a synthetic moiety having nuclease activity covalently linked to an oligonucleotide having an antisense sequence instead of relying upon recruitment of an endogenous nuclease.
  • Synthetic moieties having nuclease activity include, but are not limited to, enzymatic RNAs (as in ribozymes) , lanthanide ion comlexes, and the like (Haseloff et al . , Nature, 1988, 334 , 585; Baker et al . , J. Am . Chem . Soc , 1997, 119, 8749).
  • Aptamers are single-stranded oligonucleotides that bind specific ligands via a mechanism other than Watson- Crick base pairing. Aptamers are typically targeted to, e.g., a protein and are not designed to bind to a nucleic acid (Ellington et al . , Nature, 1990, 346, 818).
  • Molecular decoys are short double-stranded nucleic acids (including single-stranded nucleic acids designed to "fold back" on themselves) that mimic a site on a nucleic acid to which a factor, such as a protein, binds. Such decoys are expected to competitively inhibit the factor; that is, because the factor molecules are bound to an excess of the decoy, the concentration of factor bound to the cellular site corresponding to the decoy decreases, with resulting therapeutic, palliative or prophylactic effects.
  • Methods of identifying and constructing nucleic acid decoy molecules are described in, e.g., U.S. Patent
  • bioactive oligonucleotide is an RNA- DNA hybrid molecule that can direct gene conversion of an endogenous nucleic acid (Cole-Strauss et al . , Science, 1996, 273 , 1386) .
  • pulmonary administration of phosphodiester oligonucleotides is particularly advantageous. Specifically, it has been discovered in accordance with the present invention that the level of nuclease activity in lung tissue is sufficiently low to afford phosphodiester oligonucleotides longer lifetimes in lung tissue than was previously believed. Accordingly, contrary to conventional knowledge in the art (see, e.g., Wu-Pong et al . , Adv. Drug Delivery, 1996, 19, 47) , phosphodiester antisense oligonucleotides reside undegraded in the lung for a sufficiently long period of time to exert an antisense effect.
  • the present invention provides oligonucleotides, preferably phosphodiester and phosphorothioate oligonucleotides, that have at least one 2 ' -alkoxy-alkyloxy substituent, which is preferably, 2 ' -methoxyethoxy. It has been discovered that the presence of such 2 ' -alkoxy-alkyloxy substituents confer nuclease resistance, and increased binding.
  • a further preferred modification includes 2 ' -dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMA0E, as described in co-owned United States patent application Serial Number 09/016,520, filed on January 30, 1998, the contents of which are herein incorporated by reference.
  • Other preferred modifications include 2 ' -methoxy (2'-0-CH 3 ), 2 ' -aminopropoxy (2 ' -OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F).
  • the base portion of the nucleoside may be selected from a large palette of different base units available. These may be 'modified' or 'natural' bases (also reference herein as nucleobases) including the natural purine bases adenine (A) and guanine (G) , and the natural pyrimidine bases thy ine (T) , cytosine (C) and uracil (U) .
  • A purine
  • G guanine
  • T thy ine
  • C cytosine
  • U uracil
  • modified nucleobases including other synthetic and natural nucleobases 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-propynyl uracil and cytosine, 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 uracils and cytosines particularly 5-bromo, 5-trifluor
  • nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T.
  • nucleobases include adenine, guanine, cytosine, uridine, and thymine, as well as other non-naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudo uracil) , 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8 -substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine .
  • nucleobases such as
  • nucleosidic base' is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain 'universal bases' that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases.
  • a universal base is 3-nitropyrrole.
  • Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Patent 3,687,808, as well as U.S.
  • a base for any particular nucleoside of an oligonucleotide consideration is first given to the need of a base for a particular specificity for hybridization to an opposing strand of a particular target.
  • adenine might be selected however other alternative bases that can effect hybridization in a manner mimicking an 'A' base such as 2, 6-diaminopurine might be selected should other considersation, e.g., stronger hybridization (relative to hybridization achieved with adenine) , be desired.
  • the sugar portion of the nucleoside may be selected from a large palette of different sugar or sugar surrogate units available. These may be modified sugar groups, for instance sugars containing one or more substituent groups .
  • Preferred substituent groups comprise the following at the 2' position: OH; F; 0-, S-, or N- alkyl, 0-, S-, or N-alkenyl, or 0, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C x to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • n and m are from 1 to about 10.
  • substituent groups comprise one of the following at the 2' position: C x to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or 0- aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3, 0CF 3, SOCH 3, S0 2 CH 3, 0N0 2 N0 2r N 3; NH 2( heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a preferred modification includes 2 ' -methoxyethoxy (2 ' -0-CH 2 CH 2 OCH 3 , also known as 2 ' -0- (2-methoxyethyl) or 2 ' -MOE) (Martin et al . , Helv. Chim. Acta, 1995, 78, 486) i.e., an alkoxyalkoxy group.
  • a further preferred modification includes 2'- dimethylamino oxyethoxy, i.e., a 0 (CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMA0E, as described in co-owned United States patent application Serial Number 09/016,520, filed on January 30, 1998, the contents of which are herein incorporated by reference .
  • Modified Linkages (Backbones) : In addition to phosphodiester linkages, specific examples of some preferred modified oligonucleotides envisioned for this invention include those containing modified internucleosidic linkages, depicted as moiety "M" in the compounds described herein. These internucleoside linkages are also referred to as linkers, backbones or oligonucleotide backbones. For forming these nucleoside linkages, a palette of different internucleoside linkages or backbones is available.
  • modified oligonucleotide backbones for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotri- esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates , phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates , thionoalkylphosphonates , thionoalklyphosphotriesters, and boranophosphates having normal 3' -5' linkages, 2' -5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2 '-5' to 5 '-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred internucleoside linkages for oligonucleotides that do not include a phosphorus atom therein, i.e., for oligonucleosides, have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages .
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones siloxane backbones
  • sulfide, sulfoxide and sulfone backbones formacetyl and thioformacetyl backbones
  • methylene formacetyl and thioformacetyl backbones alkene containing backbones
  • sulfamate backbones methyleneimino and methylenehydrazino backbones
  • sulfonate and sulfonamide backbones amide backbones
  • others having mixed N, 0, S and CH 2 component parts .
  • both the sugar and the intersugar linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA) .
  • PNA peptide nucleic acid
  • the sugar-phosphate backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al . ,
  • the most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH 2 -NH-0-CH 2 - , - CH 2 -N(CH 3 ) -0-CH 2 - [known as a methylene (methylimino) or MMI backbone], -CH 2 -0-N(CH 3 ) -CH 2 - , - CH 2 -N(CH 3 ) -N(CH 3 ) -CH 2 - and -0- N(CH 3 ) -CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as -0-P-0-CH 2 -] of the above referenced U.S.
  • effector groups include, but are not limited to, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties.
  • a variety of chemical linkers may be used to conjugate an effector group to an oligonucleotide of the invention.
  • the 5 ' and 3 ' termini of an oligonucleotide may be modified to serve as points of chemical conjugation of, e.g., lipophilic moieties (see immediately subsequent paragraph), intercalating agents (Kuyavin et al . , WO 96/32496, published October 17, 1996; Nguyen et al., U.S.
  • oligonucleotide of the invention can be used to chemically link thereto one or more effector groups to form an oligonucleotide conjugate.
  • U.S. Patent No. 5,578,718 to Cook et al discloses methods of attaching an alkylthio linker, which may be further derivatized to include additional groups, to ribofuranosyl positions, nucleosidic base positions, or on internucleoside linkages . Additional methods of conjugating oligonucleotides to various effector groups are known in the art; see, e.g., Protocols for Oligonucleotide
  • oligonucleotide Conjugates (Methods in Molecular Biology, Volume 26) Agrawal, S., ed. , Humana Press, Totowa, NJ, 1994.
  • Another preferred additional or alternative modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more lipophilic moieties which enhance the cellular uptake of the oligonucleotide.
  • lipophilic moieties may be linked to an oligonucleotide at several different positions on the oligonucleotide.
  • Some preferred positions include the 3' position of the sugar of the 3' terminal nucleotide, the 5' position of the sugar of the 5' terminal nucleotide, and the 2' position of the sugar of any nucleotide.
  • the N 6 position of a purine nucleobase may also be utilized to link a lipophilic moiety to an oligonucleotide of the invention (Gebeyehu, G., et al . , Nucleic Acids Res . , 1987,
  • Such lipophilic moieties include but are not limited to a cholesteryl moiety (Letsinger et al . , Proc .
  • a phospholipid e.g., di- hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
  • Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides, are disclosed in U.S. Patents Nos. 5,138,045, 5,218,105 and 5,459,255, the contents of which are hereby incorporated by reference.
  • Oligonucleotide Synthesis The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis . Unsubstituted and substituted phosphodiester oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine .
  • Phosphorothioates are synthesized as per the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2- benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages.
  • the thiation wait step was increased to 68 sec and was followed by the capping step .
  • the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution.
  • Phosphinate oligonucleotides are prepared as described in U.S. Patent 5,508,270, herein incorporated by reference .
  • Alkyl phosphonate oligonucleotides are prepared as described in U.S. Patent 4,469,863, herein incorporated by reference.
  • 3 ' -Deoxy-3 ' -methylene phosphonate oligonucleotides are prepared as described in U.S. Patents 5,610,289 or 5,625,050, herein incorporated by reference.
  • Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Patent 5,366,878, hereby incorporated by reference.
  • Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively) .
  • 3 ' -Deoxy-3 ' -amino phosphoramidate oligonucleotides are prepared as described in U.S. Patent 5,476,925, herein incorporated by reference .
  • Phosphotriester oligonucleotides are prepared as described in U.S. Patent 5,023,243, herein incorporated by reference.
  • Boranophosphate oligonucleotides are prepared as described in U.S. Patents 5,130,302 and 5,177,198, both herein incorporated by reference .
  • Methylenemethylimino linked oligonucleosides also identified as MMI linked oligonucleosides, methylenedi- methylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo- nucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and PO or PS linkages are prepared as described in U.S. Patents 5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289, all of which are herein incorporated by reference.
  • Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Patents 5,264,562 and 5,264,564, herein incorporated by reference.
  • Ethylene oxide linked oligonucleosides are prepared as described in U.S. Patent 5,223,618, herein incorporated by reference .
  • PNAs Peptide nucleic acids
  • PNA Peptide nucleic acids
  • Chimeric Oligonucleotides It is not necessary for all positions in a given compound to be uniformly modified. In fact, more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.
  • the present invention also includes compounds which are chimeric compounds.
  • Chimeric' compounds or “chimeras,' in the context of this invention are compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art .
  • Chimeric antisense compounds of the invention may be formed as composite structures representing the union of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as “hybrids” or "gapmers”. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S.
  • Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types .
  • Oligonucleotides of the first type are also known in the art as "gapmers ' or gapped oligonucleotides.
  • Oligonucleotides of the second type are also known in the art as "hemimers' or "wingmers.' [2 ' -O-Me] -- [2 ' -deoxy] -- [2 ' -O-Me] Chimeric Phosphorothioate Oligonucleotides : Chimeric oligonucleotides having 2 '-O-alkyl phosphorothioate and 2'- deoxy phosphorothioate oligonucleotide segments are synthesized using 2 ' -deoxy-5 ' -dimethoxytrityl-3 ' -0- phosphoramidites for the DNA portion and 5 ' -dimethoxy- trityl-2 ' -0-methyl-3 ' -O-phosphoramidites for 5' and 3' wings .
  • the standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for DNA and twice for 2'-0-methyl.
  • the fully protected oligonucleotide was cleaved from the support and the phosphate group is deprotected in 3:1 Ammonia/Ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is done to deprotect all bases and the samples are again lyophilized to dryness .
  • (methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2 ' -0-methyl chimeric oligonucleotide with the substitution of 2 ' -0- (methoxyethyl) amidites for the 2 ' -0-methyl amidites in the wing portions.
  • Sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) is used to generate the phosphorothioate internucleotide linkages within the wing portions of the chimeric structures .
  • Oxidization with iodine is used to generate the phospho- diester internucleotide linkages for the center gap.
  • chimeric oligonucleotides are synthesized according to United States Patent 5,623,065, herein incorporated by reference.
  • the present invention also includes oligonucleotides that are substantially chirally pure with regard to particular positions within the oligonucleotides.
  • substantially chirally pure oligonucleotides include, but are not limited to, those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al . , U.S.
  • Patent No. 5,587,361 and those having substantially chirally pure (Sp or Rp) alkylphosphonate, phosphoamidate or phosphotriester linkages (Cook, U.S. Patents Nos. 5,212,295 and 5,521,302).
  • Examples of specific oligonucleotides and the target genes to which they inhibit which may be employed in formulations of the present invention include: ISIS--2302 GCCCA AGCTG GCATC CGTCA (SEQ ID NO:l) ICAM-1
  • each oligo backbone linkage is a phosphorothioate linkage (except ISIS-9605) and (ii) each sugar is 2 ' -deoxy unless represented in bold font in which case it incorporates a 2 ⁇ -O-methoxyethyl group and iii) underlined cytosine nucleosides incorporate a 5-methyl substituent on their nucleobase.
  • ISIS-9605 incorporates natural phosphodiester bonds at the first five and last five linkages with the remainder being phosphorothioate linkages .
  • a patient i.e., an animal, including a human, having, suspected of having, or predisposed to a disease or disorder
  • one or more nucleic acids including oligonucleotides
  • the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease or disorder, its severity and the overall condition of the patient, and may extend from once daily to once every 20 years.
  • treatment or “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities.
  • the patient is monitored for changes in his/her condition and for alleviation of the symptoms of the disorder or disease state .
  • the dosage of the nucleic acid may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disorder or disease state is observed, or if the disorder or disease state has been ablated. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. An optimal dosing schedule is used to deliver a therapeutically effective amount of the nucleic acid being administered via a particular mode of administration.
  • terapéuticaally effective amount refers to the amount of nucleic acid-containing formulation which is effective to achieve an intended purpose without undesirable side effects (such as toxicity, irritation or allergic response) . Although individual needs may vary, determination of optimal ranges for effective amounts of formulations is within the skill of the art. Human doses can be extrapolated from animal studies (Katocs et al . ,
  • the dosage required to provide an effective amount of a formulation will vary depending on the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy (if any) and the nature and scope of the desired effect (s) (Nies et al . , Chapter 3 In :
  • the term "high risk individual” is meant to refer to an individual for whom it has been determined, via, e.g., individual or family history or genetic testing, has a significantly higher than normal probability of being susceptible to the onset or recurrence of a disease or disorder.
  • the individual can be prophylactically treated to prevent the onset or recurrence of the disease or disorder.
  • prophylactically effective amount is meant to refer to an amount of a formulation which produces an effect observed as the prevention of the onset or recurrence of a disease or disorder.
  • Prophylactically effective amounts of a formulation are typically determined by the effect they have compared to the effect observed when a second formulation lacking the active agent is administered to a similarly situated individual.
  • nucleic acid is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
  • maintenance doses ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
  • preventative doses ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.
  • an individual may be made less susceptible to an inflammatory condition that is expected to occur as a result of some medical treatment, e.g., graft versus host disease resulting from the transplantation of cells, tissue or an organ into the individual.
  • compositions of the present invention can include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
  • the pharmaceutical formulations 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. 5.
  • the compounds of the invention encompass 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 "pharmaceutically acceptable salts" of the penetration enhancers and nucleic acids of the invention and prodrugs of such nucleic acids. “Pharmaceutically acceptable salts” are physiologically and pharmaceutically acceptable salts of the penetration enhancers and nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the oligonucleotide and nucleic acid compounds employed in the compositions of the present invention (i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto) .
  • Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, ammonium, polyamines such as spermine and sper idine, and the like.
  • suitable amines are chloroprocaine, choline, N,N' -dibenzylethylenediamine, diethanolamine, dieyelohexylamine , ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al . , "Pharmaceutical Salts," J. of Pharma Sci . , 1977,
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • the free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.
  • the oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a "prodrug" form.
  • 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 oligonucleotides of the invention are prepared as SATE [ (S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO
  • nucleoside monomers are attached to the chain one at a time in a repeated series of chemical reactions such as nucleoside monomer coupling, oxidation, capping and detritylation.
  • the stepwise yield for each nucleoside addition is above 99%. That means that less than 1% of the sequence chain failed to the nucleoside monomer addition in each step as the total results of the incomplete coupling followed by the incomplete capping, detritylation and oxidation (Smith, Anal . Chem. , 1988, 60, 381A) .
  • All the shorter oligonucleotides ranging from (n-1) , (n-2) , etc., to 1- mers (nucleotides) , are present as impurities in the n-mer olignucleotide product.
  • impurities (n-2)-mer and shorter oligonucleotide impurities are present in very small amounts and can be easily removed by chromatographic purification (Warren et al . , Chapter 9 In : Methods in
  • n-1) -mer impurities are still present in the full-length (i.e., n-mer) oligonucleotide product after the purification process.
  • the (n-1) portion consists of the mixture of all possible single base deletion sequences relative to the n-mer parent oligonucleotide.
  • Such (n-1) impurities can be classified as terminal deletion or internal deletion sequences, depending upon the position of the missing base (i.e., either at the 5' or 3 ' terminus or internally) .
  • the terminal deletion sequence impurities will bind to the same target mRNA as the full length sequence but with a slightly lower affinity.
  • impurities can be considered as part of the active drug component, and are thus considered to be bioequivalents for purposes of the present invention.
  • compositions and methods of the present invention are useful for the treatment of a wide variety of disorders including asthma, cancers of the lung, pulmonary fibrosis, and various infectious diseases of the lung, including rhinovirus, tuberculosis, bronchitis, and pneumonia .
  • T lymphocytes T cells
  • ICAM-1 has been implicated in the pathogenesis of asthma, and a monoclonal antibody to ICAM-1 attenuates eosinophilia and hyperresponsiveness (Wegner et al . ,
  • ICAM-1 are described in U.S. Patents Nos. 5,514,788 and 5,591,623, and copending U.S. patent applications Serial Nos. 09/009,490 and 09/062,416, January 20, 1998 and April 17, 1998, respectively, all to Bennett et al . , each of which are incorporated herein their entirety.
  • Adhesion molecule-mediated recruitment of eosinophils and other leukocytes has been implicated in mechanisms of asthmatic inflammation (Bochner et al . , Annu .
  • adhesion molecules of particular interest include ELAM-1 (a.k.a. E-selectin) and VCAM-1.
  • ELAM-1 a.k.a. E-selectin
  • VCAM-1 a.k.a. VCAM-1
  • Antibody to ELAM-1 prevents neutrophil accumulation in monkey lungs (Gundel et al . , J. Clin . Invest . , 1991, 88, 1407).
  • Antisense compounds targeted to the adhesions molecules ELAM-1 and VCAM-1 are described in U.S. Patents Nos. 5,514,788 and 5,591, 623.
  • ICAM-1, VCAM-1, and ELAM-1 expression woud provide a novel therapeutic class of anti-inflammatory agents with activity towards a wide variety of inflammaotry diseases, or diseases within inflammatory component such as asthma.
  • the use of neutralizing monoclonal antibodies against ICAM-1 in animal models provide ample evidence that such inhibitors if identified would have therapeutic benefit for asthma. See Wegner et al., Science 1990, 247, 456-459.
  • B7-1 and B7-2 are thought to be the primary molecules expressed on professional antigen presenting cells, (APCs) (see Li ⁇ and Linsley, Curr. Opin . Immunol . ,
  • the B7 proteins are thought to provide an essential signal for differentiation of T cells (T H 0 lymphocytes) and to contribute to the activation of memory cells .
  • Antisense compounds targeted to B7 proteins are described in copending U.S. patent application Serial No. 08/777,266, filed December 31, 1996, to Bennett et al .
  • Another molecule expressed on APCs and which stimulates T cell activation is CD40 (for a review, see Banchereau et al., Annu. Rev. Immunol . , 1994, 12, 881).
  • Antisense compounds targeted to CD40 are described in copending U.S. patent application Serial No. 09/071,433, filed May 1, 1998, to Bennett et al .
  • LFA-3 Yet another molecule expressed on APCs and which stimulates T cell activation is LFA-3 (see Liu and Linsley, Curr. Opin . Immunol . , 1992, 4, 265). Antisense compounds targeted to LFA-3 are described in copending U.S. patent application Serial No. 09/045,106, filed March 20, 1998, to Bennett et al . PECAM-1 proteins are glycoproteins which are expressed on the surfaces of a variety of cell types (for reviews, see Newman, J " . Clin . Invest . , 1997, 99, 3 and
  • PECAM-1 In addition to directly participating in cell-cell interactions, PECAM-1 apparently also regulates the activity and/or expression of other molecules involved in cellular interactions (Litwin et al . , J. Cell Biol . , 1997,
  • Antisense compounds targeted to PECAM-1 are described in copending U.S. patent application Serial No. 09/044,506, filed March 19, 1998, to Bennett et al .
  • compositions and methods of the present invention are useful for the treatment of cancers of the lung.
  • antisense oligonucleotides directed to any of a number of molecular targets involved in tumorigenesis, maintenance of the hyperproliferative state and metastasis can targeted to prevent or inhibit lung cancers, or to prevent their spread to other tissues.
  • the ras oncogenes are guanine-binding proteins that have been implicated in cancer by, e.g., the fact that activated ras oncogenes have been found in about 30% of human tumors generally; this figure approached 100% in carcinomas of the exocrine pancreas (for a review, see Downward, Trends in Biol . Sci . , 1990, 15, 469).
  • Protein Kinase C (PKC) proteins have also been implicated in tumorigenesis .
  • Antisense compounds targeted to Protein Kinase C (PKC) proteins are described in U.S. Patents Nos. 5,620,963 to Cook et al . and 5,681,747 to Boggs et al .
  • the compositions and methods of the present invention are useful for the treatment of Pulmonary Fibrosis. Phan ( Thorax, 1995, 50, 415) reviews current beliefs regarding pulmonary fibrosis, and notes that potential targets for therapy include cell adhesion and/or T cell stimulatory molecules (e.g., ICAM-1, ELAM-1, VCAM-1,
  • compositions and methods of the present invention also find use in the treatment and/or prevention of rhinovirus.
  • ICAM-1 is the cellular receptor for the major serotype of rhinovirus, which accounts for greater than 50% of common colds (Staunton et al . , Cell , 1989, 56, 849; Greve et al . ,
  • compositions and methods of the present invention also find use in the treatment of tuberculosis.
  • antisense compounds targeted to the pathogens ycobacterium tuberculosis or M. bovis can be administered to a patient in accordance with the methods of the invention.
  • bronchitis can be treated by administration in accordance with the methods of the invention of compositions of the invention containing one or more antisense compounds targeted to the appropriate pathogen (s) .
  • compositions and methods of the present invention also find use in the treatment of pneumonia, for example by administration of antisense compounds targeted to the pathogen Streptococcus pneumoniae .
  • the methods and compositions of the invention are also directed to antisense oligonucleotides targeted to genes that are implicated in other lung disorders.
  • these include, for example, viruses which infect the lung (e.g. respiratory syncytial virus, H. Influenzae, parainfluenza) , obstructive lung disorders such as pulmonary embolism or anaphylaxis, chronic obstructive pulmonary disease (COPD) , emphysema, chronic bronchitis, bronchiectasis and cystic fibrosis.
  • viruses which infect the lung e.g. respiratory syncytial virus, H. Influenzae, parainfluenza
  • obstructive lung disorders such as pulmonary embolism or anaphylaxis
  • COPD chronic obstructive pulmonary disease
  • emphysema chronic bronchitis
  • bronchiectasis cystic fibrosis.
  • the invention is drawn to the pulmonary administration of a nucleic acid, such as an oligonucleotide, having biological activity to an animal.
  • a nucleic acid such as an oligonucleotide
  • having biological activity it is meant that the nucleic acid functions to modulate the expression of one or more genes in an animal as reflected in either absolute function of the gene (such as ribozyme activity) or by production of proteins coded by such genes .
  • to modulate means to either effect an increase (stimulate) or a decrease (inhibit) in the expression of a gene.
  • Such modulation can be achieved by, for example, an antisense oligonucleotide by a variety of mechanisms known in the art, including but not limited to transcriptional arrest; effects on RNA processing (capping, polyadenylation and splicing) and transportation; enhancement or reduction of cellular degradation of the target nucleic acid; and translational arrest (Crooke et al . , Exp . Opin . Ther. Patents, 1996, 6:1).
  • compositions and methods of the invention can be used to study the function of one or more genes in the animal.
  • antisense oligonucleotides have been systemically administered to rats in order to study the role of the N- methyl-D-aspartate receptor in neuronal death, to mice in order to investigate the biological role of protein kinase C-a, and to rats in order to examine the role of the neuropeptide Yl receptor in anxiety (Wahlestedt et al . , Nature, 1993, 363:260; Dean et al . , Proc . Natl . Acad. Sci .
  • antisense knockouts i.e., inhibition of a gene by systemic administration of antisense oligonucleotides
  • antisense oligonucleotides may represent the most accurate means for examining a specific member of the family (see, generally, Albert et al . , Trends
  • compositions and methods of the invention are also useful therapeutically, i.e., to provide therapeutic, palliative or prophylactic relief to an animal, including a human, having or suspected of having or of being susceptible to, a disease or disorder that is treatable in whole or in part with one or more nucleic acids .
  • disease or disorder (1) includes any abnormal condition of an organism or part, especially as a consequence of infection, inherent weakness, environmental stress, that impairs normal physiological functioning; (2) excludes pregnancy per se but not autoimmune and other diseases associated with pregnancy; and (3) includes cancers and tumors.
  • the term "having or suspected of having or of being susceptible to” indicates that the subject animal has been determined to be, or is suspected of being, at increased risk, relative to the general population of such animals, of developing a particular disease or disorder as herein defined.
  • a subject animal could have a personal and/or family medical history that includes frequent occurrences of a particular disease or disorder.
  • a subject animal could have had such a susceptibility determined by genetic screening according to techniques known in the art (see, e.g., U.S. Congress,
  • a disease or disorder that is treatable in whole or in part with one or more nucleic acids refers to a disease or disorder, as herein defined, (1) the management, modulation or treatment thereof, and/or (2) therapeutic, palliative and/or prophylactic relief therefrom, can be provided via the administration of more nucleic acids.
  • a disease or disorder is treatable in whole or in part with an antisense oligonucleotide.
  • the compounds and method of the invention employ particles containing oligonucleotide therapeutics or diagnostics .
  • the particles can be solid or liquid, and are preferably of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs.
  • respirable size that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs.
  • particles ranging from about 5 to 20 microns in size are respirable and are expected to reach the bronchioles (Allen, Secundum Artem,
  • Liquid pharmaceutical compositions of oligonucleotide can be prepared by combining the oligonucleotide with a suitable vehicle, for example sterile pyrogen free water, or saline solution. Other therapeutic compounds may optionally be included.
  • a suitable vehicle for example sterile pyrogen free water, or saline solution.
  • Other therapeutic compounds may optionally be included.
  • compositions preferably comprise particles of oligonucleotide that are of respirable size.
  • particles can be prepared by, for example, grinding dry oligonucleotide by conventional means, fore example with a mortar and pestle, and then passing the resulting powder composition through a 400 mesh screen to segregate large particles and agglomerates .
  • a solid particulate composition comprised of an active oligonucleotide can optionally contain a dispersant which serves to facilitate the formation of an aerosol, for example lactose.
  • oligonucleotide compositions are aerosolized. Aerosolization of liquid particles can be produced by any suitable means, such as with a nebulizer. See, for example, U.S. Patent No. 4,501,729.
  • Nebulizers are commercially available devices which transform solutions or suspensions into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.
  • Suitable nebulizers include those sold by Blairex ® under the name PARI LC PLUS, PARI DURA-NEB 2000, PARI-BABY Size, PARI PRONEB Compressor with LC PLUS, PARI WALKHALER Compressor/Nebulizer System, PARI LC PLUS Reusable Nebulizer, and PARI LC Jet+ ® Nebulizer.
  • Exemplary formulations for use in nebulizers consist of an oligonucleotide in a liquid, such as sterile, pyragen free water, or saline solution, wherein the oligonucleotide comprises up to about 40% w/w of the formulation. Preferably, the oligonucleotide comprises less than 20% w/w. If desired, further additives such as preservatives (for example, methyl hydroxybenzoate) antioxidants, and flavoring agents can be added to the composition.
  • preservatives for example, methyl hydroxybenzoate
  • flavoring agents can be added to the composition.
  • Solid particles comprising an oligonucleotide can also be aerosolized using any solid particulate medicament aerosol generator known in the art .
  • Such aerosol generators produce respirable particles, as described above, and further produce reproducible metered dose per unit volume of aerosol.
  • Suitable solid particulate aerosol generators include insufflators and metered dose inhalers .
  • Metered dose inhalers suitable fore used in the art (along with the trade name, manufacturer and indication they are used for) and useful in the present invention include: Delivery Device Trade Name Manufacture
  • MDI Metered Dose Inhaler
  • Beta- adrenergic bronchodilator Atrovent- Boehringer Ingelheim Anti- cholinergic bronchodilator
  • Beta-adrenergic bronchodilator Proventil - Key Pharm. Beta-adrenergic bronchodilator
  • Beta-adrenergic bronchodilator (Rotocaps for use in Rotohaler device) Beta-adrenergic bronchodilator
  • liquid or solid aerosols are produced at a rate of from about 10 to 150 liters per minute, more preferably from about 30 to 150 liters per minute, and most preferably about 60 liters per minute.
  • alkyl includes but is not limited to straight chain, branch chain, and alicyclic hydrocarbon groups. Alkyl groups of the present invention may be substituted. Representative alkyl substituents are disclosed in United States Patent No. 5,212,295, at column 12, lines 41-50.
  • Further representative 2 ' sugar modifications amenable to the present invention include fluoro, 0-alkyl, O-alkylamino, O-alkylalkoxy, protected 0-alkylamino, O-alkylaminoalkyl, 0-alkyl imidazole, and polyethers of the formula (O-alkyl) m , where m is 1 to about 10.
  • PEGs linear and cyclic polyethylene glycols
  • PEG polyethylene glycols
  • PEG polyethylene glycols
  • Sugars having 0-substitutions on the ribosyl ring are also amenable to the present invention.
  • Representative substitutions for ring 0 include S, CH 2 , CHF, and CF 2 , see, e.g., Seerist, et al . , Abstract 21, Program & Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, Sept.
  • aralkyl denotes alkyl groups which bear aryl groups, for example, benzyl groups.
  • alkaryl denotes aryl groups which bear alkyl groups, for example, methylphenyl groups.
  • Aryl groups are aromatic cyclic compounds including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl.
  • hetero denotes an atom other than carbon, preferably but not exclusively N, 0, or S.
  • heterocycloalkyl denotes an alkyl ring system having one or more heteroatoms (i.e., non-carbon atoms) .
  • Preferred heterocycloalkyl groups include, for example, morpholino groups.
  • heterocycloalkenyl denotes a ring system having one or more double bonds, and one or more heteroatoms.
  • Preferred heterocycloalkenyl groups include, for example, pyrrolidino groups.
  • the compounds of the invention can comprise a linker connected to a solid support .
  • Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Caruthers U.S. Patents Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
  • Linkers are known in the art as short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques . Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach,
  • Solid supports according to the invention include those generally known in the art to be suitable for use in solid phase methodologies, including, for example, controlled pore glass (CPG) , oxalyl-controlled pore glass (see, e.g., Alul, et al . , Nucleic Acids Research 1991, 19, 1527, hereby incorporated by reference in its entirety) ,
  • CPG controlled pore glass
  • oxalyl-controlled pore glass see, e.g., Alul, et al . , Nucleic Acids Research 1991, 19, 1527, hereby incorporated by reference in its entirety
  • TentaGel Support an aminopolyethyleneglycol derivatized support (see, e. g. , Wright, et al . , Tetrahedron Letters
  • Some preferred embodiments of the invention comprise one or more hydroxyl protecting groups .
  • a wide variety of hydroxyl protecting groups can be employed in the methods of the invention.
  • the protecting group is stable under basic conditions but can be removed under acidic conditions.
  • protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
  • Representative hydroxyl protecting groups are disclosed by Beaucage, et al . , Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter
  • R 2 , R 3 and R 3a include dimethoxytrityl (DMT) , monomethoxytrityl, 9-phenylxanthen- 9-yl (Pixyl) and 9- (p-methoxyphenyl) anthen-9-yl (Mox) .
  • DMT dimethoxytrityl
  • Pixyl 9-phenylxanthen- 9-yl
  • Mox 9- (p-methoxyphenyl) anthen-9-yl
  • the R 2 or R 3 group can be removed from oligomeric compounds of the invention by techniques well known in the art to form the free hydroxyl.
  • dimethoxytrityl protecting groups can be removed by protic acids such as formic acid, dichloroacetic acid, trichloroacetic acid, p- toluene sulphonic acid or with Lewis acids such as for example zinc bromide. See for example, Greene and Wuts, supra .
  • amino groups are appended to alkyl or other groups, such as, for example, 2 ' -alkoxy groups (e.g., where R ⁇ is alkoxy) .
  • Such amino groups are also commonly present in naturally occurring and non-naturally occurring nucleobases. It is generally preferred that these amino groups be in protected form during the synthesis of oligomeric compounds of the invention. Representative amino protecting groups suitable for these purposes are discussed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John Wiley & Sons, New York,
  • the term "protected" when used in connection with a molecular moiety such as “nucleobase” indicates that the molecular moiety contains one or more functionalities protected by protecting groups .
  • the oligomeric compounds of the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein.
  • Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms .
  • Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warm-blooded animals, can be treated.
  • each cell of multicellular eukaryotes can be treated, as they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity.
  • organelles e.g., mitochondria and chloroplasts
  • organelles e.g., mitochondria and chloroplasts
  • single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.
  • Oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry with oxidation using iodine. Beta-cyanoethyldiisopropyl phosphoramidites were purchased from Applied Biosystems (Foster City, CA) . For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1, 2-benzodithiole-3-one-l, 1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages .
  • the 2 ' -fluoro-phosphorothioate oligonucleotides of the invention are synthesized using 5 ' -dimethoxytrityl- 3 ' -phosphoramidites and prepared as disclosed in U.S. patent application Serial No. 08/383,666, filed February 3, 1995, and U.S. Patent 5,459,255, which issued October 8, 1996, both of which are assigned to the same assignee as the instant application and which are incorporated by reference herein.
  • the 2 ' -fluoro-oligonucleotides are prepared using phosphoramidite chemistry and a slight modification of the standard DNA synthesis protocol (i.e., deprotection was effected using methanolic ammonia at room temperature) .
  • PNA antisense analogs are prepared essentially as described in U.S. Patents Nos. 5,539,082 and 5,539,083, both of which (1) issued July 23, 1996, (2) are assigned to the same assignee as the instant application and (3) are incorporated by reference herein.
  • Oligonucleotides comprising 2 , 6-diaminopurine are prepared using compounds described in U.S. Patent No. 5,506,351 which issued April 9, 1996, and which is assigned to the same assignee as the instant application and incorporated by reference herein, and materials and methods described by Gaffney et al . ( Tetrahedron, 1984, 40:3),
  • Oligonucleotides comprising 2 , 6-diaminopurine can also be prepared by enzymatic mean ⁇ (Bailly et al . , Proc . Natl . Acad. Sci .
  • the 2 ' -methoxyethoxy oligonucleotides of the invention were synthesized essentially according to the methods of Martin et al . (Helv. Chim . Acta, 1995, 78, 486) .
  • the 3' nucleotide of the 2'- methoxyethoxy oligonucleotides was a deoxynucleotide
  • 2 ' -0-CH 2 CH 2 OCH 3 _cytosines were 5-methyl cytosines, which were synthesized according to the procedures described below.
  • the resulting syrup was poured into diethylether (2.5 L) , with stirring.
  • the product formed a gum.
  • the ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL) .
  • the solution was poured into fresh ether (2.5 L) to yield a stiff gum.
  • the ether was decanted and the gum was dried in a vacuum oven (60°C at 1 mm Hg for 24 h) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield) .
  • the material was used as is for further reactions.
  • Anhydro-5-methyluridine (195 g, 0.81 M) , tris(2- methoxyethy1) borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160°C. After heating for 48 hours at 155-160°C, the vessel was opened and the solution evaporated to dryness and triturated with methanol (200 mL) . The residue was suspended in hot acetone (1 L) . The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH 3 CN (600 mL) and evaporated.
  • Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L) , cooled to -5°C and stirred for 0.5 h using an overhead stirrer.
  • P0C1 3 was added dropwise, over a 30 minute period, to the stirred solution maintained at 0- 10°C, and the resulting mixture stirred for an additional 2 hours.
  • the first solution was added dropwise, over a 45 minute period, to the later solution.
  • the resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with lx 300 mL of NaHC0 3 and 2x 300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.
  • N 4 -Benzoyl-2' -O-methoxyethyl-5 ' -O- dimethoxytrityl-5-methylcytidine 2 ' -O-Methoxyethyl-5 ' -0- dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tic showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL) .
  • O-methoxyethyl-5 ' -0-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 C1 2 (1 L) .
  • Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra- (isopropyl) phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (tic showed the reaction to be 95% complete) .
  • the reaction mixture was extracted with saturated NaHC0 3 (ix 300 mL) and saturated NaCl (3x 300 mL) .
  • Oligonucleotide Labeling Antisense oligonucleotides were labeled in order to detect the presence of and/or measure the quantity thereof in samples taken during the course of the in vivo pharmacokinetic studies described herein. Although radiolabeling by tritium exchange is one preferred means of labeling antisense oligonucleotides for such in vivo studies, a variety of other means are available for incorporating a variety of radiological, chemical or enzymatic labels into oligonucleotides and other nucleic acids. 1. Tritium Exchange: Essentially, the procedure of Graham et al . (Nucleic Acids Research, 1993,
  • oligonucleotide 21:3737 was used to label oligonucleotides by tritium exchange. Specifically, about 24 mg of oligonucleotide was dissolved in a mixture of 200 uL of sodium phosphate buffer (pH 7.8), 400 uL of 0.1 mM EDTA (pH 8.3) and 200 uL of deionized water. The pH of the resulting mixture was measured and adjusted to pH 7.8 using 0.095 N NaOH. The mixture was lyophilized overnight in a 1.25 mL gasketed polypropylene vial. The oligonucleotide was dissolved in 8.25 uL of b-mercaptoethanol, which acts as a free radical scavenger (Graham et al . , Nucleic Acids Research, 1993,
  • oligonucleotide mixture is then frozen in liquid nitrogen and transferred to a lyophilization apparatus wherein lyophilization was carried out under high vacuum, typically for 3 hours .
  • the material was then resuspended in mL of double-distilled H0 and allowed to exchange for 1 hour at room temperature. After incubation, the mixture was again quick frozen and lyophilized overnight.
  • oligonucleotide material is removed for HPLC analysis .
  • Three further lyophilizations were carried out, each with approximately 1 mL of double-distilled H 2 0, to ensure the removal of any residual, unincorporated tritium.
  • the final resuspended oligonucleotide solution is transferred to a clean polypropylene vial and assayed.
  • the tritium labeled oligonucleotide is stored at about -70BC.
  • oligonucleotides and other nucleic acids can be labeled and to separate unincorporated label from the labeled nucleic acid.
  • double-stranded nucleic acids can be radiolabeled by nick translation and primer extension
  • a variety of nucleic acids, including oligonucleotides can be terminally radiolabeled by the use of enzymes such as T4 polynucleotide kinase or terminal deoxynucleotidyl transferase (see, generally, Chapter 3 In :
  • oligonucleotides and other nucleic acids with nonradioactive labels such as, for example, enzymes, fluorescent moieties and the like (see, for example, Beck, Methods in Enzymology, 1992, 216:143; and Ruth, Chapter 6
  • Aqueous solutions of oligonucleotides were nebulized, and the resulting aerosol was delivered to an animal model (male CD-I mice) via a nose-only inhalation system.
  • the particle size was targeted for 1 to 5 ⁇ m.
  • mice were evaluated for signs of toxicity and designated tissues were collected for assessment of organ-specific effects and the oligonucleotide concentrations.
  • the male CD-I mouse was chosen as the animal model for this study since considerable scientific data is available for this species.
  • ISIS 2105 a phosphorothioate antisense 2'- deoxyribose oligonucleotide targeted to HPV, and having the sequence:
  • ISIS 17009 a phosphorothioate antisense 2'- deoxyribose oligonucleotide targeted to mouse ICAM-1, having the sequence: 5' -GGA-GTC-CAG-CAC-TAG-CAC-TG-3 ' (SEQ ID NO:ll)
  • ISIS 15163 a phosphodiester antisense 2 ' -O- methoxyethyl oligonucleotide targeted to mouse ICAM-1
  • Sterile sodium chloride (saline) for injection was used to formulate solutions of oligonucleotide, and sodium chloride for injection was used a ⁇ the control article.
  • mice were given a 30 minute nose-only exposure of solutions of I ⁇ IS-2105 having concentrations of either 10 or 100 mg/ml, with saline controls. Calculated lung doses (see infra) were 1.2 and 12 mg/kg, respectively. Animals were necropsied at 0 minutes (at the end of exposure) , 2 hours, 8 hours, and 24 hour ⁇ . Animals were generally assessed for their health, and more limited assessments were made of lung tolerability . Lung concentrations of oligonucleotide and oligonucleotide metabolites were performed by capillary gel electrophoresis (CGE) and distribution of oligonucleotide within lung tissue was determined immunohistologically.
  • CGE capillary gel electrophoresis
  • control group and the low dose group each displayed a 7% or 13% decrease, respectively, in breathing rate during exposure.
  • the high dose group displayed a 28 percent decrease in breathing rate during exposure. Exposure had no effect on body weight or organ weight.
  • Figure 1 shows the elimination of oligonucleotide from the lung of mice in this study. It can be seen that elimination appears to be monophasic in the low dose group, and biphasic in the high do ⁇ e group. However, it may be that integrity was compromised in the high dose group; i.e., the high dose may have overdosed the lung. There was a relatively long half-life for both parent compound and metabolites which, in the case of the full length oligonucleotide, is greater than 20 hours and for the total oligonucleotide is greater than 40 hour . Metabolism of parent oligonucleotide in the lung appears to be faster than clearance rate from the lung, which is consistent with observations made in other organs.
  • oligonucleotide was distributed to all cell types in the lung, including bronchiolar and alveolar epithelium, endothelial cells, and alveolar macrophages.
  • significant concentrations of oligonucleotide and metabolites were found in lung tissue (by CGE analysis) : 80 percent of the oligonucleotide was found to be intact at the end of the exposure, with 50 percent remaining intact 8 hours after the exposure, and 20 to 30 percent intact 24 hours after the exposure.
  • oligonucleotide and/or metabolite were found in plasma: 0.6 micromolar at 0 hours (52 percent full length), and 0.3 micromolar at 2 hours (38 percent full length) . Significant concentrations were found also in the liver; 30 micrograms 24 hours after the exposure; 12-16 percent of intact parent compound. From these data it can be seen that for the high dosage group, that portion of the oligonucleotide that was delivered to plasma, is cleared relatively quickly.
  • oligonucleotide may adversely affect the breathing rate, possibly by airway irritation, or as a result of the relatively high viscosity of the solution.
  • pulmonary delivery of oligonucleotide resulted in distribution to all cell types in the lung.
  • the exposure systems used were designed to nebulize the test article solution or saline only.
  • the exposure atmosphere ⁇ were generated using PARI LC PLUS nebulizers (PARI Respiratory Equipment, Inc, Richmond, VA) .
  • Filtered compressed air was used as the air ⁇ upply.
  • Airflow rate ⁇ were set and maintained at levels required to assure a consistent aerosol generation and maintain animal health. Empty ports within the generation chamber provided locations for obtaining samples for gravimetric and particle size determination or analysis.
  • Atmosphere concentration was determined both gravimetrically (development phase) and by analytical measurements (animal exposure) .
  • Glass fiber filters (Gelman #66075, Gel an sciences, Ann Arbor, MI) were placed into in-line filter holders. Airflow rates were regulated to sample a known volume of test atmosphere . Immediately after sampling, the filters were collected and the mass concentration calculated. The filter samples were then processed to extract and analyze the test material deposited on the filter. Analytical measurements were used to calculate the inhaled dose . Samples were collected during each exposure in which animals were placed in the chambers . Particle size was measured with a Mercer style cascade impactor (Chen et al., Fundam . Appl . Toxicol . ,
  • the effective cut-off diameters for the impactor ranged from 4.8 microns to 0.30 microns. Particle size was measured for each oligonucleotide tested, following the first and last exposure.
  • the Mass Median Aerodynamic Diameter (MMAD) for the three oligonucleotides ranged from 2.72 to 3.26 and the Geometric Standard Deviation (GSD) ranged from 2.44 to 2.46.
  • RMV respiratory minute volume, assumed* to be 0.03 1/min for a 30 gram mouse
  • Deposition Factor fraction that remains in lung, assumed* to be 10% with a particle size of 2 to 3 micrometers .
  • Body Weight mean body weight in grams (30 grams was used as the average)
  • the estimated pulmonary dose for the low, mid, and high dose groups was approximately 0.8, 1.5 and 3.2 mg/kg, respectively.
  • Figure 1 shows a plot of milligrams oligonucleotide collected in impinger versus time. These data show the successful nebulization of oligonucleotide; i.e., that the oligonuclotide is uniformly nebulized, and that the size of the resultant particles is not altered over time.
  • mice Microscopic observations were limited to the lungs of 5 of 5 mice in the 4 exposure-high dose ISIS 2105 group, 2 of 5 mice in the 4 exposure-mid dose ISIS 2105 group, and 1 of 4 or 1 of 5 mice in the high dose ISIS 15163 single or multiple exposure groups, respectively. These effects in the lungs were described as a multifocal inflammatory cell infiltrate that was regarded as being minimal in severity. Similar observations have been noted following intravenous administration of oligonucleotides in mice and these effects have been attributed to immune stimulation aspects that occurs in rodents administered this class of compounds . No other changes were noted in the lungs, and there were no observations of effects noted for the other tissues examined (liver, kidney, spleen, and nasal passages) .
  • each oligonucleotide and its metabolites was determined in tissue samples of lung, liver, kidney and spleen.
  • Table 1 and Table 2 show the concentrations of total oligonucleotide (parent oligonucleotide and oligonucleotide metabolites) in the lung, liver and kidney. Concentrations observed in the lung were dose-dependent and were greater in mice administered four exposures versus a single exposure.
  • oligonucleotide As can be seen, nose-only inhalation exposure of oligonucleotide was well tolerated in mice. Effects in the lung were limited to a minimal cellular infiltrate that was likely due to the general immune stimulation that occurs in mice administered this class of compounds. Lung was also the tissue with the greatest concentration of oligonucleotide. Minimal oligonucleotide concentrations were observed in the other organs evaluated, and no histologic alterations were observed in these organs. Similar observations were noted for the phosphorothioate oligonucleotides, i.e. tissue concentrations and tissue effects.
  • the 2 ' -methoxyethyl modified phosphodiester oligonucleotide (ISIS 15163) was detected in greater concentrations in lung, but histologic alterations were limited to 1 animals in each of the single and multiple exposure groups . It is intended that each of the patents, applications, printed publications, and other published documents mentioned or referred to in this specification be herein incorporated by reference in their entirety.

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Abstract

La présente invention concerne des compositions destinées à l'administration pulmonaire d'acides nucléiques, en particulier d'oligonucléotides, et des procédés associés. Dans un mode de réalisation préféré, les compositions et les procédés de l'invention sont utilisés pour effectuer l'administration pulmonaire d'un oligonucléotide antisens chez un animal dans le but de moduler l'expression d'un gène chez cet animal à des fins thérapeutiques, prophylactiques ou de recherche.
EP99923263A 1998-05-21 1999-05-20 Compositions destinees a l'administration pulmonaire d'acides nucleiques et procedes Withdrawn EP1086116A4 (fr)

Applications Claiming Priority (3)

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US8358598A 1998-05-21 1998-05-21
US83585 1998-05-21
PCT/US1999/011214 WO1999060010A1 (fr) 1998-05-21 1999-05-20 Compositions destinees a l'administration pulmonaire d'acides nucleiques et procedes

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EP1086116A1 true EP1086116A1 (fr) 2001-03-28
EP1086116A4 EP1086116A4 (fr) 2004-03-17

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AU (1) AU4007899A (fr)
WO (1) WO1999060010A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5955443A (en) * 1998-03-19 1999-09-21 Isis Pharmaceuticals Inc. Antisense modulation of PECAM-1
US6197584B1 (en) * 1998-05-01 2001-03-06 Isis Pharmaceuticals, Inc. Antisense modulation of CD40 expression
US20090324596A1 (en) * 2008-06-30 2009-12-31 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers
US10745701B2 (en) 2007-06-28 2020-08-18 The Trustees Of Princeton University Methods of identifying and treating poor-prognosis cancers
USRE47320E1 (en) 2007-11-20 2019-03-26 Ionis Pharmaceuticals, Inc. Modulation of CD40 expression

Citations (4)

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Publication number Priority date Publication date Assignee Title
WO1993019203A1 (fr) * 1992-03-16 1993-09-30 Isis Pharmaceuticals, Inc. Modulation par oligonucleotide de la proteine kinase c
WO1999011778A1 (fr) * 1997-09-02 1999-03-11 University Of Sheffield Traitement antisense de l'hypertension pulmonaire
WO2000020645A1 (fr) * 1998-10-05 2000-04-13 Isis Pharmaceuticals, Inc. Modulation de l'expression du facteur de necrose tumorale alpha (tnf-alpha) au moyen d'un oligonucleotide antisens
WO2000050050A1 (fr) * 1999-02-23 2000-08-31 Isis Pharmaceuticals, Inc. Formulation multiparticulaire

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591623A (en) * 1990-08-14 1997-01-07 Isis Pharmaceuticals, Inc. Oligonucleotide modulation of cell adhesion
US5858784A (en) * 1991-12-17 1999-01-12 The Regents Of The University Of California Expression of cloned genes in the lung by aerosol- and liposome-based delivery

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993019203A1 (fr) * 1992-03-16 1993-09-30 Isis Pharmaceuticals, Inc. Modulation par oligonucleotide de la proteine kinase c
WO1999011778A1 (fr) * 1997-09-02 1999-03-11 University Of Sheffield Traitement antisense de l'hypertension pulmonaire
WO2000020645A1 (fr) * 1998-10-05 2000-04-13 Isis Pharmaceuticals, Inc. Modulation de l'expression du facteur de necrose tumorale alpha (tnf-alpha) au moyen d'un oligonucleotide antisens
WO2000050050A1 (fr) * 1999-02-23 2000-08-31 Isis Pharmaceuticals, Inc. Formulation multiparticulaire

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
LEVESQUE L. ET AL.: "Antisense oligonucleotides targeting human Protein Kinase C-alpha inhibit phorbol ester-induced reduction of Bradykinin-evoked Calcium mobilization in A549 cells" MOLECULAR PHARMACOLOGY, vol. 51, 1997, pages 209-216, XP002247729 *
MCKAY R.A. ET AL.: "Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human Protein Kinase C-alpha expression" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 3, 15 January 1999 (1999-01-15), pages 1715-1722, XP002247728 *
NICKLIN P.L. ET AL.: "Pulmonary bioavailability of a phosphorothioate oligonucleotide (CGP 64128A): comparison with other delivery routes" PHARMACEUTICAL RESEARCH, vol. 15, no. 4, April 1998 (1998-04), pages 583-591, XP008019675 *
NYCE J.W. AND METZGER W.J.: "DNA antisense therapy for asthma in an animal model" NATURE, vol. 385, 20 February 1997 (1997-02-20), pages 721-725, XP000891493 ISSN: 0028-0836 *
PHAN S.H.: "New strategies for treatment of pulmonary fibrosis" THORAX, vol. 50, no. 4, 1 April 1995 (1995-04-01), pages 415-421, XP000601085 ISSN: 0040-6376 *
ROJANASAKUL Y. ET AL.: "Antisense inhibition of Silica-induced Tumor Necrosis Factor in alveolar macrophages" THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 7, 14 February 1997 (1997-02-14), pages 3910-3914, XP002247730 *
See also references of WO9960010A1 *

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AU4007899A (en) 1999-12-06
WO1999060010A1 (fr) 1999-11-25
EP1086116A4 (fr) 2004-03-17

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