KR20020095600A - Low adenosine anti-sense oligonucleotide, compositions, kit and method for treatment of airway disorders associated with bronchoconstriction, lung inflammation, allergy(ies) and surfactant depletion - Google Patents

Abstract

An in vivo method of selectively delivering a nucleic acid to a target gene or mRNA comprises the topical admistration, e.g. to the respiratory system, of a subject of a therapeutic amount of an oligonucleotide that is antisense to the initiation codon region, coding region, 5' or 3' inton-exon junctions or regions within 2 to 10 nucleotides of the junctions of the target gene, or antisense to a mRNA complementary to the target gene in an amount effective to reach the target polynucleotide and reducing or inhibiting expression. Additionally, a method of treating and adenosine mediated effect is described also utilizing the delivery of antisense and in order to minimize triggering adenosine receptors by their metabolism, the administered oligos have a low content of or are essentially free of adenosine. The agent, composition and formulations are used for prophlactic, preventive and therapeutic treatment of ailments associated with impaired respiration, lung allergies and/or vasoconstriction, inflammation, allergies, allergic rhynitis, asthma, impeded respiration, lung pain, cystic fibrosis and bronchoconstriction.

Description

Low adenosine anti-sense oligonucleotide, compositions, kit and method for treatment of airway disorders with low adenosine oligonucleotides, compositions, kits and bronchial contractions, pulmonary inflammation, allergy (s) and surfactant deficiency associated with bronchoconstriction, lung inflammation, allergy (ies) and surfactant depletion}

Respiratory diseases associated with various diseases and ailments are very common in the general population, especially among certain minorities such as African Americans. In some cases, respiratory diseases are accompanied by inflammation that worsens the condition of the lungs. Asthma, for example, is one of the most common diseases in industrial countries. In the United States, it is estimated that about 1% of the total health care expenses. It has been reported that the number of people with asthma and the death toll over the past decade has increased dramatically, and asthma is expected to be a significant occupational lung disease in the next decade. In industrial countries, the increased death toll from asthma is thought to be due to deficiency dependence on beta agonists in the treatment of the disease, but the underlying causes of asthma are hardly understood.

For a variety of diseases, including bronchial asthma, adenosine may be an important mediator in the lungs. The potential role of adenosine has been suggested in asthmatic subjects by showing significant bronchial contractile response to aerosolized adenosine, but not to the general public. The fine dust allergic rabbit model, an asthmatic rabbit animal model for human asthma, showed similar bronchial contraction responses to aerosolized adenosine, whereas non-asthmatic rabbits did not show any response. Recent studies in this animal model suggested that adenosine-induced bronchial contraction and bronchial hyperresponsiveness in asthmatic rabbits could be primarily mediated through stimulation of adenosine receptors. It has also been shown that adenosine may be a cause of side effects, including death, when adenosine is administered to a subject with a hypersensitive reactive airway that has not been previously diagnosed for the treatment of other diseases and conditions.

In general, although some drugs all have disadvantages, they have been used to treat respiratory diseases and diseases. Theophylline, an important drug for the treatment of asthma, is a known adenosine receptor antagonist reported to eliminate adenosine-mediated bronchial contraction in asthmatic rabbits. In addition, the selective adenosine A ₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 8-cyclopentyl-1,3-dipropylxanthine) is responsible for adenosine-mediated bronchial contraction and bronchial hyperreactivity in allergic rabbits. It has been reported to inhibit. Nevertheless, the therapeutic and prophylactic applications of the adenosine A ₁ receptor-specific antagonists currently used are of limited use because of their toxicity. For example, deophylline has been widely used for the treatment of asthma, but is associated with frequent and severe toxicity due to narrow therapeutic doses. DPCPX is much more toxic and cannot be used clinically. Although many studies have been conducted for decades, virtually no specific adenosine receptor antagonists can be used to demonstrate clinical use for the general toxicity of these drugs. Antisense oligonucleotides have received considerable theoretical attention for their potential as useful pharmacological drug formulations of human diseases. However, their practical application in real models of human disease was a bit difficult. One obstacle to their effective application is that it is difficult to find a suitable route of administration for delivering antisense oligonucleotides to their site of action. Many in vivo experiments have been conducted in which antisense oligonucleotides are directly administered to specific regions of the brain. However, these applications are inevitably limited in clinical use because of their invasive nature. Although antisense oligonucleotides have received significant therapeutic interest in their potential for use as pharmacological agents of human disease, they must be administered in large amounts, so that practical and effective applications of these drugs in human models of human disease can be avoided. Not found or there are many differences Another important concern of pharmacological application of these molecules is their route of administration. Many in vivo applications include the direct administration of antisense oligonucleotides to limited areas of the brain. However, such applications have limited clinical use because of their invasive nature. In addition, systemic administration of antisense oligonucleotides as pharmacological agents has been found to have significant problems such as a substantial inherent difficulty in targeting disease-containing tissues. That is, an indispensable dilution of antisense oligonucleotides in the circulation makes it very difficult to obtain therapeutic doses in target tissues by intravenous or oral administration. The bioavailability of orally administered antisense oligonucleotides is very low, less than about 5%. Antisense oligonucleotides have been used in the treatment of many researchers, including the inventors, who have successfully treated various diseases and disorders by direct administration of antisense nucleotide drugs into the lungs in a preliminary work. In many instances, other researchers have faced difficulties associated with delivering DNA molecules to desired targets. Therefore, the route of administration is very important for treating generalized diseases and disorders as well as localized diseases and disorders. However, to date, delivery of antisense drugs to the lung has been relatively undeveloped. As mentioned in more detail by the inventors below, the lungs provide a very good target, non-invasive, and cospecific specific route for the direct administration of antisense oligonucleotides.

Clearly, there are currently no effective therapies for the treatment of these diseases, and even if they exist, there are no therapies that are effective and can avoid harmful and serious side effects. Thus, for the treatment of adenosine mediated diseases related to lung and respiratory diseases affecting obstruction, including respiratory problems, bronchial contraction, inflammation, allergy (s), lack of surfactants or hyposecretion, etc. There is still a need for drugs that are highly effective and sufficiently selective to avoid the harmful side effects created by other therapies. Moreover, there is a clear need to make available delivery methods that require small amounts with therapeutic drug formulations and that are effective for the rapid targeted access of target genes of mRNAs and the reversal of deleterious actions that plague subjects.

This patent relates to compositions comprising oligonucleotides (oligoses) which are antisense to adenosine receptors and have low or no adenosine (A) content. These drugs are particularly suitable for the treatment of pulmonary diseases and pulmonary diseases associated with inflamed, damaged airways, including those that adversely affect the subject's lungs due to lung disease and secondary action. Examples of these diseases are allergies, asthma, respiratory retardation, allergic rhinitis, pain, cystic fibrosis and cancers such as leukemia, colon cancer and the like. The present drug formulations may be administered prophylactically or therapeutically in combination with other therapies, or may be used in place of treatments with severe negative side effects.

Summary of the Invention

The present invention generally relates to initiation codons, coding regions, 5'- and 3'-terminal gene flanking regions, 5 'and 3' intron-axon junctions of genes encoding target polypeptides involved in obstructive dysfunction, and Bronchial contraction and / or when an oligo containing an antisense or antisense to a polypeptide mRNA and containing about 0 to about 15% adenosine (A) to a target selected from the group consisting of regions within 2 to 10 nucleotides of the junction is administered to the mammal Antisense oligonucleotide (s) (oligo (s)) effective to alleviate pulmonary inflammation, allergy (s), and / or surfactant deficiency and / or low secretion; Combinations of the oligos; And oligos in mixtures; And pharmacological or veterinary compositions containing a pharmacological or veterinary acceptable carrier or diluent. Targets generally include transcription factors, stimulatory and active peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, intrinsically produced specific and nonspecific enzymes, immunoglobulins and antibodies, antibody receptors, central nervous system central nervous system (CNS) and peripheral and non-nerve receptors, CNS and peripheral and non-nerve peptide neurotransmitters, adsorption molecules, defensins, growth factors, vascular peptides and receptors, binding proteins, and malignant tumor proteins There are molecules associated with the same airway disease, cancer, etc. Examples are oligo (s) targeted to adenosine receptor (s), which (s) are generally adenosine mediated effect (s) such as airway obstruction, inflammation, allergy (s) and surfactant deficiency, etc. Is present in the composition in an amount effective to reduce it. The adenosine receptor is preferably selected from the group consisting of adenosine A ₁ , A _2b and A ₃ receptors, and in some cases an A _2a receptor. Oligos of the present invention may be used to (a) reduce adenosine-mediated bronchial contraction, respiratory retardation, inflammation, allergic (s), deficiency production of surfactants and other detrimental lung actions in subjects in need of treatment, and / or ( b) in the manufacture of drugs for treating specific diseases and disorders such as asthma, cystic fibrosis, allergic rhinitis, chronic obstructive pulmonary disease (COPD), and the like. First, the present invention provides targeted administration of one or more oligonucleotides directly to the respiratory system. Oligos can be associated with any target and can be rapidly delivered through mucosal tissue of the lung to hybridize with the desired target polynucleotides, eg, mRNA, to disrupt gene transcription and translation, thereby reducing protein expression or It is intended to interfere or stop completely. Accordingly, the present invention also relates to various forms of disease by a method by direct administration to the respiratory system, for example by inhalation, by the introduction of solutions or aerosols directly into the respiratory tract and / or the lungs. Provided is a more general method of administering oligonucleotides that are antisense to target genes and mRNAs involved.

In addition, the oligos include effects mediated by various target proteins and genes, such as pulmonary, respiratory and many other related effects such as bronchial contraction, inflammation, immune mediated responses, allergy (s) and cancer. It is suitable to reduce various airway problems or adenosine mediated effects that can be caused by various diseases. Examples of diseases and conditions that can be treated defensively, prophylactically or therapeutically with the pharmaceutical formulations of the invention include pulmonary vasoconstriction, inflammation, allergies, asthma, respiratory retardation, dyspnea syndrome, pain, cystic fibrosis, Allergic rhinitis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonay disease (COPD), bronchitis and colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, liver Cancers such as leukemia, lymphoma, carcinoma, etc., including all forms of cancer that can be translated or translated into lung (s) including breast cancer and prostate cancer as well as translational cancers. In addition, the present drug formulations are also suitable for administration before, during and after other treatments, including radiation therapy, chemotherapy, phototherapy and cancer therapy, and other forms of surgical therapy. The present drug formulations are effectively administered either in combination with other therapies, or in the prophylactic and therapeutic aspects, in the absence of known therapies, or to replace therapies that have serious negative side effects. The oligo (s) may be administered by any method known to the subject, for example into the subject's lungs, more generally via any and all systemic and local routes. These oligonucleotide (s) (oligo (s)) used are antisense to the target DNA or RNA, for example adenosine receptor DNA or RNA, and preferably contain up to about 15% adenosine (A). More preferably no adenosine. The present oligos have specific compositions with a carrier or diluent, and optionally other therapeutic agents and additives used for administration by specific route, such as the respiratory system, topically, transdermally, parenterally, by injection, and the like, and It is provided in the form of agents. The oligos are also provided as capsules or medicines and in the form of kits. Oligos of the present invention can be produced by selecting specific targeted segments of a gene or mRNA encoding adenosine receptor, as mentioned below. In one preferred embodiment, the selection is necessarily by obtaining oligos containing up to about 15% adenosine (A). As mentioned above, a target is selected, including genes related to diseases that adversely affect the lung airways, gene flanking regions, RNA, and polypeptides; Obtaining a sequence of mRNA corresponding to the target gene (s) and / or their gene blanking region and / or their juxta-membrane regions, and the sequence of mRNA (s) encoding the target polypeptide; Selecting a segment of at least one mRNA (s); In order to synthesize one or more antisense oligonucleotide (s) of the selected mRNA segment (s) and, if necessary, reduce the A content present in the oligonucleotides to 15% or less, and to be free of adenosine, one or more As may be Oligos of the present invention are produced, for example, by substitution with common base (s) or other base (s). Similarly, adenosine, for example specific A ₁ , A _2b , A ₃ receptor antagonists or A _2a receptor agonists, deophylline, enpropylin and many other adenosine receptor antagonists known in the art, eg For example, agonists with significantly reduced agonist activity for less than 0.5%, less than 0.3% and so on of adenosine are substituted with other and / or common bases.

In the following, the invention will be explained more generally in terms of concept and experimentation with reference to specific examples. Other objects, advantages and features of the present invention will be apparent to those skilled in the art from the following specification.

Detailed description of the preferred embodiment

The present invention has been driven by the inventors' desire to improve the treatment of conventional lung and other diseases that require the administration of large amounts of therapeutic agents and generally have deleterious side effects. The present invention provides that antisense oligonucleotides (oligods) targeted to adenosine receptors may impair breathing, cause inflammation and / or allergies, constrict bronchial tissue, occlude pulmonary airways, lack surfactant secretion or It is from the inventors' own finding that otherwise, general breathing can be used therapeutically in the treatment of retarded diseases or conditions. In general, many diseases and disorders are associated with or cause inflammation, constrict bronchial tissue, occlude pulmonary airways, lack surfactant secretion, or otherwise delay general breathing. The therapy is selective for specific targets that are associated with or mediate these symptoms, and the drug formulations are administered up to 1,000 times lower than those previously used. In addition, the inventors have improved the protective outcomes and lifestyle of subjects who are performing or receiving other therapies including surgical treatments such as antibody therapy, chemotherapy, radiation therapy, phototherapy and cancer surgery, It was desired to provide a treatment that could be administered both therapeutically and therapeutically. The inventors believe that the present findings may be further improved by selecting oligos with reduced adenosine content or by reducing the adenosine content of the targeted antisense oligos corresponding to the intrinsic polynucleotide sequences. The present invention presupposes the inventors' discovery that oligonucleotides are metabolized in vivo to their mononucleotides. Adenosine (A) -containing oligonucleotides degrade and release adenosine, which in turn activates adenosine receptors, causing bronchial contraction, surfactant deficiency, allergy (s), and the like. Thus, the inventors have contemplated the use of low adenosine or oligos that do not contain adenosine to avoid these side effects when administering oligos. The inventors have succeeded in this effort and in this patent, new and improved compositions, formulations and bronchial contractions, allergy (s), inflammation, dyspnea, surfactant deficiency and airway obstruction, as well as direct or indirect to the lungs Provided are methods that can provide significantly improved results when compared to conventionally known therapies for preventing and alleviating other diseases that affect.

In other embodiments, one or more nucleic acids of the invention may be formulated alone and / or with one or more surfactant components and / or carriers, and / or with other therapeutic agents and / or known agents. Thus, the compositions of the present invention can be incorporated into various formulations for systemic and topical administration. In addition, the inventors have provided a broad method for the delivery of antisense oligonucleotides (oligoses) through the respiratory system as a rapid method of initiating treatment to cope with other diseases and diseases with acute and rapid seizures of asthma. to provide. In addition, the present medications have long half-lives and can be administered in very small amounts. This makes them ideal for forms of treatment once a week. In the past, antisense oligonucleotides have received considerable theoretical interest for their potential utility as pharmacological agents in the treatment of human diseases (Wagner, R., Nature 372: 333-335 (1994)). However, it has been difficult to apply these molecules to alleviate or treat human diseases. One important consideration in the pharmacological application of these molecules is that the various routes of administration for delivery of the compound to its target have failed, avoiding invasion of the circulatory system and delivery to other untargeted tissues with excessive side effects. Most in vivo experiments with antisense oligonucleotides involved the application of oligos directly to restricted regions of the brain (Wahlestedet, C., Trends in Pharmacol. Sci. 15: 42-46 (1994); Lai, J.). et al., Neuroreport 5: 1049-1052 (1994); Standifer, K., et al., Neuron 12: 805-810 (1994); Akabayashi, A., et al., Brain Res. 21: 55- 61 (1994). Others have applied antisense oligonucleotides to spinal fluid (Tseng, L., European J. Pharmacol. 258: R 1-3 (1994); Raffa, R., et al., European J. Pharmacol. 258: R5-7 (1994); see Gillardon, F., et al., European J. Neurosci. 6: 880-884 (1994). Clearly, such applications do not have practical clinical use because of their invasive nature. Thus, systemic administration of antisense oligonucleotides poses significant problems with respect to their pharmacological application and there are considerable problems in selectively targeting disease-containing tissues. In addition, systemic administration of antisense oligonucleotides poses significant problems with respect to their pharmacological application and there are considerable problems in selectively targeting disease-containing tissues.

However, the respiratory system, in particular the lung, as the ultimate entry point into the organism is an excellent route of administration of antisense oligonucleotides. It can utilize the lung not only in the treatment of lung disease, but also as a delivery method, in particular because of the non-invasive and tissue specific properties of the lung. Thus, direct local delivery of antisense oligonucleotides to a target tissue makes these compounds therapeutically available. Fomivirsen (ISIS 2303) is an example of drug delivery locally to the eye for the treatment of cytomegalovirus (CMV) retinitis (CMV), a new drug application being filed by ISIS. Inhalation is non-invasive and direct infusion into the vitreous of the eye is invasive, but the administration of the drug through the lungs provides better benefits. The compositions and formulations of the present invention provide for bronchial contraction, dyspnea, disorders and obstructed obstruction. It is very effective in preventing and treating diseases and conditions related to, allergy (s), inflammation and surfactant deficiency. Examples of diseases and disorders suitably treated by the method of the present invention include acute respiratory distress syndrome (ARDS), asthma, administration of adenosine in the treatment of ventricular tachycardia (SVT) and various arrhythmias, And hypersensitivity subjects to stress tests, anemia, kidney damage or dysfunction by some drugs, infantile dyspnea syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), Lungs, including breast and prostate cancers, as well as cancers such as leukemia, lymphoma, carcinoma, including lung transplant rejection, lung infections and colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, liver translation cancer, etc. Diseases and disorders, including all forms of cancer, which may or may not be translated. Although the present invention will refer to adenosine receptors as targets, other targets may similarly be applied with respect to lung administration of antisense oligos. Examples provided below include bronchial stenosis in hypersensitive monkeys, an animal model of human hypersensitivity to adenosine receptor agonists, as well as complete inhibition of the adenosine receptor-related symptoms in a rabbit model for human bronchial contraction, allergy (s), and inflammation. The same level of excellence with the resulting adenosine shows the elimination of the ability of the adenosine agonist. Therefore, the pharmaceutical compositions and formulations of the present invention are suitable for preventing and alleviating the symptoms associated with the stimulation of adenosine receptors, such as adenosine A ₁ receptors. Thus, the compositions and formulations of the present invention are suitable for preventing the undesired side effects of adenosine-mediated hypersensitivity in some individuals generally seen in diseases that adversely affect respiratory activity.

The method of the present invention is intended to prevent the release of adenosine by antisense degradation by minimizing, reducing or eliminating the adenosine content of antisense compounds and used to treat airway diseases and disorders in subjects of any kind and for any cause. Can be. Examples of diseases and conditions that can be treated defensively, prophylactically and therapeutically with the compositions and formulations of the present invention include pulmonary vasoconstriction, inflammation, allergies, asthma, allergic rhinitis, respiratory retardation, acute respiratory distress syndrome ( ARDS), renal impairment and dysfunction associated with the administration of anemia and some drugs, side effects associated with administration of adenosine in ventricular tachycardia (SVT) and adenosine stress tests, infantile dyspnea syndrome (infant RDS), ARDS, pain, cysts Cancers such as sex fibrosis, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infection and leukemia, lymphoma, carcinoma, for example, colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic Translation cancers such as translation cancer, lung, breast and prostate translation cancer. The compositions and formulations are suitable for administration prior to, during and after various treatments, including radiation therapy, chemotherapy, antibody therapy, phototherapy and cancer therapy, and other forms of surgical therapy. In addition, the compositions and formulations can be administered effectively in place of therapies having serious negative side effects. In general, the term “antisense” relates to small synthetic oligonucleotides, similar to single-stranded DNA, and is applied in this patent to inhibit gene expression by inhibition of target messenger RNA (mRNA) (Milligan, JF et al., J. Med. Chem. 36 (14), 1923-1937 (1993), the relevant parts of which are incorporated herein by reference.). Although the complementary sequence (s) of oligonucleotides are also included within the four edges of the present invention, all RNAs and oligonucleotides are read in a single chain in the 5 'to 3' direction when read from left to right. It is shown in. In addition, all nucleotide bases and amino acids are indicated using the rules of the IUPAC-IUB Biochemical Nomenclature Mcommission, or by known 3-letter codes (amino acids). Nucleotide sequences are shown here as single chains in the 5 'to 3' direction when read from left to right. In addition, nucleotides and amino acids are indicated here by the three letter code in the manner prescribed by the IUPAC-IUB Biochemical Nomenclature, or in accordance with 37 CFR '1.822 (for amino acids) and general practice (see PattenIn User's Guide, 99-). 102 (1990, November) (US Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, DC 20231); US Patent No. 4,871,670 to Hudson et al. At col. 3, lines 20-43). The method utilizes antisense drug formulations to inhibit or downregulate gene expression of target genes, including those described in Tables 1 and 2 below. Generally this is obtained by hybridization of an antisense oligonucleotide with a sequence (sense) encoding a targeted messenger RNA (mRNA), as is known in the art. Innately administered drug formulations of the invention reduce the amount of mRNA and protein encoded by a target gene and / or cause changes in the growth properties or morphology of the treated cells (Milligan et al. (1993) ; Helene, C. and Toulme, J. Biochim. Biophys.Acta 1049, 99-125 (1990); Cohen, JSD, Ed., Oligodeoxynucleotide as Anti-sense Inhibitor Gene Expression; CRC Press: Boca Raton, FL (1987) Reference, where relevant parts are incorporated herein by reference). As used herein, an "antisense oligonucleotide or antisense oligo" generally comprises (1) hybridization with some segments of mRNA encoding a protein targeted under appropriate hybridization conditions, and (2) It is a short sequence of synthetic nucleotides that causes a decrease in gene expression. The terms "des-adenosine" (desA) and "des-thymidine" (desT) refer to oligonucleotides that are substantially deficient in adenosine (desA) or lacking thymidine (desT). In some cases, desA or desT sequences are naturally present, but in others it is not required to act as an agonist or to reduce unwanted activity, such as to induce a stimulatory effect of the adenosine A receptor (s), nucleotides (A) It may also be generated by substituting. In this specification, substitutions are generally carried out by substituting A with "common or other bases" known in the art at the present time, or as mentioned later. As used herein, the term "prophylaxis", "preventing", "treatment" or "treating" means a protective, prophylactic, maintenance, or therapeutic treatment, wherein the subjects to whom the treatment is prescribed are adenosine. It is about reducing the likelihood of presenting symptoms associated with receptor stimulation. The term "downregulation" induces a decrease in production, secretion or availability, thereby reducing the concentration of intracellular target products such as adenosine A ₁ , A _2b , A ₃ , bradykinin 2B, GATA-3, or other receptors. Or by increasing the concentration of adenosine A _2a receptors. The technique involves the preparation of antisense oligos targeted to mRNA associated with diseases including obstructive abnormalities; And modifications of oligonucleotides to reduce the occurrence of unwanted side effects caused by the release of adenosine upon disruption while maintaining their activity and efficiency for the intended purpose of the oligonucleotides. In this manner, selectively binds to the corresponding mRAN; And, if necessary, have no or substantially reduced activity in activating or antagonizing adenosine A ₁ , A _2b or A ₃ receptors; Or by constructing one or more antisense oligonucleotide (s) (oligoses) with reduced adenosine content through substitution with other or common bases or adenosine analogs that may act as agonists to the adenosine A _2a receptor, thereby providing a specific gene. Target. Any number of adenosine that binds to thymidine but is present by other and / or common bases such as heteroaromatic bases having less than 0.3 of adenosine base agonists or antagonist activity at the adenosine A ₁ , A _2b and A ₃ receptors. Can be substituted. Based on prior experience in the art, in addition to the "downregulation" of specific genes, it also activates adenosine receptors in the lungs and the like, with or without thymidine content, or with RNA fragments that are low or low in thymidine, and express the inhibited action of adenosine. Increasing the effectiveness of the administered drug formulation by selecting RNA segments that have substituted one or more adenosine (s) present in other nucleotide bases, the so-called oligonucleotide (s) made of so-called common bases, which lack the ability to The inventors have found that it can be made. Since adenosine (A) is a nucleotide base complementary to thymidine (T), if T was present in the RNA, the antisense oligo will have A at the same position.

According to one aspect of the invention, the oligonucleotide is a portion of an mRNA molecule encoding a protein associated with respiratory retardation, allergy (s), pulmonary inflammation, lack of pulmonary surfactant or lowering of pulmonary surfactant, airway obstruction, bronchitis, or the like Have a sequence that specifically binds to a segment. One effect of this binding is to reduce or disrupt translation of the corresponding mRNA, thus reducing the amount of target protein available to the subject's lungs. According to a preferred embodiment of the present invention, the phosphodiester residues of the antisense oligonucleotides are modified or substituted. Chemical analogs of oligonucleotides having modified or substituted phosphodiester moieties that increase the stability of the oligonucleotides in vivo, such as methylphosphonates, phosphorus esters, phosphorothioates, phosphorodithioates or Phosphoramidate, α-methoxyethyl and similar modifications are preferred. Naturally occurring phosphodiester bonds of oligonucleotides can be disrupted to some extent by intracellular nucleases. In contrast, many of the residues proposed herein have high resistance to nuclease disruption (see Milligan et al .; Cohen, JSD, supra.). According to another embodiment of the invention, by replacing phosphodiester bonds with nuclease-resistant bonds at the 3 'end of the oligonucleotide, that is, the "3'-end cap" It can be prevented by adding (Tidd, D, M. and Warenius, HM, Be. J. Cancer 60: 343-350 (1989); Shaw, JP Nucleic Acids Res. 19: 747-750 (1991) See, the relevant parts are incorporated herein by reference.). Phosphoramidate, phosphorothioate and methylphosphonate linkages all fully function in this manner for the purposes of the present invention, such as α 'modifications such as α'methoxyethyl. As the phosphodiester backbone is further extended and modified, the modified drug formulation is more stabilized and, in many cases, more RNA affinity and cell permeability are elevated (see Milligan, et al., Supra). In addition, it is known that polysubstitution of carbohydrate rings can improve the stability of nucleic acids. Thus, the number of residues that may be modified or substituted may vary depending upon the purpose of administration, target and route, and may be any number from one to all residues and in between. Many different methods are known for converting the entire phosphodiester skeleton into novel bonds (see Millikan et al. Supra). Preferred skeletal analog residues are phosphoramidate, phosphorothioate, methylphosphonate, phosphorotriester, phosphorusster, 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) and methoxymethyl (MOM), methoxyethyl (MOE), and methyleneoxy (Methyleneimino) (MOMI) residues, and their bonds. Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred because of their availability through automated oligonucleotide synthesis (Millikan et al., Supra). Suitably, the pharmaceutical formulations of the present invention may be administered in the form of pharmaceutically acceptable salts or in the form of a mixture of antisense oligonucleotides and salts thereof. According to another embodiment of the invention, a mixture of other antisense oligonucleotides or their pharmaceutically acceptable salts is administered. Single drug formulations of the present invention have the ability to reduce the expression of target mRNAs, and / or several other drug formulations have the ability to either increase or decrease the activity of one pathway. In accordance with an embodiment, the method comprises, in each target mRNA or gene, all possible deoxyribonucleotide fragments having a low thymidine (T) content, or up to about 7 or more mononucleotides, preferably up to about 60 mononucleotides, more preferably Achievement may be achieved by detecting deoxynucleotide segments having a low adenosine (A) content of about 10 to about 36 mononucleotides, most preferably about 12 to about 21 mononucleotides. This can be achieved by searching for mononucleotides in the target sequence that are low or lacking thymidine (RNA), low adenosine (gene), and have a nucleotide length of at least 7 and the nucleotides are complementary to adenosine. In most cases, this search typically finds antisense oligonucleotides of varying lengths for a typical target mRNA of about 10 to 30 such sequences that are naturally lacking or having less than 40% adenosine and an average length of about 1800 nucleotides. . Oligonucleotides having a high content of T or A can each be immobilized by substituting one or more common bases for A. The drug formulation (s) of the present invention is not particularly limited because it may be of various lengths depending on the specific target and delivery method, but is about 7 to 60 nucleotides in length, preferably about 12 to 45 nucleotides, more preferably 30 Suitable lengths up to nucleotides, most preferably up to 21. The drug formulation (s) of the present invention may be directed to any and all segments of the target RNA. Preferred groups of drug formulation (s) include those relating to mRNA regions containing junctions of introns and axons. If the drug formulation relates to an intron / axon junction, for example to the 3 'or 5' end of an antisense oligonucleotide that can be located within about 2 to 10, preferably about 3 to 5 nucleotides of the intron / axon junction To prevent splicing-out of interstitial axons during the process of turning progenitors into mature mRNAs, antisense oligonucleotides must be completely overlie or be close enough to the junction. Can be. Also preferred are antisense oligonucleotides that overlap the start codon or near the 5 'and 3' ends of the coding region. In addition, the flanking regions of the axon and spliced segments of the precursor mRNA can also be targeted. MRNA sequences of adenosine receptors and many other targets can be inferred from DNA sequences of genes that express receptors, such as adenosine receptors, enzymes, factors, or other targets associated with airway disease. For example, the genomic sequence of the human gene A ₁ adenosine receptor is known and disclosed in US Pat. No. 5,320,963 (Stiles, G., et al.). A ₃ adenosine receptors are described in humans (Jacobson, MA, et al., UK Patent Application No. 9304582.1 (1993) in rats (Zhou, F., et al., PNAS (USA) 89: 7432 (1992))). ), Cloned, identified and expressed. The sequences of adenosine A _2b receptor genes are also known (see Salvatore, CA, Luneau, CJ, Johnson, RG and Jacobson, M. Genomics (1995), hereby incorporated by reference in the relevant sections). The sequences of most examples of the remaining target genes are also known (GeneBank, NIH, see). Sequences of genes for which sequences are not yet available can be obtained by separating target segments by applying techniques known in the art. If the sequence, RNA and / or protein of a gene is known, antisense oligonucleotides can be produced according to the invention described above, in order to reduce the production of the target protein according to standard techniques. Sequences for adenosine A _2a , bradykinin and other genes, and methods of preparing oligonucleotides, are also known as sequences and methods of preparing many other target genes and mRNAs suitable for the present invention. Thus, antisense oligonucleotides that downregulate the production of target sequences related to airway disease, including adenosine A ₁ , A _2a , A _2b , A ₃ , bradykinin, GATA-3, COX-2 and many other receptors, are standard techniques. It can be prepared according to. Examples of diseases and conditions that can be treated by this method include adenosine administration, anemia, and some drugs, including acute respiratory distress syndrome (ARDS), asthma, ventricular tachycardia (SVT) and stress testing of hypersensitive individuals. Kidney damage or dysfunction, infantile respiratory distress syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infections and colon cancer, breast cancer, lung cancer, pancreatic cancer Diseases such as cancers, such as leukemia, lymphoma, carcinoma, etc., including hepatocellular carcinoma, kidney cancer, melanoma, hepatic translational cancer, etc., as well as all forms of cancer that can be translated or translated into the lung, including breast and prostate cancer. have.

The adenosine receptors mentioned above are merely to show that the technique of the present invention has excellent ability. Indeed, a large number of genes can be targeted in the same manner as the drug formulation (s) of the invention to reduce or downregulate protein expression. In accordance with an embodiment, the target disease or condition may be respiratory retardation or reduction, bronchial contraction, chronic bronchitis, pulmonary bronchial contraction and / or hypertension, chronic obstructive pulmonary disease (COPD), pulmonary transplant rejection, allergy, asthma, cystic fibrosis, If it relates to respiratory distress syndrome, cancers adversely affecting the lungs directly or by translation, the method can be applied to a list of possible target mRNAs, including the targets set forth in Tables 1 and 2 below. The antisense drug formulation (s) of the present invention has a low A content to prevent release upon in vivo degradation of the drug formulation (s). For example, if the system is a lung or respiratory system, a large number of genes are involved in several functions, including those listed in Table 1 below.

Table 1: Asthma and Inflammation Targets Associated with Lung Disease or Disease

Nf6B transcription factor interleukin-5 receptor (IL-5R) interleukin-3 receptor (IL-3R) interleukin-1β receptor (IL-1βR) tryptaseβ ₂ -adrenergic receptor kinase endotheline receptor B Bradykinin B ₂ Receptor (B2B) Interleukin-1 (IL-1) Interleukin-9 (IL-9) Interleukin-11 (IL-11) Induced Nitric Oxide Synthase Adsorption Molecule 1 (ICAM- 1; intracellular Adhesion Molecule 1) Substance P Rantes ETA receptor cyclooxygenase-2 (COX-2) monocyte activating factor neutrophil elastase muscarinic acetylcholine receptor tumor necrosis factor α (Tumor necrosis factor) phosphodiesterase IV substance P receptor chymase interleukin-2 (IL-2) interleukin-12 (IL-12) interleukin-6 (IL-6) interleukin-8 (IL- Interleukin-7 receptor (IL-7R) Interleukin-14 receptor (IL-14R) CCR-2 CC chemokine receptor CCR-4 CC chemokine receptor Interleukin-8 Receptor (IL-8R) Interleukin-4 Receptor (IL-4R) Interleukin-1β (IL-1β) Eotaxin Major Basic Protein Endocrine Receptor A Preproendocyrin ( preproendothelin) IgE (high affinity receptor) interleukin-1 receptor (IL-1R) interleukin-9 receptor (IL-9R) interleukin-11 receptor (IL-11R) cyclooxygenase (COX; Cyclooxygenase) vascular cell adsorption molecule (VCAM endothelial Leukocyte Adhesion Molecule (ELAM-1), GM-CSF, Endothelin-1 neutrophil chemitactic factor defensin 1, 2, vascular celluar adhersion molecule Platelet Activating factor 5-lipoxygenase substance P histamine receptor CCR-1 CC chemokine receptor interleukin-4 (IL-4) interleukin-5 (IL-5) interleukin-7 (IL-7) interleukin -12 Receptor (IL-12) Interleukin-1 (IL-1) Interleukin-14 (IL-14) CCR-3 CC Chemokine Receptor CCR-5 CC Chemokine Receptor

Table 1: Lung Disease or Pain (Asthma / Inflammation) Targets (Continued)

Prostanoid Receptor Neutrophil Adsorption Receptor Interleukin-15 (IL-15) Interleukin-11 (IL-11) NFAT Transcription Factor MIP-1α MCP-3 Cyclophilin (A, B, etc.) Basic Fibroblast Growth Factor CSBP / p38 MAP Kinase PDG2 Interleukin-10 (IL-10) FK506-binding protein fibronectin cMad CAM-1PECAM-1C3biE-selectin CD-34p150,95 fucosyl transferase STAT-1CD-18 / CD11aICAM2 and ICAM3CCR3 (eotaxin receptor) LTB-4 Protein Kinase C tachykinnen Receptor (tach R) Interleukin-2 Receptor (IL-2R) STAT 6NF-Interleukin-6 (NF-IL-6) Interleukin-3 (IL-3) Interleukin-13 (IL-13) Interleukin-14 (IL-14) Interleukin-16 (IL-16) Medurasin (Medullasin) GATA-3 Transcription Factor MAP Kinase Interleukin-15 Receptor (IL-15R) Interleukin-11 Receptor (IL-11R) STAT 4MCP-2MCP-4 Phospholipase A2 Metalloproteinase Trypsin Receptor Interleukin-3 (IL-3) Sporin A-binding protein α4β1 selectin α4β7 selectin LFA-1 (CD11a / CD18) LFA-1 selectin PSGL-1P-selectin L-selectin Mac-1 (CD11b / CD18) VLA-4STAT-2CD11b / CD18C5aCCR1, CCR2, CCR4, CCR5AP-1 Transcription Factors Cysteinyl Leucotriene Peptide I6B Kinases 1 and 2 (eg, Substance P, NK-1 & NK-3 Receptors) c-mas Interleukin-10 Receptor (IL- 10R) Interleukin-2 Receptor (IL-2R) Interleukin-12 Receptor (IL-12R) Interleukin-6 Receptor (IL-6R) Interleukin-13 Receptor (IL-13R) Interleukin-16 Receptor (IL-16R)

Table 1: Lung Disease or Pain (Asthma / Inflammation) Targets (Continued)

Adenosine A ₁ receptor (A ₁ R) Adenosine A _2b receptor (A _2b R) β tryptase adenosine A _2a receptor (A _2a R) Fc-epsilon receptor CD23 antigenIgE receptor Fc epsilon receptor (IgERFcξR) histidine decarboxylase Azeprostaglandin D Synthase Eosinophil Induction Neurotoxin Endothelial Nitric Oxide Synthase Neutrophil Oxidase Factor Macrophage Inflammatory Protein-1-alpha / Rantetsu Receptor Tryptase-I adenosine A ₃ receptor (A ₃ R) STAT-3IgE receptor β subunit (IgE Rβ) IgE receptor α subunit (IgE Rα) substance P receptor tryptase-1 eosinophil cationic protein eosinophil peroxidase Endothelial monocyte activator cathepsin G interleukin-8 receptor α subunit (IL-8 R α) substance P endocyrin receptor ET-B

These genes and others include cystic fibrosis, asthma, pulmonary hypertension and vasoconstriction, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infections, chronic bronchitis, dyspnea syndrome (ARDS), allergic rhinitis, lung cancer It is included in normal respiratory function as well as diseases associated with respiratory abnormality (s), including other translational diseases including lung translational cancer and diseases with inflammatory responses.

Antisense oligos on target receptors such as adenosine A ₁ , A _2a , A _2b and A ₃ receptors, CCR3 (chemokine receptors), bradykinin 2B, CAM (vascular cell adsorption molecule) and eosinophil receptors are responsible for the expression of these genes. It has been found to be effective in down-regulation. Some of these are for example "downregulated" of adenosine A ₁ , A _2a , A _2b and A ₃ receptors, CCR3 (chemokine receptors), bradykinin 2B, CAM (vascular cell adsorption molecule) and eosinophil receptors. Thereby alleviating or reducing the symptoms of respiratory diseases and / or inflammation. These drug agents can be used either alone or in combination with antisense oligos targeting other genes to validate the pathways and / or networks included therein. For better results, oligos are preferably administered directly to the laboratory animal's respiratory system, for example by inhalation or other means, so that the lungs are reached without extensive systemic dissemination. This allows the use of smaller drug dosages as compared to those administered systemically or by other generalized routes, thereby reducing the number and extent of unwanted side effects resulting from the widespread dispersal of the drug product into the body. Let's do it. It has been found that the drug agent (s) of the present invention reduce the amount of receptor protein expressed in tissues. Thus, these drug agents reduce the number of target proteins that other drugs can interact with, rather than simply interact with their targets such as receptors. In this way, the drug formulation (s) have low toxicity and provide very good efficacy. Antisense oligonucleotides for A ₁ , A _2a , A _2b and A ₃ receptors, bridicinin B2, GATA-3, CAM (vascular cell adsorption molecule), eosinophil receptors and COX-2 receptors, etc. It has been shown to be effective for downregulation of receptor proteins. Compared with the conventional treatment of adenosine-mediated bronchial contraction, a novel feature of the treatment is that it is administered directly to the lung, or that the administration can remain intact without affecting other tissues, organs or intragranular system. In addition, the receptor protein itself reduces dose and reduces toxicity, rather than merely interacting with drugs. Other proteins that may be targeted as antisense drugs for the treatment of lung disease include, but are not limited to, the CCR3 (chemokine) receptor, human A _2a adenosine receptor, human A _2b adenosine receptor, human IgE receptor β, human Fc-epsilon receptor CD23 antigen, human histidine decarboxylase, human beta tryptase, human tryptase-I, human prostaglandin D synthase, human cyclooxygenase-2, human eosinophil cationic protein, human eosinophil-derived neurotoxin, Human eosinophil peroxidase, human intracellular adsorption molecule-1 (ICAM-1), human vascular cell adsorption molecule-1 (VCAM-1), human endothelial leukocyte adsorption molecule-1 (ELAM-1), human P selectin, human endothelial Monocyte activating factor, human IL-3, human IL-4, human IL-5, human IL-6, human IL-8, human monocyte-induced neutrophil chemotactic factor, human neutrophil elastase, human neutrophil oxidase phosphorus Now, human capidin G, human defensin 1, human defensin 3, human macrophage inflammatory protein-1-alpha, human muscarinic acetylcholine receptor HM3, human fibronectin, human GM-CSF, human tumor necrosis factor α, Human leukotriene C4 synthase, human major basic protein and human endoterin 1 and the like. Although not intended to be excluded, a more extensive list of genes is provided below. Some of these are, for example, "downregulated" by A ₁ , A _2a , A _2b and A ₃ receptors, CCR3 (chemokine receptors), bradykinin 2B, VCAM (vascular cell adsorption molecule) and eosinophil receptors. Acts to alleviate or reduce the symptoms of respiratory diseases and / or inflammation. These drugs are preferably administered directly to the respiratory system, for example by inhalation or other means, so that the lungs are reached without extensive systemic dissemination. This allows the use of substantially lower drug dosages, as compared to being introduced systemically or by other generalized routes by the prior art, resulting in undesired consequences of the widespread dispersal of the drug product into the body. Reduce the number and extent of side effects. It has been found that the drug agent (s) of the present invention reduce the amount of receptor protein expressed in tissues. Thus, these drug agents reduce the number of target proteins that other drugs can interact with, rather than simply interact with their targets such as receptors. In this way, the drug formulation (s) have low toxicity and provide very good efficacy. In these latter targets and general target genes, it is particularly necessary to eliminate or reduce the adenosine content of the corresponding antisense oligonucleotides such that adenosine is not released upon disruption.

As used herein, the terms "treatment", "treating" asthma means a treatment that reduces the likelihood of developing symptoms of lung disease in a subject to whom such treatment is applied. The term "downregulation" means a decrease (ie, a decrease in concentration) of the production, secretion or availability of a protein in a target cell. The present invention is primarily concerned with the treatment of human subjects. In addition, the drug formulations and methods disclosed herein may also be used for veterinary purposes, such as the treatment of other mammals, such as pets such as cattle, horses, wildlife, zoo animals, dogs and cats. The target protein is preferably a mammal and more preferably if it is of the same species as that of the subject being prescribed. In general, "antisense" refers to the use of small synthetic oligonucleotides resembling single-stranded DNA to inhibit gene expression by inhibiting the function of target messenger RNA (mRNA) (Milligan, JF et al., J Med. Chem. 36 (14), 1923-1937 (1993). In the present invention, inhibition of gene expression of A ₁ or A ₃ adenosine receptors is preferred. Gene expression is inhibited through hybridization with coding (sense) sequences in specific messenger RNA (mRNA) by hydrogen bonding according to the Watson-Crick base pairing rules. The mechanism of antisense inhibition is that the inherently applied oligonucleotides reduce the amount of mRNA and protein of the target gene or induce changes in the growth characteristics or morphology of the cells (see Milligan et al. (1993); Helene, supra). C. and Toulme, J. Biochim. Biophys.Acta 1049, 99-125 (1990); Cohen, JSD, Ed., Oligodeoxynucleotide as Anti-sense Inhibitors Gene Expression; CRC Press: Boca Raton, FL (1987)). As used herein, "antisense oligonucleotide" (1) hybridizes with any coding sequence in mRNA encoding a target protein according to the hybrid forming conditions mentioned below, and (2) by hybridization, A ₁ or It is defined as the short sequence of synthetic nucleotides in which the gene expression of the A ₃ adenosine receptor is reduced. The aforementioned receptors are merely examples showing that the present technology is very good. Indeed, in order to effectively downregulate or eliminate protein expression, and to observe any changes that affect one or more functions in systems such as the respiratory system and other lung disease related targets, a large number of genes actually apply the method. Can be targeted in a similar manner to that. In embodiments, targets in the respiratory system include dyspnea, bronchial contraction, inflammation, allergic rhinitis, chronic bronchitis, surfactant deficiency; And chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infection, inhalation burn, acute respiratory distress syndrome (ARDS), cystic fibrosis, pulmonary fibrosis, radiation pulmonitis, tonsillitis, emphysema, It may be associated with cancers that directly affect the respiratory system, such as dental pain, oral inflammation, joint pain, esophagitis, lung cancer, esophageal cancer, or indirectly affect the respiratory system by translation. Asthma, allergies, allergic rhinitis, pulmonary bronchoconstriction and hypertension, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infections, allergies, asthmatic cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), and infants Due to its relevance to respiratory dysfunction, such as in sexual and pregnancy-related RDS, cancers that adversely affect the lungs, either directly or by translation, this function has been of great interest, and the present antisense oligonucleotides are listed in Table 1, above. It may be directed to a list of target mRNAs including the target.

First, selecting fragments of a target nucleic acid consisting of G and C and / or having at least four or more contiguous nucleic acids selected from the group having a specific form and / or having high activity; It is then composed of selected fragments, most preferably having a thymidine nucleic acid content of about 15% or less, preferably about 12%, about 10%, about 7%, about 5%, about 3%, about 1% or less Oligos of the present invention can be obtained by obtaining the first oligonucleotide of length 4 to 60 nucleotides free of thymidine. The next step consists of a sequence that is antisense to the selected fragment and has an adenosine base content of about 15% or less, preferably about 12%, about 10%, about 7%, about 5%, about 3%, about 1% Or less preferably, by obtaining a second oligonucleotide of 4 to 60 nucleotides in length, most preferably free of adenosine. If the selected fragment contains at least one thymidine base, the adenosine base binds to the thymidine base, but less than about 10%, preferably less than 1%, to the adenosine A ₁ , A _2a , A _2b and A ₃ receptors. More preferably, heteroaromatic bases having less than 0.3% adenosine base agonist activity, and, when present in the respiratory system, a general base selected from the group consisting of heteroaromatic bases having no activity on the adenosine A _2a receptor May be substituted in the antisense nucleotide fragment. Different adenosine activities in other systems can be appropriately determined in other systems. Heteroaromatic base analogues are O, halo, NH ₂ , SH, SO, SO ₂ , SO ₃ , COOH and branched and conjugated primary and secondary amino, alkyl, alkenyl, alkynyl, cycloacyl, heterocycloalkyl , Aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsuloxyl, halocycloalkyl, alkylcycloalkyl, alkenyl Cycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylallyl, alkynylaryl, arylacyl, arylalkenyl, arylacynyl, arylcycloacyl and can be substituted with O, halo, NH ₂ , 1 It can be selected from all pyrimidines and purines, which may be further substituted with primary, secondary and tertiary amines, SH, SO, SO ₂ , SO ₃ , cycloalkyl, heterocycloalkyl and heteroaryl. Pyrimidines and purines may be substituted at any position known in the art, but are preferably substituted at positions 1,2,3,4,7, and / or 8. Furthermore, pyrimidines and purines, such as deophylline, caffeine, dyphylline, etophylline, acephylline piperazine, bacifylline, enpropyline and xanthine, having the formula (1) Are preferred.

Wherein R ¹ and R ² are independently H, alkyl, alkenyl or alkynyl, and R ³ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cyclo Alkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH ₂ -alkylamino-ketoxyalkyloxy-aryl, mono and dialkylaminoalkyl-N-alkylamino-SO ₂ aryl, and the like. .

Similar variations in the sugar component are also embodiments of the present invention. The adenosine content of the reduced antisense oligos corresponding to thymidine (T) present in the target RNA prevents oligos from decaying into free adenosine in the system and tissue environment of the lung, brain, heart, kidney, etc. To prevent unwanted effects. According to an embodiment, the NF6B transcription factor may be selected as a target and its mRNA or DNA low in thymidine (T) or destimidine (desT) may be found. As a complementary strand, only desT segments of mRNA or DNA capable of producing desA antisense are selected. When numerous desT segments are found, the sequences of antisense segments can be estimated. In general, about 10-30 and more desA antisense sequences can be obtained. These antisense sequences are some or all of the desA antisense oligonucleotide sequences corresponding to the desT segments of the target mRNA, such as Table 1, Table 2 below and others related to the function of the brain, cardiovascular and kidney and many others. Can include them. When such oligonucleotides are found, it is said that the antisense oligonucleotides found are 'contain no 100% adenosine'. For each of the original desA antisense oligonucleotide sequences corresponding to a target gene, such as an NF6B transcription factor, generally about 10-30 sequences can be found in the target gene or RNA having a low thymine content (RNA). In addition, according to the present invention, selected fragment sequences may contain a small number of thymidine (RNA) nucleotides in secondary or tertiary or quaternary sequences. In some cases, a high amount of adenosine content is sufficient to make antisense oligonucleotides that are less active or even inactivated to the target. According to the present invention, it is preferred that the so-called "completely desA" sequence has an adenosine content of less than about 15%, about 12%, about 10%, about 7%, about 5%, about 2%. Most preferred is no adenosine content (0%). However, in some cases a high adenosine content is also acceptable, and oligonucleotides still do not exhibit detrimental "adenosine activity". A particular important embodiment is the replacement of adenosine nucleotides with "common or other" bases, which are base pairs with similar or equivalent affinity to two or more of the four nucleotides (A, G, C and T) that are immobilized or present in native DNA. .

In this patent, general or other bases have the practical ability to hybridize with thymidine, while at the same time binding to adenosine receptors or other molecules to exhibit unwanted side effects of adenosine in laboratory animals or cell lines. This is defined as any compound that is diminished or substantially lacking, generally adenosine analogs. In addition, it cannot be fully activated, or the ability to activate adenosine receptors such as adenosine A ₁ , A _2b and / or A ₃ receptors, most preferably A ₁ receptors, is effectively reduced, and even adenosine A _2a Adenosine analogs that can act as agonists of the receptor can be used. An example of a common base is α-deoxyribofurasol- (5-nitroindole), which selects others. Methods are also known in the art. This "fixing" step differs from the antisense found in nature and results in newer sequences that the antisense oligonucleotides can bind, preferably identically, with the target RNA. Other examples of common bases or other bases are 2-dioxyribosyl- (5-nitroindole). Other examples of common bases include 3-nitropyrrole-2'-dioxynucleoside, 5-nitro-indole, 2-dioxyribosyl- (5-nitroindole), 2-dioxyribofuranolsil- (5-nitroindole ), 2'-deoxyinosine, 2'-deoxynebularine, 6H, 8H-3,4-dihydropyrimido [4,5c] oxazine-7-one and 2-amino -6-methoxyaminopurine. In addition to these, common bases with slightly reduced hybridization capacity but capable of substituting other bases are 3-nitropyrrole 2'-deoxynucleoside, 2-dioxyribofuranosyl- (5-nitroindole) , 2'-deoxyinosine and 2'deoxynebulin, and the like (Glen Research, Sterling, VA). More specific repairs include 6H, 8H-3,4-dihydropyrimido [4,5-], the base of which is a "P" nucleotide that base pairs with guanine (G) or adenine (A). c] [1,2] oxazine-7-one, and 2-amino-methoxyaminopurine, which is a “K” nucleotide that base binds to cytidine or thymidine. Also suitable are others that are known in the art or may be used. See, eg, Loakers, D. and Brown, DM, Nucl. Acids Res. 22: 4039-4043 (1994); Ohtsuka, E. et al., J. Biol. Chem. 260 (5): 2605-2608 (1985); Lin, PKT and Brown, DM, Nucleic Acids Res. 20 (19): 5149-5152 (1992); Nichols, R. et al., Nature 369 (6480): 492-493 (1994); Rahman, MS and Humayun, NZ, Mutation Research 377 (2): 263-8 (1997); Amosova, O., et al., Nucleic Acids Res. 25 (10): 1930-1934 (1997); Loakes D. & Brown, DM, Nucleic Acids Res. 22 (20): 4039-4043 (1994) all parts related to the general bases and their preparation and use of nucleic acid binding are incorporated herein by reference. Once completely desT sequences are found in naturally occurring targets, they can be selected by performing about 1 to 3 general base substitutions, generally to obtain 100% “desA” antisense oligonucleotides. Thus, the method replaces antisense oligonucleotides, or one or more adenosine nucleotides, such as about 1 to 3, or more, with various "lower" or "substituted" bases for several droplets with or without A content. Antisense oligonucleotides that can be "fixed" by General bases are known in the art and need not be listed below. Those skilled in the art will know which bases can serve as general bases and can substitute for A. Table 2 below provides a selected number of targets to which the drug formulation of the present invention is effectively applied. But others can also be targeted.

Table 2: Cancer Targets

Cancer target Transgenic Oncogene (transforming Oncogene) Therapeutic target rassrcmycbcl-2 angiogenesis factor tumor genes DNA repair gene Thymidylate syntase thymidylate syntase dihydrodrofolate reductase thymidine kinase dioxycytidine kinase ribonucleotide reductase adsorption molecules folate pathway enzymes Telomerase HMG CoA Reductase Farnesyl Transferase Glucose-6-Phosphate Transferase

Preferred groups of targets for treating cancer are genes related to various types of cancers or genes commonly known as melanoma, or genes that regulate or are involved in the production of RNA and / or proteins. For example, there are transgenic oncogenes including but not limited to ras, src, myc and BCL-2. Other targets, although not absolute, primarily relate to chemotherapeutic agents of current cancers, such as several enzymes, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxytidine kinase, ribonucleotide reductase, and the like. Things. Conventional cancer therapies contain the unsolved problem of selectively killing only cancer cells while preserving normal cells from the destructive effects of treatments such as combination therapy, radiation therapy, etc., and thus the present technology is particularly useful for the treatment of cancer diseases. The present technology provides the ability to selectively reduce or enhance the required pathway or target. This approach offers significant advantages over standard therapies for cancer because it allows selection of pathways including primary, secondary and possibly tertiary targets that are generally not expressed simultaneously in normal cells. Thus, the present drug preparations are selectively toxic in tumor cells expressing all three targets, for example, while being significantly less affected or tolerated by normal cells capable of expressing only one or both of the targets. It may be administered to a subject to increase the. Preferred groups of targets for treating cancer are genes related to various types of cancers or genes commonly known as melanoma, or genes that regulate or are involved in the production of RNA and / or proteins. For example, there are transgenic oncogenes including but not limited to ras, src, myc and BCL-2. Other targets, although not absolute, primarily relate to chemotherapeutic agents of current cancers, such as several enzymes, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxytidine kinase, ribonucleotide reductase, and the like. Things.

In one embodiment, at least one of the mRNAs to which the oligos of the invention targets are transcription factors, stimulating and activating factors, intracellular and extracellular receptors and common peptide transporters, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenous Specific and nonspecific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral and non-nerve receptors, CNS and peripheral and non-nerve peptide transporters, adsorbent molecules, defensins, growth factors, vascular functions Proteins such as peptides and receptors, binding proteins and the like, or correspond to oncogenes and various genes associated with various diseases and disorders. Examples of target proteins include, in particular, eotaxin, major basic protein, preproendocyrin, eosinophil cationic protein, P-selectin, STAT 4, MIP-1α, MCP-2, MCP-3, STAT 6, c-mas, NF-IL-6, cyclophilin, PDG2, cyclosporin A-binding protein, FK5-binding protein, fibronectin, LFA-1 (CD11a / CD18), PECAM-1, C3bi, PSGL-1, CD-34 , Substance P, p150, 95, Mac-1 (CD11b / 18), VLA-4, CD-18 / CD11a, CD11b / CD18, C5a, CCR1, CCR2, CCR4, CCR5, and LTB-4. However, others may also be suitable examples. In another embodiment, at least one of the mRNAs to which the oligo targets is sympathetic receptor, parasympathetic receptor, GABA receptor, adenosine receptor, bradykinin receptor, insulin receptor, glucagon receptor, prostaglandin receptor, thyroid receptor, androgen receptor Intracellular and extracellular receptors and peptide transporters such as anabolic receptors, estrogen receptors, progesterone receptors, coagulation related receptors, pituitary receptors, pituitary peptide transporters, and histamine receptors (HisR) . But others can also be considered. The sympathetic and parasympathetic receptors encoded are acetylcholinesterase receptor (AcChaseR), acetylcholine receptor (AcChR), atropine receptor, muscaric receptor, epinephrine receptor (EpiR), dopamine receptor (DOPAR) and norepinephrine Receptor (NEpiR) and the like. Other examples of receptors encoded are adenosine A ₁ receptors, adenosine A _2b receptors, adenosine A ₃ receptors, endocrine receptors A, endocrine receptors B, IgE high affinity receptors, muscaric acetylcholine receptors, substance P receptors, Histamine receptor, CCR-1 CC chemokine receptor, CCR-2 CC chemokine receptor, CCR-3 CC chemokine receptor (eotaxin receptor), interleukin-1β receptor (IL-1βR), interleukin-1 receptor (IL-1R), interleukin -1β receptor (IL-1βR), interleukin-3 receptor (IL-3R), CCR-4 CC chemokine receptor, cysteininyl leukotriene receptor, prosteoid receptor, 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, sympathetic stimulatory receptor, parasympathetic stimulatory receptor, 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 clotting cascades, pituitary receptors, pituitary gland Peptide transporters, and histamine receptors (HisR). Others not listed here can also be considered. Enzymes encoded for the development of oligos of the present invention include synthetase, kinases, oxidases, phosphatase, reductases, polysaccharides, triglycerides, lipids and protein hydrolases, esterases, erratases, and Polysaccharides, triglycerides, lipid and protein synthase and the like. Examples of target enzymes include tryptase, inducible nitric oxide synthase, cyclooxygenase (Cox), MAP kinase, eosinophil peroxidase, β2-adrenergic receptor kinase, leukotrien c-4 synthase, 5- Lipooxygenase, phosphodiesterase IV, metalloproteinase, tryptase, CSBP / p35 MAP kinase, neutrophil elastase, phospholipase A ₂ , cyclooxygenase 2 (Cox-2), pew Cosyl transferase, chimase, frontin kinase C, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, and ribonucleide reductase. However, any enzyme related to the disease or condition is also suitable as a target of the present invention. Coding factors suitable for applying the present invention are Nf6B transcription factor, granulocyte macrophage colony stimulating factor (GM-CSF), AP-1 transcription factor, GATA-3 transcription factor, monocyte activator, neutrophil chemotactic factor, granulocyte / large Phagocytic colony-stimulating-factor (G-CSF), NFAT transcription factor, platelet activating factor, tumor necrosis factor α (TNFα), and basic fibroblast growth factor (BFGF). Although not mentioned in detail, the present invention also includes other factors. Adsorbent molecules suitable for use in the present invention include intracellular adsorption molecules 1 (ICAM-1), 2 (ICAM-2), and 3 (ICAM-3), vascular cell adsorption molecules (VCAM), endothelial leukocyte adsorption molecules- 1 (ELAM-1), neutrophil adsorption receptor, luminescent CAM-1, and the like. In addition, other known and unknown factors (at this point) may be targeted here. Among cytokines, lymphokines and chemokines, interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 ( IL-5), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-11 (IL-11), CCR-5 CC chemokines, and Rantes. However, others that are known to be included in the specific disease or disorders to be treated or whose general activity is known, such as inflammation, may also be targeted. Examples of defensins for practical application of the invention include defensin 1, defensins 2, and defensins 3, and the selectins include α4β1 selectin, α4β7 selectin, LFA-1 selectin, E-selectin, P-selectin, L- There is a selectin. Although not an absolute list, examples of oncogenes are ras, src, myc, and bcBCL. However, others are also suitable for use in the present invention.

Drug agents administered in accordance with the present invention are preferably designed with antisense of target genes and / or mRNAs associated with the origin of the species being administered. To treat humans, drug agents are preferably designed with antisense of human genes or RNA. Drug formulations of the invention include oligonucleotides that are antisense of naturally occurring DNA and / or RNA sequences; Their fragments that are up to one (1) base long than the target sequence, preferably seven nucleotides in length; At least about 0.02%, preferably at least about 0.1%, more preferably at least about 1%, most preferably at least about 4% adenosine nucleotides, at most about 30%, preferably at most about 15%, more preferably Oligos with less than about 0%, most preferably less than about 5% adenosine nucleotides, or are completely deficient in adenosine; And oligos in which one or more of the adenosine nucleotides have been substituted with a so-called general base, which is capable of base binding with thymidine nucleotides, but which cannot substantially induce adenosine receptor activity. Examples of human sequences and fragments of the antisense oligonucleotides of the invention are preferably fragments of the following fragments and shorter fragments, and for the entire gene or for each of the sequences preferably 7, 10, 15, 18 to 21, 24, There are, but are not limited to, mRNAs encoding sequences of introns and intron-axon junctions, including up to 27, 30, n-1, where n is the total number of nucleotides of the sequences. Such fragments may be selected starting at any portion of the long oligo, eg, at the middle 5'-end, 3'-end or any other position of the original sequence. Fragments of low adenosine content, ie up to about 30%, preferably up to 15%, more preferably up to 10%, even more preferably up to 5%, most preferably by selection or in the present invention Particularly important are fragments without adenosine nucleotides by substitution with a common base accordingly. The drug formulations of the present invention comprise, as the most preferred group, sequences substituted with a general base (B) and one or more adenosine present in the sequence, as exemplified herein. Similarly, as described above, shorter all of the B-containing fragments designed by substituting B (s) for adenosine (s) (A (s)) contained in sequences, their fragments or their segments. Also includes fragments.

Some of the examples of antisense oligonucleotide sequence fragments target the initiation codon of each gene, and in some cases, adenosine is a generic or other base, denoted as "B" lacking binding capacity with adenosine A ₁ and / or A ₃ receptors. Is substituted with adenosine analogues. Indeed, such substitutional nucleotides act as "spacers". Many examples shown below provide many such sequences and fragments that overlap the initiation codon, wherein the number of nucleotides, n, is up to about 7, about 10, about 12, about 15, about 18, about 21 and about 28, about Up to 35, up to about 40, up to about 50, up to about 60 are preferred.

Human Receptor-Related Antisense Polynucleotides

Human Enzyme-Related Antisense Polynucleotides

Human Factor Related Antisense Oligonucleotides

Human Adenosine A1 Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human adenosine A _2a Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human adenosine A _2b Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human adenosine A ₃ Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human IgE Receptor β Nucleic Acid and Antisense Oligonucleotide Fragments

Human Fc-ε Receptor CD23 Antigen (IgE Receptor) Nucleic Acids and Antisense Oligonucleotide Fragments

Human IgE Receptor α Subunit Nucleic Acid and Antisense Oligonucleotide Fragments

Human IgE Receptor (Fc epsilon R) Nucleic Acids and Antisense Oligonucleotide Fragments

Human High Affinity IgE Receptor Oligonucleotide Fragments

Human histidine decarboxylase nucleic acid and antisense oligonucleotide fragments

Human Beta Tryptase Nucleic Acid and Antisense Oligonucleotide Fragments

Human Tryptase-I Nucleic Acid and Antisense Oligonucleotide Fragments

Human Prostaglandin D Synthase Nucleic Acid and Antisense Oligonucleotide Fragments

Human Cyclooxygenase-2 Nucleic Acid and Antisense Oligonucleotide Fragments

Human eosinophil cationic protein nucleic acid and antisense oligonucleotide fragments

Human eosinophil-derived neurotoxin nucleic acid and antisense oligonucleotide fragments

Human eosinophil peroxidase nucleic acid and antisense oligonucleotide fragments

Human intracellular adsorption molecule-1 (ICAM-1) nucleic acids and antisense oligonucleotide fragments

Human vascular cell adsorption molecule 1 (VCAM-1) nucleic acid and oligonucleotide fragments

Human Endocrine Leukocyte Adsorption Molecule (ELAM-1) Nucleic Acid and Antisense Oligonucleotide Fragments

Human P Selectin Fragments

Human Endocrine Monocyte Activator Factor Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL3 * Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL3 Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL-4 Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL4 Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL5 * Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL-5 Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

Human IL-6 Receptor Fragments

Human IL-6 Nucleic Acids and Antisense Oligonucleotide Fragments

Human monocyte-induced neutrophil chemotactic factor nucleic acid and antisense oligonucleotide fragments

Human neutrophil elastase (medulacin) nucleic acid and antisense oligonucleotide fragments

Human neutrophil oxidase factor nucleic acid and antisense oligonucleotide fragments

Human Kaepsin G Nucleic Acids and Antisense Oligonucleotide Fragments

Human Defensin 1 Nucleic Acid and Antisense Oligonucleotide Fragments

Human Defensin 2 Nucleic Acid and Antisense Oligonucleotide Fragments

Human Macrophage Inflammatory Protein-1-alpha / Rantetsu Receptor Nucleic Acid and Antisense Oligonucleotide Fragments

Lantetsu antisense oligonucleotide fragments

Human Muscarinic Acetylcholine Receptor HM1 * Nucleic Acids and Antisense Oligonucleotide Fragments

Human Muscarinic Acetylcholine Receptor HM3 * Nucleic Acid and Antisense Oligonucleotide Fragments

Human Fibronectin * Antisense Oligonucleotide Fragments

Human Interleukin-1 (IL-1) Nucleic Acids and Antisense Oligonucleotide Fragments

Human Interleukin-1 Receptor (IL-1R) Nucleic Acids and Antisense Oligonucleotide Fragments

Human Interleukin-8 * Fragments Antisense Oligonucleotide Fragments

Human IL-8 Receptor Alpha Antisense Oligonucleotide Fragments

Interleukin-11 (IL-11) Nucleic Acid and Antisense Oligonucleotide Fragments

Human GM-CSF Nucleic Acid and Antisense Oligonucleotide Fragments

Human Tumor Necrosis Factorα Antisense Oligonucleotide Fragments

Human Rucotriene C4 Synthase Nucleic Acid and Antisense Oligonucleotide Fragments

Human Entocillin-1 Nucleic Acid and Antisense Oligonucleotide Fragments

Endocrine Receptor ET-B Nucleic Acids and Antisense Oligonucleotide Fragments

Endocrine ETA Receptor Nucleic Acid and Antisense Oligonucleotide Fragments

Endocrine Receptor A Nucleic Acids and Antisense Oligonucleotide Fragments

Substance P Antisense Nucleic Acid and Oligonucleotide Antisense Oligonucleotide Fragments

Substance P Receptor Nucleic Acid and Antisense Oligonucleotide Fragments

Cimase antisense nucleic acid and oligonucleotide antisense oligonucleotide fragments

Endocrine nitric oxide synthase nucleic acid and antisense oligonucleotide

Inducible nitric oxide synthase nucleic acid and antisense oligonucleotide fragments

NF-κB nucleic acid and antisense oligonucleotide fragments

Human major basic protein nucleic acid and antisense oligonucleotide fragments

Human eosinophil major basic protein nucleic acid and antisense oligonucleotide fragments

BP-1 Nucleic Acids and Antisense Oligonucleotide Fragments

C / EBP Nucleic Acid and Antisense Oligonucleotide Antisense Oligonucleotide Fragments

Bradykinin Receptor Nucleic Acids and Antisense Oligonucleotide Fragments

β2-adrenergic receptor kinase nucleic acid and antisense oligonucleotide fragments

CCR-2 CC Chemokine Receptor Nucleic Acid and Antisense Oligonucleotide Fragments

CCR-4 CC Chemokine Receptor Nucleic Acid and Antisense Oligonucleotide Fragments

CD-34 Nucleic Acids and Antisense Oligonucleotide Fragments

Eotaxin Antisense Nucleic Acid and Oligonucleotide Fragments

FK-506 Binding Protein Nucleic Acids and Oligonucleotide Fragments

Wherein B is substituted for adenosine, more preferably adenosine, and is "equal or" common "base and adenosine A _2a receptor antagonist or only minimal antagonist, adenosine A _2b receptor antagonist, adenosine A ₃ receptor antagonist, or adenosine A ₁ receptor antagonist Similarly, adenosine (A) is as described above with "other," equivalent "and / or" common "bases having adenosine activity of less than 0.3 in a small proportion, preferably adenosine receptor (s). It can always be substituted.

In one preferred embodiment, the bond between neighboring mononucleotides is a phosphodiester bond. According to another preferred embodiment, at least one mononucleotide phosphodiester residue of the antisense oligonucleotide (s) is methylphosphonate, phosphoester, phosphorothioate, phosphodithioate, boranophosphate, formacetal, Thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxyamine, 2'-0-methyl, methylene (methimiino), Methyleneoxy (methylimino), phosphoramidate residues and their bonds. Oligos having one or more phosphodiester residues substituted with one or more other residues generally last longer because these residues are more resistant to hydrolysis than phosphodiester residues. In some cases, up to about 10%, up to about 30%, up to about 50%, up to about 75%, or all phosphodiester residues (100%) may be substituted. In general, the multiple target antisense oligonucleotides (oligos) of the present invention have at least about 7 mononucleotides, in some cases at least about 60 mononucleotides, preferably about 10 to about 36 mononucleotides, more preferably about 12 to about 21 mononucleotides. However, depending on the length of the target polymer, other lengths are also suitable. Examples of MTA oligos of the present invention are provided in Table 3 below, which contains 94 sequences (SEQ ID NOS .: 2316 to 2410).

Table 3: MTA Oligos, Targeted and Target Locations

The MTA oligos in Table 3 are suitable for use with two or more targets listed in Table 4 below.

Table 4: Targets of the MTA Oligos of Table 3

Whether existing targets or those which will be revealed later, the mRNA sequence of the target protein can be inferred from the nucleotide sequence of the gene expressing the protein. Sequences for many target genes of different systems are currently known (GenBank database, NIH reference, all related sequences are incorporated herein by reference). Sequences of genes that are not yet available can also be obtained by separating target segments by applying techniques known in the art. If the sequence of a gene, its RNA and / or protein is known, antisense oligonucleotides can be made as described above, used to identify the target by in vivo administration, and the target protein production or specific according to standard techniques. The reduction in function can be checked. As already mentioned above, the antisense oligonucleotides may be of suitable length, for example about 7 to 60 nucleotides in length, depending on the particular target to which it binds and the method of delivery thereof. Antisense oligonucleotides relate to mRNA regions or junctions that contain junctions between introns and axons. If antisense oligonucleotides are directed to intron / axon junctions, e.g. at the 3 'or 5' end of an antisense oligonucleotide located within the 10, 5, 3 or 2 nucleotides of the intron / axon junction, the mRNA matured at the precursor mRNA In order to prevent axons caught in the middle of the process from being 'spliced out', the junction can be completely covered or close enough to the junction. Also preferred are antisense oligonucleotides that overlap the initiation codon, more generally antisense oligonucleotides that target the coding region of the target mRNA. In practical application of the invention, the antisense oligonucleotides administered may be related to the origin of the species administered. When dealing with humans, human antisense can be used if desired. However, antisense oligos for intrinsic sequences of other species may also be included.

Through the cell membrane and in the cell A ₁ or A ₃ and specificity mRNA encoding the adenosine receptor coupled to, and by that to interfere with its translation (translation), A ₁ or A ₃ to the effective in reducing expression of the adenosine receptors Pharmaceutical compositions containing such antisense oligonucleotides are another aspect of the present invention. Such compositions are provided in suitable pharmaceutically acceptable carriers, such as sterile pyrogen-free saline solutions. To cross cell membranes, antisense oligonucleotides may be formulated with a hydrophobic carrier such as liposomes, and the liposomes may be supported on a pharmaceutically acceptable water soluble carrier. The oligonucleotides may be combined with substances capable of inactivating mRNA such as ribozyme. Such oligonucleotides may be administered to a subject to inhibit activation of a target such as an adenosine receptor, which subject requires such treatment for the reasons mentioned herein. Pharmaceutical formulations may also contain chimeric molecules consisting of antisense oligonucleotides attached to substances known to internalize in the cell. These oligonucleotide conjugates utilize cell uptake pathways to increase cell concentration of oligonucleotides. Examples of polymers used in this method include transferrin, asialoglycoprotein (attached to oligonucleotides via polylysine) and streptavidin. In pharmaceutical formulations, antisense compounds may be included in lipid particles or vesicles such as liposomes or microcrystalline. As long as antisense is included, the particles can be of any suitable structure, such as a single lamellar structure or a plurality of lamellar structures. Particular preference is given to such particles and becyclos with positively charged lipids such as N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammoniumethylsulfate or "DOTAP". The preparation of such lipid particles is known (US Patent Nos. 4,880,635 (Janoff et al.); 4,906,477 (Kurono et al.); 4,911,928 (Wallach); 4,917,951 (Wallack); 4,920,016 (Allen et al.); 4,921,757 (4) wheatley et al.).

The active composition can be injected into the subject by a method of delivering the antisense nucleotide composition to the lung. Although antisense compounds that can be introduced into the subject's lungs by appropriate methods are disclosed herein, it is desirable to produce and add an aerosol consisting of antisense compounds and composed of respirable particles for inhalation by the patient. Respirable particles can be liquid or solid. The particles may optionally contain other therapeutic ingredients. Particles composed of antisense compounds for practical application of the present invention should contain particles of respirable size: particles of a size small enough to pass through the bronchi and alveoli through the mouth and larynx upon inhalation. Generally particles in the range of about 0.5 to about 10 microns in size are respirable. Since non-breathable particles in the aerosol must accumulate or be swallowed in the neck, it is desirable to minimize the amount of non-breathable particles in the aerosol. In the case of nasal administration, a particle size of 10 to 500 μm is preferred to maintain in the nasal cavity. Accordingly, particles within about 4, about 10, about 25, about 50, about 75, about 100, about 250, about 500 and other specific ranges are preferred. However, other things can also be considered within the scope of the present invention.

Liquid pharmaceutical compositions of the activating compounds for producing aerosols can be prepared by mixing an antisense compound with sterile pyrogenic water and a suitable vesicle. Optionally, other therapeutic compound may be further included. Solid particle compositions containing respirable dry particles of the micronized antisense compound are ground to a microscopic composition through a 400 mesh screen, followed by pulverizing the dry antisense compound with a mortar and pestle to destroy or separate large chunks. It is prepared by passing through. Solid particle compositions composed of antisense compounds may optionally further contain a dispersant, as the case may be, to facilitate preparation of the aerosol formulation. Suitable dispersants include lactose which can be mixed with the antisense compound in a suitable proportion (eg, by weight ratio of 1: 1). In addition, other therapeutic compounds may be included.

The dose of antisense compound administered depends on the condition being treated, the condition of the subject, the particular formulation, the route of administration, the time of administration to the subject, and the like. Generally, intracellular concentrations of oligonucleotides are required from about 0.01, about 0.05, about 0.02, about 1 to about 5 μM, about 50 μM, about 100 μM, more preferably about 0.2 to about 0.5 μM. When administered to a subject such as a human, the dosage may be from about 0.01, about 0.1 or about 1 mg / kg to about 50 mg / kg, up to about 100 mg / kg, up to about 150 mg / kg or more It can be used generally according to the administration route known in the art. Depending on the solubility of the particular agent of the activating compound administered, the daily dose may be divided into one or several unit doses. Administration of antisense compounds can be carried out therapeutically (as cure treatment) or prophylactically. Aerosols of liquid particles composed of antisense compounds can be prepared by any suitable method such as a nebulizer (see U.S. Patent No. 4,501,729). Nebulizers convert a solution or suspension of the active ingredient into a therapeutic aerosol mist by acceleration of compressed gas, generally air or oxygen, or by ultrasonic agitation through a good venturi orifice. Commercially available devices. Formulations suitable for use with the nebulizer consist of the active ingredient in a liquid carrier, the active ingredient being contained in an amount of up to 40% w / w, but preferably up to 20% in the formulation. In general, the carrier is water or dilute aqueous alcohol solution, preferably made of isotonic body fluid with the addition of sodium chloride, for example. Optional additives include preservatives such as methylhydroxybenzoate (if the formulation is not produced aseptically), antioxidants, flavors, volatile oils, buffers and surfactants.

In one preferred embodiment, the pharmaceutical composition comprises nucleic acid (s) comprising one or more of the antisense oligo (s) mentioned above and one or more surfactants. Surfactants or surfactant components suitable for enhancing the uptake of the antisense oligonucleotides of the present invention are not only natural and synthetic, but also surfactant proteins A, surfactant proteins B, surfactant proteins C, surfactant proteins. D and surfactant protein E, 2-saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; Phosphatidyl acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dihydroandrotesterone, dolicholes, sulfatidyl acid, glycerol-3 Phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CPD) diacylglycerol, CDP choline, choline, choline phosphate and Truncated form; And nonionic block copolymers of omega-3-fatty acid, polyacid, polyenoic acid, lecithin, palmitic acid, ethylene or propylene oxide, polyoxy propylene, monomers and polymers, polyoxy Poly (vinyl amine), Brij 35, Trypton X-100 and synthetic surfactants ALEC, Exosurf, of ethylene monomers and polymers, monomers with dextran and / or alkanoyl side chains, and polymers Natural and artificial lamellae constructs which are natural carriers for the Survan and Atovaquone and the like.

These surfactants may be used alone or as part of a plurality of surfactant components in the formulation, or added and covalently attached to the 5 'and / or 3' ends of the antisense oligo (s). In addition, aerosols of solid particles composed of the active compound may also be prepared as any solid particle drug aerosol generator. An aerosol generator for administering a solid particle drug to a subject produces the aforementioned respirable particles and generates a volume of aerosol containing a pre-measured amount of drug at a rate suitable for human administration. One exemplary form of a solid particle aerosol derivative is an insufflator. Preferred formulations for administration by blowing include finely divided powders which can be carried by the blower or transferred to the nasal cavity in the manner of snuff. In the blower, the powder (amount determined to be effective for carrying out the therapies mentioned herein) is usually contained in capsules or medicines made of gelatin or plastic which are spontaneously penetrated or opened, and the powder is supplied via an inhalation device. Delivered by air intake or by hand pump. The powder used in the blower consists of the active ingredient alone or a powder mixture consisting of the active ingredient, a suitable powder diluent such as lactose, and an optional surfactant. The active ingredient is generally contained in 0.1 to 100 w / w of the formulation. A second exemplary form of aerosol generator includes a metered dose inhaler. A metered dose inhaler is generally a dispenser containing an active ingredient suspension formulation or a pharmaceutical formulation in a liquefied propellant. To produce microparticle sprays containing the active ingredient, during use these devices release the formulation through a valve suitable for delivering a measured volume (typically 10 to 150: 1). Suitable propellants include any chlorofluorocarbon compounds such as dichlorodifluoromethane, trifluoromethane, dichlorotetrafluoroethane and mixtures thereof. Optionally, the formulation may further contain one or more cosolvents, for example surfactants such as ethanol, oleic acid or sorbitan triolate, antioxidants and suitable flavoring agents. Aerosols formed from solid or liquid particles are, for example, at a rate of about 10 liters, about 30 liters, about 70 liters, about 100 liters, about 150 liters, about 150 liters, more preferably about 30 to about 15 liters per minute Can be produced by the aerosol generator most preferably at a rate of about 60 liters per minute. Aerosols containing higher amounts of drug may be administered faster than is known in the art.

The disclosures of all relevant scientific publications and the related patents cited in this patent are hereby intended to be incorporated in the relevant literature, in particular with respect to the manufacturing methods and techniques which make the invention possible. The following examples are provided to illustrate the invention but are not intended to be limiting thereof.

In the following examples, μM is micromolar, ml is milliliters, μm is micrometers, mm is millimeters, cm is centimeters, ° C is degrees Celsius, μg is micrograms, mg is in milligrams, g is in grams, kg is in kilos Gram, m is mole, and h is time.

Example 1 Design and Synthesis of Antisense Oligonucleotides

Designing antisense oligonucleotides for A ₁ and A ₃ adenosine receptors may require interpretation of the complex secondary structure of the target A ₁ receptor mRNA and target A ₃ receptor mRNA. After forming this structure, it can be interpreted as giving functional activity or stability to mRNA, and antisense oligonucleotides are designed for target regions of mRNA that can overlap with the start codon optimally. Other targeting sites can be readily used. As in the demonstration of the specificity of the antisense effect, other nucleotides, which are not entirely complementary to the target mRNA but contain the same nucleotide composition based on w / w, are included as controls in the antisense experiments.

The mRNA secondary structure of the adenosine A ₁ receptor was analyzed and used as shown above to design phosphorothioate antisense oligonucleotides. The synthesized antisense oligonucleotide is named HAdA ₁ AS and has the sequence 5'-GATGGA GGG CGG CAT GGC GGG-3 '(SEQ ID NO: 1). As a control, mismatched phosphorothioate antisense nucleotides designated HAdA ₁ MM1 were synthesized as SEQ ID NO: 5'-GTA GCA GGC GGG GAT GGG GGC-3 '(SEQ ID NO: 2). Each oligonucleotide has the same base content and general sequence structure. Searching for similarity in GENBANK (Release 85.0) and EMBL (Release 40.0) shows that the antisense oligonucleotides are specific at adenosine A ₁ receptors in humans and rabbits, and the mismatched control is a substance for hybridization with any known gene sequence. indicated that it could not be (candidate).

The secondary structure of adenosine A ₃ receptor mRNA can be similarly analyzed and used as shown above to design the two phosphothioate antisense oligonucleotides. The first antisense oligonucleotide synthesized (HAdA ₃ AS1) has the sequence 5'-GTT GTT GGG CAT CTT GCC-3 '(SEQ ID NO: 3). As a control, a mismatched phosphorothioate antisense oligonucleotide (HAdA ₃ MM1) having the sequence 5'-GTA CTT GCG GAT CTA GGC-3 '(SEQ ID NO: 4) is synthesized. In addition, a second phosphorothioate antisense oligonucleotide (HAdA ₃ AS2) is designed and synthesized and has the sequence 5'-GTG GGC CTA GCT CTC GCC-3 '(SEQ ID NO: 5). Its control oligonucleotide (HAdA ₃ MM2) has the sequence 5 'GTC GGG GTA CCT GTC GGC-3' (SEQ ID NO: 6). Phosphothioate oligonucleotides were synthesized with an Applied Biosystem Model 396 Olgonucleotide Sunthesizer and purified using NENSORB chromatography (Dupont, MD).

Example 2: Adenosine A _One In Vivo Testing of Receptor Antisense Oligos]

Antisense oligonucleotides (SEQ ID NO: 1) for the human A ₁ receptor described above were tested for efficacy in an in vitro model using lung adenocellular HTB-54. It has been demonstrated that HTB-54 lung adenomas express A ₁ adenosine receptors using receptor probes designed and synthesized in standard Northern blotting experiments and laboratories. HTB-54 human lung adenoma cells (106/100 mm tissue culture dish) were exposed to 5.0 μM HAdA ₁ AS or HAdA ₁ MM1 for 24 hours and incubated for 12 hours before being replaced with fresh medium and oligonucleotides. After exposure to oligonucleotides for 24 hours, cells were harvested and their RNA extracted by standard procedures. A 21-mer probe corresponding to the mRNA region targeted by antisense (hence, having the same sequence as antisense but not phosphorothioated) was synthesized to HAdA ₁ AS-treated, GAdA ₁ MM1-treated and untreated It was used as a Northern blot probe of RNA prepared from HTB-54 cells. These blots clearly showed that HAdA ₁ AS effectively reduced the amount of human adenosine receptor mRNA by more than about 50%, while HAdA ₁ MM1 did not. These results show that HAdA ₁ AS may be a useful material for anti-asthma drugs because it reduces the intracellular mRNA of adenosine A ₁ receptors associated with asthma.

Example 3: In Vivo Efficacy of Adenosine A1 Receptor Antisense Oligos

Due to the accidental homology between the rabbit and human DNA sequences within the adenosine A ₁ gene that overlaps the initiation codon, phosphorothioate antisense oligonucleotides designed for use with the human adenosine A ₁ receptor are the first to be used in rabbit models. It is possible. Berkeley Biologicals extract of 312 antigen unit / ml House dust mite (D.farinae) mixed with neonatal New Zealand white Pasteurella-free rabbits with approximately 10% kaolin , Berkeley, Calif.) And were intraperitoneally immunized within 24 hours of birth. The first month is immunized once a week and the next two months once every two weeks. At 3-4 months of age, 8 immunized rabbits were anesthetized by intramuscular administration of a mixture of ketamine hydrochloride (44 mg / kg) and acepromazine maleate (0.4 mg / kg) intramuscularly. Relaxed. The tusks were laid flat in a comfortable position in a small, fluffy molded animal box and then incubated in a 4.0 mm intratracheal tube (Mallinkrodt, Inc., Glen Falls, NY). An external diameter 2.4 mm polyethylene catheter with a latex balloon was placed in the esophagus and maintained the same distance from the mouth (about 16 cm) during this experiment. Endotracheal tube is attached to a heated Fleisch pneumotachograph (Size 00; DOM Medical, Richmond, VA), and the flow is a Goul carrier amplier (Model 11-4113). Measured using a Validine dfferential pressure transducer (model DP-45161927; Validyne Engineering Corp., Northridge, Calif.), Powered by Gould Electronic, cleveland, OH. An esophageal balloon was attached to one side of the partial pressure inducer and the outlet of the endotracheal tube was connected to the opposite side of the partial pressure inducer to record the pressure of the bronchus. Flow is recorded to provide a continuous tidal volume, and measurements of overall lung resistance (RL) and dynamic compliance (C _dyn ) are measured using an autorespiratory analyzer (Model 6; Buxco, Shron, CT) were used to calculate at isovolumetric and flow zero points, respectively. Animals were randomly selected and the first day pretreatment value of PC ₅₀ was obtained for aerosolized adenosine. Antisense (HAdA ₁ AS) or mismatched control (HAdA ₁ MM) oligonucleotides were dissolved in sterile physiological saline at a concentration of 5000 μg (5 mg) per 1.0 mL. The animals were then administered aerosolized antisense or mismatched oligonucleotides (5000 μg in a volume of about 1 mL) through the endotracheal tube twice daily for two days. Aerosols of saline, adenosine, or antisense or mismatched oligonucleotides are made by ultrasonic nebulizers (DeVilbiss, Somerset, PA), and 80% of the aerosol droplets formed are smaller than 5 μm in diameter. In the first step of the experiment, four randomly selected allergic rabbits received antisense oligonucleotides and four mismatched control oligonucleotides. On the third morning, the PC ₅₀ value (from the baseline value, the concentration of aerosolized adenosine required to reduce dynamic compliance in the bronchial airways by 50%, mg / ml) was obtained, and the PC obtained from animals prior to exposure to oligonucleotides. The value of ₅₀ was compared. After a week, the animals crossed each other. That is, rabbits first administered antisense oligonucleotides were administered with mismatched control oligonucleotides, and rabbits first administered mismatched control oligonucleotides were administered antisense oligonucleotides. Treatment methods and measurements are the same as those used in the first method of this experiment. It is noteworthy that in 6 of 8 animals treated with antisense oligonucleotides, adenosine-mediated bronchial stenosis could not be obtained up to 20 mg / ml, the limit of solubility of adenosine. For the purposes of the calculation, the value of PC ₅₀ for these animals was fixed at 20 mg / ml. Therefore, the values provided represent the minimum values for the effectiveness of the antisense. The actual effect is higher. The results of this experiment are shown in Table 5 below.

Table 5: PC from asthma rabbit ₅₀ Adenosine A on Value _One Effect of Receptor Antisense Oligos

Discrepancy control A1 receptor antisense oligo Oligonucleotides Before treatment Oligonucleotides After treatment Oligonucleotides Before treatment Oligonucleotides After treatment 3.56 ± 1.02 5.16 ± 1.03 2.36 ± 0.68 > 19.5 ± 0.34 ** The results are expressed as mean (m = 8) ± SEM. Significance was calculated by repaeted-measures analysis of variance (ANOVA) and Tukey's protected test. ** All other Significant difference from group, p <0.01

In the two experimental methods, animals treated with antisense oligonucleotides showed that the dose of aerosolized adenosine required to reduce lung dynamic compliance by up to 50% was increased in order of magnitude. It was observed that the mismatched control oligonucleotides had no effect on the value of PC ₅₀ . No toxicity was observed in any animal inhaled with antisense or control oligonucleotides.

These results clearly show that the lung has exceptional potential as a target for antisense oligonucleotide-based therapeutic intervention in lung disease. Moreover, they show that in a model system very similar to human asthma, downregulation of adenosine A ₁ receptors can eliminate adenosine-mediated bronchial contraction mainly in the asthma airway. Since the tissue involved in this response is near the point of contact with the aerosolized oligonucleotide and the model is stimulated similar to a critical human disease, bronchial hypersensitivity is a good model for antisense intervention in allergic rabbit models of human asthma. .

Example 4: A _One Specificity of Adenosine Receptor Antisense Oligonucleotides]

As a conclusion of the crossover experiment of Example 3, the number of adenosine A ₁ receptors in airway smooth muscle from all rabbits was quantitatively analyzed. In addition, as a control for the specificity of antisense oligonucleotides, unaffected adenosine A ₂ receptor was quantified. Airway smooth muscle tissue was dissected from each rabbit, and the cell membrane fragments were analyzed by Kleinstein et al. (Kleinstein, J. and Glossmann, H., Naunyn-Schmiedeberg's Arch.Pharmacol. Related parts of the above documents are hereby incorporated by reference in their entirety. Crude plasma membrane preparations were stored at 70 ° C. until testing. Protein content was determined by Bradford's method (M. Bradford, Anal. Biochem. 72, 240-254 (1976), the relevant parts of which are incorporated herein by reference in their entirety). The frozen plasma membrane was dissolved at room temperature, 0.2U / ml adenosine deaminase was added, and incubated at 37 ° C. for 30 minutes to remove intrinsic adenosine. Binding of [ ³ H] DPCPX (A ₁ receptor-specific) or [ ³ H] CGS-21680 (A ₁ receptor-specific) is described by Ali et. al. Method (Ali, S. et al., J. Pharmacol. Exp. Ther. 268, Am. J. Physiol 266, L271-277 (1994), relevant parts of which are incorporated herein by reference in their entirety. ) Was measured as described above. When analyzed by the specific binding of the A ₁ -specific antagonist DPCPX, animals treated with adenosine A ₁ antisense oligonucleotides in the crossover experiments reduced nearly 75% in the number of A ₁ receptors compared to the control. Analyzes with specific binding of A ₂ receptor-specific agonist 2- [p- (2-carboxyethyl) -phenethylamino] -5 '-(N-ethylcarboxamido) adenosine (CGS-21680) There was no change in the number of adenosine A ₂ receptors. This is shown in Table 6 below.

Table 6: Adenosine A _One Specificity of Action of Receptor Oligonucleotide Antisense

Discrepancy control Oligonucleotides A _One Antisense Oligonucleotides A ₁ -specific combination 1105 ± 48 ** 293 ± 18 A ₂ -specific combination 302 ± 22 442 ± 171 The results are mean (m = 8) ± SEM. Significance was calculated by ANOVA (repaeted-measures analysis of variance) and Tukey's protected test (**), meaning significant differences from discordant controls. p <0.01

The results show the effect of antisense oligonucleotides in treating airway disease. Since the antisense oligos described above eliminate the receptor system that causes adenosine-mediated bronchial contraction, it is not necessary to remove adenosine from the receptor system. However, it is desirable to remove adenosine from these oligonucleotides in order to reduce the dosage required to achieve a similar effect. Other antisense oligonucleotides that target mRNA of proteins involved in inflammation are described above. To prevent the release of adenosine during degradation, adenosine was removed from the nucleotides.

Example 5 Antisense Oligonucleotides Associated with Other Target Nucleic Acids

This work was performed to show that the present invention can be widely applied to antisense oligonucleotides (“oligodes”) that are broadly specific for nucleic acid targets. The following experimental studies were performed, demonstrating that the method of the present invention is designed as disclosed herein and is suitable for widespread use of antisense oligos that target any and all adenosine receptor mRNAs. For this purpose various antisense oligos were prepared with adenosine receptor mRNAs exemplified by adenosine A ₁ , A _2b , A ₃ receptor mRNAs. Antisense Oligo I was disclosed above (SEQ. ID NO: 1). Additional five antisense phosphorothioate oligos were designed and synthesized as described above.

1-Oligo II, which targets adenosine A1 receptor but differs from oligo I (SEQ. ID NO: 7)

Oligo V that targets 2-adenosine A _2b receptor (SEQ. ID NO: 10)

3- oligos III (SEQ. ID NO: 8) and IV (SEQ. ID NO: 9) that target different regions of the adenosine A ₃ receptor

4- oligo I-PD (SEQ. ID NO: 1681) (phosphodiester oligo of the same sequence as oligo I)

Such antisense oligos are designed for treatment in a selected species, as described above, and are generally specific to that species unless the fragments of the target mRNA of another species contain similar sequences. All antisense oligos were prepared as follows, and as described herein above in vivo with antisense oligos in a rabbit model for bronchial stenosis, inflammation, and allergies with respiratory disorders and respiratory failure as in the case of diseases such as asthma I did my test.

Example 6: Design and Sequence of Other Antisense Oligos

Six oligos and their effects were studied in rabbit models and the results of this study were recorded and reviewed below. Five of these oligos were selected for the study to supplement the data for oligo I (SEQ ID NO: 1) provided in Examples 1-4 above. This oligo is antisense for one region of adenosine A ₁ receptor mRNA. The oligos tested were antisense oligos I (SEQ ID NO: 1) and II (SEQ ID NO: 7), which targeted other regions of adenosine A ₁ receptor mRNA, and oligo V (SEQ ID NO: 7) that targeted adenosine A _2b receptor mRNA. 8), identical to oligos III and IV (SEQ ID NOs: 9 and 10) that target the other two regions of adenosine A ₃ receptor mRNA. The sixth oligo (oligo I-PD) is a phosphodiesterated version of oligo I (SEQ ID NO: 1). The design and synthesis of these antisense oligos was performed according to Example 1 above.

(I) antisense oligo I

Antisense oligonucleotide I mentioned in Examples 1-4 above is targeted to human A1 adenosine receptor mRNA (EPI 2010). Antisense oligo I is 21 nucleotides in length, overlaps the initiation codon, and has the sequence 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ. ID No 1).

As described above, Nyce, J.W. As shown in & Metzger, WJ, Nature, 385: 721 (1977) (the relevant part of which is incorporated herein by reference in its entirety), Oligo I is responsible for adenosine-induced bronchial stenosis in allergic rabbits. It has been shown above to eliminate and reduce allergen-induced airway disorders and bronchial hyperresponsiveness (BHR).

(II) antisense oligo II

Phosphorothioate antisense oligos (SEQ. ID NO: 7) were designed according to the present invention to target the rabbit adenosine A ₁ receptor mRNA regions +936 to +956 against the start codon (start point). Antisense Oligo II is 21 nucleotides in length and has the sequence 5'-CTC GTC GCC GTC GCC GGC GGG-3 '(SEQ. ID NO: 7).

(III) antisense oligo III

Phosphorothioate antisense oligos (SEQ. ID NO: 8) different from those provided in Example 1 above were designed according to the invention to target the antisense A ₃ receptor mRNA regions +3 to +22 relative to the initiation codon (starting point). It became. Antisense Oligo III is 20 nucleotides in length and has the sequence 5'-GGG TGG TGC TAT TGT CGG GC-3 '(SEQ. ID NO: 8).

(IV) antisense oligo IV

Another phosphorothioate antisense oligo (SEQ. ID NO: 9) was designed in accordance with the present invention to target the adenosine A ₃ receptor mRNA region +386 to +401 relative to the initiation codon (start point). Antisense Oligo IV is 15 nucleotides in length and has the sequence 5'-GGC CCA GGG CCA GCC-3 '(SEQ. ID NO: 9).

(V) antisense oligo V

Phosphorothioate antisense oligos (SEQ. ID NO: 10) were designed according to the present invention to target adenosine A _2b receptor mRNA regions -21 to -1 relative to the initiation codon (starting point). Antisense oligo V is 21 nucleotides in length and has the sequence 5'-GGCCGG GCC AGC CGG GCC CGG-3 '(SEQ. ID NO: 10).

(VI) A _One Raise discrepancies

Two different mismatched oligonucleotides with the following sequence were used as controls for the antisense oligo I (SEQ. ID NO: 1) described in Example 5 above:

A ₁ MM2 5′-GTA GGT GGC GGG CAA GGC GGG-3 ′ (SEQ. ID NO: 2421);

A ₁ MM3 5'-GAT GGA GGC GGG CAT GGC GGG-3 '(SEQ.ID NO: 2422)

Antisense oligo I and two mismatched antisense oligos had the same base content and general sequence structure. Similarity searches in GENBANK (Release 85.0) and EMBL (Release 40.0) show that antisense oligo I is specific for adenosine A ₁ receptor genes in rabbits as well as humans, and mismatched controls hybridize with the gene sequences of other human or animal known It did not appear to be a substance.

(VII) antisense oligo A1-PD (oligo VI)

Phosphodiester antisense oligos (Oligo VI; SEQ. ID NO: 2420) having the same nucleotide sequence as oligo I were designed as disclosed herein above. Antisense oligo I-PD is 21 nucleotides in length, overlaps the initiation codon, and has the sequence 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ. ID NO: 2420).

(VIII) control

5.0 ml of aerosolized sterile saline was administered to each rabbit according to the same method as the antisense oligos in (II), (III) and (IV) above.

Example 7: Synthesis of Antisense Oligos

Phosphorothioate antisense oligos having the sequence described in (a) above were synthesized with an Applied Biosystem Model 396 Oligonucleotide Synthesizer and purified using NENSORB chromatography (DuPont, DE). TETD (tetraethylthiuram disulfide) was used during the synthesis as a sulfurizing agent. Antisense oligonucleotide II (SEQ. ID NO: 7), antisense oligonucleotide III (SEQ. ID NO: 8), and antisense oligonucleotide IV (SEQ. ID NO: 9) were each synthesized and purified in the same manner. .

Example 8: Preparation of Allergic Rats

Within 24 hours of birth of a newborn New Zealand White Pasteurella-deficient rabbit, 312 antigen units / ml House dust mite (D. farinae) extract (Berkeley Biologicals, Berkeley, CA) mixed with 10% kaolin as described above ) Intraperitoneally immunized with 0.5 ml (Metzger, WJ, in Late Phase Allergic Reactions, Sorsch, W., Ed., CRC Handbook, pp.347-362, CRC Press, Boca RAton (1990); Ali, S., Metzger, WJ and Mustafa, SJ, Am. J. Resp. Crit.Care Med. 149: 908 (1994), the relevant parts of which are incorporated herein by reference in their entirety. Immunization was repeated once a week for the first month and once every two weeks until the next four months. Preferably such rabbits produce allergen-specific IgE antibodies and generally respond to aeroallergen infection in both early and late asthma reactions, and bronchial hypersensitivity (BHR) was seen. Allergen (312 unit dust mite allergen as described above) was administered monthly to the peritoneum to continuously stimulate and maintain allergen-specific IgE antibody and BHR. As described by Alo et al., At 4 months, an aerosol was administered to prepare immunized rabbits (Ali., S., Metzger WJ and Mustafa, SJ, Am. J. Resp. Crit. Care Med). 149 (1994), the relevant parts of which are incorporated herein by reference in their entirety.

Dose-response study

Example 9: Experimental design

Aerosols of either adenosine (0-20 mg / ml), or antisense oligonucleotides or two mismatched oligonucleotides (5 mg / ml) were prepared using an ultrasonic nebulizer (Model 646, DeVilbiss, Somerset, PA), respectively. The ultrasonic nebulizer produced an aerosol droplet, of which 80% was smaller than 5 μm in diameter. An equal volume of aerosol was administered directly to the lungs through an endotracheal tube. Animals were randomly selected and administered aerosolized adenosine. On the first day, the pretreatment value in response to adenosine was calculated as the dose of adenosine causing a 50% loss of compliance (PC ₅₀ adenosine). One of the aerosolized antisense or mismatched antisense oligos was then administered to the animals for 2 minutes twice a day for two days through a tube in the trachea (5 mg / ml) (total dose, 20 mg). Post-treated PC ₅₀ values were recorded on the third morning (post-treated infections). The results of this study are mentioned in Example 21 below.

Example 10 Crossover Experiment

In some experiments using antisense oligo I (SEQ ID NO: 1) and the corresponding mismatched control oligonucleotide A ₁ MM2, animals were crossed after 2 weeks. That is, those previously administered with the mismatched control A ₁ MM2 receive antisense oligo I and those previously treated with the antisense oligo I receive the mismatched control A ₁ MM2 oligo. The number of animals in each group is as follows. Since one animal died in the second arm of the experiment due to technical difficulties, the discrepancy A ₁ MM2 (control 1) was n = 7 and for n discrepancy A ₁ MM3 n = 4 (control 2) and A ₁ AS antisense oligo I was n = 8. A ₁ MM3 oligo-treated animals were analyzed separately and the crossover experiments were not. The treatment methods and measurements applied after the crossover experiments were the same as those used in the first stage of the experiment.

In 6 of 8 animals treated with antisense oligo I (SEQ. ID NO: 1), no PC ₅₀ values were obtained at adenosine doses up to 20 mg / ml, which is the limit of solubility of adenosine. Therefore, the value of PC ₅₀ for these animals was assumed to be 20 mg / ml for calculation purposes. Therefore, a given value represents the minimum value in the effect of the antisense oligonucleotides of the present invention. According to the method described above, 0.5 mg or 0.05 mg (total dosage 2.0 mg or 0.2 mg) of antisense oligo I (SEQ ID NO: 1) or A ₁ MM2 oligo was added to another group of allergic rabbits (n for each group). = 4). The results of this study are mentioned in Example 22 below.

Example 11: Prescription of Antisense Oligo

Each antisense oligo is separated and dissolved in an aqueous solution, respectively, and four 5 mg using nebulizer through the endotracheal tube, as described for antisense oligo I (SEQ. ID NO: 1) in (e) above. In doses (total dose 20 mg). The results for antisense oligo I and its discrepancy control 3 are shown in Example 19 and Nyce & Metzger, Nature 385; As described in Table 1 of 721-725 (1997), it was confirmed that the mismatched control was identical to saline. Because of this finding, saline was used as a control in lung function studies using antisense oligos II, III and IV (SEQ. ID NO: 7, 8 and 9).

Example 12 Adenosine A _One Specificity of Oligo I to Receptor (Receptor Binding Study)]

Airway smooth muscle tissue from rabbits administered 20 mg oligo I (SEQ ID NO: 1) at four divided doses over 48 hours as above was incised into primary, secondary and tertiary bronchus. Ali et al. (Ali., S., et al., Am. J. Resp. Crit. Care Med. 149: 908 (1994), parts relating to the manufacture of cell membrane segments are hereby incorporated by reference in their entirety. Cell membrane segments were prepared according to the method of. Protein content was measured using the Bradford method, the plasma membrane was incubated for 30 minutes at 37 ℃ with adenosine deaminase of 0.2 U / ㎖ to remove the intrinsic adenosine (Bradford, MM Anal. See Biochem. 72, 240-254 (1976), the relevant parts of which are incorporated herein by reference in their entirety. Binding of [ ³ H] DPCPX, [ ³ H] NPC17731 or [ ³ H] CGS-21680 was measured as described by Jarvis et al. (Jarvis et al. See, Jarvis, MF, et al, Pharmaco. Exptl. See Ther. 251, 888-893 (1989), the relevant parts of which are incorporated herein by reference in their entirety. The results of this study are mentioned in Table 8 and in Example 20 below.

Example 13: Pulmonary Function Measurement (Compliance C _DYN And resistance)]

At 4 months of age, the immunized animals were anesthetized and relaxed by intramuscular injection of 1.5 ml of a mixture of ketamine hydrochloride (35 mg / kg) and acepromazine malate (1.5 mg / kg). After induction of anesthesia, the allergic rabbit was laid comfortably in a softly molded animal box. Ointment was applied to eyes and eyes closed to prevent drying. It was then intubated with a 4.0 mm medium high-low cuffed Murphy 1 endotracheal tube (Mallinckrodt, Glen Fall, NY), as described by Zavala and Rhodes (Zavala and Rhodes, Proc. Soc. Exp. Biol. Med. 144: 509-512 (1973), the relevant parts of which are incorporated herein by reference in their entirety. A OD 2.4 mm polyethylene catheter (Becton Dickinson, ClayAdams, Parsippany, NJ) with thin latex abundance was passed through the esophagus and maintained the same distance from the mouth (about 16 cm) during the experiment. Endotracheal tubes are attached to a heated Fleisch pneumotach (Size 00; DEM Medical, Richmond, VA) and a Gould carrier amplifier (Model 11-4113, Gould Electronics, Cleveland, OH). Flow (v) was measured using a Validine partial pressure inductor (Model DP-45-16-1927, Validyne Engineering, Northridge, Calif.) Powered by. The esophageal balloon was attached to one side of the ballidin partial pressure inducer and the other side to the outlet of the endotracheal tube to obtain transpulmonary pressure (P _tp ). Flow was recorded to provide continuous tidal volume and the total lung resistance (Rt) and dynamic compliance (C _dyn ) at back volume and flow zero were measured. Eight channels and Ould of ranked the flow, volume and pressure at 2000W high high-frequency recorder, was _calculated C _dyn using P _tp difference in the total volume and a zero flow, P _tp and in a closed volume of the middle period P _tp R _t was calculated as the ratio of and V. As previously described by Giles et al., The calculations were made automatically using a Buksco automated pulmonary mechanical breathing analyzer (Model 6, Buxco Electronics, Shron, CT; automated pulmonary mechinics respiratory analyzer) (Giles et al. Arch. Int. Pharmacodyn. Ther. 194: 213-232 (1971), relevant portions of which are incorporated herein by reference in their entirety. The results obtained by administering Oligo II to allergic rabbits are shown in Example 26 below and discussed.

Example 14 Measurement of Bronchial Hypersensitivity (BHR)

Baseline hypersensitivity was measured by aerosolized histamine administered to each allergic rabbit. An aerosol of saline or histamine was prepared for 30 seconds and then administered using a Devilbiss nebulizer (devilbiss, Somerset, PA) for 2 minutes each. 80% of the aerosol droplets made with ultrasonic nebulizers were less than 5 microns in diameter. Histamine aerosol was administered at increasing concentrations (from 0.156 to 80 mg / ml) and pulmonary function was measured after each administration. The B4R was then determined by calculating the concentration of histamine (mg / ml) (PC ₅₀ histamine) required to reduce C _dyn 50% from baseline.

Example 15 Cardiovascular Effect of Antisense Oligo I

The measurement of cardiac output and other cardiovascular parameters using Cardiomax ^™ utilizes a thermal dilution principle that monitors the temperature change of the blood exiting the heart after cold saline is introduced in known amounts. Cold saline was injected rapidly into the right atrium through the right jugular vein at one time, and the corresponding change in the temperature of the mixed input and blood of the aortic arch was recorded by a temperature-sensitive small probe through the carotid artery. As mentioned in (d) above, allergic rabbits were treated with oligo I (EPI 2010; SEQ. ID NO: 1) of aerosol and after 12 hours, animals were treated with 80% ketamine and 20% xyazine (Xylazine). Anesthesia at 0.3 mg / kg. As clearly shown in Neyce & Metzger (1997), spura (the appropriate disclosure of which is incorporated herein by reference in its entirety), this time point is shown in SEQ. Consistent with previous data indicating efficacy of ID NO: 1. Then, a thermocouple was inserted into the left carotid artery into each rabbit, advanced 6.5 cm, and tightly tightened with silk suture. The right jugular vein was plumbed, tipped into polyethylene tube length and tightened.

Then, sterile saline was added at 20 ° C. to obtain a thermal dilution curve using Cardiomax ^™ II (Columbus Instrument, Ohio) to determine the exact position of the thermocouple probe. After confirming the exact location of the thermocouple, the femoral artery and vein were isolated. The femoral vein was used as a portal for drug administration and the femoral artery was used for blood pressure and pulse measurements. Once constant baseline cardiovascular parameters were established, blood pressure, pulse rate, cardiac output, total peripheral resistance and cardiac stretch were measured with Cardiomax ^™ .

Example 16: Activity Period of Oligo I (SEQ. ID NO: 1)

As mentioned in (f) above, using the nebulizer through the endotracheal tube, the initial increasing log beginning with 0.156 mg / ml until compliance is reduced to 50% (PC _{50 adenosine} ) to find the reference point. Doses were administered to eight allergic rabbits. Then, as described above, six of the rabbits were administered four times a 5 mg aerosolized amount of (SEQ. ID NO: 1). Two rabbits received the same amount of saline solution as a control. From 18 hours after the last dose, the value of PC _{50 adenosine} was tested again. After this point, measurements were continued for all animals daily up to 10 days. The results of this study are discussed in Example 25 below.

Example 17 Adenosine A with Antisense Oligo V _2b Reduction in the number of receptors]

As described above, 2.0 mg of respirable antisense oligo V (SEQ ID NO: 10) was administered to Sprague Dawley rats three times a day using an inhalation chamber. Twelve hours after the last dose, lung cell tissues were dissected and analyzed for adenosine A _2b receptor binding using [311] -NECA as described in Nyce & Metzger (1997). The same amount of saline was administered to the control group. The results using Student's paired t test had a significance of p <0.05 and are discussed in Example 28 below.

Example 18 Comparison of Oligo I with Corresponding Phosphodiester Oligo VI (SEQ. ID NO: 1681)

Oligo I (SEQ. ID NO: 1) reduced the effects of adenosine and eliminated responsiveness to adenosine amounts up to 20 mg adenosine / 5.0 ml (limit of adenosine solubility). Oligo VI (SEQ. ID NO: 1681), a sequence of oligonucleotides of the phosphodiester type, was completely ineffective when tested in the same manner. All of these compounds differed only in the presence or absence of phosphorothioate residues in oligo I (SEQ. ID NO: 1) and had the same sequence and were aerosolized and administered as described above and in Nyce & Metzger (1997). . The paired t test of the students differed significantly at p <0.001 and the results are discussed in Example 29 below.

Results obtained for antisense oligo I (SEQ. ID NO: 1)

Example 19: Result of Predecessor

The nucleotide sequence and other data of antisense oligo I (SEQ. ID NO: 1) specific for adenosine A ₁ receptor have been provided above. In addition, experimental data showing the effectiveness of oligo I in downregulation of receptor number and activity were also provided above. More detailed information on the properties and activities of antisense oligo I is provided in Nyce, JW and Metzger, WJ, Nature 385: 721 (1997), and the sections relevant to the following results are incorporated herein by reference in their entirety. Meanwhile, a publication by Nyce & Metzger (1997) provided data indicating the following antisense oligo I (SEQ. ID NO: 1) characteristics:

(1) As shown in Table 5 below, antisense oligo I decreases the number of adenosine A ₁ receptors in bronchial smooth muscle of allergic rabbits depending on the dose.

(2) Antisense Oligo I alleviates adenosine-induced bronchial stenosis and allergen-induced bronchial stenosis.

(3) Oligo I alleviates bronchial hyperresponsiveness, as measured by standard measurement PC ₅₀ histamine for assessing bronchial hyperresponsiveness. As shown in Table 5, this result clearly explains the anti-inflammatory activity of antisense oligo I.

(4) As anticipated, since antisense oligo I was designed to target the adenosine A ₁ receptor, antisense oligo I is specific for the adenosine A ₁ receptor as a whole and very closely related adenosine A ₂ receptor at any dose. Or has no effect on the associated bradykinin B ₂ receptor. This is shown in Table 5.

(5) In contrast to the above effects of oligo I, the mismatched control molecules MM2 and MM3 (SEQ. ID NO: 1682 and SEQ. ID NO: 1683) have the same base composition as antisense oligo I (SEQ. ID NO: 1) and It has a molecular weight but differs from the antisense oligo I due to a mismatch of about 6 and 2, respectively. The minimal inconsistency that could continue to maintain the same base composition still remaining showed no sign of any of the targeted receptors (A ₁ , A ₂ , or B ₂ ).

Since there is no prior art for the use of antisense oligonucleotides such as oligo I, which target adenosine A ₁ receptors, these results are unpredictable. The results shown in this patent may intentionally show the effectiveness of the claimed drug formulations and methods for treating diseases or conditions associated with pulmonary airways, such as bronchial stenosis, inflammation, allergies, and the like.

Example 20 Oligo I Effectively Reduces Response to Adenosine Infection

Receptor binding experiments were described in Example 12 above, and the results shown in Table 5 below are membranes isolated from airway smooth muscle of antisense- and mismatched-treated allergic rabbits of A ₁ adenosine receptor and B ₂ bradykinin receptor. Shows the binding properties of adenosine A ₁ -selective ligand [ ³ H] DPCPX and bradykinin B ₂ -selective ligand [ ³ H] NPC 17731.

Table 5: Binding Properties of Three Antisense Oligos

process A _One Receptor B ₂ Receptor Kd B _max Kd B _max Adenosine A _One Receptor 20 mg2 mg0.2 mg A ₁ MM1 20 mg2 mg B ₂ A (bradykinin receptor) 20 mg2 mg0.2 mg B ₂ MM (control) 20 mg2 mg0.2 mg saline 0.36 ± 0.029 nM0.38 ± 0.030 nM0.37 ± 0.030 nM (control) 0.34 ± 0.027 nM0.37 ± 0.033 nM0.36 ± 0.028 nM0.39 ± 0.035 nM0.40 ± 0.028 nM0.39 ± 0.031 nM0.41 ± 0.035 nM0.37 ± 0.029 nM0.37 ± 0.041 19 ± 1.52 fmoles * 32 ± 2.56 fmoles * 49 ± 3.43 fmoles52.0 ± 3.64 fmoles51.8 ± 3.88 fmoles45.0 ± 3.15 fmoles44.3 ± 2.90 fmoles47.0 ± 3.76 fmoles42.0 ± 2.94 fmoles40.0 ± 3.20 fmoles43. 0 ± 3.14 fmoles46.0 ± 5.21 0.39 ± 0.031 nM0.41 ± 0.028 nM0.34 ± 0.024 nM0.35 ± 0.024 nM0.38 ± 0.028 nM0.38 ± 0.027 nM0.34 ± 0.024 nM0.35 ± 0.028 nM0.41 ± 0.029 nM0.37 ± 0.030 nM0. 36 ± 0.025 nM 0.39 ± 0.047 nM 14.8 ± 0.99 fmoles15.5 ± 1.08 fmoles15.0 ± 1.0614.0 ± 1.0 fmoles14.6 ± 1.02 fmoles8.7 ± 0.62 fmoles11.9 ± 0.76fmoles ** 15.1 ± 1.05 fmoles14.0 ± 0.98 fmoles14.8 ± 0.99 fmoles15. 1 ± 1.35 fmoles ¹ Total oligos administered in equal amounts over 4 hours over 48 hours. Treatments and analyzes were performed as mentioned in the method. Significance was determined by repeated-measures analysis of variance (ANOVA), and tucky. Protection t was calculated through the test. N was significantly different from 4-6 * mismatched control- and saline-treated groups p <0.001; ** significant difference from mismatched control muscle- and saline-treated groups p <0.05

Example 21: Dose-response Effect of Oligo I

Antisense oligo I (SEQ ID NO: 1) has been found to reduce the effects of adenosine administration to animals in a dose dependent manner over the dosage ranges tested as shown in Table 6 below.

Table 6: Dose-response effects of antisense oligo I

Total dose (mg) PC ₅₀ Adenosine (mg Adenosine) Antisense Oligo I 0.22.020 A ₁ MM2 Oligo (Control) 0.22.020 8.32 ± 7.214.0 ± 7.219.5 ± 0.342.51 ± 0.463.13 ± 0.713.25 ± 0.34 The results were studied by the student's paired t test and the statistical difference was found to be p = 0.05.

The anti-adenosine A1 receptor oligo, oligo I (SEQ ID NO: 1), specifically acts on the A ₁ receptor but does not act on the adenosine A ₂ receptor. As described above in Example 9 and Nyce & Metzger (1997), supra (administered 5 mg doses four times at 8-12 hour intervals via nebulizer or endotracheal tube), antisense oligo I (SEQ ID NO: 1) or treatment of adenosine A ₁ and adenosine A ₂ receptors as determined in treatment with mismatched control oligo (SEQ ID NO: 1682; A ₁ MM2), incised bronchial smooth muscle tissue, and Nyce & Metzger (1997) The same result comes from the numbers.

Example 22 Specificity of Oligo I (SEQ ID NO: 1) for Target Gene Product

Oligo I (SEQ ID NO: 1) is specific for adenosine A ₁ receptor but its mismatched control has no activity. 1 shows the results obtained from the crossover experiments described in Example 10 and Nyce & Metzger (1997), supra. Administration of two mismatched controls (SEQ ID NO: 1682 and SEQ ID NO: 1683) had no effect on PC _{50 adenosine} values. Conversely, administration of antisense oligo I (SEQ ID NO: 1) resulted in a 7-fold increase in PC _{50 adenosine} values. These results clearly show that antisense oligo I (SEQ ID NO: 1) reduces (relaxes sensitivity) extracellularly to adenosine administered compared to saline controls. The results provided in Table 6 above clearly confirm that the effect of antisense oligo I is dose dependent (see column 3 of Table 5). In addition, oligo I is particularly specific for the adenosine A ₁ receptor (cf. line 3 in Table 5), and therefore does not show any activity at the very closely related A ₂ receptor or the bradykinin B ₂ receptor (cf. Table 6 above). Line 8-10). Furthermore, the results shown in Table 6 show that antisense oligo I (SEQ ID NO: 1) reduces the sensitivity of adenosine in a dose dependent manner and in an antisense oligo-dependent manner. Because neither of the two mismatched control oligonucleotides (A ₁ MM2; SEQ ID NO: 1682 and A ₁ MM3 SEQ ID NO: 1683) had any effect on the PC _{50 adenosine} value or the reduction of the number of adenosine A ₁ receptors. to be.

Example 23 Effect on Aeroallergen-Induced Bronchial Stenosis and Inflammation

Oligo I (SEQ ID NO: 1) has been shown to significantly reduce the histamine-induced effect in rabbit models when compared to mismatched oligos. Effects of antisense oligo I (SEQ IDNO: 1) and mismatch oligos (A ₁ MM2, SEQ ID NO: 1682 and A ₁ MM3 SEQ ID NO: 1682) on allergen-induced airway obstruction and bronchial hypersensitivity Evaluated at. The effect of antisense oligo I (SEQ ID NO: 1) on allergen-induced airway obstruction was evaluated.

As calculated from the plotted curve area, antisense oligo I significantly inhibited allergen-induced airway obstruction compared to the inconsistent control (55%, P <0.05; repeated ANOVA, and Turkey's t). black). Mismatched oligo A ₁ MM2 (control) had no effect on the effect on allen-induced airway obstruction. The effect of antisense oligo I (SEQ ID NO: 1) on allergen-induced BHR was determined as above. As calculated from the PC _{50 histamine} value, antisense oligo I (SEQ ID NO: 1) significantly inhibited allergic-induced BHR compared to the inconsistent control (61%, p <0.05; repeated ANOVA, Turkish t test). A ₁ MM mismatched control was observed to have no effect on allergen-induced BHR.

These results show that antisense oligo I (SEQ ID NO: 1) is effective in preventing from allergen-induced bronchial stenosis (indoor fine dust). In addition, antisense oligo I (SEQ ID NO: 1) is a dust mite-induced bronchial hyperreactivity, as shown by its effect on histamine sensitivity, which exhibits anti-inflammatory activity against antisense oligo I (SEQ ID NO: 1). It has been found to be a potential inhibitor of. These results show that antisense oligo I (SEQ ID NO: 1) is effective in preventing from allergen-induced bronchial stenosis (indoor fine dust). In addition, antisense oligo I (SEQ ID NO: 1) is a dust mite-induced bronchial hypersensitivity, as shown by its effect on histamine sensitivity, which exhibits anti-inflammatory activity against antisense oligo I (SEQ ID NO: 1). Has been found to be a potential inhibitor of

Example 24 Antisense Oligo I Has No Toxic Side Effects

Oligo I (SEQ ID NO: 1) was shown to have no side effects that are toxic to the recipient. When 2.0 or 20 mg of Oligo I (SEQ ID NO: 1) was administered, no change in arterial blood pressure, cardiac output, heart rate, pulse rate, total terminal resistance or cardiac contractility (dPdT) was observed. In addition, cardiac output (CO), pulse rate (SV), mean arterial blood pressure (MAP), pulse rate (HR), total terminal resistance (TRP), and contractility (dPdT) were measured using a Cardiomax ^TM instrument (Columbus Instrument, Ohio). The measurement results were evaluated.

These results demonstrated that oligo I (SEQ ID NO: 1) did not have any detrimental effect on critical cardiovascular parameters. More specifically, this oligo does not cause hypotension. This result is particularly important because other phosphorothioate antisense oligonucleotides have been shown to induce hypotension in some model systems in the past. Moreover, adenosine A ₁ receptors play an important role in isotonic conduction in the heart. It can be expected to deplete adenosine A ₁ receptors by antisense oligo I (SEQ ID NO: 1), resulting in detrimental extrapulmonary activity in response to downregulation of the receptors. However, this is not so. Antisense Oligo I (SEQ ID NO: 1) does not cause any deleterious pulmonary effect, and administration of small amounts of this antisense oligo does not produce unexpected and unwanted side effects. This shows that when oligo I (SEQ ID NO: 1) is administered directly to the lungs, it does not reach the heart in a significant amount causing a deleterious effect. This is in contrast to conventional adenosine receptor antagonists such as theophylline, which can be released from the lungs and cause harmful and even life-threatening effects.

Example 25 Long-Term Sustained Effect of Oligo I

Oligo I (SEQ ID NO: 1) showed long-lasting effects, as evidenced by the value of PC ₅₀ and resistance obtained by administering oligo I (SEQ ID NO: 1) prior to adenosine infection. As described above, for the value of PC ₅₀ of adenosine antisense oligo I, the persistence of the effect was measured when the same dose was administered four times, 5 mg each via an endotracheal tube, via a nebulizer. The effect of drug was significant from day 1 to day 8 after administration. When the effect of antisense oligo I (SEQ ID NO: 1) disappeared, the animals were administered saline aerosol (control) and the PC _{50 adenosine} value was again measured for all animals. Saline-treated animals showed a baseline PC ₅₀ adenosine value (n = 6).

As described above, 6 allergic rabbits administered with 20 mg of antisense oligo I (SEQ ID NO: 1) were measured for airway resistance, and the persistence of the effect was measured. The duration of the average calculated effect was 8.3 days in both PC _{50 adenosine} (p <0.05) and resistance (p <0.05). These results show that antisense oligo I (SEQ ID NO: 1) has a very long lasting effect, which is completely unexpected.

Example 26 Antisense Oligo II

It has been found that antisense oligo IIs targeted to other regions of adenosine A ₁ receptor mRNA have a very high activity against adenosine A ₁ -mediated effects. As described above, the effect of administration of antisense oligo II (SEQ ID NO: 7) was measured in compliance and resistance values when 20 mg of antisense oligo II or saline (control) was administered to two groups of allergic rabbits. It was. Compliance and resistance values were measured as described in Example 13 after administration of adenosine or saline.

The effect of the antisense oligos of the present invention was statistically significant, p <0.05 for compliance with paired t-test and p <0.01 for resistance, showing differences from controls.

These results showed that antisense oligo II (SEQ ID NO: 7) targeting the adenosine A ₁ receptor effectively maintains compliance and decreases resistance following adenosine treatment.

Example 27 Antisense Oligos III and IV

As described above, when 20 mg of antisense oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) were administered to allergic rabbits by the effect of reducing the number of inflammatory and inflammatory cells, It has been shown that antisense oligo III (SEQ ID NO: 8) and antisense oligo IV (SEQ ID NO: 9) actually target specifically adenosine A ₃ receptors. After placing inflammatory cells in the bronchial lavage solution for 3 hours, the number of inflammatory cells was determined by measuring at least 100 viable cells per lavage solution. The effects of antisense oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) on whole cells in granulocytes and bronchial lavage fluid were evaluated after exposure to testite allergens. These results showed that antisense oligo III (SEQ ID NO: 8) and antisense oligo IV (SEQ ID NO: 9) are very useful as anti-inflammatory drugs in asthmatic lungs after exposure to dust mite allergens. As is known in the art, the primary inflammatory cells of asthma and granulocytes, especially eosinopil, when administered antisense oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9), their number is about 40, respectively. The numbers of% and 66% decreased. Moreover, antisense oligos IV (SEQ. ID NO: 9) and III (SEQ. ID NO: 8) also reduced the total number of cells by about 40% and 80% in bronchial lavage, respectively. It is also an important indicator of anti-inflammatory activity by the anti-adenosine A ₃ drug formulation of the present invention. Inflammation is known to underlie bronchial hypersensitivity and allergen-induced bronchial stenosis in asthma. Antisense Oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9), which target adenosine A ₃ receptors, are both important new anti-inflammatory drug formulations that can be designed to specifically target various lung receptors. This can be a representative example of a class.

Example 28 Antisense Oligo V

Antisense oligo V (SEQ. ID NO: 10) targeting adenosine A _2b adenosine receptor mRNA has been shown to be very effective in reducing adenosine A _2b -mediated effects and reducing the number of adenosine A _2b receptors by less than half.

Example 29 Unexpected Excellence of Oligo I-DS Substituted with Phosphodiester-Residue (SEQ. ID NO: 1681)

As described above, oligo I (SEQ. ID NO: 1) and I-DS (SEQ. ID NO: 1681) were separately isolated and administered to allergic rabbits, and then rabbits were administered adenosine. Phosphodiester oligo I-DS (SEQ. ID NO: 1681) was statistically less effective in reducing the effects of adenosine, while oligo I (SEQ. ID NO: 1) was 20 mg. As shown in PC _{50 adenosine} , it has an excellent effect.

Example 30 Antisense Oligo VI

For this task, an additional antisense phosphorothioate oligo (Oligo IV) was designed that targets the adenosine A ₁ receptor. As described in the patent application, this antisense oligo is designed for the treatment of selected species and is generally specific to that species unless the fragments of adenosine receptor mRNAs of the other species selected are of similar sequence. Antisense oligos were prepared as follows and tested in vivo in a rabbit model for bronchial contraction, inflammation and lung allergy, with dyspnea and respiratory disability as in the case of diseases such as asthma, as described in the above application. . The effect on oligos and its rabbit model was studied and the results of this study were reported and discussed below. Oligos (antisense oligo VIs) of the present invention were selected in this study to supplement the results of SEQ ID NO: I (oligo I), which is an antisense of the adenosine A ₁ receptor mRNA provided in the above patent application. This additional oligo is named antisense oligo VI and, unlike oligo I, targets another region of adenosine A ₁ receptor mRNA. The design and synthesis of this antisense oligo was carried out according to the method described in the above patent application, in particular in Example 1.

Antisense Oligo VI is a phosphorothioate designed to target the coding region of rabbit adenosine A ₁ receptor mRNA region +964 to +984 relative to the start codon (starting point). Oligo VI is prepared and has 20 nucleotides in length, as described above in this application. Oligo VI is an adenosine A ₁ receptor gene and has the sequence 5'-CGC CGG CGG GTG CGG GCC GG-3 '(SEQ. ID NO: _).

Phosphorothioate antisense oligo VIs having the sequences described in (5) above were synthesized with an Applied Biosystem Model 396 oligonucleotide synthesizer and purified using NENSORB chromatography (DuPont, DE). TETD (tetraethylthiuram disulfide) was used as a vulcanizing agent during the synthesis.

Example 31 Preparation of Allergic Rabbit

Within 24 hours of birth of a newborn New Zealand White Pasteurella-deficient rabbit, peritoneal with 0.5 ml of 312 antigen units / ml D. farinae extract (Berkeley Biologicals, Berkeley, CA) mixed with 10% kaolin as described above Immunization (Metzger, WJ, in Late Phase Allergic Reactions, Dorsch, W., Ed., CRC Handbook, pp.347-362, CRC Press, Boca RAton (1990); Ali, S., Metzger, WJ and Mustafa , SJ, Am. J. Resp. Crit. Care Med. 149: 908 (1994), the relevant parts of which are incorporated herein by reference in their entirety.

Immunized once a week for the first month and once every two weeks until the next four months. These rabbits preferentially produce allergen-specific IgE antibodies, generally respond to aeroallergen infection in both early and late asthma reactions, and bronchial hypersensitivity (BHR) is shown. Allergens (312 unit fine dust allergens described above) are administered intraperitoneally each month to continuously stimulate and maintain allergen-specific IgE antibodies and BHR. At 4 months of age, sensitized rabbits were prepared for aerosol administration as described by Ali et al. (1994).

Example 32 Preparation of Adenosine Aerosol

Adenosine aerosol (20 mg / ml) was prepared by an ultrasonic nebulizer (Model 646, DeVilbiss, Somerset, PA) producing aerosol droplets. 80% of the aerosol droplets are smaller than 5 μm in diameter. An equal volume of aerosol was administered directly to the lungs through the endotracheal tube to all three rabbits.

The animals were then dosed with aerosolized adenosine and the day 1 pretreatment value for sensitivity to _adenosine was calculated as the amount of _adenosine (PC _{50 adenosine} ) that produced a 50% loss of compliance. The aerosolized antisense (5 mg / 1.0 mL) was then administered to these animals via an endotracheal tube (20 mg total) twice a day for two days. Post-treatment PC ₅₀ values were recorded on the third morning (post-treatment dose). The results of these studies are mentioned in (9) below.

Example 33 Antisense Oligo Formulations

Each of the antisense oligos was individually dissolved in an aqueous solution and administered in 5 mg aliquots four times using an atomizer through an endotracheal tube, as described for antisense oligo I in (e) above (total dose). 20 mg).

Example 34 Oligo VI Reduces Response to Adenosine Administration Similarly or Better to Oligo I

Adenosine VI was tested and prepared in three allergic rabbits, as described in (7) and this patent application. Oligo VI targets the portion of the coding region of oligo I and other A ₁ receptors. All of these target sequences were randomly selected from many possible coding region target sequences. Three rabbits were treated as above Oligo I. Briefly, 5 mg of Oligo VI was sprayed onto rabbits for two days at 8-hour intervals twice a day. Then, a PC ₅₀ adenosine study was performed on the third morning and compared with pretreatment PC ₅₀ values. The experimental procedure is described in more detail in Nyce and Metzger (Nyce & Metzger, Nature 385: 721-725 (1997)).

The results obtained in three rabbits are shown in Table 7 below.

Table 7: PC before and after aerosolized adenosine treatment ₅₀ Adenosine

Processing time PC ₅₀ Adenosine (mg) Before treatment After treatment 3.0 ± 2.1> 20.0 * Maximum amount adenosine can be achieved by insolubility in saline

All three animals treated with Oligo VI completely reduced the sensitivity to adenosine to the measurable level of the drug formulation, as shown in Table 7 above. That is, administration of oligo VI alleviated adenosine-induced bronchial stenosis in three allergic rabbits. Thus, the actual effect of oligo IV is greater than can be measured in the experimental system used.

In comparison to the results from oligo I, oligo IV was found to exhibit as much or better effect than oligo I.

Example 34 Conclusion

The results of the work described and the work discussed in the examples indicate that all antisense oligonucleotides designed according to the disclosure of the above-mentioned applications are very effective in neutralizing or reducing the effects mediated by the receptors targeted by them. It is clearly shown. That is, each of the two antisense oligos targeting adenosine A ₁ receptor mRNA, and all of them, one antisense oligo that targets adenosine A _2b receptor mRNA, and two antisense oligos that target A ₃ receptor mRNA, have specificity that they target The effects of extracellular incorporation of adenosine mediated by receptors can be alleviated. In addition, the activity of the antisense oligos of the invention is specific to the target and cannot substantially inhibit other targets. In addition, the results show that the administration of the drug formulation of the present invention is very low or rarely present with side effects or toxicity. This shows 100% success due to the administration of drug formulations that are highly effective and specific for the treatment of bronchial contraction and / or inflammation. The invention can be broadly applied in the same manner to all gene (s) and corresponding mRNAs encoding proteins involved in or contained airway disease. Comparing the phosphodiester and phosphodiester bonds with the same oligonucleotide type substituted with phosphorothioester bonds, the phosphothioester oligonucleotides have unexpected superiority over the phosphodiester antisense oligos.

Example 35 In Vivo Reaction of Adenosine Treatment with and without Oligo I Pretreatment

After pretreatment with or without antisense oligo I (SEQ. ID NO: 1), two hypersensitive monkeys (sensitive to ascaris) were administered inhaled adenosine. PC ₄₀ adenosine equal to the mg adenosine amount that caused a 40% reduction in dynamic compliance in hypersensitivity airways was calculated from the collected data. Oligo I (SEQ. ID NO: 1; EPI 2010) was then administered by inhalation at 10 mg / day for 2 days. On day 3, PC adenosine was measured again. Oligonucleotide Ⅰ-treated PC ₄₀ adenosine value and oligonucleotide were compared for the PC ₄₀ to perform after the adenosine treatment, in turn Ⅰ (it Did not show in the drawings). Experimental results performed in two animals showed that the sensitization of adenosine was completely eliminated by administering the oligos of the invention in one animal and substantially reduced in the second animal.

Example 36 Expansion of Experimental Results

The method of the present invention also applies antisense oligonucleotides targeting various genes, mRNAs and their corresponding proteins described above, in the same manner as mentioned above, for the treatment of various diseases of the lung. These examples include human A _2a adenosine receptor, human A _2b adenosine receptor, human IgG receptor β, human Fc-epsilon receptor CD23 antigen (IgE receptor), human IgE receptor, α subunit, human IgE receptor, Fc epsilon R, human histidine Decarboxylase, human beta tryptase, human tryptase-I, human prostaglandin D synthase, human cyclooxygenase-2, human eosinophil cationic protein, human eosinophil-induced neurotoxin, human eosinophil fur Oxidase, human intracellular adsorption molecule 1 (CAM-1), human vascular cell adsorption molecule (VCAM-1), human endothelial leukocyte adsorption molecule (ELAM-1), human P selectin, human endothelial monocyte activator, human IL3, human IL4, human IL5, human IL6, human monocyte-induced neutrophil chemotactic factor, human neutrophil elastase (medulacin), human neutrophil oxidase factor, human capidine G, human defensin 1, human defensin 3, human macrophage inflammatory protein-1-alpha, human muscarinic acetylcholine receptor HM1, human muscarinic acetylcholine receptor HM3, human fibronectin, human interleukin 8, human GM-CSF, human tumor necrosis factor α, human leucine There are courtesy C4 synthase, major human proteins, and many others.

The above embodiments illustrate the invention, but are not limited thereto. In the same category of claims contained in the invention, the invention can be described by the following claims.

The present invention can effectively prevent and treat the respiratory disease by reducing the expression of the respiratory disease-related mRNA and protein by using the antisense oligonucleotide of the mRNA related to the respiratory disease.

Claims (92)

Oligonucleotide (s) (oligo (s)) effective when alleviated to bronchitis and / or inflammation, allergy (s), or surfactant deficiency or low secretion when administered to stray animals; Combinations of the oligos; And mixtures of the oligos; And a pharmaceutically or veterinary acceptable carrier or diluent, The oligo contains from about 0 to about 15% adenosine (A), and the initiation codon, coding region, 5 'terminal and 3' terminal gene flanking region, 5 'of the gene encoding the target polypeptide involved in obstructive dysfunction. And an antisense to a target selected from the group consisting of 3 ′ intron-axon junctions and regions within 2 to 10 nucleotides of the junction, or antisense to the polypeptide mRNA. The pharmaceutical composition of claim 1, wherein the oligo does not contain adenosine (A). 2. The method of claim 1, wherein the target is initiation codon, coding region, 5 'terminal and 3' terminal gene flanking of oncogene (s) and gene (s) encoding target polypeptide (s) associated with respiratory failure. Antisense for said oncogene mRNA and said polypeptide mRNA, or selected from the group consisting of regions, 5 'and 3' intron-axon junctions and regions within 2 to 10 nucleotides of said junction; Combinations of the oligos; And mixtures of the oligos, The polypeptide is selected from the group consisting of peptide factors and transporters, antibodies, cytokines and chemokines, enzymes, binding proteins, adsorption molecules, their receptors and melanoma related proteins. Pharmaceutical composition. 4. The method of claim 3, wherein the target is a tumor gene (s) and initiation codons of the gene (s) and the coding region, the 5 'and 3' terminal gene flanking regions, which encode target peptides involved in obstructive dysfunction. Antisense for oncogene mRNAs and polypeptide mRNAs, or selected from the group consisting of 'and 3' intron-axon junctions and regions within 2 to 10 nucleotides of the junctions; Combinations of the oligos; And mixtures of the oligos, The polypeptides are transcription factors, stimulatory and activating peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, intrinsically produced specific and nonspecific enzymes, immunoglobulins And antibodies, antibody receptors, central nervous system (CNS) and peripheral and non-nerve receptors, CNS and peripheral and non-nerve transporters, adsorption molecules, defensins, growth factors, vascular peptides and Pharmaceutical composition, characterized in that it is selected from the group consisting of receptors, binding proteins and melanoma related proteins. The method of claim 4, wherein the encoded polypeptide (s) comprises adenosine receptors A ₁ , A _2a , A _2b and A ₃ , bradykinin receptors B ₁ and B ₂ , Nf6B transcription factor, interleukin-8 receptor (IL-8R). ), Interleukin-5 receptor (IL-5R), interleukin-4 receptor (IL-4R), interleukin-3 receptor (IL-3R), interleukin-1β (IL-1β), interleukin-1β receptor (IL-1βR) , Eotaxin, tryptase, major basic protein, β ₂ -adrenergic receptor kinase, endocylin receptor A, endocyrin receptor B, preproendocyrin, bradykinin B2 receptor, IgE high affinity receptor, interleukin 1 (IL-1), interleukin 1 receptor (IL-1R), interleukin 9 (IL-1), interleukin 9 receptor (IL-9R), interleukin 11 (IL-11), interleukin 11 receptor (IL-11R), induction Sex nitrogen monoxide synthase, cyclooxygenase-1 (COX-1), cyclo-oxygenase-2 (COX-2), intracellular adsorption protein 1 (ICAM-1), vascular cell adsorption molecule (VCAM), Lantetsu, Endo Lean leukocyte adsorption molecule (ELAM-1), monocyte activating factor, neutrophil chemotactic factor, neutrophil elastase, defensin 1, 2 and 3, muscarinic acetylcholine receptor, platelet activating factor, tumor necrosis factor α, 5- Lipooxygenase, phosphodiesterase IV, substance P, substance P receptor, histamine receptor, dentifrice, CCR-1 CC chemokine receptor, CCR-2 CC chemokine receptor, CCR-3 CC chemokine receptor, CCR- 4 CC chemokine receptor, CCR-5 CC chemokine receptor, prostanoid receptors, GATA-3 transcription factor, neutrophil adsorption receptor, MAP kinase, interleukin-9 (IL-9), NFAT transcription factor, STAT 4, MIP-1α , MCP-2, MCP-3, MCP-4, cyclophylline, phospholipase A2, basic fibroblast growth factor, metalloproteinase, CSBP / p38 MAP kinase, tryptose receptor, PDG2, interleukin-3 (IL -3), interleukin-1β (IL-β), cyclosporin A-binding protein, FK5- Binding protein, α4β1 selectin, fibronectin, α4β7 selectin, luminescent CAM-1, LFA-1 (CD11a / CD18), PECAM-1, LFA-1 selectin, C3bi, PSGL-1, E-selectin, P-selectin, CD- 34, L-selectin, p150,95, Mac-1 (CD11b / CD18), fucosyl transferase, VLA-4, CD18 / CD11a, CD11b / CD18, ICAM2 and ICAM3, C5a, CCR3 (eotaxin receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, AP-1 transcription factors, protein kinase C, cysteinyl leukotriene receptor, tachykinen receptor (tachR; tachychinnen receptors), I6B kinase 1 and 2, STAT6, c-mas and NF-interleukin-6 (NF-IL-6). The method of claim 1, wherein the one or more adenosine (A) binds but antagonizes thymidine base and is an agonist at the adenosine A ₁ , A _2a , A _2b and A ₃ receptors or the antagonist activity is less than about 0.3 of the adenosine base. Pharmaceutical composition, characterized in that substituted with a common base selected from the group consisting of heteroaromatic bases. 7. The heteroaromatic base of claim 6 wherein the heteroaromatic base is O, halo, NH ₂ , SH, SO, SO ₂ , SO ₃ , COOH and branched and conjugated primary and secondary amino, alkyl, alkenyl, alkynyl, cyclo Alkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsuloxyl, halocycloalkyl, alkylcycloalkyl, Alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, akynylaryl, arylacyl, arylalkenyl, arylalkynyl, arylcycloalkyl, and can be substituted with O, halo, NH ₂ Characterized in that it is selected from the group consisting of pyrimidines and purines which may be further substituted with primary, secondary and tertiary amines, SH, SO, SO ₂ , SO ₃ , cycloalkyl, heterocycloalkyl and heteroaryl Pharmaceutical composition. According to claim 7, wherein the pyrimidines and purines are substituted in positions 1, 2, 3, 4, 7 and 8, the pyrimidines and purines are theophylline (caffeine, di) having the formula Pharmaceutical, characterized in that it is selected from the group consisting of dyphylline, etophylline, acephylline piperazine, bamifylline, enprofylline and xanthine Composition. [Formula 1] Wherein R ¹ and R ² are independently H, alkyl, alkenyl or alkynyl, R ³ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH ₂ -alkylamino-ketoxyalkyloxy-aryl, and mono and dialkylaminoalkyl-N-alkylamino-SO ₂ -aryl.) The method of claim 8, wherein the general base is 3-nitropyrrole-2′-dioxynucleoside, 5′-nitro-indole, 2-dioxyribosyl- (5-nitroindole), 2-dioxyribofuranosyl -(5-nitroindole), 2'-deoxyinosine, 2'-deoxynebularine, 6H, 8H-3,4-dihydropyrimido [4,5-c] oxazine-7 Pharmaceutical composition, characterized in that it is selected from the group consisting of -one or 2-amino-6-methoxyaminopurine. The pharmaceutical composition of claim 1, wherein when present in the oligo, at least one methylated cytosine (s) ( ^m C) is substituted with C in at least one CpG dinucleotide. The method of claim 1, wherein at least one mononucleotide (s) of the oligo (s) is methylphosphonate, 5'-N-carbamate, phosphorus ester, phosphorothioate, phosphorodithioate, borano Phosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxyamine, methylene (methylimino ( MMI)), methoxymethyl (MOM), methoxyethyl (MOE), methyleneoxy (methylimino (MOMI)), 2'-0-methyl, phosphoramidate, C-5 substituted residues, or their Pharmaceutical composition, characterized in that bound or modified by one or more of the combinations. 12. The pharmaceutical composition of claim 11, wherein the mononucleotide residues are bound by phosphorothioate residues. The pharmaceutical composition of claim 1, wherein the antisense oligo consists of about 7 to about 60 mononucleotides. The pharmaceutical composition of claim 1, wherein the antisense oligo comprises fragments 1, 3, 5, 7 and 8 to 2313 (Seq. ID. Nos .: 1 to 2419). The agent according to claim 1, wherein the antisense oligo is operatively bound or combined with a drug agent selected from the group consisting of internalized or up-taken drug agents and cell-targeted drug agents. Pharmaceutical composition. 16. The pharmaceutical composition of claim 15, wherein the intracellular transduction or receptive drug formulation is selected from the group consisting of transferrin, asialoglycoprotein and streptavidin. The pharmaceutical composition of claim 1, wherein the oligo is efficiently bound to a prokaryotic or eucaryotic vector. The pharmaceutical composition of claim 1, wherein the oligo (s) are hybridized to ribonucleic acid. A cell comprising the oligo of claim 1. The pharmaceutical composition of claim 1, wherein the carrier or diluent is selected from the group consisting of gas, liquid and solid carriers or diluents. The pharmaceutical formulation of claim 20 further comprising a therapeutic agent selected from the group consisting of other therapeutic agents, surfactants, flavoring and coloring agents, fillers, volatile oils, buffers, dispersants, RNA inerts, antioxidants, flavors, propellants and preservatives. A pharmaceutical composition, characterized in that it contains. The pharmaceutical composition of claim 21 containing one or more oligo (s), surfactants, and carriers or diluents of oligos and surfactants. The pharmaceutical composition of claim 21, wherein the drug agent is an RNA inert agent consisting of an enzyme, optionally a ribozyme. The pharmaceutical composition of claim 1, wherein the antisense oligo is present in the composition in an amount of about 0.01 to about 99.99 w / w. The pharmaceutical composition of claim 1, which is a systemic or topical agent. Oral, intranasal, intrapulmonary, intrarectal, intrauterine, intratumoral, cranial, nasal, intramuscular, subcutaneous, intravascular, intravesical, inhaled, transdermal, intradermal, intraorbital, transplanted, iontophoretic, intraocular, vaginal, arterial The formulation of claim 25, which is selected from the group consisting of intra, ear, intravenous, intramuscular, inboard, intratracheal, lymphatic, graft, slow release and fully coded formulations. 27. The formulation of claim 26, wherein the carrier is an oral formulation selected from the group consisting of solid and liquid carriers. 28. The method of claim 27, wherein the powder, dragee, pill, capsule, spray, aerosol, solution, suspension and emulsion, optionally water-in-oil and oil-in-water An oral preparation selected from the group consisting of emulsions. The formulation of claim 25, wherein the carrier is a topical formulation selected from the group consisting of creams, gels, ointments, sprays, aerosols, patches, solutions, suspensions and emulsions. 27. The formulation according to claim 26, wherein the carrier is an injection formulation selected from the group consisting of water-soluble and alcoholic solutions and suspensions, oily solutions and suspensions, and water-in-oil and oil-in-water emulsions. 27. The formulation according to claim 26, wherein the rectal formulation is a suppository. 27. The formulation of claim 26, wherein the carrier is a transdermal formulation selected from the group consisting of water soluble and alcoholic solutions and suspensions, oily solutions and suspensions, and water-in-oil and oil-in-water emulsions. 33. The iontophoretic transdermal formulation of claim 32, wherein the carrier is selected from the group consisting of water-soluble and alcoholic solutions and suspensions, oily solutions and suspensions, and water-in-oil and oil-in-water emulsions, further comprising a transdermal delivery promoter. Formulation characterized in that. 27. The formulation of claim 26, which is provided in an implant, capsule or medicine. The pharmaceutical composition of claim 20, wherein the carrier is selected from the group consisting of water-soluble and alcoholic solutions and suspensions, oily solutions and suspensions, and water-in-oil and oil-in-water emulsions. The formulation of claim 20, wherein the carrier contains a hydrophobic carrier. The formulation of claim 36, wherein the carrier contains a lipid vesicle, any liposomes, or particles, any microcrystals. 38. The formulation of claim 37, wherein the carrier contains liposomes and the liposomes contain antisense oligos. 27. The formulation of claim 26 which is a respirable or inhalable formulation, any aerosol. A pharmaceutical composition according to claim 1 in the form of a single or plural unit. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a bulk. Delivery device; The oligo (s) of claim 1 in a separate container; And Guidelines for Adding Carriers and Using Kits Kit comprising a. 43. The kit of claim 42, wherein the formulation is a respirable formulation and the delivery device contains a nebulizer delivering a single metered amount of the formulation. 44. The kit of claim 43, wherein the nebulizer comprises an insufflator and the composition is provided in a capsule or medicine that can be penetrated or opened. 45. The kit of claim 44, wherein said delivery device is a pressurized inhaler and said composition contains a dry formulation of a suspension, solution or oligo. 46. The method of claim 45, wherein in a separate container, other therapeutic agents, surfactants, antioxidants, flavoring agents, fillers, volatile oils, dispersants, antioxidants, propellants, preservatives, buffers, RNA inactivators, cell-derived or receptors and a drug agent selected from the group consisting of an uptake agent and a colorant. 47. The formulation of claim 46, wherein the separate container contains one or more oligos, one or more surfactants and a carrier or diluent, and any other therapeutic agents. 43. The kit of claim 42, wherein the device is a transdermal delivery device, the kit further contains a transdermal delivery agent, transdermal carrier or diluent, and instructions for preparing a transdermal delivery agent. 43. The kit of claim 42, wherein the device is an iontophoretic delivery device and the kit further comprises instructions for preparing the iontophoretic drug preparation and the iontophoretic preparation. By reaching and hybridizing to the target polynucleotide (s), the antisense of the polypeptide in an amount effective to reduce the production or availability of the target mRNA, increase disruption, or reduce the amount of target polypeptide present in the lung. A method of delivering antisense oligonucleotide (s) (oligo (s)) to one or more target polynucleotide (s) selected from the group consisting of administering one or more oligo (s) to a subject's respiratory system. Reaching target polynucleotides and hybridizing thereby reducing or inhibiting transcription and / or expression of polynucleotide (s), alleviating bronchial contraction and / or pulmonary inflammation, allergic (s) and / or low production of surfactants A target poly associated with bronchial contraction and / or pulmonary inflammation, allergic (s) and / or low production of a surfactant, comprising administering to the subject the composition of claim 1 comprising an amount of oligo (s) effective for A method of delivering antisense oligonucleotides (oligos) to nucleotides in vivo. The composition of claim 51, wherein the administered composition comprises an amount of the oligo (s) and alleviates bronchial contraction and / or pulmonary inflammation, allergic (s) and / or low production of surfactant when administered to a mammal. Administered under conditions effective to do so. The method of claim 51, wherein the composition is administered to the respiratory system of the subject. The method of claim 53, wherein the composition is administered directly to the subject's lung (s). The composition of claim 51, wherein the administered composition comprises an amount of the oligo (s) and is effective in reducing the production or availability of the target mRNA, increasing disruption, or reducing the amount of polypeptide present in the lung. Characterized in that administered under conditions. 53. The method of claim 51, wherein the drug formulation is administered as a respirable aerosol. 52. The method of claim 51, wherein pulmonary occlusion and / or bronchial contraction and / or pulmonary inflammation, allergy (s) and / or low production of surfactants are associated with pulmonary vasoconstriction, inflammation, allergies, asthma, respiratory distress, respiratory distress syndrome ( RDS), pain, cystic fibrosis (CF), allergic rhinitis (AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), lung transplant rejection, lung infection, bronchitis and cancer or A method characterized by being associated with a disease. 58. The method of claim 57, wherein the disease or condition is associated with allergy (s), The oligo may comprise initiation codons, coding regions, 5′-terminal and 3′-terminal gene flanking regions, 5 ′ of immunoglobulin (s) and antibody (s), the gene (s) encoding receptors of the immunoglobulin and antibodies. And a 3 'intron-axon junction, and an antisense to a target selected from the group consisting of regions within 2 to 10 nucleotides of the junction, or immunoglobulin (s) and antibody (s), immunoglobulins and receptors for antibodies Antisense; Combination of said oligo (s); And a mixture of said oligos. The method of claim 57, wherein the disease or condition is associated with melanoma or cancer, The oligo may comprise initiation codons, coding regions, 5'- and 3'-terminal gene flanking regions, 5 'and 3' intron-axon junctions of genes encoding oncogene (s) and / or melanoma related proteins, And antisense for a target selected from the group consisting of 2 to 10 nucleotide regions of the junction, or antisense for oncogene (s) and / or melanoma related protein mRNA; Combination of said oligo (s); And mixtures of the oligos, Said oligo (s) is administered in an amount effective to reduce the amount of protein mRNA or melanoma-related protein, or to reduce the growth of melanoma cells, or to provide beneficial properties to melanoma cells. The method of claim 51, wherein the composition is administered transdermally or systemically. 61. The composition of claim 60, wherein the composition is orally, intracavitarily, intranasally, anus, vaginally, transdermally, intranasally, intravenously, subcutaneously, intramuscularly, intratumorally, intranasally To, sustained release by intraocular, intracranial, tracheal, intravascular, intravesical, by implantation, inhalation, intradermal, intrapulmonary, intraotically, slow release. And by a pump. The method of claim 51, wherein the subject is a mammal, not a human. 52. The method of claim 51, wherein the mammal is a human. The method of claim 51, wherein the oligo is administered in an amount of about 0.005 to about 150 mg / kg body weight. The method of claim 51, wherein the oligo is prepared by the following steps: (a) selecting fragments of the target nucleic acid having at least four contiguous nucleic acids selected from the group consisting of G and C; (b) obtaining a primary oligonucleotide of 4 to 60 nucleotides in length that comprises a selected fragment and has a C and G nucleic acid content of about 15% or less; And (c) obtaining secondary oligonucleotides of 4 to 60 nucleotides in length having a sequence that is antisense to the selected fragment and an adenosine (A) base content of 15% or less. 65. The method of claim 64, wherein the oligo does not contain adenosine. 53. The method of claim 51, wherein the target is an initiation codon, coding region, 5'-end and 3'-end gene flanking region, 5 ', and oncogene (s) or gene encoding a target polypeptide associated with airway dysfunction. 3 ′ intron-axon junction, and an antisense to mRNA of the polypeptide or tumor gene, or selected from the group consisting of regions within 2 to 10 nucleotides of the junction; Combination of said oligo (s); And mixtures of the oligos, The polypeptides may be transcription factors, stimulatory and activating factors, interleukins, interleukin receptors, chemokines, chemokine receptors, intrinsically produced specific and non-specific enzymes, immunoglobulins, antibody receptors. , Central nervous system (CNS) and peripheral and non-neural receptors, CNS and peripheral and non-neural peptide transporters, adsorption molecules, defensines, growth factors, The vascular peptides, peptide receptors and binding proteins, and melanoma related proteins. The method of claim 51, wherein the one or more adenosine (A) in the oligo (s) binds to thymidine base but is less than 0.3 of the base agonist activity or antagonist activity of adenosine at the adenosine A ₁ , A _2a , A _2b and A ₃ receptors. Characterized in that it is substituted by a general base selected from the group consisting of heteroaromatic bases. 69. The method of claim 68, wherein the heteroaromatic bases are O, halo, NH ₂ , SH, SO, SO ₂ , SO ₃ , COOH and branched and conjugated primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl , Heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsuloxyl, halocycloalkyl, alkylcycloalkyl, al May be substituted by kenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylacyl, arylalkenyl, arylalkynyl, arylcycloalkyl, O, halo, NH ₂ , Characterized in that it is selected from the group consisting of pyrimidines and purines which may be further substituted with primary, secondary and tertiary amines, SH, SO, SO ₂ , SO ₃ , cycloalkyl, heterocycloalkyl and heteroaryl Way. 69. The method of claim 68, wherein the pyrimidines and purines are substituted in positions 1, 2, 3, 4, 7 and 8, and the pyrimidines and purines are theophylline, caffeine, di A method selected from the group consisting of dyphylline, etophylline, acephylline, piperazine, bamifylline, enprofylline and xanthine. [Formula 1] Wherein R ¹ and R ² are independently H, alkyl, alkenyl or alkynyl, R ³ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH ₂ -alkylamino-ketoxyalkyloxy-aryl, and mono and diaalkylaminoalkyl-N-alkylamino-SO ₂ -aryl.) The method of claim 70, wherein the general base is 3-nitropyrrole-2'-dioxynucleoside, 5'-nitro-indole, 2-dioxyribosyl- (5-nitroindole), 2-dioxyribofurano Sil- (5-nitroindole), 2'-dioxyinosine, 2'-dioxynebulaline, 6H, 8H-3,4-dihydropyrimido [4,5-c] oxazine-7-one Or 2-amino-6-methoxyaminopurine. 52. The method of claim 51, further comprising replacing methylated cytosine (s) ( ^m C) with C in one or more CpG dinucleotides when present in the oligo. 52. The method of claim 51, wherein the nucleotide residue (s) of one or more of the oligo (s) is selected from methylphosphonate, phosphoroester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioform. Cetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxyamine, methylene (methylimino (MMI)), methoxymethyl ( MOM), methoxyethyl (MOE), methyleneoxy (methylimino (MOMI)), 2'-0-methyl, phosphoramidate, C-5 substituted residues, or combinations thereof And further comprising. 52. The method of claim 51, wherein the antisense oligo effectively binds to or raises the drug formulation from a group selected from the group of internalized and up-taken and cell target drug formulations. Further comprising mixing. 75. The method of claim 74, wherein said intracellular migration or recipient drug formulation is selected from the group consisting of transferrin, asialoglycoprotein, and streptavidin. 75. The method of claim 74, wherein the cellular target drug formulation is a vector, any prokaryotic vector, or an eucaryotic vector. A method of treating a disease or condition associated with a selection target associated with a disease or condition that affects the degree of obstruction comprising performing the method of claim 56. 78. The treatment of claim 77, wherein the amount of oligo (s) administered is an amount effective to reduce the production and availability of said mRNA present in the lung or to increase disruption or to reduce the amount of polypeptide. Way. 80. The amount of claim 78, wherein the amount of oligo (s) administered is an amount effective to reduce or increase disruption of the production and availability of the mRNA present in the subject's lungs, or to reduce the amount of surfactant. Treatment method characterized in that. 5. The method according to claim 4, wherein the oligo (s) comprises initiation codons, coding regions, 5′-terminal and 3′-terminal gene mapping of genes encoding adenosine A ₁ , A _2a , A _2b and / or A ₃ receptors. Antisense, or adenosine A ₁ , A _2a , A _2b and / or A ₃ receptors for a target selected from the group consisting of a region, 5 ′ and 3 ′ intron-axon junctions, and regions within 2 to 10 nucleotides of the junction Pharmaceutical composition characterized in that it is antisense against the mRNA of. 81. The pharmaceutical composition of claim 80, wherein all nucleotide binding moieties are phosphorothioates. The pharmaceutical composition of claim 1, wherein the oligo is DNA. The method of claim 1, wherein the oligo is RNA. The pharmaceutical composition of claim 1, wherein the oligo is between about 7 and about 60 mononucleotides. 81. The method of claim 80, wherein said oligo (s) are fragment (s) SEQ. ID NOS .: selected from the group consisting of 1, 3, 5, 7, 8 and / or 11 to 2419, Optionally at least one mononucleotide residue is methylphosphonate, 5'-N-carbamate, phosphorus ester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thio Hetero, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxyamine, methylene (methylimino (MMI)), methoxymethyl (MOM), Pharmaceutical compositions characterized by being substituted, bound or modified by methoxyethyl (MOE), methyleneoxy (methylimino (MOMI)), 2'-0-methyl, phosphoramidate, or combinations thereof. . 52. The method of claim 51, wherein said oligo is administered topically to the subject's respiratory tract, respiratory tract, or lung epithelium. The pharmaceutical composition of claim 1, wherein the oligo has a particle size in the range of about 5-10 μm or 10-500 μm. The pharmaceutical composition of claim 1, further comprising a propellant. 51. The method of claim 50, wherein the oligo has a particle size in the range of about 5-10 탆 or 10-500 탆. 51. The method of claim 50, further comprising adding a propellant. The method of claim 51, wherein the oligo has a particle size in the range of about 5-10 μm or 10-500 μm. 53. The method of claim 51 further comprising adding a propellant.