WO2000062736A2 - Oligonucleotide antisens a faible teneur en adenosine, compositions, kit et procede pour le traitement d'affections des voies aeriennes associees a la bronchoconstriction, a l'inflammation pulmonaire, aux allergies et a la depletion de surfactant - Google Patents

Oligonucleotide antisens a faible teneur en adenosine, compositions, kit et procede pour le traitement d'affections des voies aeriennes associees a la bronchoconstriction, a l'inflammation pulmonaire, aux allergies et a la depletion de surfactant Download PDF

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WO2000062736A2
WO2000062736A2 PCT/US2000/008020 US0008020W WO0062736A2 WO 2000062736 A2 WO2000062736 A2 WO 2000062736A2 US 0008020 W US0008020 W US 0008020W WO 0062736 A2 WO0062736 A2 WO 0062736A2
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oligo
composition
receptor
receptors
group
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WO2000062736A3 (fr
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Jonathan W. Nyce
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East Carolina University
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Priority to BR0006019-4A priority Critical patent/BR0006019A/pt
Priority to IL14005400A priority patent/IL140054A0/xx
Priority to EP00919668A priority patent/EP1168919A4/fr
Priority to CA002330022A priority patent/CA2330022A1/fr
Priority to AU40317/00A priority patent/AU4031700A/en
Priority to JP2000611873A priority patent/JP2003515525A/ja
Publication of WO2000062736A2 publication Critical patent/WO2000062736A2/fr
Publication of WO2000062736A3 publication Critical patent/WO2000062736A3/fr
Priority to HK02102615.9A priority patent/HK1042017A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/08—Bronchodilators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base

Definitions

  • This patent relates to a composition
  • oligonucleotides that are anti-sense to adenosine receptors, and contain low amounts of or no adenosine (A).
  • oligos oligonucleotides
  • A adenosine
  • These agents are suitable for the treatment, among others, of pulmonary diseases associated with inflammation, impaired airways, including lung disease and diseases whose secondary effects afflict the lungs of a subject. Examples of these diseases are allergies, asthma, impeded respiration, allergic rhynitis, pain, cystic fibrosis, and cancers such as leukemias, e.g. colon cancer, and the like.
  • the present agent may be administered prophylactically or therapeutically in conjunction with other therapies, or may be utilized as a substitute for therapies that have significant, negative side effects.
  • asthma Respiratory ailments, associated with a variety of diseases and conditions, are extremely common in the general population, and more so in certain ethnic groups, such as African Americans. In some cases they are accompanied by inflammation, which aggravates the condition of the lungs. Asthma, for example, is one of the most common diseases in industrialized countries. In the United States it accounts for about 1 % of all health care costs. An alarming increase in both the prevalence and mortality of asthma over the past decade has been reported, and asthma is predicted to be the preeminent occupational lung disease in the next decade. While the increasing mortality of asthma in industrialized countries could be attributable to the depletion reliance upon beta agonists in the treatment of this disease, the underlying causes of asthma remain poorly understood.
  • Adenosine may constitute an important mediator in the lung for various diseases, including bronchial asthma. Its potential role was suggested by the finding that asthmatics respond favorably to aerosolized adenosine with marked bronchoconstriction whereas normal individuals do not.
  • An asthmatic rabbit animal model the dust mite allergic rabbit model for human asthma, responded in a similar fashion to aerosolized adenosine with marked bronchoconstriction whereas non-asthmatic rabbits showed no response. More recent work with this animal model suggested that adenosine- induced bronchoconstriction and bronchial hyperresponsiveness in asthma may be mediated primarily through the stimulation of adenosine receptors.
  • Adenosine has also been shown to cause adverse effects, including death, when administered therapeutically for other diseases and conditions in subjects with previously undiagnosed hyper reactive airways.
  • a handful of medicaments have been available for the treatment of respiratory diseases and conditions, although in general they all have limitations.
  • Theophylline an important drug in the treatment of asthma, is a known adenosine receptor antagonist which was reported to eliminate adenosine-mediated bronchoconstriction in asthmatic rabbits.
  • a selective adenosine A, receptor antagonist, 8-cyclopentyl-l, 3-dipropylxanthine (DPCPX) was also reported to inhibit adenosine- mediated bronchoconstriction and bronchial hyperresponsiveness in allergic rabbits.
  • adenosine A, receptor-specific antagonists are, nevertheless, limited by their toxicity.
  • Theophylline for example, has been widely used in the treatment of asthma, but is associated with frequent, significant toxicity resulting from its narrow therapeutic dose range.
  • DPCPX is far too toxic to be useful clinically.
  • Anti-sense oligonucleotides have received considerable theoretical consideration as potential useful pharmacological agents in human disease. Their practical application in actual models of human disease, however, has been somewhat elusive.
  • Anti-sense oligonucleotides have been used in therapy by many, including the present inventor, who in his previous work successfully treated various diseases and conditions by direct administration of these agents to the lung. In many instances, other workers have had to face the difficulties associated with the delivery of DNA molecules to a desired target. Thus, the route of administration may be of extreme importance for treating generalized diseases and conditions as well as those which are localized. In contrast, up to the present time, the delivery of anti-sense agents to the lung has been relatively undeveloped. As described by the present inventor in more detail below, the lung is an excellent target for the direct administration of anti-sense oligonucleotides and provides a non-invasive and a tissue-specific route.
  • the present invention generally relates to a pharmaceutical or veterinary composition, comprising an anti-sense oligonucleotide(s) (oligo(s)) which is (are) effective for alleviating bronchoconstriction and/or lung inflammation, allergy(ies), and ⁇ or surfactant depletion and ⁇ or hyposecretion, when administered to a mammal, the oligo containing about 0 to about 15% adenosine (A) and being anti-sense to a target selected from the group consisting of the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of a gene encoding a target polypeptide associated with lung airway dysfunction or anti-sense to the polypeptide mRNA; combinations of the oligos; and mixtures of the oligos; and a pharmaceutically or veterinarily
  • the targets are typically molecules associated with airway disease, cancer, etc., such as transcription factors, stimulating and activating peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, endogenously produced specific and non-specific enzymes, immunoglobulins and antibodies, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors, binding proteins, and malignancy associated proteins, among others.
  • transcription factors such as transcription factors, stimulating and activating peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, endogenously produced specific and non-specific enzymes, immunoglobulins and antibodies, antibody receptors,
  • oligo(s) targeted to adenosine rece ⁇ tor(s) are typically present in the composition in an amount effective to reduce adenosine mediated effect(s), such as airway obstruction, inflammation, allergy(ies), and sufactant depletion, among others.
  • the adenosine receptor is preferably selected from the group consisting of the adenosine A réelle A 2b , and A 3 receptors, and in some instances even adenosine A 2a receptors.
  • the oligo of the invention may be applied to the preparation of a medicament for (a) reducing adenosine-mediated bronchoconstriction, impeded respiration, inflammation, allergy(ies), depletion production of surfactant, and other detrimental pulmonary effects in a subject in need of treatment, and/or for (b) treating specific diseases and conditions such as asthma, cystic fibrosis, allergic rhynitis, COPD, etc.
  • this invention provides the targeted administration of one or more oligonucleotides directly into the repiratory system.
  • the oligos may be directed to any target and are intended for fast delivery through the mucosal tissue of the lungs for hybridization to a desired target polynucleotide, e. g.
  • this invention also provides a more general method for administering oligonucleotides that are anti-sense to targeted genes and mRNAs associated with any type of diseases, by direct administration into the respiratory system, e. g. by inhalation, by introduction of a solution or aerosol into the respiratory airways, and/or directly into the lung.
  • the present oligos are suitable for reducing effects mediated by a variety of target proteins and genes, for example adenosine-mediated effects, including pulmonary, respiratory, and other associated effects, e. g. bronchoconstriction, inflammation, immune mediated reactions, allergy(ies) and other airway problems, which may be caused by different conditions, including cancer.
  • adenosine-mediated effects including pulmonary, respiratory, and other associated effects, e. g. bronchoconstriction, inflammation, immune mediated reactions, allergy(ies) and other airway problems, which may be caused by different conditions, including cancer.
  • diseases and conditions which may be treated preventatively, prophylactically and therapeutically with the agent of this invention, are pulmonary vasoconstriction, inflammation, allergies, asthma, impeded respiration, respiratory distress syndrome, pain, cystic fibrosis, allergic rhynitis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), bronchitis, and cancers such as leukemias, lymphomas, carcinomas, and the like, e.g.
  • the present agents are also suitable for administration before, during and after other treatments, including radiation, chemotherapy, antibody therapy, phototherapy and cancer, and other types of surgery.
  • the present agent is effectively administered prophylactically and therapeutically in conjunction with other therapies, or by itself for conditions without known therapies or as a substitute for therapies that have significant negative side effects.
  • the oligo(s) may be administered by any means known to a subject, e. g.
  • oligonucleotide(s) employed are anti-sense to to a target DNA or RNA, e. g. an adenosine receptor DNA or RNA, and preferably consist essentially of up to about 15% adenosine (A), and more preferably contain no adenosine.
  • the oligos are provided in the form of specific compositions and formulations, with a carrier or diluent, and optionally with other therapeutic agents and additives which are used for administration by specific routes, e.g. into the respiratory system, topically, transdermally, parenterally, by implantation, and the like.
  • the oligo is also provided as a capsule or cartridge, and in the form of a kit.
  • the oligos of the invention may be produced by selection of specific targeted segments of the gene or mRNA encoding the adenosine receptor as described below. In one preferred embodiment, the selection is made to obtain oligos that consisting essentially of less than about 15% adenosine (A).
  • a universal base(s) or other base(s) for one or more A to reduce the proportion of A present in the oligonucleotide to less than about 15%, and down to no adenosine.
  • alternative and/or universal bases may be substituted for adenosine, e. g. specific adenosine Al, A2b and A3 receptor antagonists or A2a receptor agonists, theophilline, enprophylline, and many other adenosine receptor antagonists known in the art as well as agonists with significantly reduced agonist activity with respect to adenosine, e. g. less than 0.5%, less than 0.3%, and the like.
  • oligos adenosine receptor targeted anti-sense oligonucleotides
  • oligos may be utilized therapeutically in the treatment of diseases or conditions which impair respiration, cause inflammation and/or allergy(ies), constrict bronchial tissue, obstruct the lung airways, depletion surfactant secretion, or otherwise impede normal breathing.
  • oligonucleotides are metabolized in vivo to their mononucleotides.
  • Adenosine (A)-containing oligonucleotides break down and release adenosine which, in turn, activates adenosine receptors, thereby causing bronchoconstriction, inflammation, surfactant depletion, allergy(ies), and the like.
  • one or more nucleic acids of the invention may be formulated alone, and/or with one or more surfactant components and/or with a carrier, and/or with other therapeutic agents and/or formulation agents known in the art.
  • compositions of this invention may be incorporated into a variety of formulations for systemic and topical administration.
  • the inventor also provides a broad method for delivery of anti-sense oligonucleotides (oligos) through the respiratory system, as a fast means of starting treatment to address acute attacks of asthma and other diseases and conditions that have a rapid onset.
  • the present agents have long halflives and may be administered at very low doses. This makes them ideal for once a week type therapies.
  • anti-sense oligonucleotides received considerable theoretical consideration as being potentially useful as pharmacologic agents for the treatment of human disease. Wagner, R., Nature 372: 333-335 (1994).
  • the systemic administration of anti-sense oligonucleotides poses significant problems with respect to their pharmacologic application, not the least of which is the difficulty in selectively targeting disease-involved tissues.
  • the systemic administration of anti-sense oligonucleotides also poses significant problems with respect to their pharmacologic application, not the least of which is the difficulty in selectively targeting disease- involved tissues.
  • the respiratory system and in particular the lung, as the ultimate port of entry into the organism, however, is an excellent route of administration for anti-sense oligonucleotides. This is so not only for the treatment of lung disease, but also when utilizing the lung as a means for delivery, particularly because of its non-invasive and tissue-specific nature. Thus, local delivery of antisense oligonucleotides directly to the target tissue enables the therapeutic use of these compounds.
  • Fomivirsen (ISIS 2302) is an example of a local drug delivery into the eye to treat cytomegalovirus (CMV) retinitis, for which a new drug application has been filed by ISIS.
  • composition and formulations of this invention are highly efficacious for preventing and treating diseases and conditions associated with bronchoconstriction, difficult breathing, impeded and obstructed lung airways, allergy(ies), inflammation and surfactant depletion, among others.
  • diseases and conditions which are suitably treated by the present method are diseases and conditions, including Acute Respiratory Distress Syndrome (ARDS), asthma, adenosine administration e.g.
  • ARDS Acute Respiratory Distress Syndrome
  • asthma adenosine administration e.g.
  • SVT Supra Ventricular Tachycardia
  • other arrhythmias and in stress tests to hyper-sensitized individuals, ischemia, renal damage or failure induced by certain drugs, infantile respiratory distress syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), lung transplantation rejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, including colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic metastases, etc., as well as all types of cancers which may metastasize or have metastasized to the lung(s), including breast and prostate cancer.
  • SVT Supra Ventricular Tachycardia
  • COPD chronic obstructive pulmonary disease
  • the invention will be described with respect to the adenosine receptors as targets, but is similarly applicable to any other target with respect to the pulmonary administration of anti-sense oligos.
  • the examples provided below show a complete inhibition of such adenosine receptor associated symptoms in a rabbit model for human bronchoconstriction, allergy(ies) and inflammation as well as the elimination of the ability of the adenosine receptor agonist par excellence, adenosine, to cause bronchoconstriction in hyper-responsive monkeys, which are animal models for human hyperresponsiveness to adenosine receptor agonists.
  • compositions and formulations of the invention are suitable for preventing and alleviating the symptoms associated with stimulation of adenosine receptors, such as the adenosine A, receptors.
  • adenosine receptors such as the adenosine A, receptors.
  • the compositions and formulations of this invention are also suitable for prevent the untoward side effects of adenosine-mediated hyperresponsiveness in certain individuals, which are generally seen in diseases affecting respiratory activity.
  • the method of the present invention may be used to treat airway diseases and conditions in a subject of any kind and for any reason, with the intention that the adenosine content of anti-sense compounds be minimized, reduced or eliminated so as to prevent its liberation upon anti-sense degradation.
  • diseases and conditions which may be treated preventatively, prophylactically and therapeutically with the compositions and formulations of this invention, are pulmonary vasoconstriction, inflammation, allergies, asthma, allergic rhynitis, impeded respiration, Acute Respiratory Distress Syndrome (ARDS), renal damage and failure associated with ischemia as well as the administration of certain drugs, side effects associated with adenosine administration e.g.
  • SupraVentricular Tachycardia SVT and in adenosine stress tests, infantile Respiratory Distress Syndrome (infantile RDS), ARDS, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), lung transplantation rjejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, e.g. colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, metastatic cancer such as hepatic metastases, lung, breast and prostate metastases, among others.
  • SVT SupraVentricular Tachycardia
  • infantile RDS infantile Respiratory Distress Syndrome
  • ARDS ARDS
  • pain cystic fibrosis
  • pulmonary hypertension pulmonary vasoconstriction
  • emphysema chronic ob
  • compositions and formulations are suitable for administration before, during and after other treatments, including radiation, chemotherapy, antibody therapy, phototherapy and cancer, and other types of surgery.
  • the present compositions and formulations may also be administered effectively as a substitute for therapies that have significant negative side effects.
  • anti-sense oligonucleotides generally refers to small, synthetic oligonucleotides, resembling single-stranded DNA, which in this patent are applied to the inhibition of gene expression by inhibition of a target messenger RNA (mRNA). See, Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993), the relevant portion of which is hereby incorporated in its entirety by reference.
  • RNAs and oligonucleotides are represented in this patent by a single strand in the 5' to 3' direction, when read from left to right, although their complementary sequence(s) is (are) also encompassed within the four corners of the invention.
  • nucleotide bases and amino acids are represented utilizing the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission, or by the known 3-letter code (for amino acids). Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right.
  • nucleotide and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR ' 1.822 and established usage. See, e.g., Patentln User Manual, 99-102 (Nov. 1990) (U.S. Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, D.C. 20231); U.S. Patent No. 4,871,670 to Hudson et al. at col. 3, lines 20-43.
  • the present method utilizes anti-sense agents to inhibit or down-regulate gene expression of target genes, including those listed in Tables 1 and 2 below.
  • mRNA messenger RNA
  • the exogenously administered agents of the invention decrease the levels of mRNA and protein encoded by the target gene and/or cause changes in the growth characteristics or shapes of the thus treated cells. See, Milligan et al. (1993); Helene, C. and Toulme, J. Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S.
  • anti-sense oligonucleotide or asnti-sense oligo is generally a short sequence of synthetic nucleotide that (1) hybridizes to any segment of a mRNA encoding a targeted protein under appropriate hybridization conditions, and which (2) upon hybridization causes a decrease in gene expression of the targeted protein.
  • desA and des-thymidine refer to oligonucleotides substantially lacking either adenosine (desA) or thymidine (desT).
  • des A or des T sequences are naturally occurring, and in others they may result from substitution of an undesirable nucleotide (A) by another lacking its undesirable activity, such as acting as an agonist or having a triggering effect at the adenosine A receptor(s).
  • substitution is generally accomplished by substitution of A with a "universal or alternative base", presently known in the art or to be ascertained at a later time.
  • the terms “prevent”, “preventing”, “treat” or “treating” refer to a preventative, prophylactic, maintenance, or therapeutic treatment which decreases the likelihood that the subject administered such treatment will manifest symptoms associated with adenosine receptor stimulation.
  • the term “down-regulate” refers to inducing a decrease in production, secretion or availability and, thus, a decrease in concentration, of intracellular target product, be it a receptor e. g. adenosine A réelle A 2b , A 3 , bradykinin 2B, GATA-3, or other receptors, or an increase in concentration of the adenosine A 2a receptor.
  • the present technology relies on the design of anti-sense oligos targeted to mRNAs associated with ailments involving lung airway pathology(ies), and on their modification to reduce the occurrence of undesirable side effects caused by their release of adenosine upon breakdown, while preserving their activity and efficacy for their intended purpose.
  • the inventor targets a specific gene to design one or more anti-sense oligonucleotide(s) (oligos) that selectively bind(s) to the corresponding mRNA, and then reduces, if necessary, their content of adenosine via substitution with an alternative or a universal base, or an adenosine analog incapable of significantly, or having substantially reduced ability for, activating or antagonizing adenosine A, A 2b or A 3 receptors or which may act as an agonist at the adenosine A 2a , receptor.
  • adenosines present may be substituted by an alternative and/or universal base, such as heteroaromatic bases, which binds to a thymidine base but has less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A,, A 2a , A 2b and A 3 receptors.
  • an alternative and/or universal base such as heteroaromatic bases, which binds to a thymidine base but has less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A,, A 2a , A 2b and A 3 receptors.
  • adenosine (A) is a nucleotide base complementary to thymidine (T)
  • T thymidine
  • the anti-sense oligonucleotide has a sequence which specifically binds to a portion or segment of a mRNA molecule which encodes a protein associated with impeded breathing, allergy(ies), lung inflammation, depletion of lung surfactant or lowering of lung surfactant, airway obstruction, bronchitis, and the like.
  • the phosphodiester residues of the anti-sense oligonucleotide are modified or substituted.
  • the naturally occurring phosphodiester linkages of oligonucleotides are susceptible to some degree of degradation by cellular nucleases. Many of the residues proposed herein, on the contrary, are highly resistant to nuclease degradation. See, Milligan et al.; Cohen, J. S. D., supra.
  • the oligonucleotides may be protected from degradation by adding a "3'-end cap" by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide.
  • a "3'-end cap” by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide.
  • Phosphoramidates, phosphorothioates, and methylphosphonate linkages all function adequately in this manner for the purposes of this invention, as do ⁇ ' modifications, such as ⁇ ' methoxy ethyl, and the like.
  • ⁇ ' modifications such as ⁇ ' methoxy ethyl, and the like.
  • the more extensive the modification of the phosphodiester backbone the more stable the resulting agent, and in many instances the higher their RNA affinity and cellular permeation. See, Milligan, et al., supra.
  • a plurality of substitutions to the carbohydrate ring are also known to improve stability of nucleic acids.
  • the number of residues which may be modified or substituted will vary depending on the need, target, and route of administration, and may be from 1 to all the residues, to any number in between.
  • Preferred backbone analogue residues include phosphoramidate, phosphorothioate, methylphosphonate, phosphorotriester, phosphotriester, thioformacetal, phosphorodithioate, phosphoramidate, formacetal, triformacetal, thioether, carbamate, boranophosphate, 3'-thioformacetal, 5'-thioether, carbonate, C 5 -substituted nucleotides, 5'-N- carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, 2'-0 methyl, sulfoxide, sulfide, hydroxylamine, methylene(methylimino) (MMI), methoxymethyl (MOM), and methoxyethyl(MO
  • Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred due to their availability through automated oligonucleotide synthesis. See, Millikan et al, supra.
  • the agent of this invention may be administered in the form of their pharmaceutically acceptable salts, or as a mixture of the anti-sense oligonucleotide and its salt. In another embodiment of this invention, a mixture of different anti-sense oligonucleotides or their pharmaceutically acceptable salts is administered.
  • a single agent of this invention has the capacity to attenuate the expression of a target mRNA and/or various agents to enhance or attenuate the activity of a pathway.
  • the present method may be practiced by identifying all possible deoxyribonucleotide segments which are low in thymidine (T) or deoxynucleotide segments low in adenosine (A) of about 7 or more mononucleotides, preferably up to about 60 mononucleotides, more preferably about 10 to about 36 mononucleotides, and still more preferably about 12 to about 21 mononucleotides, in a target mRNA or a gene, respectively.
  • T thymidine
  • A adenosine
  • RNA thymidine
  • gene a nucleotide which is complementary to adenosine
  • this search typically results in about 10 to 30 such sequences, i.e. naturally lacking or having less than about 40% adenosine, anti-sense oligonucleotides of varying lengths for a typical target mRNA of average length, i.e., about 1800 nucleotides long.
  • Those with high content of T or A, respectively, may be fixed by substitution of a universal base for one or more As.
  • the agent(s) of this invention may be of any suitable length, including but not limited to, about 7 to about 60 nucleotides long, preferably about 12 to about 45, more preferably up to about 30 nucleotides long, and still more preferably up to about 21, although they may be of other lengths as well, depending on the particular target and the mode of delivery.
  • the agent(s) of the invention may be directed to any and all segments of a target RNA.
  • One preferred group of agent(s) includes those directed to an mRNA region containing a junction between an intron and an exon.
  • the agent may either entirely overlie the junction or it may be sufficiently close to the junction to inhibit the splicing- out of the intervening exon during processing of precursor mRNA to mature mRNA, e.g. with the 3' or 5' terminus of the anti-sense oligonucleotide being positioned within about, for example, within about 2 to 10, preferably about 3 to 5, nucleotide of the intron/exon junction. Also preferred are anti-sense oligonucleotides which overlap the initiation codon, and those near the 5' and 3' termini of the coding region. The flanking regions of the exons may also be targeted as well as the spliced segments in the precursor mRNAs.
  • the mRNA sequences of the adenosine receptors and of many other targets are derived from the DNA base sequence of the gene expressing either receptors, e. g. the adenosine receptors, the enzymes, factors, or other targets associated with airway disease.
  • receptors e. g. the adenosine receptors
  • the sequence of the genomic human A, adenosine receptor is known and is disclosed in U.S. Patent No. 5,320,963 to Stiles, G., et al.
  • the A 3 adenosine receptor has been cloned, sequenced and expressed in rat (see, Zhou, F., et al., P.N.A.S. (USA) 89: 7432 (1992)) and human (see, Jacobson, M.
  • the sequence of the adenosine A 2b receptor gene is also known. See, Salvatore, C. A., Luneau, C. J., Johnson, R. G. and Jacobson, M., Genomics (1995), the relevant portion of which is hereby incorporated in its entirety by reference.
  • the sequences of many of the remaining exemplary target genes are also known. See, GenBank, NIH. The sequences of those genes whose sequences are not yet available may be obtained by isolating the target segments applying technology known in the art.
  • an anti-sense oligonucleotides may be produced according to this invention as described above to reduce the production of the targeted protein in accordance with standard techniques.
  • the sequences for the adenosine A 2a bradykinin, and other genes as well as methods for preparation of oligonucleotides are also known as those of many other target genes and mRNAs for which this invention is suitable.
  • anti-sense oligonucleotides that downregulate the production of target sequences associated with airway disease, including the adenosine A,, A 2o , A 2b , A 3 , bradykinin, GATA- 3, COX-2, and many other receptors, may be produced in accordance with standard techniques.
  • diseases and conditions which are suitably treated by the present method are diseases and conditions, including Acute Respiratory Distress Syndrome (ARDS), asthma, adenosine administration e.g.
  • ARDS Acute Respiratory Distress Syndrome
  • asthma adenosine administration e.g.
  • SVT SupraVentricular Tachycardia
  • COPD chronic obstructive pulmonary disease
  • pulmonary infections and cancers such as leukemias, lymphomas, carcinomas, and the like, including colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic metastases, etc., as well as all types of cancers which may metastasize or have metastasized to the lung(s), including breast and prostate cancer.
  • a large number of genes may be targeted in a similar manner by the present agent(s), to reduce or down-regulate protein expression.
  • the target disease or condition is one associated with impeded or reduced breathing, bronchoconstriction, chronic bronchitis, pulmonary bronchoconstriction and/or hypertension, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, allergy, asthma, cystic fibrosis, respiratory distress syndrome, cancers, which either directly or by metastasis afflict the lung
  • the present method may be applied to a list of potential target mRNAs, which includes the targets listed in Table 1 and Table 2 below, among others.
  • the anti-sense agent(s) of the invention have a low A content to prevent its liberation upon in vivo degradation of the agent(s). For example, if the system is the pulmonary or respiratory system, a large number of genes is involved in different functions, including those listed in Table 1 below.
  • IL-8 R Interleukin-8 Receptor
  • IL-5R Interleukin-5 Receptor
  • IL-4R Interleukin-4 Receptor
  • Interleukin-3 Receptor IL-3R
  • Interleukin- 1 ⁇ IL- 1 ⁇
  • B2BR Bradykinin B2 Receptor IgE (High Affinity Receptor)
  • Interleukin- 1 Interleukin 1 Receptor (IL-1 R)
  • IL-1 R Interleukin 1 Receptor
  • Interleukin-9 Interleukin-9 Receptor (IL-9 R)
  • Interleukin- 11 Interleukin- 11 Receptor (IL- 11 R) Inducible Nitric Oxide Synthase Cyclooxygenase (COX)
  • ICM-1 Intracellular Adhesion Molecule 1
  • Cyclophillin (A, B, etc.) Phospholipase A2 Basic Fibroblast Growth Factor Metalloproteinase CSBP/p38 MAP Kinase Tryptase Receptor PDG2 Interleukin-3 (IL-3)
  • Interleukin- 10 Cyclosporin A - Binding Protein FK506-Binding Protein ⁇ 4 ⁇ l Selectin Fibronectin ⁇ 4 ⁇ 7 Selectin
  • CCR3 (Eotaxin Receptor) CCR1, CCR2, CCR4, CCR5
  • Tachykinnen Receptors (tach R) I6B Kinase 1 & 2 lnterleukin-2 Receptor (IL-2R) (e.g., Substance P, NK-1 & NK-3 Receptors)
  • NF-Interleukin-6 Interleukin- 10 Receptor (IL-1 OR)
  • Interleukin-3 IL-3
  • IL-2R Interleukin-2 Receptor
  • Interleukin- 13 IL-13
  • Interleukin- 12 Receptor IL-12R
  • Interleukin- 14 Interleukin-6 Receptor (IL-6R)
  • IL-6R Interleukin-6 Receptor
  • Interleukin- 16 Interleukin- 13 Receptor (IL-13R)
  • IL-16 Interleukin- 13 Receptor
  • Adenosine A 2b Receptor (A 2b R) Adenosine A 3 Receptor (A 3 R) ⁇ Tryptase STAT-3
  • Adenosine A 2a Receptor (A 2a R) IgE Receptor ⁇ Subunit (IgE R ⁇ )
  • Anti-sense oligos to the target receptors e. g. the adenosine A,, A 2a , A 2b , and A 3 receptors, CCR3 (chemokine receptors), bradykinin 2B, CAM (vascular cell adhesion molecule), and eosinophil receptors, among others, have been shown to be effective in down-regulating the expression of their genes. Some of these act to alleviate the symptoms or reduce respiratory ailments and/or inflammation, for example, by "down regulation" of the adenosine A,, A 2a , A 2b , and/or A 3 receptors and CCR3, bradykinin 2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors.
  • the target receptors e. g. the adenosine A,, A 2a , A 2b , and A 3 receptors, CCR3 (chemokine receptors), bradykinin 2B, CAM (vascular cell adhesion
  • agents may be utilized by the present method alone or in conjunction with anti-sense oligos targeted to other genes to validate pathway and/or networks in which they are involved.
  • the oligos are preferably administered directly into the respiratory system, e.g., by inhalation or other means, of the experimental animal, so that they may reach the lungs without widespread systemic dissemination.
  • the agent(s) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue.
  • a receptor lower the number of target proteins that other drugs may interact with.
  • the present agent(s) afford(s) extremely high efficacy with low toxicity.
  • Anti-sense oligonucleotides to the A, , A 2b , A 3 , bradykinin B2, GATA- 3, CAM (vascular cell adhesion molecule), eosinophil receptors, and COX-2 receptors, among others, have been shown to be effective in the down-regulation of the respective receptor proteins in the cell.
  • One novel feature of this treatment is that administration is direct to the lungs, or in situ to other tissues, organs or systems of the body.
  • a receptor protein itself is reduced in amount, rather than merely interacting with a drug, and toxicity is reduced.
  • Other proteins that may be targeted with anti-sense agents for the treatment of lung conditions include, but are not limited to: CCR3 (chemokine) receptors, human A 2a adenosine receptor, human A 2b adenosine receptor, human IgE receptor ⁇ , human Fc-epsilon receptor CD23 antigen, human histidine decarboxylase, human beta tryptase, human tryptase-I, human prostaglandin D synthase, human cyclooxigenase-2, human eosinophil cationic protein, human eosinophil derived neurotoxin, human eosinophil peroxidase, human intercellular adhesion molecule-1 (ICAM-1), human vascular cell adhesion molecule-1 (VCAM-1), human endothelial leukocyte adhesion molecule-1 (ELAM-1), human P
  • genes are provided below. Some of these act to alleviate the symptoms or reduce respiratory ailments and/or inflammation, for example, by "down regulation" of the adenosine A,, A 2a , A 2b , and or A 3 receptors and CCR3, bradykinin 2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors. These agents are preferably administered directly into the respiratory system, e.g., by inhalation or other means, so that they may reach the lungs without widespread systemic dissemination.
  • agent(s) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue
  • targets e g a receptor
  • the present agent(s) afford(s) extremely high efficacy with low toxicity In these latter targets, and m target genes in general, it is particularly imperative to eliminate or reduce the adenosine content of the corresponding anti-sense oligonucleotide to prevent their breakdown products from liberating adenosine
  • the term “treat” or “treating” asthma refers to a treatment which decreases the likelihood that the subject administered such treatment will manifest symptoms of the lung disease
  • the term “downregulate” refers to inducing a decrease in production, secretion or availability (and thus a decrease in concentration) of the targeted intracellular protein
  • the present invention is concerned primarily with the treatment of human subjects
  • the agents and methods disclosed here may also be employed for veterinary purposes, such as is the case in the treatment of other mammals, such as cattle, horses, wild animals, zoo animals, and domestic animals, e g dogs and cats
  • Targeted proteins are preferably mammalian and more preferably of the same species as the subject being treated
  • anti-sense refers to the use of small, synthetic oligonucleotides, resembling single-stranded DNA, to inhibit gene expression by inhibiting the function of the target messenger RNA (mRNA) Milligan, J F et al , J Med Chem 36(14), 1923-1937 (19
  • the oligos of this invention may be obtained by first selecting fragments of a target nucleic acid having at least 4 contiguous nucleic acids selected from the group consisting of G and C and/or having a specific type and/or extent of activity, and then obtaining a first oligonucleotide 4 to 60 nucleotides long which comprises the selected fragment and has a thymidine (T) nucleic acid content of up to and including about 15%, preferably, about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no thymidine.
  • T thymidine
  • the latter step may be conducted by obtaining a second oligonucleotide 4 to 60 nucleotides long comprising a sequence which is anti-sense to the selected fragment, the second oligonucleotide having an adenosine base content of up to and including about 15%, preferably about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no adenosine.
  • an adenosine base may be substituted in the corresponding anti-sense nucleotide fragment with a universal base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have less than about bout 10%, preferably less than about 1%, and more preferably less than about 0.3% of the adenosine base agonist activity at the adenosine A,, A 2a , A 2b and A 3 receptors, and heteroaromatic bases which have no activity at the adenosine A 2a receptor, when validating in the respiratory system.
  • Other adenosine activities in other systems may be determined in other systems, as appropriate.
  • the analogue heteroaromatic bases may be selected from all pyrimidines and purines, which may be substituted by O, halo, NH 2 , SH, SO, S0 2 , S0 3 , COOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloal
  • pyrimidines and purines may be substituted at all positions as is known in the art, but preferred are those which are substituted at positions 1, 2, 3, 4, 7 and/or 8. More preferred are pyrimidines and purines such as theophylline, caffeine, dyphylline, etophylline, acephylline piperazine, bamifylline, enprofylline and xantine having the chemical formula
  • R 1 and R 2 are independently H, alkyl, alkenyl or alkynyl and R 3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH 2 -alkylamino-ketoxyaIkyloxy-aryl, mono and dialkylaminoalkyl-N-alkylamino- S0 2 aryl, among others. Similar modifications in the sugar are also embodiments of this invention.
  • Reduced adenosine content of the anti-sense oligos corresponding to the thymidines (T) present in the target RNA serves to prevent the breakdown of the oligos into products that free adenosine into the system, e.g. the lung, brain, heart, kidney, etc., tissue environment and, thereby, to prevent any unwanted effects due to it.
  • the Nf6B transcription factor may be selected as a target, and its mRNA or DNA searched for low thymidine (T) or desthymidine (desT) fragments. Only desT segments of the mRNA or DNA are selected which, in turn, will produce des A anti-sense as their complementary strand.
  • RNA desT segments When a number of RNA desT segments are found, the sequence of the anti- sense segments may be deduced. Typically, about 10 to 30 and even larger numbers of desA anti-sense sequences may be obtained.
  • These anti-sense sequences may include some or all desA anti-sense oligonucleotide sequences corresponding to desT segments of the mRNA of the target, such as anyone of those shown in Table 1 above, in Table 2 below, and others associated with functions of the brain, cardiovascular and renal systems, and many others. When this occurs, the anti-sense oligonucleotides found are said to be 100% A- free. For each of the original desA anti-sense oligonucleotide sequences corresponding to the target gene, e.g.
  • the NF6B transcription factor typically about 10 to 30 sequences may be found within the target gene or RNA which have a low content of thymidine (RNA).
  • the selected fragment sequences may also contain a small number of thymidine (RNA) nucleotides within the secondary or tertiary or quaternary sequences. In some cases, a large adenosine content may suffice to render the anti-sense oligonucleotide less active or even inactive against the target.
  • these so called “non-fully desA" sequences may preferably have a content of adenosine of less than about 15%, about 12%, about 10%, about 7%, about 5%, and about 2% adenosine. Most preferred is no adenosine content (0%). In some instances, however, a higher content of adenosine is acceptable and the oligonucleotides still fail to show detrimental "adenosine activity".
  • a particular important embodiment is that where the adenosine nucleotide is "fixed” or replaced by a "Universal or alternative” base that may base-pair with similar or equal affinity to two or more of the four nucleotide present in natural DNA: A, G, C, and T.
  • a universal or alternative base is defined in this patent as any compound, more commonly an adenosine analogue, which has substantial capacity to hybridize to thymidine, while at the same time having reduced, or substantially lacking, ability to bind adenosine receptors or other molecules through which adenosine may exert an undesirable side effect in the experimental animal or in a cell system.
  • adenosine analogs which completely fail to activate, or have significantly reduce ability for activating, adenosine receptors, such as the adenosine A,, A 2b and or A 3 receptors, most preferably A, receptors, and those that may even act as agonists of the adenosine A 2a , receptor, may be used.
  • a universal base is ⁇ -deoxyribofuranosol-(5-nitroindole), and an artisan will know how to select others. This "fixing" step generates further novel sequences, different from those anti-sense to the ones found in nature, that permits the anti-sense oligonucleotide to bind, preferably equally well, with the target RNA.
  • Other examples of universal or alternative bases are 2-deoxyribosyl-(5- nitroindole).
  • universal bases are 3 - nitropyrrole - 2' - deoxynucleoside, 5 - nitroindole, 2 - deoxyribosyl - (5 - nitroindole), 2-deoxyribofuranosyl - (5-nitroindole), 2' - deoxyinosine, 2' -deoxynebularine, 6H, 8H-3,4-dihydropyrimido [ 4, 5 - c] oxazine - 7 - one and 2 - amino - 6 -methoxy aminopurine.
  • Universal bases which may be substituted for any other base although with somewhat reduced hybridization potential, include 3 - nitropyrrole 2' - deoxynucleoside 2 - deoxyribofuranosyl - (5 - nitroindole), 2' - deoxyinosine and 2' - deoxynebularine (Glen Research, Sterling, VA).
  • More specific mismatch repairs may be made using "P" nucleotide, 6H, 8H - 3, 4 - dihydropyrimido [4,5 - c] [1, 2] oxazin - 7 - one, which base pairs with either guanine (G) or adenine (A) and "K" nucleotide, 2 - amino - 6 - methoxyaminopurine, which base pairs with either cytidine (C) or thymidine (T), among others.
  • G guanine
  • A adenine
  • K 2 - amino - 6 - methoxyaminopurine
  • C cytidine
  • T thymidine
  • Others which are known in the art or will become available are also suitable. See, for example, Loakes, D. and Brown, D. M., Nucl. Acids Res. 22:4039-4043 (1994); Ohtsuka, E.
  • the present method provides either anti-sense oligonucleotides to different targets which are low in, or devoid of, A content, as well as anti-sense oligonucleotides where one or more adenosine nucleotides, e. g.
  • Oncogenes Targets ras thymidylate synthetase src thymidylate synthetase myc dihydrofolate reductase bcl-2 thymidine kinase deoxycytidine kinase ribonucleotide reductase Angiogenesis factors Adhesion Molecules Oncogenes Folate Pathway Enzymes
  • a group of preferred targets for the treatment of cancer are genes associated with any of different types of cancers, or those generally known to be associated with malignancies, whether they are regulatory or involved in the production of RNA and/or proteins. Examples are transforming oncogenes, including, but not limited to, ras, src, myc, and BCL-2, among others. Other targets are those to which present cancer chemotherapeutic agents are directed to, such as various enzymes, primarily, although not exclusively, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase, and the like.
  • the present technology is particularly useful in the treatment of cancer ailments given that traditional cancer therapies are fraught with the unresolved problem of selectively killing cancer cells while preserving normal living cells from the devastating effects of treatments such as chemotherapy, radiotherapy, and the like.
  • the present technology provides the ability of selectively attenuating or enhancing a desired pathway or target. This approach provides a significant advantage over standard treatments of cancer because it permits the selection of a pathway, including primary, secondary and possibly tertiary targets, which are not generally expressed simultaneously in normal cells.
  • the present agent may be administered to a subject to cause a selective increase in toxicity within tumor cells that, for instance, express all three targets while normal cells that may expresses only one or two of the targets will be significantly less affected or even spared.
  • a group of preferred targets for the treatment of cancers are genes associated with different types of cancers, or those generally known to be associated with malignancies, whether they are regulatory or involved in the production of RNA and/or proteins. Examples are transforming oncogenes, including, but not limited to, ras, src, myc, and BCL-2, among others. Other targets are those to which present cancer chemotherapeutic agents are directed to, such as various enzymes, primarily, although not exclusively, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase, and the like.
  • At least one of the mRNAs to which the oligo of the invention is targeted encodes a protein such as transcription factors, stimulating and activating factors, intracellular and extracellular receptors and peptide transmitters in general, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously produced specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral nervous and non- nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensines, growth factors, vasoactive peptides and receptors, and binding proteins, among others; or the mRNA is corresponding to an oncogene and other genes associated with various diseases or conditions.
  • a protein such as transcription factors, stimulating and activating factors, intracellular and extracellular receptors and peptide transmitters in general, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously produced specific and non-
  • target proteins are eotaxin, major basic protein, preproendothelin, eosinophil cationic protein, P-selectin, STAT 4, MlP-l ⁇ , MCP-2, MCP-3, MCP-4, STAT 6, c-mas, NF-IL-6, cyclophillins, PDG2, cyclosporin A-binding protein, FK5-binding protein, fibronectin, LFA-1 (CDl la/CD18), PECAM-1, C3bi, PSGL-l.CD-34, substance P, pl50,95, Mac-1 (CDl lb/CD18), VLA-4, CD-18/CDl la, CDl lb/CD18, C5a, CCR1, CCR2, CCR4, CCR5, and LTB-4, among others.
  • eotaxin major basic protein
  • preproendothelin eosinophil cationic protein
  • P-selectin STAT 4
  • MlP-l ⁇ MCP-2,
  • At least one of the mRNAs to which the oligo is targeted encodes intracellular and extracellular receptors and peptide transmitters such as sympathomimetic receptors, parasympathetic receptors, GABA receptors, adenosine receptors, bradykinin receptors, insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, estrogen receptors, progesterone receptors, receptors associated with the coagulation cascade, adenohypophyseal receptors, adenohypophyseal peptide transmitters, and histamine receptors (HisR), among others.
  • intracellular and extracellular receptors and peptide transmitters such as sympathomimetic receptors, parasympathetic receptors, GABA receptors, adenosine receptors, bradykinin receptors, insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors,
  • the encoded sympathomimetic receptors and parasympathomimetic receptors include acetylcholinesterase receptors (AcChaseR) acetylcholine receptors (AcChR), atropine receptors, muscarinic receptors, epinephrine receptors (EpiR), dopamine receptors (DOPAR), and norepinephrine receptors (NEpiR), among others.
  • Further examples of encoded receptors are adenosine
  • IgE high affinity receptor muscarinic acetylcholine receptors, substance P receptor, histamine receptor, CCR-1 CC chemokine receptor, CCR-2 CC chemokine receptor, CCR-3 CC chemokine receptor (Eotaxin Receptor), interleukin- l ⁇ receptor (IL-l ⁇ R), interleukin-1 receptor (IL-1R), interleukin- l ⁇ receptor (IL-l ⁇ R), interleukin-3 receptor (IL-3R), CCR-4 CC chemokine receptor, cysteinyl leukotriene receptors, prostanoid receptors, GATA-3 transcription factor receptor, interleukin-1 receptor (IL-1R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL-5R), interleukin-8 receptor (IL-8R), interleukin-9 receptor (IL-9R), interleukin-11 receptor (IL-11R), bradykinin B2 receptor, sympathomimetic receptors, parasympathomimetic receptor
  • the encoded enzymes for development of the oligos of the invention include synthetases, kinases, oxidases, phosphatases, reductases, polysaccharide, triglyceride, and protein hydrolases, esterases, elastases, and , polysaccharide, triglyceride, lipid, and protein synthases, among others.
  • target enzymes are tryptase, inducible nitric oxide synthase, cyclooxygenase (Cox), MAP kinase, eosinophil peroxidase, ⁇ 2-adrenergic receptor kinase, leukotriene c-4 synthase, 5-lipooxygenase, phosphodiesterase IV, metalloproteinase, tryptase, CSBP/p38 MAP kinase, neutrophil elastase, phospholipase A 2 , cyclooxygenase 2 (Cox-2), fucosyl transferase, chymase, protein kinase C, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, and ribonucleotide reductase, among others.
  • Suitable encoded factors for application of this invention are, among others, Nf6B transcription factor, granulocyte macrophage colony stimulating factor (GM- CSF), AP-1 transcription factor, GATA-3 transcription factor, monocyte activating factor, neutrophil chemotactic factor, granulocyte/macrophage colony-stimulating-factor (G-CSF), NFAT transcription factors, platelet activating factor, tumor necrosis factor ⁇ (TNF ⁇ ), and basic fibroblast growth factor (BFGF). Additional factors are also within the invention even though not specifically mentioned.
  • Suitable adhesion molecules for use with this invention include intracellular adhesion molecules 1 (ICAM-1), 2 (ICAM-2) and 3 (ICAM-3), vascular cellular adhesion molecule (VCAM), endothelial leukocyte adhesion molecule-1 (ELAM-1), neutrophil adherence receptor, mad CAM-1, and the like. Other known and unknown factors (at this time) may also be targeted herein.
  • lymphokines and chemokines preferred are interleukin-1 (IL-1), interleukin- l ⁇ (IL-l ⁇ ), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin- 11 (IL-l l),CCR-5 CC chemokine, and Rantes.
  • IL-1 interleukin-1
  • IL-l ⁇ interleukin-3
  • IL-4 interleukin-4
  • IL-5 interleukin-5
  • IL-8 interleukin-8
  • IL-9 interleukin-9
  • IL- 11 interleukin-l l
  • CCR-5 CC chemokine interleukin- 11
  • defensins for the practice of this invention are defensin 1, defensin 2, and defensin 3, and of selectins are ⁇ 4 ⁇ l selectin, ⁇ 4 ⁇ 7 selectin, LFA-1 selectin, E-selectin, P- selectin, and L-selectin.
  • selectins are ⁇ 4 ⁇ l selectin, ⁇ 4 ⁇ 7 selectin, LFA-1 selectin, E-selectin, P- selectin, and L-selectin.
  • oncogenes although not an all inclusive list, are ras, src, myc, and bcBCL. Others, however, are also suitable for use with this invention.
  • the agents administered in accordance with this invention are preferably designed to be anti- sense to target genes and/or mRNAs related in origin to the species to which it is to be administered.
  • the agents are preferably designed to be anti-sense to a human gene or RNA.
  • the agents of the invention encompass oligonucleotides which are anti-sense to naturally occurring DNA and/or RNA sequences, fragments thereof of up to a length of one (1) base less than the targeted sequence, preferably at least about 7 nucleotides long, oligos having only over about 0.02%, more preferably over about 0.1 %, still more preferably over about 1%, and even more preferably over about 4% adenosine nucleotides, and up to about 30%, more preferably up to about 15%, still more preferably up to about 10% and even more preferably up to about 5%, adenosine nucleotide, or lacking adenosine altogether, and oligos in which one or more of the adenosine nucleotides have been replaced with so-called universal bases, which may pair up with thymidine nucleotides but fail to substantially trigger adenosine receptor activity.
  • These fragments may be selected from any portion of the longer oligo, for example, from the middle, 5'- end, 3'- end or starting at any other site of the original sequence.
  • fragments of low adenosine nucleotide content that is, those fragments containing less than or about 30%, preferably less than or about 15%, more preferably less than or about 10%, and even more preferably less than or about 5%, and most preferably those devoid of adenosine nucleotide, either by choice or by replacement with a universal base in accordance with this invention.
  • the agent of the invention includes as a most preferred group sequences and their fragments where one or more adenosines present in the sequence have been replaced by a universal base (B), as exemplified here.
  • anti-sense oligonucleotide sequence fragments target the initiation codon of the respective gene, and in some cases adenosine is substituted with a universal or alternative base adenosine analogue denoted as "B", which lacks ability to bind to the adenosine A, and/or A 3 receptors. In fact, such replacement nucleotide acts as a "spacer". Many of the examples shown below provide one such sequence and many fragments overlapping the initiation codon, preferably wherein the number of nucleotides n is about 7, about 10, about 12, about 15, about 18, about 21 and up to about 28, about 35, about 40, about 50, about 60.
  • CTCTGTTTCC TCTTCCCCTT TCTCCTCGTA TGTGTGTTTA CCTAAACTAT ATGCCATAAA CCTCAAGTTA TTCA
  • TD NO: 451) '- AA AGC TGA GAT GGA GGG -3' (FRAG 442) (SEQ. ID NO: 452) '- AA AGC TGA GAT GGA GG -3' (FRAG 443) (SEQ. ID NO: 453) '- AA AGC TGA GAT GGA G -3' (FRAG 444) (SEQ. ID NO: 454) '- AA AGC TGA GAT GGA -3' (FRAG 445) (SEQ. ID NO: 455) '- AA AGC TGA GAT GG -3' (FRAG 446) (SEQ. ID NO: 456) '- AA AGC TGA GAT G -3' (FRAG 447) (SEQ.
  • TD NO: 771) A GGG CGG CAT GGC GGG CAC AGG -3' (FRAG 762) (SEQ. ID NO: 772) - A GGG CGG CAT GGC GGG CAC AG-3' (FRAG 763) (SEQ. ID NO: 773) '- A GGG CGG CAT GGC GGG CAC A-3' (FRAG 764) (SEQ. ID NO: 774) - A GGG CGG CAT GGC GGG CAC-3' (FRAG 765) (SEQ. ID NO: 775) '- A GGG CGG CAT GGC GGG CA-3' (FRAG 766) (SEQ.
  • GBC BGG C-3' (FRAG. NO. 1665) (SEQ. ID NO:1680)
  • ATTTGCTCTCCTATTACTTTCTGTGTCCATTTTTTCATTAACCGAGCTGT (FRAG 992) (SEQ. ID NO: 1002)
  • BTTTGCTCTCCTBTTBCTTTCTGTGTCCBTTTTTTCBTTBBCCGBGCTGT (FRAG 993) (SEQ. ID NO: 1003)
  • TCC-3' (FRAG. NO: 1708) (SEQ. ID NO:1719)
  • TCTTCCATCT CAGATCCCAC CCAATGACCC CCGCATCAAG AACCAGCGTG ACTGCATCCC TTTCTTCCGC TCGGCACCCT CATGCCCCCA AAACAAGAAC AGAGTCCGCA ACCAGATCAA CGCGCTCACC TCCTTTGTGG ACGCCAGCAT GGTGTATGGC AGTGAGGTCT CCCTCTCGCT GCGGCTCCGC AACCGGACCA ACTACCTGGG GCTGCTGGCC ATCAACCAGC GCTTTCAAGA CAACGGCCGG GCCCTGCTGC CCTTCGACAA CCTGCACGAT GACCCCTGTC TCCTCACCAA CCGCTCGGCG CGCATCCT GCTTCCTGGC AGGTCAGACA GGGAGGAAGG TGGTGTCTTC CCAGGAAACA GCCATCCCTG GGGTCCCAAC TGGGAAGCAA TGGTGGGATG TGGTGAAGGT ACATGGTTTG GGACCTCAGT ATTAGGCACA CCATAAGCAT
  • GGTATTAGGC TATGAATCAG CGCCACGTGC AAAGGCTTGG GAGCCAAGCC ATGTGGTCTT GCACCCCAGG CAAGAAAAGT CAGCTGGAGG GTTTACAGCA CTTTCTACTG TTTCCCAGCC CTCCCTCCCC TCCCTCACCA
  • VCAM-1 Human Vascular Cell Adhesion Molecule 1 (VCAM-1) Nucleic Acid and Oligonucleotide Fragments
  • HSVCAM1AS2 5'-CTC TGC CTT TGT TTG GGT TCG-3' (FRAG. NO:1075) (SEQ. ID NO:1083) HSVCAM1AS3: 5'-CTT CCT TTC TGC TTC TTC C-3' (FRAG. NO:1076) (SEQ. ID NO: 1084) HSVCAM1AS4: 5'-CTG TGT CTC CTG TCT CCG CTT TTT TCT TC-3' (FRAG. NO:1077) (SEQ. ID NO: 1085) HSVCAM1AS5: 5'-GTC TTT GTT GTT TTC TCT TCC TTG-3' (FRAG. NO:1078) (SEQ.
  • AACTGTGAAG CAATCATGAC TTCAAGAGTT CTTTTCACCC AAAGGTTTAG GCTTGAAATA CTTTCCTGGG GAGATAAAAC ACAAAATGAA TTAAAGAAGG AAATCGTGGG TAGCTAGTTA CATTATTCTA CCATGATGTT TAAGGCAGCA TCCTAAGATT TTGGGCAAAG GACACTAGTG CAATAATCTT TATTTCAGAG TTTAATCAAA TAAATAAACA AATTTTAAGA CTTTCATTAT TTAGGTCAAA GAGAAAAGAC AGGTTTTAGC TACAATACAA TAAGAGCTTG TACAGATGTG GTTTTTATTA GAAGGCCTTT TGCATATCTG TGTTTCATGG CCCGAGGCTG CCCTTATAAA GCGTTCTGCA CTTACCGTTT TGGGAAGCAG TTGTTCAAAC ACAGGATCTC TCAGGTGGGT ATCACTGCTG CCTCTGTC AGGTCAGTAT AGGAGTTTTG ATGTGAAGTC AGCCAAGAAC
  • HUMIL3AAS1 5'-CTC TGT CTT GTT CTG GTC CTT CGT GGG GCT CTG-3' (FRAG.NO:1089)(SEQ.ID NO:1098)
  • HUMIL3AAS2 5'-TGT CGC GTG G GTG CGG CCG TGG CC-3' (FRAG. NO:1090) (SEQ. ID NO:1099)
  • GGC GGB CCB GGB GTT GGB GCB GGB GCB GGB CGG GCB GGC GGC TCB TGT TTG GBT CGG CBG GBG GCB CTC
  • FRAG. NO:1091100 5'-CTC TGT CTT GTT CTG GTC CTT CGT GGG GCT CTG-3'
  • CC-3' (FRAG. NO: 1750) (SEQ. ID NO: 1763)
  • GBG GTG CC-3' (FRAG. NO: 1751) (SEQ. ID NO: 1764)
  • AAG CCA CCC CAT TGG GAG ATG CCA AGG CAC CAG GCT G (FRAG. NO:1138) (SEQ. ID NO:1147)
  • 5'-GTGCGTGGGCCTCC-3' (FRAG. NO:1183) (SEQ. ID NO:1192) 5'-GCACGCCTCT TGCCACCTCC TGCGCAGGGC AGCGCCTTGG GGCCAGCGCC GCTCCCGGCG CGGCCAGCAG
  • GCBCCTTCBC BCBGBGC-3' (FRAG. NO:1783) (SEQ. ID NO: 1796)
  • Human Neutrophil Elastase Medullasin
  • CTTTTGAAGT AT-3' (FRAG.NO: ) (SEQ. ID NO: 2472)
  • TTGGAGTGC-3' (FRAG. NO: ) (SEQ. ID N ⁇ :3011)
  • AAACTCTGTG GTCGT-3' (FRAG. NO:1222) (SEQ. ID NO:1231)

Abstract

L'invention concerne un procédé in vivo de libération sélective d'un acide nucléique sur un gène cible ou ARNm, qui consiste à appliquer localement, par exemple sur le système respiratoire d'un sujet, une dose thérapeutique d'un oligonucléotide (oligo) antisens par rapport à la région du codon initiateur, à la région codante, aux jonctions intron-exon 3' ou 5' dans 2 à 10 nucléotides des jonctions du gène, ou antisens par rapport à un ARNm complémentaire du gène, en quantité efficace pour que le polynucléotide cible soit atteint, et à réduire ou à inhiber l'expression. Par ailleurs, un procédé de traitement d'un effet à médiation adénosinique, consiste à administrer localement à un sujet un oligo antisens, en dose efficace pour le traitement de maladie respiratoire, pulmonaire ou des voies aériennes. Afin de minimiser le déclenchement des récepteurs d'adénosine par leur métabolisme, les oligos administrés présentent une faible teneur en adénosine ou en sont sensiblement exempts. L'invention porte sur une composition et des préparations pharmaceutiques qui comprennent l'oligo antisens par rapport à un récepteur d'adénosine, des gènes et des ARNm les codant, des régions adjacentes génomiques et d'ARNm, des limites introniques et exoniques et tous les segments apparentés au plan régulatoire et fonctionnel des gènes et des ARNm codant pour les polypeptides, leurs sels et leurs mélanges. Diverses préparations contiennent un véhicule requis, et éventuellement d'autres additifs et des agents bioactifs. Les agents à faible teneur en adénosine ou exempts d'adénosine (des-A) utilisés pour la mise en oeuvre du procédé de l'invention, peuvent être préparés par la sélection d'un ou plusieurs gènes cibles, d'une ou plusieurs régions adjacentes génomiques, d'un ou plusieurs ARN et/ou d'un ou plusieurs polypeptides associés à une ou plusieurs maladies ou un ou plusieurs états affectant les voies aériennes pulmonaires, par l'obtention de la séquence du ou des ARN correspondant au(x) gène(s) et/ou à la ou aux régions adjacentes génomiques, et/ou des ARN codant pour le(s) polypeptide(s) cibles, par la sélection d'au moins un segment de l'ARNm qui peut être jusqu'à 60 % exempt de thymidine (T) et par la synthèse d'un ou plusieurs oligonucléotides antisens par rapport aux segments d'ARNm qui sont exempts d'adénosine (A), par le remplacement de A par une base universelle, lorsqu'elle est présente dans l'oligonucléotide. L'agent peut être préparé par la sélection de séquences nucléotidiques cibles à fragments de G et C, qui présentent une faible teneur en T, et par le remplacement éventuel de A dans les oligonucléotides antisens par une base universelle ou de remplacement. L'agent, la composition et les préparations sont utilisés pour le traitement prophylactique, préventif et thérapeutique d'affections associées à une respiration altérée, aux allergies pulmonaires et/ou à l'inflammation et à la déplétion de surfactant pulmonaire ou à l'hypoproduction de surfactant, comme la vasoconstriction pulmonaire, l'inflammation, les allergies, la rhinite allergique, l'asthme, les troubles de la respiration, la douleur pulmonaire, la fibrose kystique du poumon, la bronchoconstriction. Le traitement de l'invention est conçu pour être administré conjointement avec d'autres traitements, par exemple avant, pendant et après d'autres traitements, dont, entre autres, le rayonnement, la chimiothérapie, la thérapie par anticorps et la chirurgie. Ledit agent peut également être administré efficacement à des fins thérapeutiques ou prophylactiques, seul pour des états sans thérapies connues ou en tant que substitut aux thérapies à effets secondaires indésirables. Le traitement de l'invention peut être administré directement dans le système respiratoire d'un sujet, de manière que l'agent atteigne directement les poumons, ou par d'autres modes d'a
PCT/US2000/008020 1999-04-06 2000-03-24 Oligonucleotide antisens a faible teneur en adenosine, compositions, kit et procede pour le traitement d'affections des voies aeriennes associees a la bronchoconstriction, a l'inflammation pulmonaire, aux allergies et a la depletion de surfactant WO2000062736A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
BR0006019-4A BR0006019A (pt) 1999-04-06 2000-03-24 Oligonucleotìdeo de anti-sentido de adenosina baixo, composições, kit & método para tratamento de distúrbios de vias aéreas associados com broncoconstricção, inflamação pulmonar, alergia(s) & depleção de surfactante
IL14005400A IL140054A0 (en) 1999-04-06 2000-03-24 Low adenosine anti-sense oligonucleotide, compositions, kit and method for treatment of airway disorders associated with bronchoconstriction, lung inflammation, allergy (ies) and surfactant depletion
EP00919668A EP1168919A4 (fr) 1999-04-06 2000-03-24 Oligonucleotide antisens a faible teneur en adenosine, compositions, kit et procede pour le traitement d'affections des voies aeriennes associees a la bronchoconstriction, a l'inflammation pulmonaire, aux allergies et a la depletion de surfactant
CA002330022A CA2330022A1 (fr) 1999-04-06 2000-03-24 Oligonucleotide antisens a faible teneur en adenosine, compositions, kit et procede pour le traitement d'affections des voies aeriennes associees a la bronchoconstriction, a l'inflammation pulmonaire, aux allergies et a la depletion de surfactant
AU40317/00A AU4031700A (en) 1999-04-06 2000-03-24 Low adenosine anti-sense oligonucleotide, compositions, kit and method for treatment of airway disorders associated with bronchoconstriction, lung inflammation,allergy(ies) and surfactant depletion
JP2000611873A JP2003515525A (ja) 1999-04-06 2000-03-24 気管支収縮、肺炎、アレルギーおよびサーファクタント枯渇と関連する気道障害を治療するための、低アデノシンアンチセンスオリゴヌクレオチド、その組成物、キットならびにその方法
HK02102615.9A HK1042017A1 (zh) 1999-04-06 2002-04-08 低腺苷反義寡核苷酸、組合物、試劑盒及與支氣管緊縮、肺炎、過敏及表面活性劑缺失相關的通氣途徑疾病的治療方法

Applications Claiming Priority (2)

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US12795899P 1999-04-06 1999-04-06
US60/127,958 1999-04-06

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WO2000062736A2 true WO2000062736A2 (fr) 2000-10-26
WO2000062736A3 WO2000062736A3 (fr) 2001-10-11

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EP (1) EP1168919A4 (fr)
JP (1) JP2003515525A (fr)
CN (1) CN1330513A (fr)
AU (1) AU4031700A (fr)
BR (1) BR0006019A (fr)
CA (1) CA2330022A1 (fr)
HK (1) HK1042017A1 (fr)
IL (1) IL140054A0 (fr)
WO (1) WO2000062736A2 (fr)

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US6737040B1 (en) 1998-08-04 2004-05-18 Diadexus, Inc. Method and antibody for imaging breast cancer
EP1165783A4 (fr) * 1999-03-26 2002-09-11 Human Genome Sciences Inc 47 proteines humaines secretees
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US7671182B2 (en) * 2003-03-31 2010-03-02 Council Of Scientific & Industrial Research Gene variants of signal transducer and activator of transcription-6 (STAT 6) variants and process of detection the same
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WO2006086821A1 (fr) * 2004-10-20 2006-08-24 Antisense Therapeutics Ltd MODULATION ANTISENS DE L'EXPRESSION DE L'INTÉGRINE α4
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EP2068889A1 (fr) * 2006-08-10 2009-06-17 Arubor Corp Thérapie locale de troubles inflammatoires des voies aériennes inférieures avec des inhibiteurs de cytokine proinflammatoire
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WO2010111503A2 (fr) * 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. Inhibition à médiation par l'interférence arn de l'expression du gène de la chaîne alpha du récepteur à haute affinité pour les ige (fcεr1α) faisant appel à de courts acides nucléiques interférents (ansi)
WO2020063262A1 (fr) * 2018-09-30 2020-04-02 Shenzhen Sunny Bio-Technology.Co., Ltd Application de la 3'-désoxyinosine dans la préparation d'un médicament, d'un aliment ou d'un produit concernant la santé destinés à de multiples maladies

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BR0006019A (pt) 2001-03-13
CN1330513A (zh) 2002-01-09
EP1168919A4 (fr) 2002-03-06
WO2000062736A3 (fr) 2001-10-11
JP2003515525A (ja) 2003-05-07
EP1168919A2 (fr) 2002-01-09
CA2330022A1 (fr) 2000-10-26
AU4031700A (en) 2000-11-02
HK1042017A1 (zh) 2002-08-02
IL140054A0 (en) 2002-02-10

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