[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2002085309A2 - Composition, preparations et trousses pour le traitement de maladie respiratoire et pulmonaire au moyen d'oligonucleotides antisens et d'un agent bronchodilateur - Google Patents

Composition, preparations et trousses pour le traitement de maladie respiratoire et pulmonaire au moyen d'oligonucleotides antisens et d'un agent bronchodilateur Download PDF

Info

Publication number
WO2002085309A2
WO2002085309A2 PCT/US2002/013143 US0213143W WO02085309A2 WO 2002085309 A2 WO2002085309 A2 WO 2002085309A2 US 0213143 W US0213143 W US 0213143W WO 02085309 A2 WO02085309 A2 WO 02085309A2
Authority
WO
WIPO (PCT)
Prior art keywords
agents
receptor
composition
adenosine
receptors
Prior art date
Application number
PCT/US2002/013143
Other languages
English (en)
Other versions
WO2002085309A9 (fr
Inventor
Jonathan W Nyce
Yukui Li
Anthony Sandrasagra
Evan Katz
Jonathan Pabalan
Douglas Aguilar
Shoreh Miller
Lei Tang
Syed Shahabuddin
Original Assignee
Epigenesis Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Epigenesis Pharmaceuticals, Inc. filed Critical Epigenesis Pharmaceuticals, Inc.
Priority to AU2002305236A priority Critical patent/AU2002305236A1/en
Publication of WO2002085309A2 publication Critical patent/WO2002085309A2/fr
Priority to US10/627,930 priority patent/US20040049022A1/en
Publication of WO2002085309A9 publication Critical patent/WO2002085309A9/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/331—Universal or degenerate base

Definitions

  • This patent relates to a composition
  • a composition comprising a carrier, oligonucleotides (oligos) that are anti-sense to adenosine receptors, and contain low amounts of or no adenosine (A), and bronchodilating agents.
  • oligos oligonucleotides
  • A adenosine
  • bronchodilating agents oligonucleotides
  • These agents and the composition and formulations provided are suitable for the treatment of respiratory tract, pulmonary and malignant diseases associated with bronchoconstriction, respiratory tract inflammation and allergies, impaired airways, including lung disease and diseases whose secondary effects afflict the lungs of a subject, such as allergies, asthma, impeded respiration, allergic rhinitis, pain, cystic fibrosis, pulmonary fibrosis, RDA, COPD, and cancers, among others.
  • the present agents and composition may be adrninistered preventatively, prophylactically or therapeutic
  • ARDS Acute Respiratory Distress Syndrome
  • Asthma is a condition characterized by variable, in many instances reversible obstruction of the airways. This process is associated with lung inflammation and in some cases lung allergies. Many patients have acute episodes referred to as “asthma attacks,” while others are afflicted with a chronic condition. The asthmatic process is believed to be triggered in some cases by inhalation of antigens by hypersensitive subjects. This condition is generally referred to as “extrinsic asthma.” Other asthmatics have an intrinsic predisposition to the condition, which is thus referred to as “intrinsic asthma,” and may be comprised of conditions of different origin, including those mediated by the adenosine rece ⁇ tor(s), allergic conditions mediated by an immune IgE-mediated response, and others.
  • ARDS Acute Respiratory Distress Syndrome
  • ARDS occurs as a medical emergency and may be caused by other conditions that directly or indirectly cause the blood vessels to "leak" fluid into tire lungs.
  • ARDS the ability of the lungs to expand is severely decreased and produces extensive damage to the air sacs and lining or endothelium of the lung. ARDS' most common symptoms are labored, rapid breathing, nasal flaring, cyanosis blue skin, lips and nails caused by lack of oxygen to the tissues, breathing difficulty, anxiety, stress, tension, joint stiffness, pain and temporarily absent breathing. ARDS is commonly diagnosed by testing for symptomatic signs, for example by a simple chest auscultation or examination with a stethoscope that may reveal abnormal symptomatic breath sounds. A preliminary diagnosis of ARDS may be confirmed with chest X-rays and the measurement of arterial blood gas.
  • ARDS appears to be associated with other diseases, such as acute myelogenous leukemia, with acute tumor lysis syndrome (ATLS) developed after treatment with, e. g. cytosine arabinoside.
  • ATLS acute tumor lysis syndrome
  • ARDS appears to be associated with traumatic injury, severe blood infections such as sepsis, or other systemic illness, high dose radiation therapy and chemotherapy, and inflammatory responses which lead to multiple organ failure, and in many cases death.
  • premies premature babies
  • the lungs are not quite developed and, therefore, the fetus is in an anoxic state during development.
  • lung surfactant a material critical for normal respiration
  • premies often hyper-express the adenosine Ai receptor and/or underexpress the adenosine A 2a receptor and are, therefore, susceptible to respiratory problems including bronchoconstriction, lung inflammation and ARDS, among others.
  • RDS Respiratory Distress Syndrome
  • Preterm infants exhibiting RDS are currently treated by ventilation and administration of oxygen and surfactant preparations.
  • BPD bronchopulmonary dysplasia
  • Adenosine administered by inhalation is known to cause bronchoconstriction in asthmatics, possibly due to mast cell degranulation and histamine release, effects which have not been observed in normal subjects.
  • Adenosine infusion has caused respiratory compromise, for example, in patients with COPD.
  • adenosine As a consequence of the untoward side effects observed in many patients, caution is recommended in the prescription of adenosine to patients with a variety of conditions, including obstructive lung disease, emphysema, bronchitis, etc, and complete avoidance of its administration to patients with or prone to bronchoconstriction or bronchospasm, such as asthma.
  • the administration of adenosine must be discontinued in any patient who develops severe respiratory difficulties. It would be of great help if a formulation were to be made available for joint use when adenosine administration is required.
  • Allergic rhinitis afflicts one in five Americans, accounting for an estimated $4 to 10 billion in health care costs each year, and occurs at all ages. Because many people mislabel their symptoms as persistent colds or sinus problems, allergic rhinitis is probably underdiagnosed.
  • IgE combines with allergens in the nose to produce chemical mediators, induction of cellular processes, and neurogenic stimulation, causing an underlying inflammation. Symptoms include nasal congestion, discharge, sneezing, and itching, as well as itchy, watery, swollen eyes.
  • allergic rhinitis sufferers often develop sinusitis, otitis media with effusion, and nasal polyposis, and may exacerbate asthma, and is associated with mood and cognitive disturbances, fatigue and irritability.
  • Degranulation of mast cells results in the release of preformed mediators that interact with various cells, blood vessels, and mucous glands to produce the typical rhinitis symptoms.
  • Most early- and late-phase reactions occur in the nose after allergen exposure. The late-phase reaction is seen in chronic allergic rhinitis, with hypersecretion and congestion as the most prominent symptoms. Repeated exposure causes a hypersensitivity reaction to one or many allergens. Sufferers may also become hyperreactive to nonspecific triggers such as cold air or strong odors.
  • Nonallergic rhinitis may be induced by infections, such as viruses, or associated with nasal polyps, as occurs in patients with aspirin idiosyncrasy.
  • infections such as viruses
  • nasal polyps such as viruses
  • pregnancy, hypothyroidism, and exposure to occupational factors or medications can cause rhinitis, as well.
  • NARES syndrome a non-allergic type of rhinitis associated with eosinophils in the nasal secretions, typically occurs in middle-aged individuals and is accompanied by loss of smell.
  • Saline is often recommended to improve nasal stuffiness, sneezing, and congestion, and saline sprays usually relieve mucosal irritation or dryness associated with various nasal conditions, minimize mucosal atrophy, and dislodge encrusted or thickened mucus, while causing no side effects, and may be tried first in pregnant patients. Also, if used immediately before intranasal corticosteroid dosing, saline helps prevent local irritation.
  • Anti-histamines often serve as a primary therapy.
  • Terfenadine and astemizole, two non-sedating anti-histarnines have been associated with a ventricular arrhythmia known as Torsades de Points, usually in interaction with other medications such as ketoconazole and erythromycin, or secondary to an underlying cardiac problem.
  • Torsades de Points a ventricular arrhythmia known as Torsades de Points
  • loratadine another nonsedating anti-histamine, and cetirizine have not been associated with serious adverse cardiovascular events, the most common side effect of cetirizine being drowsiness.
  • Claritin may be effective in relieving sneezing, runny nose, and nasal, ocular and palatal itching in a low percentage of patients, although not approved for this indication or asthma.
  • Aiiti-hista ines are typically combined with a decongestant to help relieve nasal congestion.
  • Sympathomimetic medications are used as vasoconstrictors and decongestants, the three most common decongestants being pseudoephedrine, phenylpropanolamine and phenylephrine. These agents, however, cause hypertension, palpitations, tachycardia, restlessness, insomnia and headache.
  • Topical decongestants are recommended for a limited period of time, as their overuse results in nasal dilatation.
  • Anti-cholinergic agents such as Cromolyn
  • Cromolyn have a role in patients with significant rhinorrhea or for specific entities such as "gustatory rhinitis", which is usually associated with ingestion of spicy foods, and have been used on the common cold.
  • the Cromolyn spray produces sneezing, transient headache, and even nasal burning.
  • Topical and nasal spray corticosteroids such as Vancenase are effective agents in the treatment of rhinitis, especially for symptoms of congestion, sneezing, and runny nose, but often cause irritation, stinging, burning, sneezing, local bleeding and septal perforation.
  • Topical steroids are generally more effective than Cromolyn Sodium, particularly in the treatment of NARES, but side effects limit their usefulness except for temporary therapy in patients with severe symptoms.
  • Immunotherapy while expensive and inconvenient, often can provide substantial benefits, especially the use of drugs that produce blocking antibodies, alter cellular histamine release, and result in decreased IgE.
  • Presently available treatments such as propranolol, verapamil, and adenosine, may help to minimize symptoms.
  • Verapa il is most commonly used but it has several shortcomings, since it causes or exacerbates systemic hypotension, congestive heart failure, bradyarrhythmias, and ventricular fibrillation.
  • verapamil readily crosses the placenta and has been shown to cause fetal bradycardia, heart block, depression of contractility, and hypotension.
  • Adenosine has several advantages over verapamil, including rapid onset, brevity of side effects, theoretical safety, and probable lack of placental transfer, but may not be administered to a variety of patients.
  • Chronic obstructive pulmonary disease COPD
  • COPD chronic obstructive pulmonary disease
  • Emphysema is characterized by abnormal permanent enlargement of the air spaces distal to the terminal bronchioles, accompanied by destruction of their walls and without obvious fibrosis.
  • Chronic bronchitis is characterized by chronic cough, mucus production, or both, for at least three months for at least two successive years where other causes of chronic cough have been excluded.
  • COPD characteristically affects middle aged and elderly people, and is one of the leading causes of morbidity and mortality worldwide. In the United States it affects about 14 million people and is the fourth leading cause of death. Both morbidity and mortality, however, are rising. The estimated prevalence of this disease in the United States has risen by 41% since 1982, and age adjusted death rates rose by 71% between 1966 and 1985. This contrasts with the decline over the same period in age-adjusted mortality from all causes (wliich fell by 22%), and from cardiovascular diseases (which fell by 45%).
  • COPD COPD
  • the disease is rare in lifetime non-smokers, in whom exposure to environmental tobacco smoke will explain at least some of the airways obstruction.
  • Other proposed etiological factors include airway hyper- responsiveness or hypersensitivity, ambient air pollution, and allergy.
  • the airflow obstruction in COPD is usually progressive in people who continue to smoke. This results in early disability and shortened survival time. Stopping smoking reverts the decline in lung function to values for non-smokers. Many patients will use medication chronically for the rest of their lives, with the need for increased doses and additional drugs during exacerbations.
  • Pulmonary fibrosis, interstitial lung disease (ILD), or interstitial pulmonary fibrosis include more than 130 chronic lung disorders that affect the lung by damaging lung tissue, and producing inflammation in the walls of the air sacs in the lung, scarring or fibrosis in the interstitium (or tissue between the air sacs), and stiffening of the lung, thus the name of the disease. Breathlessness during exercise may be one of the first symptoms of these diseases, and a dry cough may be present. Neither the symptoms nor X-rays are often sufficient to tell apart different types of pulmonary fibrosis. Some pulmonary fibrosis patients have known causes and some have unknown or idiopathic causes. The course of this disease is generally unpredictable. Its progression includes thickening and stiffening of the lung tissue, inflammation and difficult breathing. Some people may need oxygen therapy as part of their treatment.
  • Cancer is one of the most prevalent and feared diseases of our times. It generally results from the carcinogenic transformation of normal cells of different epitheha. Two of the most damaging characteristics of carcinomas and other types of malignancies are their uncontrolled growth and their ability to create metastases in distant sites of the host, particularly a human host. It is usually these distant metastases that may cause serious consequences to the host since frequently the primary carcinoma is removed by surgery.
  • the treatment of cancer presently relies on surgery, irradiation therapy and systemic therapies such as chemotherapy, different irnmunity- boosting medicines and procedures, hyperthermia and systemic, radioactively labeled monoclonal antibody treatment, immunotoxins and chemotherapeutic drugs.
  • Adenosine may constitute an important mediator in the lung for various diseases, including bronchial asthma, COPD, CF, RDS, rhinitis, pulmonary fibrosis, and others. Its potential role was suggested by the finding that asthmatics respond favorably to aerosolized adenosine with marked bronchoconstriction whereas normal individuals do not.
  • adenosine-induced bronchoconstriction and bronchial hyperresponsiveness in asthma may be mediated primarily through the stimulation of adenosine receptors.
  • Adenosine has also been shown to cause adverse effects, including death, when administered therapeutically for other diseases and conditions in subjects with previously undiagnosed hyper reactive airways.
  • Adenosine is a purine involved in intermediary metabolism, and may constitute an important natural mediator of many of diseases.
  • Adenosine plays a unique role in the body as a regulator of cellular metabolism. It can raise the cellular level of AMP, ADP and ATP which are the energy intermediates of the cell.
  • Adenosine can stimulate or down regulate the activity of adenylate cyclase and hence regulate cAMP levels.
  • cAMP plays a role in neurotransmitter release, cellular division and hormone release.
  • Adenosine's major role appears to be to act as a protective injury autocoid. In any condition in which ischemia, low oxygen tension or trauma occurs adenosine appears to play a role.
  • adenosine has been postulated to contribute to the over activity of the brain excitatory amino acid neurotransmitters, and hence various pathological states.
  • Adenosine has also been implicated as a primary determinant underlying the symptoms of bronchial asthma and other respiratory diseases, the induction of bronchoconstriction and the contraction of airway smooth muscle.
  • adenosine causes bronchoconstriction in asthmatics but not in non-asthmatics.
  • Other data suggest the possibility that adenosine receptors may also be involved in allergic and inflammatory responses by reducing the hyperactivity of the central dopaminergic system.
  • Adenosine is said to inhibit the production of super-oxide by stimulated neutrophils. Recent evidence suggests that adenosine may also play a protective role in stroke, CNS trauma, epilepsy, ischemic heart disease, coronary by-pass, radiation exposure and inflammation. Overall, adenosine appears to regulate cellular metabolism through ATP, to act as a carrier for methionine, to decrease cellular oxygen demand and to protect cells from ischemic injury. Adenosine is a tissue hormone or inter- cellular messenger that is released when cells are subject to ischemia, hypoxia, cellular stress, and increased workload, and or when the demand for ATP exceeds its supply.
  • Adenosine is a purine and its formation is directly linked to ATP catabolism. It appears to modulate an array of physiological processes including vascular tone, hormone action, neural function, platelet aggregation and lymphocyte differentiation. It also may play a role in DNA formation, ATP biosynthesis and general intermediary metabolism. It is suggested that it regulates the formation of cAMP in the brain and in a variety of peripheral tissues. Adenosine regulates cAMP formation through two receptors A ! and A 2 . Via Ai receptors, adenosine reduces adenylate cyclase activity, while it stimulates adenylate cyclase at A 2 receptors.
  • the adenosine Ai receptors are more sensitive to adenosine than the A 2 receptors.
  • the CNS effects of adenosine are generally believed to be Ai-receptor mediated, where as the peripheral effects such as hypotension, bradycardia, are said to be A 2 receptor mediated.
  • Anti-sense oligonucleotides have received considerable theoretical consideration as potential useful pharmacological agents in human disease. Their practical application in actual models of human disease, however, has been somewhat elusive. One important impediment to their effective application has been a difficulty in finding an appropriate route of administration to deliver them to their site of action. Many in vivo experiments were conducted by administering anti-sense oligonucleotides directly to specific regions of the brain.
  • anti-sense oligonucleotides have received considerable theoretical consideration for their potential use as pharmacological agents in human disease, finding practical and effective applications for these agents in actual models of human disease, however, have been few and far between, particularly because they had to be administered in large doses.
  • Another important consideration in the pharmacologic application of these molecules is their route of administration.
  • Many in vivo applications have involved the direct administration of anti-sense oligonucleotides to limited regions of the brain. Such applications, however, have limited clinical utility due to their invasive nature.
  • anti-sense oligonucleotides as pharmacological agents has been found to have also significant problems, not the least of which being an inherent difficulty in targeting disease-involved tissues. That is, the necessary dilution of the anti- sense oligonucleotide in the circulatory system makes extremely difficult to attain a therapeutic dose at the target tissue by intravenous or oral adminisfration.
  • the bioavailability of orally administered anti-sense oligonucleotides is very low, of the order of less than about 5%.
  • Anti-sense oligonucleotides have been used in therapy by many, including the present inventor, who in his previous work successfully treated various diseases and conditions by direct administration of these agents to the lung.
  • the route of administration may be of extreme importance for treating generalized diseases and conditions as well as those that are localized.
  • the delivery of anti-sense agents to the lung has been relatively undeveloped.
  • the lung is an excellent target for the direct administration of anti-sense oligonucleotides and provides a non-invasive and a tissue-specific route.
  • Theophylline an important drug in the treatment of asthma, is a known adenosine receptor antagonist that was reported to eliminate adenosine-mediated bronchoconstriction in asthmatic rabbits.
  • a selective adenosine Ai receptor antagonist, 8-cyclopentyl-l, 3-dipropylxanthine (DPCPX) was also reported to inhibit adenosine-mediated bronchoconstriction and bronchial hyperresponsiveness in allergic rabbits.
  • DPCPX 8-cyclopentyl-l, 3-dipropylxanthine
  • Theophylline for example, has been widely used in the treatment of asthma, but is associated with frequent, significant toxicity resulting from its narrow therapeutic dose range.
  • DPCPX is far too. toxic to be useful clinically.
  • glucocorticosteroids are beclomethasone and corticoid 21-sulfopropionates.
  • a bronchodilator are an older ⁇ l adrenergic agonist such as albuterol, and a newer one such as sahneterol.
  • glucocorticosteroids are taken daily either by inhalation or orally, they attenuate inflammation.
  • the ⁇ l adrenergic agonists on the other hand, primarily alleviate bronchoconstriction.
  • glucocorticosteroids are not useful in general for acute settings, bronchodilators are used in acute care, such as in the case of asthma attacks.
  • many asthma patients require daily use of both types of agents, a glucocorticosteroid to contain pulmonary inflammation, and a bronchodilator to alleviate bronchoconstriction.
  • fluticasone propionate a glucocorticoid steroid was combined with ⁇ l adrenergic agonists in one therapeutic formulation said to have greater efficiency in the treatment of asthma.
  • glucocorticosteroids particularly when taken for prolonged periods of time, have extremely deleterious side effects that, although somewhat effective, make their chronic use undesirable, particularly in children.
  • the present invention generally relates to a pharmaceutical or veterinary composition, comprising an anti- sense oligonucleotide(s) (oligo(s)) and bronchodilating agents that are effective for alleviating bronchoconstriction, respiratory tract inflammation and allergies, surfactant depletion or hyposecretion, abnormal adenosine or adenosine receptor levels, or ubiquinone depletion, when administered to a subject.
  • an anti- sense oligonucleotide(s) oligo(s)
  • bronchodilating agents that are effective for alleviating bronchoconstriction, respiratory tract inflammation and allergies, surfactant depletion or hyposecretion, abnormal adenosine or adenosine receptor levels, or ubiquinone depletion, when administered to a subject.
  • the composition comprises a first active agent contains between about 0 to about 15% adenosine (A) and is anti-sense to a target comprising the initiation codon, the coding region, the 5'-end or the 3'-end genomic flanking regions, the 5' or 3' infron-exon junctions, and/or regions within 2 to 10 nucleotides of the junctions of at least one gene encoding or regulating expression of a polypeptide associated with lung airway dysfunction or cancer, or anti-sense to the corresponding mRNA; combinations, multiple target anti-sense oligos (MTAs), or mixtures thereof; a second active agent comprising a bronchodilating agent(s); and a pharmaceutically or veterinarily acceptable carrier or diluent.
  • A adenosine
  • the oligo and bronchodilating agent of the invention may be applied to the preparation of a medicament for
  • oligos may be directed to any pre-selected target, and in one embodiment they are intended for fast delivery through the mucosal tissue of the lungs or otherwise for hybridization to a desired target polynucleotide to prevent gene franscription and franslation, such that protein expression will be reduced, or completely stopped.
  • the targets for the first active agent(s) are typically molecules associated with airway, lung and cancer disease, and include transcription factors, stimulating and activating peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, endogenously produced specific and non-specific enzymes, immunoglobulins and antibodies, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and peripheral nervous and non-nervous system peptide fransmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors, binding proteins, and malignancy associated proteins, among others.
  • the bronchodilating agents are known in the art as are their dosages.
  • the oligos and bronchodilating agents are provided for separate and joint administration in the form of individual compositions and formulations, with a carrier or diluent, and optionally with other therapeutic agents and formulation additives for administration by specific routes, e. g. into the respiratory system, topically, fransdermally, parenterally, by implantation, and the like.
  • the oligo and the bronchodilating agent are also provided in a capsule, cartridge or blister, and in the form of a kit.
  • Figure 1 illustrates the inhibition of HT-29 SF cells by DHEA.
  • Figures 2A and 2B illustrate the effects of different amounts of DHEA on cell cycle distribution in HT-29 SF cells.
  • Figures 3A and 3B illustrate the reversal of DHEA-induced growth inhibition in HT-29 cells treated with CON: Confrol; MVA: Mevalonic Acid; SQ: Squaline; CH: Cholesterol; DN: Deoxyribonucleodies; RN: Ribonucleosides.
  • Figures 4A, 4B, 4C and 4D illustrate the reversal of DHEA-induced GI arrest in HT-29 SF cells for different durations of treatment with DHEA.
  • This invention arose from a desire by the inventor to improve on prior treatments for respiratory, pulmonary, malignant, and other diseases, including his own prior discovery that specifically targeted anti-sense oligonucleotides (oligos) may be utilized preventatively, prophylactically and therapeutically in the treatment of diseases that impair the respiratory tract, including bronchoconstriction, inflammation, allergies, wheezing, dirninished lung function, constricted bronchial tissue, obstructed lung airways, depleted surfactant secretion, or otherwise impeded normal breathing.
  • diseases that impair the respiratory tract, including bronchoconstriction, inflammation, allergies, wheezing, dirninished lung function, constricted bronchial tissue, obstructed lung airways, depleted surfactant secretion, or otherwise impeded normal breathing.
  • diseases are associated with or cause inflammation, constrict bronchial tissue or the lung airways, deplete secretion of surfactant, augment allergies, or otherwise impede normal breathing.
  • This treatment combines the selectivity of antisense oligonucleotides for specific targets associated with or mediating these symptoms, with the bronchodilating effects afforded by different classes of molecules.
  • the oligos are generally administered in low doses and, therefore, evidence minor to no detrimental side effects.
  • the inventor wanted to provide a treatment that would improve the outcome and life style of patients undergoing other procedures or being administered other therapies, including antibody therapy, chemotherapy, radiation, phototherapy, and surgery such as cancer and other surgeries, that could be effectively aclministered preventatively, prophylactically or therapeutically.
  • the present invention is premised on the discovery by the inventor that the combination of these two very different classes of medicaments would provide a potentiating effect never before seen of their individual beneficial activities.
  • ARDS' most common symptoms are labored, rapid breathing, nasal flaring, cyanosis blue skin, lips and nails caused by lack of oxygen to the tissues, breathing difficulty, anxiety, stress, tension, joint stiffness, pain and temporarily absent breathing.
  • ARDS is currently diagnosed by mere symptomatic signs, e. g. chest auscultation with a stethoscope that may reveal abnormal symptomatic breath sounds, and confirmed with chest X-rays and the measurement of arterial blood gas.
  • ARDS in some instances, appears to be associated with other diseases, such as acute myelogenous leukemia, acute tumor lysis syndrome (ATLS) developed after treatment with, e. g.
  • ATLS acute tumor lysis syndrome
  • ARDS cytosine arabinoside, etc.
  • ARDS is associated with traumatic injury, severe blood infections such as sepsis or other systemic illness, high-dose radiation therapy and chemotherapy, and inflammatory responses which lead to multiple organ failure and in many cases death.
  • premies premature babies
  • the lungs are not quite developed and, therefore, the fetus is in an anoxic state during development.
  • lung surfactant a material critical for normal respiration
  • premies often hyper-express the adenosine Al receptor and/or underexpress the adenosine A2a receptor and are, therefore, susceptible to respiratory problems including bronchoconstriction, lung inflammation and ARDS, among others.
  • RDS Respiratory Distress Syndrome
  • Preterm infants exhibiting RDS are currently treated by ventilation and a ⁇ nrinisfration of oxygen and surfactant preparations.
  • BPD bronchopulmonary dysplasia
  • Rhinitis may be seasonal or perennial, allergic or non-allergic.
  • Non-allergic rhinitis may be induced by infections, such as viruses, or associated with nasal polyps, as occurs in patients with aspirin idiosyncrasy. Medical conditions such as pregnancy or hypothyroidism and exposure to occupational factors or medications may cause rhinitis.
  • NARES syndrome is a non-allergic type of rhinitis associated with eosinophils in the nasal secretions, which typically occurs in middle-age and is accompanied by some loss of sense of smell. When cholinergic pathways are stimulated they produce typical secretions that are identified by their glandular constituents so as to implicate neurologic stimulation.
  • saline sprays are generally used to relieve mucosal irritation or dryness associated with various nasal conditions, minimize mucosal atrophy, and dislodge encrusted or thickened mucus. If used immediately before infranasal corticosteroid dosing, saline sprays may help prevent drug-induced local irritation.
  • Anti-liistamines such as terfenadine and astemizole, two non- sedating anti-l ⁇ stamines, are also employed to treat this condition, but have been associated with a ventricular arrhythmia known as Torsades de Points, usually in interaction with other medications such as ketoconazole and erythromycin, or secondary to an underlying cardiac problem.
  • Loratadine, another non-sedating anti-histamine, and cetirizine have not been associated with an adverse impact on the QT interval, or with serious adverse cardiovascular events. Cetirizine, however, produces extreme drowsiness and has not been widely prescribed.
  • Non- sedating anti-Mstamines e. g.
  • Claritin may produce some relieving of sneezing, runny nose, and nasal, ocular and palatal itching, but have not been tested for asthma or other more specific conditions.
  • Terfenadine, loratadine and astemizole exhibit extremely modest bronchodilating effects, reduction of bronchial hyper- reactivity to histamine, and protection against exercise- and antigen-induced bronchospasm. Some of these benefits, however, require higher-man-currently-recommended doses.
  • the sedating-type anti-Mstarnines help induce night sleep, but they cause sleepiness and compromise performance if taken during the day. When employed, anti- histamines are typically combined with a decongestant to help relieve nasal congestion. Sympathomimetic medications are used as vasoconstrictors and decongestants.
  • Anti-cholinergic agents are given to patients with significant rhinorrhea or for specific conditions such as "gustatory rhinitis", usually caused by ingestion of spicy foods, and may have some beneficial effects on the common cold.
  • Cromolyn for example, if used prophylactically as a nasal spray, reduces sneezing, rhinorrhea, and nasal pruritus, and blocks both early- and late-phase hypersensitivity responses, but produces sneezing, transient headache, and even nasal burning.
  • Topical corticosteroids such as Vancenase are somewhat effective in the treatment of rhinitis, especially for symptoms of congestion, sneezing, and runny nose.
  • corticosteroid nose sprays may cause irritation, stinging, burning, or sneezing, as well.
  • Local bleeding and septal perforation can also occur sometimes, especially if the aerosol is not aimed properly.
  • Topical steroids generally are more effective than cromolyn sodium, particularly in the treatment of NARES, and also to reduce some symptoms of rhinitis.
  • Their side effects limit their usefulness except for temporary therapy in patients with severe symptoms. These agents are sometimes used for shrinking nasal polyps when local therapy fails.
  • Immunotherapy while expensive and inconvenient, often provides benefits, especially for inpatients who experience side effects from other medications.
  • This effect is useful in IgE-mediated diseases, e. g., hypersensitivity in atopic patients with recurrent middle ear infections.
  • IgE-mediated diseases e. g., hypersensitivity in atopic patients with recurrent middle ear infections.
  • a runny nose is more than a nuisance.
  • the disorder often results in impaired quality of life and sets the stage for more serious ailments, including psychological problems.
  • rhinitis is mostly treated with propranolol, verapamil, and adenosine, all of which have Food and Drug A ⁇ ninisfration-approved labeling for acute termination of supravenfricular tachycardia (SVT).
  • SVT supravenfricular tachycardia
  • Iprafropium bromide Three treatments compared over a five year period found that iprafropium bromide had no significant effect on the decline in the functional effective volume of the patient's lungs whereas smoking cessation produced a slowing of the decline in the functional effective volume of the lungs. Iprafropium bromide, however, produced serious adverse effects, such as cardiac symptoms, hypertension, skin rashes, and urinary retention. Short and long acting inhaled ⁇ l adrenergic agonists achieve short-term bronchodilation and provide some symptomatic relief in COPD patients, but show no meaningful maintenance effect on the progression of the disease.
  • Short acting ⁇ l adrenergic agonists improve symptoms in subjects with COPD, such as increasing exercise capacity and produce some degree of bronchodilation, and even an increase in lung function in some severe cases.
  • the maximum effectiveness of the newer long acting inhaled ⁇ l adrenergic agonists was found to be comparable to that of short acting ⁇ l adrenergic agonists.
  • Salmeterol was found to improve symptoms and quality of life, although only producing modest or no change in lung function. In asthmatics, however, ⁇ l adrenergic agonists have been linked to an increased risk of death, worsened control of astlima, and deterioration in lung function.
  • Theophyllines appear to have some bronchodilatory effect in COPD patients whereas they have some common adverse effects, although they have a small therapeutic range given that blood concentrations of 15-20 mg/1 are required for optimal effects.
  • Adverse effects include nausea, diarrhea, headache, irritability, seizures, and cardiac arrhythmias, and they occur at highly variable blood concentrations and, in many people, they occur within the therapeutic range.
  • the theophyllines' doses must be adjusted individually according to smoking habits, infection, and other treatments, wliich is cumbersome. Although theophyllines have been claimed to have an anti-inflammatory effect in asthma, especially at lower doses, none has been reported in COPD, although their bronchodilating short- term effect appears to be statistically different from placebo .
  • Oral corticosteroids show some improvement in baseline functional effective volume in stable COPD patients whereas systemic corticosteroids have been found to be harmful at least producing some osteoporosis and inducing overt diabetes.
  • the longer term use of oral corticosteroids may be useful in COPD, but it usefulness must be weighed against their substantial adverse effects.
  • Inhaled corticosteroids have been found to have no real short- term effect in airway hyper-responsiveness to histamine, but a small long-term effect on lung function, e. g., in pre- bronchodilator functional effective volume.
  • Fluticasone tteatment of COPD patients showed a significant reduction in moderate and severe (but not mild) exacerbations, and a small but significant improvement in lung function and six minute walking distance.
  • Oral prednisolone, inhaled beclomethasone or both had no effects in COPD patients, but lung function improved oral corticosteroids.
  • Mucolytics have a modest beneficial effect on the frequency and duration of exacerbations but an adverse effect on lung function.
  • Neither N-acetylcysteine nor other mucolytics have a significant effect in people with severe COPD (functional effective volume ⁇ 50%) in spite of evidencing greater reductions in frequency of exacerbation.
  • N-acetylcysteine produced gastrointestinal side effect.
  • pulmonary fibrosis and other ILDs may vary from person to person, they have one common link: they affect parts of the lung.
  • bronchiolitis When inflammation involves the walls of the bronchioles (small airways), it is called bronchiolitis, when it involves the walls and air spaces of the alveoli (air sacs), it is called alveolitis, and when it involves the small blood vessels (capillaries) of the lungs, it is called vasculitis.
  • the inflammation may heal, or it may lead to permanent scarring of the lung tissue, in which case it is called pulmonary fibrosis.
  • Pulmonary fibrosis is caused by, or takes the form of, occupational and environmental exposure to irritants such as asbestos, silica and metal dusts, bacteria and animal dusts, gases and fumes, asbestosis and silicosis, infections that produce lung scarring, of which tuberculosis is one example, connective tissue or collagen diseases such as Rheumatoid Arthritis,
  • Systemic Sclerosis and Systemic Lupus Erythematosis idiopathic pulmonary fibrosis and, although not as common, pulmonary fibrosis of genetic/familial origin and certain medicines.
  • Many of the diseases are often named after the occupations with which they are associated, such as Grain handler's lung, Mushroom worker's lung, Bagassosis, Detergent worker's lung, Maple bark stripper's lung, Malt worker's lung, Paprika splitter's lung, and Bird breeder's lung.
  • IPF pulmonary fibrosis
  • Glucocorticosteroids are usually administered to treat inflammation present in pulmonary fibrosis, with inconclusive results. Other drugs, however, are not usually added until it is clear that the steroids are not effective in reversing the disease. Glucocorticosteroids are also used in combination with other drugs when a diagnosis is first established., for example oxygen therapy prescribed in severe cases. The adminisfration of influenza and pneumococcal pneumonia vaccines is often recommended in pulmonary fibrosis and more generally for all lung diseases to prevent infection.
  • Pulmonary fibrosis may also be caused by other specific diseases, such as sarcoidosis, a disease whose cause is unknown, that is characterized by the formation of granulomas or areas of inflammatory cells. The disease may attack any organ of the body, but most frequently attacks the lungs, and is generally diagnosed when a chest x-ray shows enlarged lymph glands in the center of both lungs or evidence of lung tissue thickening.
  • sarcoidosis is a minor problem, and symptoms including dry cough, shortness of breath, mild chest pain, fatigue, weakness and weight loss-may appear infrequently and stop even without medication. For others, it is a serious, disabling disease that affects African-americans more than members of any other race, although almost everybody may develop the disease, most common in young adults 20 to 40. Histiocytosis X, also associated with pulmonary fibrosis, seems to begin in the bronchioles or small airways of the lungs and their associated arteries and veins, and is generally followed by destruction of the bronchioles and narrowing and damaging of small blood vessels. It is diagnosed by a bronchoalveolar lavage test involving the removal and identification of cells from the lower respiratory tract. Symptoms of this disease include a dry cough
  • Asbestosis and silicosis are two occupational lung diseases whose causes are known. Asbestosis is caused by small needle-like particles of asbestos inhaled into the lungs, and cause lung scarring or pulmonary fibrosis that may lead to lung cancer. Silicosis is a dust disease that comes from breathing in free crystalline silica dust, and is produced by all types of mining in which the ore, e. g. gold, lead, zinc, copper, iron, anthracite (hard) coal, and some bituminous (soft) coal, are extracted from quartz rock. Workers in foundries, sandstone grinding, tunneling, sandblasting, concrete breaking, granite carving, and china manufacturing also encounter silica.
  • the ore e. g. gold, lead, zinc, copper, iron, anthracite (hard) coal, and some bituminous (soft) coal
  • oligonucleotides When oligonucleotides are metabolized in vivo, those that contain adenosine (A) break down and release amounts of this purine that, in turn, activates adenosine receptors and cause bronchoconstriction, inflammation, surfactant depletion, and respiratory tract allergies, among other symptoms.
  • A adenosine
  • the inventor thus, conceived of employing low adenosine and adenosine free oligos to avoid these side effects upon their adminisfration.
  • compositions, formulations and methods employing these oligos and bronchodilating agents that afford greatly improved results when compared with previously known treatments for bronchoconstriction, allergies, inflammation, breathing difficulties, surfactant depletion and blockage of airways, as well as for other conditions which affect the lung directly or indirectly.
  • one or more nucleic acids of the invention may be formulated alone, with one or more bronchodilating agents and/or with one or more surfactant components and a carrier, and optionally with other therapeutic agents and formulation agents.
  • the compositions of this invention may be incorporated into a variety of formulations for systemic and topical at ⁇ minisfration that are described below in some detail.
  • a preferred method for the delivery of anti-sense oligonucleotides (oligos) and bronchodilating agents is through the respiratory system, as a fast means of jump-starting treatment to address acute attacks of asthma and other diseases that have a rapid onset while providing long term relief and improving a patient's quality of life.
  • the oligos have long half-lives, and are generally administered at very low doses, which makes them ideal for once-a-week type therapies.
  • the respiratory system and in particular the lung, as the ultimate port of entry into the organism, however, is an excellent route of administration for anti-sense oligonucleotides. This is so not only for the treatment of lung disease, but also when utilizing the lung as a means for delivery, particularly because of its non-invasive and tissue- specific nature. Thus, local delivery of antisense oligonucleotides directly to the target tissue enables the therapeutic use of these compounds.
  • Fomivirsen (ISIS 2922) is an example of a local drug delivery into the eye to treat cytomegalovirus (CMV) retinitis, for which a new drug application has been filed by ISIS.
  • composition and formulations of this invention are highly efficacious for preventing and treating diseases and conditions associated with bronchoconstriction, difficult breathing, impeded and obstructed lung airways, allergy(ies), inflammation and surfactant depletion, among others.
  • diseases and conditions include Acute Respiratory Distress Syndrome (ARDS), asthma, adenosine administration, e. g.
  • SVT Supra Ventricular Tachycardia
  • other arrhythmias and in stress tests to hyper-sensitized individuals, ischemia, renal damage or failure induced by certain drugs, infantile respiratory distress syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), lung transplantation rejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, including colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic metastases, etc., as well as all types of cancers which may metastasize or have metastasized to the lung(s), including breast and prostate cancer.
  • SVT Supra Ventricular Tachycardia
  • COPD chronic obstructive pulmonary disease
  • the invention will be described with respect to the oligos by employing adenosine receptors as targets, but is similarly applicable to any other target with respect to the pulmonary administration of anti-sense oligos.
  • the examples provided below show complete inhibition of various adenosine receptor and bradykinin associated symptoms in a rabbit model for human bronchoconstriction, allergies and inflammation as well as the elirnination of the ability of the adenosine receptor agonist par excellence, adenosine, to cause bronchoconstriction in hyper-responsive monkeys, which are animal models for human hyper-responsiveness to adenosine receptor agonists.
  • compositions and formulations of the invention are suitable for preventing and alleviating the symptoms associated with stimulation of, for example, adenosine receptors, such as the adenosine Al, A2b and A3 receptors.
  • adenosine receptors such as the adenosine Al, A2b and A3 receptors.
  • the compositions and formulations of this invention are also suitable for prevent the untoward side effects of adenosine-mediated hyperresponsiveness in certain individuals, which are generally seen in diseases affecting respiratory activity.
  • the method of the present invention may be used to treat airway, lung and cancer diseases and conditions of any kind, origin and for any reason, with the intention that the reduced adenosine content anti-sense compounds will produce a minimum liberation of adenosine upon its degradation.
  • diseases and conditions which may be treated preventatively, prophylactically and therapeutically with the compositions and formulations of this invention, are pulmonary vasoconstriction, inflammation, allergies, asthma, allergic rhinitis, impeded respiration, Acute Respiratory Distress Syndrome (ARDS), renal damage and failure associated with ischemia as well as the aiirninistration of certain drugs, side effects associated with adenosine administration e. g.
  • SVT Supra Ventricular Tachycardia
  • infantile RDS infantile Respiratory Distress Syndrome
  • ARDS infantile Respiratory Distress Syndrome
  • COPD chronic obstructive pulmonary disease
  • lung transplantation rejection pulmonary infections
  • cancers such as leukemias, lymphomas, carcinomas, and the like, e. g. colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, metastatic cancer such as hepatic metastases, lung, breast and prostate metastases, among others.
  • compositions and formulations are suitable for adminisfration before, during and after other freatments, including radiation, chemotherapy, antibody therapy, phototherapy and cancer, and other types of surgery.
  • the present compositions and formulations may also be administered effectively as a substitute for therapies that have significant negative side effects.
  • anti-sense oligonucleotides generally refers to small, synthetic oligonucleotides, resembling single-stranded DNA, which in this patent are applied to the inhibition of gene expression by inhibition of a target messenger RNA (mRNA). See, Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993), the relevant portion of which is hereby incorporated in its entirety by reference.
  • RNAs and oligonucleotides are represented in this patent by a single strand in the 5' to 3' direction, when read from left to right, although their complementary sequence(s) is (are) also encompassed within the four corners of the invention.
  • nucleotide bases and amino acids are represented utilizing the recommendations of the IUPAC-IUB Biochemical Nomenclature Commission, or by the known 3-letter code (for amino acids). Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right.
  • nucleotide and amino acids are represented herein in the manner recommended by the IUPAC- IUB Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR ' 1.822 and established usage. See, e. g., Patentln User Manual, 99-102 (Nov. 1990) (U.S. Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, D.C. 20231); U.S. Patent No. 4,871,670 to Hudson et al. at col. 3, lines 20-43.
  • the present method utilizes anti-sense agents to inhibit or down-regulate gene expression of target genes, including those listed in Tables 1 and 2 below.
  • mRNA messenger RNA
  • the exogenously administered agents of the invention decrease the levels of mRNA and protein encoded by the target gene and/or cause changes in the growth characteristics or shapes of the thus treated cells. See, Milligan et al. (1993); Helene, C. and Toulme, J. Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S. D.,
  • anti-sense oligonucleotide or anti-sense oligo is generally a short sequence of synthetic nucleotide that (1) hybridizes to any segment of a mRNA encoding a targeted protein under appropriate hybridization conditions, and which (2) upon hybridization causes a decrease in gene expression of the targeted protein.
  • the terms “desAdenosine” (desA), “desUridine” (desU), and “des-thymidine” (desT) refer to oligonucleotides substantially lacking either adenosine (desA), uridine (desU) or thymidine (desT).
  • the des A, desU or des T sequences are naturally occurring, and in others they may result from substitution of an undesirable nucleotide (A) by another lacking its undesirable activity, such as acting as an agonist or having a triggering effect at the adenosine A receptor(s).
  • the substitution is generally accomplished by substitution of A with a "universal or alternative base", presently known in the art or to be ascertained at a later time.
  • the terms "prevent”, “preventing”, “treat” or “treating” refer to a preventative, prophylactic, maintenance, or therapeutic treatment which decreases the likelihood that the subject aclministered such freatment will manifest symptoms associated with adenosine receptor stimulation.
  • down-regulate refers to inducing a decrease in production, secretion or availability and, thus, a decrease in concentration, of intracellular target product, be it a receptor, e. g. adenosine A b A 2 , A 3 , bradykinin 2B, GATA-3, or other receptors, or an increase in concentration of the adenosine A 2a receptor.
  • a receptor e. g. adenosine A b A 2 , A 3 , bradykinin 2B, GATA-3, or other receptors, or an increase in concentration of the adenosine A 2a receptor.
  • the present technology relies on the design of anti-sense oligos targeted to mRNAs associated with ailments involving lung airway ⁇ athology(ies), and on their modification to reduce the occurrence of undesirable side effects caused by their release of adenosine upon breakdown, while preserving their activity and efficacy for their intended purpose.
  • the anti- sense oligos target a specific gene to design one or more anti-sense oligonucleotide(s) (oligos) that selectively bind(s) to the corresponding mRNA, and then reduces, if necessary, their content of adenosine via substitution with an alternative or a universal base, or an adenosine analog incapable of significantly, or having substantially reduced ability for, activating or antagonizing adenosine Ai, A 2b or A 3 receptors or which may act as an agonist at the adenosine A 2a , receptor.
  • adenosines present may be substituted by an alternative and or universal base, such as heteroaromatic bases, wliich binds to a thymidine or uridine base but has less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A A 2a , A 2b and A 3 receptors.
  • an alternative and or universal base such as heteroaromatic bases, wliich binds to a thymidine or uridine base but has less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A A 2a , A 2b and A 3 receptors.
  • RNA that are devoid, or have a low content, of thymidine (T) or uridine (U) or, alternatively, substitute one or more adenosine(s) present in the designed oligonucleotide(s) with other nucleotide bases, so called universal bases, which bind to thymidine but lack the ability to activate adenosine receptors and otherwise exercise the constricting effect of adenosine in the lungs, etc.
  • T thymidine
  • U uridine
  • universal bases which bind to thymidine but lack the ability to activate adenosine receptors and otherwise exercise the constricting effect of adenosine in the lungs, etc.
  • adenosine is a nucleotide base complementary to thymidine (T) or uridine (U)
  • T or U thymidine
  • U uridine
  • the anti-sense oligonucleotide has a sequence wliich specifically binds to a portion or segment of a mRNA molecule which encodes a protein associated with impeded breathing, allergies, lung inflammation, depletion of lung surfactant or lowering of lung surfactant, airway obstruction, bronchitis, and the like.
  • One effect of this binding is to reduce or even prevent the franslation of the corresponding mRNA and, thereby, reduce the available amount of target protein in the subject's lung.
  • the phosphodiester residues of the anti-sense oligonucleotide are modified or substituted.
  • oligonucleotides with modified or substituted phosphodiester residues e. g., to the methylphosphonate, the phosphofriester, the phosphorothioate, the phosphorodithioate, or the phosphoramidate, ⁇ ' methoxy ethyl and similar modifications, which increase the in vivo stability of the oligonucleotide are particularly preferred.
  • the naturally occurring phosphodiester linkages of oligonucleotides are susceptible to some degree of degradation by cellular nucleases. Many of the residues proposed herein, on the contrary, are highly resistant to nuclease degradation. See, Milligan et al.; Cohen, J. S. D., supra.
  • the oligonucleotides may be protected from degradation by adding a "3'-end cap" by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide.
  • a "3'-end cap” by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide.
  • Phosphoramidates, phosphorothioates, and methylphosphonate linkages all function adequately in this manner for the purposes of this invention, as do ⁇ ' modifications, such as o ⁇ methoxy ethyl, and the like.
  • ⁇ ' modifications such as o ⁇ methoxy ethyl, and the like.
  • the more extensive the modification of the phosphodiester backbone the more stable the resulting agent, and in many instances the higher their RNA affinity and cellular permeation. See, Milligan, et al., supra.
  • a plurality of substitutions to the carbohydrate ring are also known to improve stability of nucleic acids.
  • the number of residues which may be modified or substituted will vary depending on the need, target, and route of administration, and may be from 1 to all the residues, to any number in between.
  • Preferred backbone analogue residues include phosphoramidate, phosphorothioate, methylphosphonate, phosphorofriester, phosphofriester, thioformacetal, phosphorodithioate, phosphoramidate, formacetal, friformacetal, thioether, carbamate, boranophosphate, 3 -thioformacetal, 5'-thioether, carbonate, C 5 -substituted nucleotides, 5'-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, 2'- O methyl, sulfoxide, sulfide, hydroxylamine, meti ⁇ ylene(memylimino) (MMI), methoxymethyl (MOM), and methoxye
  • Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred due to their availability through automated oligonucleotide synthesis. See, Millikan et al, supra.
  • the agent of this invention may be administered in the form of their pharmaceutically acceptable salts, or as a mixture of the anti- sense oligonucleotide and its salt. In another embodiment of this invention, a mixture of different anti-sense oligonucleotides or their pharmaceutically acceptable salts is administered.
  • a single oligo of this invention has the capacity to attenuate the expression of a target mRNA and/or various agents to enhance or attenuate the activity of a pathway whereas the bronchodilating agent facilitates the opening of the airways and promotes an increase in the pulmonary function of the patient.
  • the present method may be practiced by identifying all possible deoxyribonucleotide or ribonucleotide segments which are low in thymidine (T) or uridine (U), or deoxynucleotide segments low in adenosine (A) of about 7 or more mononucleotides, preferably up to about 60 mononucleotides, more preferably about 10 to about 36 mononucleotides, and still more preferably about 12 to about 21 mononucleotides, in a target mRNA or a gene, respectively.
  • T thymidine
  • U uridine
  • A adenosine
  • RNA thymidine
  • the first active agent(s) of this invention may be of any suitable length, including but not limited to, about 7 to about 60 nucleotides long, preferably about 12 to about 45, more preferably up to about 30 nucleotides long, and still more preferably up to about 21, although they may be of other lengths as well, depending on the particular target and the mode of delivery.
  • the agent(s) of the invention may be directed to any and all segments of a target RNA.
  • One preferred group of agent(s) includes those directed to an mRNA region containing a junction between an intton and an exon.
  • the first agent may either entirely overlie the junction or it may be sufficiently close to the junction to inhibit the splicing-out of the intervening exon during processing of precursor mRNA to mature mRNA, e. g. with the 3' or 5' terminus of the anti-sense oligonucleotide being positioned within about, for example, within about 2 to 10, preferably about 3 to 5, nucleotide of the intron/exon junction. Also preferred are anti-sense oligonucleotides which overlap the initiation codon, and those near the 5' and 3' termini of the coding region.
  • flanking regions of the exons may also be targeted as well as the spliced segments in the precursor mRNAs.
  • the mRNA sequences of the adenosine receptors and of many other targets are derived from the DNA base sequence of the gene expressing either receptors, e. g. the adenosine receptors, the enzymes, factors, or other targets associated with airway disease.
  • receptors e. g. the adenosine receptors
  • the enzymes, factors, or other targets associated with airway disease e.g. the adenosine receptors
  • the sequence of the genomic human Ai adenosine receptor is known and is disclosed in U.S. Patent No. 5,320,963 to Stiles, G., et al.
  • the A 3 adenosine receptor has been cloned, sequenced and expressed in rat (see, Zhou, F., et al., P.N.A.S. (USA) 89: 7432 (1992)) and human (see, Jacobson, M. A., et al., U.K. Patent Application No. 9304582.1 (1993)).
  • the sequence of the adenosine A 2b receptor gene is also known. See, Salvatore, C. A., Luneau, C. J., Johnson, R. G. and Jacobson, M., Genomics
  • an anti-sense oligonucleotides may be produced according to this invention as described above to reduce the production of the targeted protein in accordance with standard techniques.
  • sequences for the adenosine A 2a bradykinin, and other genes as well as methods for preparation of oligonucleotides are also known as those of many other target genes and mRNAs for which this invention is suitable.
  • anti-sense oligonucleotides that downregulate the production of target sequences associated with airway disease including the adenosine A A 2a , A 2b , A 3 , bradykinin, GATA-3, COX-2, and many other receptors, may be produced in accordance with standard techniques.
  • bronchodilating agents examples include ubiquinones, glucocorticosteroids, adenosine receptor antagonists such as theophyllines, anti-cholinergics, anti-histamines, and ⁇ l adrenergic agonists, amongst others.
  • ⁇ l adrenergic agonists examples include ephedrine, isoproterenol, isoetharine, epinephrine, metaproterenol, terbutaline, fenoterol, procaterol, albuterol, salmeterol, pirbuterol, formoterol, biloterol, bambuterol, salbutamol, and seretide, among others.
  • glucocorticosteroids such as beclomethasone, corticoid 21- sulfopropionates, (16 alpha)-16,17-alkylidene bis (oxy)-3-arylpregna-2,4-trien-20-ones, hydrocortisone esters, cyproterone thiopivalate (CTP), hydrocortisone, dexamethasone trimethyl acetate, alkane sulfonic acids of decinine, ⁇ -hydroxyprednisolone, 18,18-difluorosteroids, preparing 17-alpha-hydroxy corticoid 21-phosphate, 21-phosphate corticoids having unprotected hydroxyl radicals at least at the 17-al ⁇ ha- and 21 -position, 16-alpha-methylated ⁇ - 17(20)-corticoids, 21-(L-ascorbyl-2-phosphoryl) dexamethasone, 21-(L-ascorbyl-2-phosphoryl) hydrophos
  • the daily dosage of the bronchodilators and the optional anti-inflammatory glucocorticosteroid to be administered to a subject will vary with the overall treatment programmed, the agent employed, the type of formulation, the route of adminisfration and the state of the patient.
  • a large number of bronchodilators and anti- inflammatory glucocorticosteroids are known in the art, and are commercially available. Their use is widespread and their broad range of dosages are in the public domain. See, e. g. US. Patent 5,270,350 for salmeterol.
  • Examples 36 to 48 show aerosolized preparations in accordance with the invention for delivery with a device for respiratory or nasal aclminisfration, or adrninisfration by inhalation.
  • liquid preparations are preferred.
  • bioactive agents there exist FDA recommended amounts for supplementing a person's dietary intake with additional bioactive agents, such as in the case of vitamins and minerals.
  • additional bioactive agents such as in the case of vitamins and minerals.
  • the pharmacopeia's recommendations cover a very broad range of dosages, from which the medical artisan may draw guidance. Amounts for the exemplary agents described in this patent may be in the range of those currently being recommended for daily consumption, below or above those levels.
  • the treatment may typically begin with a low dose of a bronchodilator in combination with a n anti-sense oligo, and optionally other therapeutic agents, including glucocorticoid steroids, mucolytics, anti- histamines, anti-cholinergic agents, and other bioactive agents as appropriate, and then a titration up of the dosage for each patient.
  • a bronchodilator in combination with a n anti-sense oligo
  • other therapeutic agents including glucocorticoid steroids, mucolytics, anti- histamines, anti-cholinergic agents, and other bioactive agents as appropriate, and then a titration up of the dosage for each patient.
  • Higher and smaller amounts, including initial amounts, however, may be administered within the confines of this invention as well.
  • Other agents that may be incorporated into the present composition are one or more of a variety of therapeutic agents that are aclministered to humans and animals.
  • agents suitable for incorporation into the present composition and formulations are analgesics, pre-menstrual medications, menopausal agents, anti-aging agents, anti-anxyolytic agents, mood disorder agents, anti-depressants, anti-bipolar mood agents, anti-schizophrenic agents, anti-cancer agents, alkaloids, blood pressure controlling agents, hormones, anti- inflammatory agents, muscle relaxants, steroids, soporific agents, anti-ischemic agents, anti-arrhythmic agents, contraceptives, vitamins, minerals, ttanquilizers, neurofransmitter regulating agents, wound healing agents, anti- angiogenic agents, cytokines, growth factors, anti-metastatic agents, antacids, anti-Wstaminic agents, anti-bacterial agents, anti-viral agents, anti-gas agents, appetite suppressants, sun screens, emollients, skin temperature lowering products, radioactive phosphorescent and fluorescent contrast diagnostic and imaging agents, libido altering agents, bilido alter
  • hormones are female and male sex hormones such as premarin, progesterone, androsterones and their analogues, thyroxine and glucocorticoids, among the libido altering agents are Viagra and other NO-level modulating agents, among the analgesics are over-the-counter medications such as ibuprofen, oruda, aleve and acetaminophen and controlled substances such as morphine and codeine, among the anti-depressants are fricyclics, MAO inhibitors and epinephrine, ⁇ -amino butyric acid (GABA), dopamine and serotonin level elevating agents, e. g.
  • Prozac, Amytryptilm, Wellbufrin and Zoloft, among the skin renewal agents are Retin-A
  • hair growth agents such as Rogaine
  • anti-inflammatory agents are non-steroidal anti-inflammatory drugs (NSAIDs) and steroids
  • soporifics are melatonin and sleep inducing agents such as diazepam, cytoprotective, anti- ischemic and head injury agents such as enadoline, and many others. Examples of agents in the different groups are provided in the following list.
  • analgesics examples include Acetorninophen, Anilerdine, Aspirin, Buprenorphine, Butabital, Butorpphanol, Choline Salicylate, Codeine, Dezocine, Diclofenac, Diflunisal, Dihydrocodeine, Elcatoninin, Etodolac, Fenoprofen, Hydrocodone, Hydromorphone, Ibuprofen, Ketoprofen, Ketorolac, Levorphanol,
  • anti-anxiety agents examples include Alprazolam, Bromazepam, Buspirone, Chlordiazepoxide, Chlormezanone, Clorazepate, Diazepam, Halazepam, Hydroxyzine, Ketaszolam, Lorazepa , Meprobamate,
  • Oxazepam and Prazepa are examples of anti-anxiety agents associated with mental depression.
  • anti-anxiety agents associated with mental depression are Chlordiazepoxide, Amitriptyline, Loxapine Maprotiline and Perphenazine, among others.
  • anti-inflammatory agents are non-rheumatic Aspirin, Choline Salicylate, Diclofenac, Diflunisal, Etodolac, Fenoprofen, Floctafenine, Flurbiprofen, Ibuprofen, Indomethacin, Ketoprofen, Magnesium Salicylate, Meclofenamate, Mefenamic Acid, Nabumetone, Naproxen, Oxaprozin, Phenylbutazone, Piroxicam, Salsalate, Sodium Salicylate, Sulindac, Tenoxicam, Tiaprofenic Acid, Tolmetin.
  • anti-inflammatories for ocular tteatment are Diclofenac, Flurbiprofen, Indomethacin, Ketorolac, Rimexolone (generally for post-operative treatment).
  • anti-inflammatories for non-infectious nasal applications are Beclomethaxone, Budesonide, Dexamethasone, Flunisolide, Triamcinolone, and the like.
  • soporifics anti-insomnia/sleep inducing agents
  • soporifics are Alprazolam, Bromazepam, Diazepam, Diphenhydramine, Doxylamine, Estazolam, Flurazepam, Halazepam, Ketazolam, Lorazepam, Nitrazepam, Prazepam Quazepam, Temazepam, Triazolam, Zolpidem and Sopiclone, among others.
  • sedatives are Diphenhydramine, Hydroxyzine,
  • Methotrimeprazine Promethazine, Propofol, Melatonin, Trimeprazine, and the like.
  • Agents used in the freatment of head trauma (Brain
  • Enadoline HCl e. g. for freatment of severe head injury, orphan status, Warner Lambert.
  • cytoprotective agents and agents for the tteatment of menopause and menopausal symptoms are Ergotamine, Belladonna Alkaloids and Phenobarbitals.
  • agents for the treatment of menopausal vasomotor symptoms are Clonidine, Conjugated Estrogens and Medroxyprogesterone, Estradiol, Esttadiol Cypionate, Esttadiol Valerate, Estrogens, conjugated Estrogens, esterified Esttone, Esttopipate and Ethinyl
  • esttadiol examples include Progesterone, Progesrin, Gonadotrophic Releasing Hormone, oral contraceptives, Danazol, Luprolide Acetate and Vitamin B6.
  • agents for the freatment of emotional/psychiatric treatments include Tricyclic Antidepressants including Amittiptyline HCl (Elavil), Amittiptyline HCl, Perphenazine (Triavil) and Doxepin HCl (Sinequan).
  • Tricyclic Antidepressants including Amittiptyline HCl (Elavil), Amittiptyline HCl, Perphenazine (Triavil) and Doxepin HCl (Sinequan).
  • ttanquilizers, anti-depressants and anti-anxiety agents are Diazepam (Valium), Lorazepam (Ativan), Alprazolam
  • Xanax SSRI's (selective Serotonin reuptake inhibitors), Fluoxetine HCl (Prozac), Sertaline HCl (Zoloft), Paroxetine HCl (Paxil), Fluvoxamine Maleate (Luvox), Venlafaxine HCl (Effexor), Serotonin, Serotonin Agonists (Fenfluramine), and other over the counter (OTC) medications.
  • anti-migraine agents are Imiteex and the like.
  • the active agents of this invention are provided within broad amounts of the composition.
  • the active agents may be contained in the composition in amounts of about 0.001%, about 1%, about 2%, about 5%, about 10%, about 20%, about 40%, about 90%, about 98%, about 99.999% of the composition.
  • the amount of each active agent may be adjusted when, and if, additional agents with overlapping activities are included as discussed in this patent.
  • the dosage of the active compounds may vary depending on age, weight and condition of the subject. Treatment may be initiated with a small dosage, e. g. less than the optimal dose, of the first active agent of the invention, and optionally other bioactive agents described above. This may be similarly done with the second active agent, until a desirable level is attained.
  • the subject may be stabilized at a desired level of these products and then administered the first active compound.
  • the dose may be increased until a desired and/or optimal effect under the circumstances is reached.
  • the active agent is preferably administered at a concentration that will afford effective results without causing any unduly harmful or deleterious side effects, and may be aiiministered either as a single unit dose, or if desired in convenient subunits administered at suitable times throughout the day.
  • the therapeutic or diagnostic agent(s) is (are) administered in amounts which are known in the art to be effective for the intended application.
  • the dose of one of the other or of both agents may be adjusted to attain a desirable effect without exceeding a dose range that avoids untoward side effects.
  • other analgesic and anti-inflammatory agents when added to the composition, they may be added in amounts known in the art for their intended application or in doses somewhat lower that when administered by themselves.
  • diseases and conditions which are suitably treated by the present method are diseases and conditions, including Acute Respiratory Distress Syndrome (ARDS), asthma, adenosine administration e. g. in the treatment of SupraVenfricular Tachycardia (SVT) and other arrhythmias, and in stress tests to hyper-sensitized individuals, ischemia, renal damage or failure induced by certain drugs, infantile respiratory distress syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, including colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic metastases, etc., as well as all types of cancers which may metastasize or have metastasized to the lung(s), including breast and prostate cancer
  • first, second and other agents employed here will vary depending on the disease they applied to treat and how advanced it is, the state of the patient, the route of administration and type of formulation employed, as an artisan will appreciate and manufacture in accordance with known procedures and components.
  • the active compounds may be administered as a once-a-week dose, once-a-day doe, or several times a day.
  • the compositions and method of preventing and treating respiratory, cardiac, cardiovascular and neoplastic diseases, among others, including aging, may be used to treat adults and infants, as well as non-human animals afflicted with the described conditions.
  • the present invention is concerned primarily with the tteatment of human subjects, it may also be employed, for veterinary purposes in the tteatment of other mammalian subjects, such as dogs and cats as well as for large domestic and wild animals.
  • the terms "high” and “low” levels of "adenosine” and “adenosine receptors” as well as “adenosine depletion” are intended to encompass both, conditions where adenosine levels are higher than, or lower (even depleted) when compared to previous adenosine levels in the same subject, and conditions where adenosine levels are within the normal range but, because of some other condition or alteration in that patient, a therapeutic benefit would be achieved in the patient by decreasing or increasing adenosine or adenosine receptor levels or hypersensitivity.
  • this treatment helps regulate (titrate) the patient in a custom tailored manner.
  • the adenosine receptors discussed above are mere examples of the high power of the inventor's technology. In fact, a large number of genes may be targeted in a similar manner by the present agent(s), to reduce or down-regulate protein expression.
  • the target disease or condition is one associated with impeded or reduced breathing, bronchoconstriction, chronic bronchitis, pulmonary bronchoconstriction and/or hypertension, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, allergy, asthma, cystic fibrosis, respiratory distress syndrome, cancers, which either directly or by metastasis afflict the lung
  • the present method may be applied to a list of potential target mRNAs, which includes the targets listed in Table 1 and Table 2 below, among others.
  • the anti-sense agent(s) of the invention have a low A content to prevent its liberation upon in vivo degradation of the agent(s). For example, if the system is the pulmonary or respiratory system, a large number of genes is involved in different functions, including those listed in Table 1 below.
  • IL-8 R NFKB Transcription Factor Interleukin-8 Receptor
  • Interleukin-5 Receptor IL-5R
  • Interleukin-4 Receptor IL-4R
  • Interleukin-3 Receptor IL-3R
  • Interleukin- I ⁇ IL-1/3
  • Interleukin- 1 ⁇ Receptor IL- 1 ⁇ R
  • Endothelin Receptor B Preproendothelin
  • B2BR Bradykinin B2 Receptor
  • IgE High Affinity Receptor
  • IL-1 R Interleukin 1 Receptor
  • Interleukin-9 Interleukin-9 Receptor (IL-9 R)
  • Interleukin-11 IL-11
  • Interleukin- 11 Receptor IL-11 R
  • ICM-1 Intracellular Adhesion Molecule 1
  • VCAM Vascular Cellular Adhesion Molecule Substance P
  • Interleukin-2 IL-2
  • Interleukin-4 IL-4
  • Interleukin- 12 Interleukin-12
  • Interleukin-5 Interleukin-5
  • IL-6 Interleukin-6
  • IL-7 Interleukin-7
  • Interleukin-8 IL-8
  • IL-12R Interleukin-12 Receptor
  • Interleukin-7 Receptor IL-7R
  • Interleukin- 1 IL-1
  • Interleukin-14 Receptor IL-14R
  • Interleukin- 15 Interleukin- 15 Receptor (IL-15R)
  • IL-15R Interleukin- 15 Receptor
  • Interleukin-l l Interleukin-l l
  • IL-1 IR Interleukin- 11 Receptor
  • ICAM2 and ICAM3 C5a CCR3 (Eotaxin Receptor) CCR1, CCR2, CCR4, CCR5
  • Tachykinnen Receptors (tach R) I ⁇ B Kinase 1 & 2
  • Interleukin-2 Receptor (IL-2R) (e.g., Substance P, NK-1 & NK-3 Receptors) STAT 6 c-mas
  • NF-Interleukin-6 Interleukin- 10 Receptor (IL-1 OR)
  • Interleukin-3 Interleukin-3
  • IGFR Interleukin-2 Receptor
  • Interleukin-13 Interleukin-13
  • Interleukin-12R Interleukin-12 Receptor
  • Interleukin-14 Interleukin-6 Receptor
  • IL-6R Interleukin-6 Receptor
  • IL-16 Interleukin- 16
  • IL-13R Interleukin-13 Receptor
  • Adenosine A 2b Receptor A 2b R
  • Adenosine A 3 Receptor A 3 R
  • Adenosine A 2a Receptor A 2a R
  • IgE Receptor j8 Subunit IgE R ⁇
  • Alpha/Rantes Receptor Endothelin Receptor ET-B H2A histone family member N Tubulin, beta polypeptide ELL gene (11-19 lysine-rich leukemia gene) 7-dehydrocholesterol reductase
  • ADP-ribosylation factor-like 7 Karyopherin alpha 2 (RAG cohort 1, importin alpha 1)
  • Myosin X ESTs (AA459692) Epithelial protein lost in neoplasm beta CD44 antigen (homing function and Indian blood group system)
  • Coagulation factor III thromboplastin, tissue factor
  • Ubiquitin carrier protein Tubulin, alpha 1 (testis specific)
  • Transglutaminase 2 C polypeptide, prote -glutamine-gamnia-glutamylttansferase) Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican)
  • Proteasome prosome, macropain
  • 26S subunit non-ATPase
  • Low density lipoprotein receptor familial hypercholesterolemia
  • Plectin 1 intermediate filament binding protein
  • CD44 antigen (homing function and Indian blood group system) Programmed cell death 5 Hexokinase 1
  • Vascular endothelial growth factor Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)
  • Diphtheria toxin receptor heparin-binding epidermal growth factor-like growth factor
  • Fnl4 for type I teansmenmbrane protein Nef-associated factor 1 High-mobility group (nonhistone chromosomal) protein isoforms I and Y
  • Interferon stimulated gene (20kD) Steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1)
  • Prostaglandin-endoperoxide synthase 2 prostaglandin G/H synthase and cyclooxygenase
  • Laminin, alpha 3 (nicein (150kD), kalinin (165kD), BM600 (150kD), epilegrin)
  • Laminin, gamma 2 (nicein (lOOkD), kalinin (105kD), BM600 (lOOkD), Herlitz junctional epidermolysis bullosa)) Homo sapiens mRNA; cDNA DKFZp586Pl 622 (from clone DKFZp586Pl 622) ESTs, Weakly similar to /prediction (AA284245)
  • Anti-sense oligos to target receptors e. g. the adenosine A A 2a , A 2b , and A 3 receptors, CCR3 (chemokine receptors), bradykinin 2B, VCAM (vascular cell adhesion molecule), and eosinophil receptors, among others, have been shown to be effective in down-regulating the expression of their genes. Some of these act to alleviate the symptoms or reduce respiratory ailments and/or inflammation, for example, by "down regulation" of the adenosine Ai, A 2a , A 2b , and/or A 3 receptors and CCR3, bradykinin 2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors.
  • target receptors e. g. the adenosine A A 2a , A 2b , and A 3 receptors, CCR3 (chemokine receptors), bradykinin 2B, VCAM (vascular cell adhesion molecule), and e
  • agents may be utilized by the present method alone or in conjunction with anti-sense oligos targeted to other genes to validate pathway and/or networks in which they are involved.
  • the oligos are preferably administered directly into the respiratory system, e. g., by inhalation or other means, of the experimental animal, so that they may reach the lungs without widespread systemic dissemination.
  • This permits the use of low agent doses as compared with those administered systemically or by other generalized routes and, consequently, reduces the number and degree of undesirable side effects resulting from the agent's widespread distribution in the body.
  • the agent(s) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue.
  • a receptor protein itself is reduced in amount, rather than merely interacting with a drug, and toxicity is reduced.
  • Other proteins that may be targeted with anti-sense agents for the freatment of lung conditions include, but are not limited to: CCR3 (chemokine) receptors, human A 2a adenosine receptor, human A 2b adenosine receptor, human IgE receptor ⁇ , human
  • Fc-epsilon receptor CD23 antigen human histidine decarboxylase, human beta tryptase, human teyptase-I, human prostaglandin D synthase, human cyclooxigenase-2, human eosinophil cationic protein, human eosinophil derived neurotoxin, human eosinophil peroxidase, human intercellular adhesion molecule-1 (ICAM-1), human vascular cell adhesion molecule-1 (VCAM-1), human endothelial leukocyte adhesion molecule-1 (ELAM-1), human P selectin, human endothelial monocyte activating factor, human IL-3, human EL-4, human IL-5, human IL-6, human IL-8, human monocyte-derived neutrophil chemotactic factor, human neutrophil elastase, human neutrophil oxidase factor, human cathepsin G, human defensin 1, human defensin 3, human
  • genes are provided below. Some of these act to alleviate the symptoms or reduce respiratory ailments and/or inflammation, for example, by "down regulation" of the adenosine A], A 2a , A 2b , and/or A 3 receptors and CCR3, bradyldnin 2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors. These agents are preferably administered directly into the respiratory system, e. g., by inhalation or other means, so that they may reach the lungs without widespread systemic dissemination.
  • agent(s) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue. These agents, thus, rather than merely interacting with their targets, e. g. a receptor, lower the number of target proteins that other drugs may interact with. In tiiis manner, the present agent(s) afford(s) extremely high efficacy with low toxicity.
  • the term "treat” or “treating” asthma refers to a tteatment which decreases the likelihood that the subject administered such tteatment will manifest symptoms of the lung disease.
  • the term “downregulate” refers to inducing a decrease in production, secretion or availability (and thus a decrease in concentration) of the targeted inttacellular protein.
  • the present invention is concerned primarily with the tteatment of human subjects.
  • agents and methods disclosed here may also be employed for veterinary purposes, such as is the case in the tteatment of other mammals, such as cattle, horses, wild animals, zoo animals, and domestic animals, e. g. dogs and cats.
  • Targeted proteins are preferably mammalian and more preferably of the same species as the subject being treated.
  • anti-sense refers to the use of small, synthetic oligonucleotides, resembling single- stranded DNA, to inhibit gene expression by inhibiting the function of the target messenger RNA (mRNA). Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993).
  • RNA inhibition of gene expression of the Ai or A 3 adenosine receptor is desired.
  • Gene expression is inhibited through hybridization to coding (sense) sequences in a specific messenger RNA (mRNA) target by hydrogen bonding according to Watson- Crick base pairing rules.
  • mRNA messenger RNA
  • the mechanism of anti-sense inhibition is that the exogenously applied oligonucleotides decrease the mRNA and protein levels of the target gene or cause changes in the growth characteristics or shapes of the cells. Id. See, also Helene, C. and Touhne, J., Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S. D.,
  • anti-sense oligonucleotide is defined as a short sequence of synthetic nucleotide that (1) hybridizes to any coding sequence in an mRNA which codes for the targeted protein, according to hybridization conditions described below, and (2) upon hybridization causes a decrease in gene expression of the Ai or A 3 adenosine receptor.
  • the receptors discussed above are mere examples of the high power of the present technology.
  • a large number of genes may be targeted in a similar manner by practicing the present methods, to significantly down- regulate or obliterate protein expression and observe any changes wrought to one or more functions within a system, e. g. the respiratory system and other lung disease associated targets.
  • the targets may be associated with difficulties of breathing, bronchoconstriction, inflammation, allergic rhinitis, chronic bronchitis, surfactant depletion, and others associated with diseases and conditions such as chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, inhalation burns, Acute Respiratory Distress Syndrome (ARDS), cystic fibrosis, pulmonary fibrosis, radiation pulmonitis, tonsilitis, emphysema, dental pain, oral inflammation, joint pain, esophagitis, cancers afflicting the respiratory system either directly such as lung cancer, esophageal cancer, and the like, or indirectly by means of metastases, among others.
  • COPD chronic obstructive pulmonary disease
  • ARDS Acute Respiratory Distress Syndrome
  • cystic fibrosis pulmonary fibrosis
  • radiation pulmonitis tonsilitis
  • emphysema dental pain
  • oral inflammation joint pain
  • the present anti-sense oligonucleotides may be directed to a list of target mRNAs, which includes the targets listed in Table 1 above, among others.
  • the first active agent of this invention may be obtained by first selecting fragments of a target nucleic acid having at least 4 contiguous nucleic acids selected from the group consisting of G and C and/or having a specific type and/or extent of activity, and then obtaining a first oligonucleotide 4 to 60 nucleotides long which comprises the selected fragment and has a thymidine (T) or uridine (U) nucleic acid content of up to and including about 15%, preferably, about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no thymidine or uridine.
  • T thymidine
  • U uridine
  • the latter step may be conducted by obtaining a second oligonucleotide 4 to 60 nucleotides long comprising a sequence which is anti-sense to the selected fragment, the second oligonucleotide having an adenosine base content of up to and includ ⁇ ig about 15%, preferably about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no adenosine.
  • an adenosine base may be substituted in the corresponding anti-sense nucleotide fragment with a universal base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have less than about bout 10%, preferably less than about 1%, and more preferably less than about 0.3% of the adenosine base agonist activity at the adenosine Ai, A 2a , A 2b and A 3 receptors, and heteroaromatic bases which have no activity at the adenosine A 2a receptor, when validating in the respiratory system.
  • Other adenosine activities in other systems may be dete ⁇ riined in other systems, as appropriate.
  • the analogue heteroaromatic bases may be selected from all pyrimidines and purines, which may be substituted by O, halo, NH 2 , SH, SO, S0 2 , S0 3 , COOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloal
  • pyrimidines and purines may be substituted at all positions as is known in the art, but preferred are those where the purines are substituted at positions 1, 2, 3, 6 and/or 8, and the pyrimidines at positions 2, 3, 4, 5 and/or 6. More preferred are pyrimidines and purines such as those having the chemical formula
  • R 1 and R 2 are independently H, alkyl, alkenyl or alkynyl and R 3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH 2 - alkylamino-ketoxyalkyloxy-aryl, or mono or dialkylammoalkyl-N-alkylarnino-S0 2 aryl, and R4 and R5 are independently RI and together are R3, and the pyrimidines and purines optionally comprise theophylline, caffeine, dyphylline, etophylline, acephylline piperazine, bamifylline, enprofylline or xantine, among others.
  • Reduced adenosine content of the anti-sense oligos corresponding to the ymidines (T) present in the target RNA serves to prevent the breakdown of the oligos into products that free adenosine into the system, e. g. the lung, brain, heart, kidney, etc., tissue environment and, thereby, to prevent any unwanted effects due to it.
  • the NfkB transcription factor may be selected as a target, and its mRNA or DNA searched for low thymidine (T), uridine (U), desuridine (desU) or desthymidine (desT) fragments.
  • desT or desU segments of the DNA or mRNA are selected which, in turn, will produce desA anti-sense as their complementary strand.
  • sequence of the anti-sense segments may be deduced. Typically, about 10 to 30 and even larger numbers of desA anti-sense sequences may be obtained.
  • These anti-sense sequences may include some or all desA anti-sense oligonucleotide sequences corresponding to desT or desU segments of the mRNA of the target, such as anyone of those shown in Table 1 above, in Table 2 below, and others associated with functions of the brain, cardiovascular and renal systems, and many others.
  • the anti-sense oligonucleotides found are said to be 100% A-free.
  • the target gene e. g. the NFKB franscription factor
  • typically about 10 to 30 sequences may be found within the target gene or RNA which have a low content of thymidine (RNA).
  • the selected fragment sequences may also contain a small number of thymidine (DNA) or uridine (RNA) nucleotides within the secondary or tertiary or quaternary sequences.
  • these so called “non-fully desA" sequences may preferably have a content of adenosine of less than about 15%, about 12%, about 10%, about 7%, about 5%, and about 2% adenosine. Most preferred is no adenosine content (0%). In some instances, however, a higher content of adenosine is acceptable and the oligonucleotides still fail to show detrimental "adenosine activity".
  • a particular important embodiment is that where the adenosine nucleotide is
  • a universal or alternative base is defined in this patent as any compound, more commonly an adenosine analogue, which has substantial capacity to hybridize to thymidine, while at the same time having reduced, or substantially lacking, ability to bind adenosine receptors or other molecules through wliich adenosine may exert an undesirable side effect in the experimental animal or in a cell system.
  • adenosine analogs which completely fail to activate, or have significantly reduce ability for activating, adenosine receptors, such as the adenosine A 1; A 2b and/or A 3 receptors, most preferably Ai receptors, and those that may even act as agonists of the adenosine A 2a , receptor, may be used.
  • a universal base is 2'-deoxyribofuranosyl-(5-niteoindole), and an artisan will know how to select others. This "fixing" step generates further novel sequences, different from those anti-sense to the ones found in nature, that permits the anti-sense oligonucleotide to bind, preferably equally well, with the target RNA.
  • Other examples of universal or alternative bases are 2 - deoxyribosyl - (5-nifroindole).
  • universal bases are 3 - nifropyrrole - - deoxynucleoside, 5 - nifro-indole, T - deoxyribosyl - (5 - nitroindole), 2'-deoxyribofuranosyl - (5-nitroindole), 2' - deoxyinosine, 2' -deoxynebularine, 6H, 8H-3,4- dihydropyrimido [ 4, 5 - c] oxazine - 7 - one and 2 - amino - 6 -methoxy aminopurine.
  • Universal bases which may be substituted for any other base although with somewhat reduced hybridization potential, include 3 - nifropyrrole 2' - deoxynucleoside 2 - deoxyribofuranosyl - (5 - nitroindole), 2' - deoxyinosine and 2' - deoxynebularine (Glen Research, Sterling, VA).
  • More specific mismatch repairs may be made using "P" nucleotide, 6H, 8H - 3, 4 - dihydropyrimido [4,5 - c] [1, 2] oxazin - 7 - one, which base pairs with either guanosine (G) or adenosine (A) and "K" nucleotide, 2 - amino - 6 - memoxyaminopurine, which base pairs with either cytidine
  • the present method provides either anti-sense oligonucleotides to different targets which are low in, or devoid of, A content, as well as anti-sense oligonucleotides where one or more adenosine nucleotides, e. g.
  • Universal bases are known in the art and need not be listed herein in further detail. An artisan will know which bases may act as universal bases, and replace them for A. Table 2 below provides a selected number of targets to which the agents of the invention are effectively applied. Others, however, may also be targeted.
  • Oncogenes Targets ras thymidylate synthetase src thymidylate synthetase myc dihydrofolate reductase bcl-2 thymidine kinase deoxycytidine kinase ribonucleotide reductase
  • Beta-2-Adernergic Receptor Beta Catenin
  • B-cell translocation gene 1 BCG1 cyclin-dependent kinase 2 (CDK2) cyclin-dependent kinase 2 (CDK2) cyclin-dependent kinase 3 (CDK3) cyclin-dependent kinase 4 (CDK4) cyclin-dependent kinase 5 (CDK5) c-ets-1 proto-oncogene checkpoint kinase Chkl (CHK1) type IV collagenase hepatocyte growth factor receptor (c-met)
  • MYB proto-oncogene protein MYB
  • a group of preferred targets for the treatment of cancer are genes associated with any of different types of cancers or generally known to be associated with malignancies, whether they are regulatory or involved in the production of RNA and/or proteins.
  • Examples are fransforming oncogenes, including, but not limited to, ras, src, myc, and BCL-2, among others.
  • Other targets are those to wliich present cancer chemotherapeutic agents are directed to, such as various enzymes, primarily, although not exclusively, thymidylate synthetase, dihydrofolate reductase, uiyrnidine kinase, deoxycytidine kinase, ribonucleotide reductase, and the like.
  • the present technology is particularly useful in the freatment of cancer ailments given that traditional cancer therapies are fraught with the unresolved problem of selectively killing cancer cells while preserving normal living cells from the devastating effects of treatments such as chemotherapy, radiotherapy, and the like.
  • the present technology provides the ability of selectively attenuating or enhancing a desired pathway or target. This approach provides a significant advantage over standard treatments of cancer because it permits the selection of a pathway, including primary, secondary and possibly tertiary targets, which are not generally expressed simultaneously in normal cells.
  • the present agent may be administered to a subject to cause a selective increase in toxicity within tumor cells that, for instance, express all three targets while normal cells that may expresses only one or two of the targets will be significantly less affected or even spared.
  • a group of preferred targets for the treatment of cancers are genes associated with different types of cancers, or those generally known to be associated with malignancies, whether they are regulatory or involved in the production of RNA and/or proteins. Examples are transforming oncogenes, including, but not limited to, ras, src, myc, and BCL-2, among others. Other targets are those to which present cancer chemotherapeutic agents are directed to, such as various enzymes, primarily, although not exclusively, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase, and the like.
  • At least one of the mRNAs to which the oligo of the invention is targeted encodes a protein such as transcription factors, stimulating and activating factors, inttacellular and extracellular receptors and peptide transmitters in general, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously produced specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensines, growth factors, vasoactive peptides and receptors, and binding proteins, among others; or the mRNA is corresponding to an oncogene and other genes associated with various diseases or conditions.
  • a protein such as transcription factors, stimulating and activating factors, inttacellular and extracellular receptors and peptide transmitters in general, interleukins, interleukin receptors, chemokines, chemokine receptors, end
  • target proteins are eotaxin, major basic protein, preproendothelin, eosinophil cationic protein, P-selectin, STAT 4, MTP-l ⁇ , MCP-2, MCP-3, MCP-4, STAT 6, c-mas, NF-IL-6, cyclophillins, PDG2, cyclosporin A-binding protein, FK5-binding protein, fibronectin, LFA-1 (CDl la/CD18), PECAM-1, C3bi, PSGL-1, CD-34, substance P, pl50,95, Mac-1 (CDl lb/CD18), VLA-4, CD-18/CDl la, CDllb/CD18, C5a, CCR1, CCR2, CCR4, CCR5, and LTB-4, among others.
  • eotaxin major basic protein
  • preproendothelin eosinophil cationic protein
  • P-selectin STAT 4
  • MTP-l ⁇ MCP-2
  • MCP-3 M
  • At least one of the m NAs to which the oligo is targeted encodes inttacellular and extracellular receptors and peptide transmitters such as sympathoinimetic receptors, parasympathetic receptors, GABA receptors, adenosine receptors, bradyldnin receptors, insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, esttogen receptors, progesterone receptors, receptors associated with the coagulation cascade, adenohypophyseal receptors, adenohypophyseal peptide transmitters, and histamine receptors (HisR), among others.
  • inttacellular and extracellular receptors and peptide transmitters such as sympathoinimetic receptors, parasympathetic receptors, GABA receptors, adenosine receptors, bradyldnin receptors, insulin receptors, glucagon receptors, prostaglan
  • the encoded sympathomimetic receptors and parasympamomimetic receptors include acetylcholinesterase receptors (AcChaseR) acetylcholine receptors (AcChR), ateopine receptors, muscarinic receptors, epinephrine receptors (EpiR), dopamine receptors (DOPAR), and norepinephrine receptors (NEpiR), among others.
  • encoded receptors are adenosine Aj receptor, adenosine A 2b receptor, adenosine A 3 receptor, endothelin receptor A, endothelin receptor B, IgE high affinity receptor, muscarinic acetylcholine receptors, substance P receptor, histamine receptor, CCR-1 CC chemokine receptor, CCR-2 CC chemokine receptor, CCR-3 CC chemokine receptor (Eotaxin Receptor), mterleukin-l ⁇ 3 receptor (IL-l ⁇ R), interleukin-1 receptor (IL-1R), interleukin- la receptor (IL-loR), mterleukin-3 receptor (IL-3R), CCR-4 CC chemokine receptor, cysteinyl leukottiene receptors, prostanoid receptors, GATA-3 transcription factor receptor, interleukin-1 receptor (IL-1R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL-5R), interleukin
  • bradykinin B2 receptor insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, esttogen receptors, progesterone receptors, receptors associated with die coagulation cascade, adenohypophyseal receptors, and Mstamine receptors (HisR). Others are also contemplated even though not hsted herein.
  • the encoded enzymes for development of the ohgos of the invention include synthetases, kinases, oxidases, phosphatases, reductases, polysaccharide, triglyceride, and protein hydrolases, esterases, elastases, and , polysaccharide, triglyceride, lipid, and protein synthases, among others.
  • target enzymes are tryptase, inducible nitric oxide synthase, cyclooxygenase (Cox), MAP kinase, eosinophil peroxidase, /32-adrenergic receptor kinase, leukottiene c-4 synthase, 5-lipooxygenase, phosphodiesterase IV, metalloproteinase, tryptase, CSBP/ ⁇ 38 MAP kinase, neutrophil elastase, phospholipase A 2 , cyclooxygenase 2 (Cox- 2), fucosyl transferase, chymase, protein kinase C, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, and ribonucleotide reductase, among others.
  • Suitable encoded factors for application of this invention are, among others, NfkB transcription factor, granulocyte macrophage colony stimulating factor (GM- CSF), AP-1 transcription factor, GATA-3 transcription factor, monocyte activating factor, neutrophil chemotactic factor, granulocyte/macrophage colony-stimulating-factor (G-CSF), NFAT transcription factors, platelet activating factor, tumor necrosis factor . (TNF ⁇ ), and basic fibroblast growth factor (BFGF). Additional factors are also within the invention even though not specifically mentioned.
  • Suitable adhesion molecules for use with this invention include intracellular adhesion molecules 1 (ICAM-1), 2 (ICAM-2) and 3 (ICAM-3), vascular cellular adhesion molecule (VCAM), endothelial leukocyte adhesion molecule-1 (ELAM-1), neutrophil adherence receptor, mad CAM-1, and the like.
  • ICM-1 intracellular adhesion molecules 1
  • IAM-2 vascular cellular adhesion molecule
  • ELAM-1 endothelial leukocyte adhesion molecule-1
  • neutrophil adherence receptor mad CAM-1
  • Other known and unknown factors may also be targeted herein.
  • cytokines lymphokines and chemokines preferred are interleukin-1 (IL-1), terleukin-1 J QL-l ⁇ ), interleukin-3 (IL-
  • interleukin-4 interleukin-4
  • interleukin-5 IL-5
  • interleukin-8 IL-8
  • interleukin-9 IL-9
  • interleukin-11 IL-11
  • CCR- 5 CC chemokine and Rantes.
  • Other examples include H2A histone family, member N, Tubulin, beta polypeptide, ELL gene (11-19 lysine-rich leukemia gene) 7-dehydrocholesterol reductase, ADP-ribosylation factor-like 7, Karyopherin alpha 2 (RAG cohort 1, importin alpha 1), EST (AI038433), EST (AI122689), EST (AI092623), ESTs (AI095492), ESTs (AI138216), ESTs (AI128305), ESTs (AI125228), ESTs (AI041482), ESTs (AI051839), Homo sapiens mRNA; cDNA DKFZp434A1716, ESTs
  • ESTs 25058 mRNA sequence ESTs (R49144), Squamous cell carcinoma antigen 1, ESTs (AA425700), Myosin X, ESTs (AA459692), Epithelial protein lost in neoplasm beta, CD44 antigen (homing function and Indian blood group system), Coagulation factor III (thromboplastin, tissue factor), ESTs (AA909635), Adducin 1 (alpha), 5' Nucleotidase (CD73), ESTs, Moderately similar to semaphorin C [M.musculus] (AA293300), ESTs (AA278764), ESTs (AA678160), Calmodulin 2 (phosphorylase kinase, delta), ESTs (R42770), Chloride intracellular channel 1, High-mobility group (nonhistone chromosomal) protein 17, Ubiquitin carrier protein, Tubulin, alpha 1 (testis specific), Transglutaminase 2 (C polypeptide, prote
  • [Hsapiens] (T74688), Ribonucleotide reductase M2 polypeptide, Larninin, gamma 2 (nicein (lOOkD), kalinin (105kD), BM600 (lOOkD), Herlitz junctional epidermolysis bullosa)), Homo sapiens mRNA; cDNA DKFZp586P1622 (from clone DKFZp586P1622), ESTs, Weakly similar to /prediction (AA284245), and Lactate dehydrogenase A. Others, however, may also be targeted, as they are known to be involved in specific diseases or conditions to be treated, or for their generic activities, such as inflammation.
  • defensins for the practice of this invention are defensin 1, defensin 2, and defensin 3, and of selectins are a ⁇ l selectin, ⁇ 4 7 selectin, LFA-1 selectin, E-selectin, P-selectin, and L-selectin.
  • selectins are a ⁇ l selectin, ⁇ 4 7 selectin, LFA-1 selectin, E-selectin, P-selectin, and L-selectin.
  • oncogenes although not an all inclusive list, are ras, src, myc, and bcBCL. Others, however, are also suitable for use with this invention.
  • the agents administered in accordance with this invention are preferably designed to be anti-sense to target genes and/or mRNAs related in origin to the species to which it is to be administered.
  • the agents are preferably designed to be anti-sense to a human gene or RNA.
  • the agents of the invention encompass oligonucleotides which are anti-sense to naturally occurring DNA and or RNA sequences, fragments thereof of up to a length of one (1) base less than the targeted sequence, preferably at least about 7 nucleotides long, oligos having only over about 0.02%, more preferably over about 0.1%, still more preferably over about 1%, and even more preferably over about 4% adenosine nucleotides, and up to about 30%, more preferably up to about 15%, still more preferably up to about 10% and even more preferably up to about 5%, adenosine nucleotide, or lacking adenosine altogether, and oligos in which one or more of the adenosine nucleotides have been replaced with so-called universal bases, which may pair up with thyrnidine nucleotides but fail to substantially trigger adenosine receptor activity.
  • Examples of human sequences and fragments, which are not limiting, of anti-sense oligonucleotide of the invention are the following fragments as well as shorter segments of the fragments and of the full gene or mRNA coding sequences, exons and intron-exon junctions encompassing preferably 7, 10, 15, 18 to 21, 24, 27, 30, n-1 nucleotides for each sequence, where n is the sequence's total number of nucleotides. These fragments may be selected from any portion of the longer oligo, for example, from the middle, 5'- end, 3'- end or starting at any other site of the original sequence. Of particular importance are fragments of low adenosine nucleotide content, that is, those fragments containing less than or about 30%, preferably less than or about 15%, more preferably less than or about
  • the agent of the invention includes as a most preferred group sequences and their fragments where one or more adenosines present in the sequence have been replaced by a universal base (B), as exemplified here.
  • B universal base
  • also encompassed are all shorter fragments of the B-containing fragments designed by substitution of B(s) for adenosine(s) (A(s)) contained in the sequences, fragments thereof or segments thereof, as described above.
  • A(s) adenosine(s)
  • anti-sense oligonucleotide sequence fragments target the initiation codon of the respective gene, and in some cases adenosine is substituted with a universal or alternative base adenosine analogue denoted as "B", wliich lacks ability to bind to the adenosine Ai and/or A 3 receptors. In fact, such replacement nucleotide acts as a "spacer". Many of the examples shown below provide one such sequence and many fragments overlapping the initiation codon, preferably wherein the number of nucleotides n is about 7, about 10, about 12, about 15, about 18, about 21 and up to about 28, about 35, about 40, about 50, about 60.
  • GCCCCTCTCC TCTCCTTCCT CTGCTTCTCG CTCTCCTTTG TGGGGCCCTC CCTGCTGCTC TTGGTTTTGG GCTTTTTTTC TCTTCCTCCT TTTTCGTGCG TGGGCCTCC GCACGCCTCT TGCCACCTCC TGCGCAGGGC AGCGCCTTGG GGCCAGCGCC GCTCCCGGCG CGGCCAGCAG GGCAGCCAGC AGCGCGCAGC CGACGGCCAG CATGCTTCCT CCTCGGCTAC CACTCCATGG TCCCGCAGAG GCGGACAGGC GCBCGCCTC TTGCCBCCTC CTGCBGGG CBGCGCCTTG GGGCCBGCGC CGCTCCCGGC GCGGCCBGCB GGGCBGCCBG CBGCGCGCBG CCGBCGGCCB GCBTGCTTCC TCCTCGGCTB CCBCTCCBTG GTCCCGCBGB GGCGGBCBGG C GCTGCCCGGC GGGGTGTGCG CTTGGC
  • GAGCCCCAGG CAGAGGCGTC TCCCTTATGC CCCACTCTGA AGTGTTTGTT AGTAAACACC
  • AGAACGCCAT TGTTGTTACT GCTGAATTTT ATTTTGGGCT GTACATATTT AGATGCTTAA GGTAAAAATG ATAAAGCCCT CAAGCCACTG TGTGGGTTTG GGTCCAAGTG TTCCTTCTTG CTGCCTCTCT AACACGCCTG GTTAAAATAA TCCCTTTGGA TGGTGCTGAG AAGCACCTGA ACCAAGTGGG TCCCCAAATA ACAATGGCGT GCAAGTGTCT GGTTCCCAGA AGTTGGTGAC TAGGTAAGCA GCTTCAGGGA GAGGGGGCTG ATTCCCAGAC AGTCGCCTGT TCCTGCGGGG ATGGCTGA GGCTTGGGGA ATGTGGGCAG GAGGATATGC CATTTGATTC TGTTGCACAC GTTCTTTTCC CTTCTTTCTG TATGTCTGGT CATTCTGCTA TTCTGT
  • AACTGCATCA TCTACTTTAA TGGTGAGGTA CCACAGCTTG TGCTGTACAT GGGCATCCTG CTGTCCCATG CCAACTCCAT GATGAACCCT ATCGTCTATG CCTATAAAAT AAAGAAGTTC AAGGAAACCT ACCTTTTGAT CCTCAAAGCC TGTGTGGTCT GCCATCCCTC TGATTCTTTG GACACAAGCA TTGAGAAGAA TTCTGAGTAG TTATCCATCA GAGATGACTC TGTCTCATTG ACCTTCAGAT TCCCCATCAA CAAACACTTG AGGGCCTGTA TGCCTGGGCC AAGGGATTTT TACATCCTTG ATTACTTCCA CTGAGGTGGG AGCATCTCCA GTGCTCCCCA ATTATATCTCTC CCCCACTCCA CTACTCTCTT CCTCCACTTC ATTTTTCCTT TGTCCTTTCT CTCTAATTCA GTGTTTTGGA GGCCTGACTTCT CTCTAATTCA GTGTTTTGGA GGCCTGACTTCT
  • TTTTGTTCCT CTGCTTCTCC CGTTTGCCTC CTTATCATGA GATCTTTTTG CTAAGCTGGC AGAAAGATTG CATAGTCAGT GCTTCCAGCT CTGCTCCCAC CTGATCCTGC ACTGTCCTCT GGTCCCTGAA TGAATGAACT CTGATACCCA ATCTTGTCTC GAGCCTTCTC TATGCCACTC ATGGCTCCTC TTCTGCTCTT TCCATCTTTT TGCTGAGAGT TCTGAGCTCT GTACTTCCTC TTGGCCCATC TCACTTCCTG AAACACCCCT GAAGAGGGTT GCTTATCTTG ATGGAACTCA AAAAGCCAAA AAGCTGCAGG CAGAGGCGTT GAGGACATCT GTTTGGGGAA CTAAGAGCAG CAGCACTTTC AGATTCAGTC CATATAGAGC TGTCCTACAG CATTCTGGAA ACTTGAGGAT GTGCGGTGCA TAAAGGCT GGAAGTGACC CACCTGTGAT GAGCCCTTTC TAAG
  • GGCAGCCTGT CCATAGACCT CTGTCCCCAA CTGGCAAGTC AGGAAACTCC AGATTAAGGA GCCCCAATGT GGTTGAACAG CCAGGTGCAC AGATGAGTCA ACCACACAGC CAGGCCAGGG AGGGCCTTCA CTCAAGAGCC TACAGCCAGT TCACAGCCAA GCCAGGGCTA GCGCCAGGCC ACCCATAAAC TGATCTGAGA CTCTGTTTCC CTGTCTCCAT GATGATGGGA TCAGGCTTGA TTGCTGGTTT GTAGGCTTGT TATGAATCAA GTCACAGGGA AGAGGAGCTG ATGGGCTGGG GGGACGTCCT CTGGCCCTCC TGTCTCTTCC CCAGATCCAC TGGGCCCACT CTTATCTGTT CTCTTCTGAA GGAAGGGTTT TAAGGCTTCA AAAAAAAATG TTTTGAAAGT CCCTGCCCTT TCCAGCTCCT ACCGTCTCAG CCCTGGGAGT GTAAAGTGCT GCAG
  • GGTGAACTCA CGCACAGCCA AGGACTCCAA AATCACAACA GCATTACTGT TCTTATTTGC TGCCACACCT GAGCCAGCCT GCTCCTTCCC AGGAGTGGAG GAGGCCTGGG GGGAGGGAGA GGAGTGACTG AGCTTCCCTC CCGTGTGTTC TCCGTCCCTG CCCCAGCAAG ACAACTTAGA TCTCCAGGAG AACTGCCATC CAGCTTTGGT GCAATGGCTG AGTGCACAAG TGAGTTGTTG CCCTGGGTTT CTTTAATCTA TTCAGCTAGAGA ACTTTGAAGG ACAATTTCTT GCATTAATAA AGGTTAAGCC CTGAGGGGTC CCTGATAACA ACCTGGAGAC CAGGATTTTA TGGCTCCCCT CACTGATGGA CAAGGAGGTC TGTGCCAAAG AAGAATCCAA TAAGCACATA TTGAGCACTT GCTGTATATG CAGTATTGAG CACTGTATATG CAGTATTGAG CACTGTAG
  • CTGTTGGTGG ATACTGGCCA AGGAAATATC CCAGTGGAGC CTCGAGATGA AGAACATGAG GCCCCCGTTT AGAACCAAGG ATCAGAGGGG GCTCTGTAAG ACCCAGGGGA GTCAGGTGCA CTGGAGCGCGCG GGCATGCAGA AAACAGCCTG AGCTCCACCT CGGCTTCTCC TTGTCCTGGC TGGTTGTCCT TAACCCCTGT CTCCTTCTGG ACCAGTTTTT GTCCTTCCCT TGTGACCGCT GAGGGGTAAC AGCCTCTTTC CACTTTCTTT CAGCGCCGAC ATGCTCAATG TCACCTTGCA AGGGCCCACT CTTAACGGGA CCTTTGCCCA GAGCAAATGC CCCCAAGTGG AGTGGCTGGG CTGGCTCAAC ACCATCCAGC CCCCCTTCCT CTGGGTGCTG TTCGTGCTGG CCACCCTAGA GAACATCTTT GTCCTCAGCG TCTTCTGCCT GCACAAGAGC AGCTGCACGG
  • CTGTCTGTTT GTGAGGACTC CGTGCCCACC ACGGCCTCTT TCAGCGCCGA CATGCTCAAT GTCACCTTGC AAGGGCCCAC TCTTAACGGG ACCTTTGCCC AGAGCAAATG CCCCCAAGTG GAGTGGCTGG GCTGGCTCAA CACCATCCAG CCCCCCTTCC TCTGGGTGCT GTTCGTGCTG GCCACCCTAG AGAACATCTT TGTCCTCAGC GTCTTCTGCC TGCACAAGAG CAGCTGCACG GTGGCAGAGA TCTACCTGGG GAACCTGGCC GCAGCAGACC TGATCCTGGC CTGCGGGCTG CCCTTCTGGG CCATCACCAT CTCCAACAAC TTCGACTGGC TCTTTGGGGA GACGCTCTGC CGCGTGGTGA ATGCCATTAT CTCCATGAAC CTGTACAGCA GCATCTGTTT CCTGATGCTG GTGAGCATCG ACCGCTACCT GGCCCTGGTG AAAACCATGT CCATGGGCCG GATGCG
  • GCACAGAGCC CATGCCTGCC CCCCTGGATG GGAGTGATGT GAAACTTGAA GGGCGGTCAG AGCAAGGGTC GGGAATGGAA GGCCCTTGGG AAAAAAGGCC CTTTCAACTA GGGGCACAGA GGAGGCCCTG GGCTGAGAAC TTGACAGCAC CTTGTAATTG GTAAGCCAAG CCCGAAGGGA CTGGAAATAC TCAGATGTGT CTGTCTCCCT TATTAGGTTC AAAGTCCCTC AAGACCCTGT CTCCATCACA GTGCTCCAGT CCAGACCCCT CCTCTGAGCT CCAGACCCTG CTGGACCCAA CCAGCCCTAT GGGGTCGCAT CCCCACCTGC CTGGAATTCT CCAAAGAACC TCCCCTTTAA CAGTTCCAGC CTTTAACAGT TCCAGTCTAA ACACATGACC TTTCTCCTCT AAATCAGCCC CCCATCTCTG CCTTTGCAGG AGATGGAAGC CATGACACCT GCCT
  • CTCTGTAAGA CCCAGGGGAG TCAGGTGCAC TGGAGCGCGG GCTGCAGAAA ACAGCCTGAG CTCCACCTCG GCTTCTCCTT GCCCTGGCTG GTTGTCCTTA ACCCCTGTCT CCTTCTGGAC CAGTTTTTGT CCTTCCCTTG TGACCTGAGG GGTAACAGCC TCTTTTCCAC TTTCTTTCAG CGCCGACATG CTCAATGTCA CCTTGCAAGG GCCCACTCTT AACGGGACCT TTGCCCAGAG CAAATGCCCC CAAGTGGAGT GGCTGGGCTG GCTCAACACC ATCCAGCC CCTCTG GGTGCTGTTC GTGCTGGCCA CCCTAGAGAA CATCTTTGTC CTCAGCGTCT TCTGCCTGCA CAAGAGCACGGTGG CAGAGATCTA CCTGGGGAAC CTGGCCGCAG CAGACCTGAT CCTGAT CCTGGCCTGC GGGCTGCCCT TCTGGGCCAT CACCATCT
  • AGAACTCCAT GGGCACACTG CGGACCTCCA TCTCCGTGGA ACGCCAGATT CACAAACTGC
  • AGGACTGGGC AGGGAGCAGA CAGTGAGCAA ACGCCAGCAG GGCTGCTGTG AATTTGTGTA AGGATTGAGG GACAGTTGCT T GCCCTTCAAA GATGAGCTGT TCCCGCCGCC ACTCCAGCTC TGGCTTCTGG GCTCCGAGGA GGGGTGGGGA CGGTGGGGAC ATCAGGCTGC CCCGCAGTAC CAGGGAGCGA CTGAAGTGCC CATGCCGCTT GCTCCGGAGA AGGTGGGTGC CGGGCAGGGG CTGCTCCAGC CGCCTCACCT CTGCTGGGAG GACAAACTGT CCCAGCACAG AGGGAGGGAG GGAGGGCAGG CAGCGGAG AAGTTTCCCT GTGGTCGTGG GGAGTT GCCCTTCAAA GATGAGCTGT TCCCGCCGCC ACTCCAGCTC TGGCTTCTGG GC
  • TTCCCAGGCC ACTTTGTGGT CAGCCGGGAG GGACGTTTTT GCCGTCCCAC GACTCCAACG GGCAGCCGGG CCTACGCAAA CATGGAAATC TTCCAAGAGC CTCCCTGGCC CCCAGGGCTC AGAGGGTGGC AGAGCGGAGA GCGAAGGTGG CCGCAGCCTT CCCGGCCCCA CAGCCAGCCT GGCTCCAGCT GGGCAGGAGT GCAGCTCA GCTGGAGGCG AGGGGGAAGT GCCCAGGAGG CTGATGACAT CACTACCCAG CCCTTCAAAG ATGAGCTGTT CCCGCCGCCA CTCCAGCTCT GGCTTCTGGG CTCCGAGGAG GGGTGGGGAC GGTGGTGACG GTGGGGACAT CAGGCTGCCC CGCAGTACCA GGGAGCGACT GAAGTGCCCA TGCCGCTTGC TCCGGAGAAG GTGGGTGCCG GGCAGGGGCT GCTCCAGCCG CCTCACCA GGGAGCG
  • CATCTTCTCC ATGAAGACCA CTGAATGAAC ACCTTTTCAT CCAGCCTTAA TTTCTTGCTC CATAACTACT CTATCCCACG ATGCAGTATT GTATCATTAA TTATTAGTGT GCTTGTGACC TCCTTATGTA TTCTCAATTA CCTGTATTTG TGCAATAAAT TGGAATAATG TAACTTGATT TCTTATCTGT GTTTGTGTTG GCATGCAAGA TTTAGGTACT TATCAAGATA ATGGGGAATT AAGGCATCAA TAAAATGATG CCAAAGACCA AGAGCAGTTT CTGAAGTCCT CCTTTTCATC AGCTCTTTAT CAAACAGAAC ACTCTATAAA CAACCCATAG CCAGAAAACA GGATGTAGGA ACAATCACCA GCACACTCTA TAAACAACCC ATAGCCAGAA AACAGAATGT AAGGACAATC ACCAGCCATC TTTTGTCAAT AATTGATGGA ATAGTTGA AAGGAACTGG AGCATGAGTC
  • CTGGAGGCTC GCAAGAAAGC CAAGAACAAG CAGCTGGGCC ATGAGGAAGA CTACGCCCTG GGCAAGGACT GCATCATGCA TGGCTACATG TCCAAGATGG GCAACCCCTT CCTGACCCAG TGGCAGCGGC GGTACTTCTA CCTGTTCCCC AACCGCCTCG AGTGGCGGGG CGAGGGCGAG GCCCCGCAGA GCCTGCTGAC CATGGAGGAG ATCCAGTCGG TGGAGGAGAC GCAGATCAAG GAGCGCAAGT GCCTGCTCCT CAAGATCCGC GGTGGGAAAC AGTTCATTTT GCAGTGCGAT AGCGACCCTG AGCTGGTGCA GTGGAAGAAG GAGCTGCGCG ACGCCTACCG CGAGGCCCAG CAGCTGGTGC AGCGGGTGCC CAAGATGAAG AACAAGCCGC GCTCGCCCGT GGTGGAGCTG AGCAAGGTGC CGCTGGTCCA GCGGCAGT GCCAACGGCC TCTGACCCGC
  • CCTTGAGCCT CAGTCACTGA GCTAAGCTCC CTTCGGAGGA AAAGGAGGTC CTGTCCGAAG GTCCCTCTTG TTGCAGTAGC ACCCCTCACC CCTACCCAAC TCAAGACACA CGGCTCACTT TTCAGGGCCC CACCCAGTCT CAGGGCCACT TCCTCTATGG CCTTTTCAAG AACACTGGCT CTAGTTCTCA GGGTCCTGAA CCCATCATTT TATGGGAGCA GAGAACAGGT CTACATAAGA CCCCCACTTT CCCGTTTTAA CTGATATCTC CTGCTTCAGG GGCTGGCCCT CATGCAGGGT TCCCTGAATT AGGAAGTGTG AACCCTGTCC CCTGAGTCCT CCCTGGGCTG TTCAGTCCCC AGCAATTCCA GGGGTCGTAG AAATTGTGTC TGTTTCCTGA GAAAGCTCTT TCATGAGTTA AGCCTGAGCC CTCAAATGCC ACAAGTGGCC CATGAAAAGG GAGATGGGTA GAGT
  • GGTATTAGGC TATGAATCAG CGCCACGTGC AAAGGCTTGG GAGCCAAGCC ATGTGGTCTT GCACCCCAGG CAAGAAAAGT CAGCTGGAGG GTTTACAGCA CTTTCTACTG TTTCCCAGCC CTCCCTCCCC TCCCTCACCA TGACTAAGAG ACCACTCGGT CCTAGCCTCC AGACACCCCA CAATACTCCT CTGAGCCTGA GGCCAGGCAG CATGCTCTGC TTCTACCAAT AAAGCACTGC CGGAATTC CATATGTATG GGAATACTGT ATTTCAGGCA TTATAAGGAA TGAAATTATA GGCCGGGCAT TGTGGCTAAC CCTTGTAATC CTAGCACTTT GAGAGGCTGA AGTGGGCAGA TCACTTGAGC TTCAGAGTTC GAGACCAGCA TGGACAACAT GGTGAAACCC AGTCTCTACC AAAAACACAA AAATATTAGC TGGGTGTGGT GGTGCATGCC TGTAGTCCCA G
  • TGCCACTCCG CTCCAGTCTT GGTGACAGAA TGAGACTCCA TCTCAAAAAT AAATAAATAA ATAAATAAAA TAAATGAAAT GAAATTATAA GAAATTACCA CTTTTTCATG TAAGAAGTGA TCATTTCCAT TATAAGGGAA GGAATTTAAT CCTACCTGCC ATTCCACCAA AGCTTACCTA GTGCTAAAGG ATGAGGTGTT AGTAAGACCA ACATCTCAGA GGCCTCTCTG TGCCAATAGC CTTCCTTCCT TTCCCTTCCA AAAACCTCAA GTGACTAGTT CAGAGGCCTG TCTGGAATAA TGGCATCATC TAATATCACT GGCCTTCTGG AACCTGGGCA TTTTCCAGTG TGTTCCATAC TGTCAATATT CCCCCAGCTT CCTGGACTCC TGTCACAAGC TGGAAAAGTG AGAGGATGGA CAGGGATTAA CCAGAGCT CCCTCCCTGGAAAGTG AGAGGATGGA CAGGGATTAA
  • CTCTCGGCCA CCTTTGATGA GGGGACTGGG CAGTTCTAGA CAGTCCCGAA GTTCTCAAGG CACAGGTCTC TTCCTGGTTT GACTGTCCTT ACCCCGGGGA GGCAGTGCAG CCAGCTGCAA GGTGAGTTGC C CTGCTTTAAA ATCTCTCGGC CACCTTTGAT GAGGGGACTG GGCAGTTCTA GACAGTCCCG AAGTTCTCAA GGCACAGGTC TCTTCCTGGT TTGACTGTCC TTACCCCGGG GAGGCAGTGC AGCCAGCTGC AAGCCCCACA GTGAAGAACA TCTGAGCTCA AATCCAGATA AGTGACATAA GTGACCTGCT TTGTAAAGCC ATAGAGATGG CCTGTCCTTG GAAATTTCTG TTCAAGACCA AATTCCACCA GTATGCAATG AATGGGGAAA AAGACATCAA CAACAATGTG GAAAGCCC CCTGTGCCAC CTCCAGTCCA GTGACACAGG ATGACCTTCA G
  • GTCTGCCCCT CAATTCCAGC CTGCTCAACA CACAAGGAAA CAAAGCCCTG ACAATCAGAG TGACTCCCTG GTGACTAAGC TCCCAGTCCT GGATGCATAT TTGTTTAGCA GTTCTGACAG CATTTGACCC AGCCCTCTCT CTGCATATCC CATCAGAACC TTCTTTTTTTTTTTTTTTTCTT TGAGACTGAG TCTTGCTCTG TCGGAAGCGA CTCCTGCC TCAGCCTCCC AAATACCTGG AATTATAGGC GTAAGCCATC ATGCCTGGCT AATTTTTGTA TTTCATGG AGATGGGGTT TTGCCATGTT GGTCAAATTG GTCTCACACT CCTGACCTCA TGTGATCCAC CTGCCTCAGC CTCCCAAACT GCTGGGATGA CAGGTGTAAG CCACCATGCT AGGCTCAGAA ATTTCCTTTT ATAAAAATGT CATTAAGGAT CTTGGCTGCA CAATATCGTT ACCAGCTTCC TTTA
  • TAAGCCCTGT TACAGGGGCT GCACCCCAGA TACAACCTGA CCTGTGTCCA AGGCGGGCAA CTCAACCCTT AGATATTGAA TGGGTCCCAT GGCACCAATG CTTAAACACC AGCAGCCCTC ACAACCACAG ATCGTGTTTT AAGGATGAGG AGGTAGTTCT CTGGATGCAC AGGCTTCAAT CCAAATGGGC TCATGACGCC GCAGCACACA CCCAGTCTGC AGCCTGAAGA GTTGGAGCAT TGCATTCACA GAAAGCATCC AGACATGATC ATGGGCTCAG GGATACACCT GTTCTCCGAT GTGTACCAGT GAAGGATGGA AACTCCTATG CCTCCCAGAA AGCACCACTC AAGCTTTTGC TGAATGCTTC TCTGAAGGCC CACAAGGCTG AGAGGCTGTGTG CAACACCAGC AGTAAAGTGA ATGCCCAGAC TCCCACCTCC TTTCTTGGGT GGCCATCTGG AAAGGCCACT CCCACCCTGA
  • AAATTAGGAC ACCTCATCCC AAAAGACCTT TAAATAGGGG AAGTCCACTT GTGCACGGCT GCTCCTTGCT ATAGAAGACC TGGGACAGAG GACTGCTGTC TGCCCTCTCT GGTCACCCTG CCTAGCTAGA GGATCTGTAA GTACTACAAA ACTTAAACTT TACACTGAGT TTTCATCATT GAAGCTATGC CTCCAATCTG ACCTCTGACT GTGGGGCCGC CCCAGAGGGA CCCAGCGGGT GAATCCCTGC TAGGAACGTC TGTCCGGACC TCTGGTGACT GCTGGGGACG ATGGCTTCCA GCTAACTTAA TAGAGAAACT CAAGCAGTTT CCTTCTAAAT ACACATGTCA CATGTCCTGG TTGACATGTC CAGTAAGAAG ACTATCACAG GTCTTTGGAA CATTCTTTTG AGAGAAACCT ATTTAGGTCC TTGGTCTGTT TTTCAATCAG GTTGTTTGAT TTTTGCTATT GAGTTG
  • GAAGAGAGGC TACCTTTGTGTG GGGAGGGGAC AGTTTAATGC CCAGAAGCGG TAAATAAGGA ATCCTCTGGG GAGTGGTAAT GATCTGGATG CTGGCTACAG GATGTGTTGG TTGTAAAAAT GCATTTTTTT ATATCTAGCT TTTTCCATGT GTATATTATA CTTCAAAGAA GTTCAGTTAA TAATTTCTCA TGTCACTGTA GAGTAGCTCA GTTAGCCCCA GCAAGCCTCT GGCTTAATCT TGTTTTACCT TAAGCCATCA GTCATTTACA AGTAGGAAAA TTCACAGGGA AAGTTAGAGT ATAAAATCCA GAATGAAGGT TTACTGGGTA AGAGTCTC CATTTTCCAA AGCCCGTTTA TTTCTTGATT CCAGTTCTTA AGAAGTCTCA GCATTGTGTC TTTTTCATGT ATCTTACAAG AAGACAGCAT GTGCTTCTAA CACCTGATAC ATTGTATCTA CCAGCACTTG GTAAACAGAA AA
  • TTGCCAGTAG TAATAGTTCA ACCACCAC CAGCTTTTAT TAAAATTTTT AATAACACTC AAGTATTGGC AGAAAGAAAT AATCTTGGGT TAACTATAAC TAGAATATTG ACTCTTCCTC TGTGGAAGAA TCAGCCAATC ACATTTGTTT ACATCAGTTC CCCTGAAGAA GAAAAATACA CTGATGTTGC AGCAAGACAA ATTTAAGCTA GATGTAAATA ACTTCCTTTA GCCTGTAATG CTAGGCTAAT TACATATTGG AACTATTTTT TCAGGGAAGA ATTGTGTAGG GTTTCAGGGA AGAATTCTGA AGAAAATATA GAGCTGAAAT GATCTTGCAG CTCACTGAAA CTGCAGGGTT TAGATCCACA CTGATACTCG TTCTATTATC ACTGTAATGA AGGCTGATGG AATAAGTAAAAAAATGTTGT ATTAGTATGT TTTTACACTT ATTTGCAAGGCATAAATA
  • CTCTGGGTCC TGCTGATATG GTATTTTTGA GATTTGGCCT AAAACATCAT TGCCCTGGTT TCCTTATTTA CCAAACAGGG CCAATGGTAG TGACTAATCA GAAAATGATA ATGCCTGGTG CACAAAATGT GTCTAGATGA GCCCATGCAC AAGGACACAT GTTTCTGGAA CTGTTCCTTA TTCCTTTCCT AAAAGAAAGG AGGGAAAGTC TCCATACTAA GACTACTAGG GCAGGGGACA AAGTGCTAGA GTCAGAAGAT TCATCTGAGG ACAGAAGAAT AGGGGTGAAG GCTCTAGTCA CTTCATTGGC TACCATGCTC TAAATAGTTA CCTGTGCCCT TTTTCTAACT ATTAGAACCC AAAAAGCCTA TAAATTCTCTCTCTCTCT GTGTATATAT ATACATATAC ACACACACAT AGACACACAC AC CACCTAA ACACACACAT AGAGATTTAT GACTTTAC TTATCCTT GTAAATGC
  • ATAAAACAAC CAAGAAAGAC CTCCACTACC CTGGGAAGGA AACTGGTTGG TATTAAGTAG GACACCACAT AAAACAGGTG TTATTGAGAG GAGAAGAACC AAAATGTAAC TGAGGTTCAA CAAGACATTA TTTATGCAAT GGCAATGAGA AAAATAAAAA ACACAGTATA ACCATGCTGT ATTGCTATAA GTCATGTTAC ACACTGGGAG ATGGCTTCAG GGGTATTTGG TTTTTACTTT TTGTTTGGGA GGTTTTTCAA AAAAATTTAG TTAGAATAAG TCCTTTGAGA AACATCACAG TAGGTTAAAC AAAGTTAGGT TAAATTAGGC TCCTAAGTTT GACTTCTCAG CAAACTTCTA CTGAATGTTC TGACTGTAAG CCCAGGATTG CATGACAAAA CCTCTAGTCT GAAGTTACTC ACCTTGACAG GTTGGTTCTG GAGATGACCA GTTTCCAAAT GGTCCACAGG TGGTTTCTTC AA
  • GAGGCAGCAT AGCCGTCTTG TCACTCCCTA CCTGTGTAAC AGAGGGCTGC CTTTAGTTTG TGGCAGGCGT CATCGTTCCA TTTGCCTGCA TCTTTGTTTC TCTTGATATA GATCTCCACG CAGTCCTCCT TGTTCTTCTT GTTGTTGGGC TCACCATCTC CCCAGTTCTC TGCTTCTTCA GTAAGAGATT TGTTGGTTCC CACCCACGTC CATATTCCTC CTATCTTCCG GATTCCTATC CAGTAGTAAG AACGACTGAA AGGCAGAGTC TTCTCCAGAT ACTCAATTTC CGCCTTGTTT TGTATGGCAA CTAAATCTGT GTAATTGTCT CGGCAGAATC TTCTAGCCCT TTGCCAGTTC ATGGGTTTTT CAGAATAATG GTAAGTCCAG CAGTTC CATGATGTGC CAGGAAATCT GCAAGACATC AGTGACCT ATGCAGACTT ACATAATGTT ACAGCTAAAA AGAAA
  • CAAGCGATTC TTCTGCCTCA GCCTCTTGAG TAGCTGGGAC TATAGGCACG CACCACTATG CCCAGCTAAT TTTTGTATTT TTAGTAGAGT TGGGGTTTCG CCATGTTGGC CAGGATGGTC TTGATCTCTT GACCTCGTGA TCCACCCGCC TCCACCTCCC AAAGTGCTGG GATTACAGGC GTGAGTCACC ATGCCCAGCA CTTGTGTGGA TGTTTTAAGC
  • CCAGCTACTC AGGAGGCTGA GGCAGGAGAA TTGCTTGAAC GCAGGAGGTG GAGGTTGCAG TGAGCCAAGA TCATGCCACT GCACTCCAGT CTAGGCAACA GAATGAGACT CCATCTCAAA ATTAAAAAAA AAAAAAGTAA AAAGAAAAGA TAAGAAATAT AGTACCAGCC CCTATCTCAG AGTTCCTAGC TTAGAAAAAT TCCCAGAATA TAATAAGTGC AATGTAAGGG TCAGCTATCT TCATTATTAT TATCTATCAT AAATGAAATT ACACAATAAA GCTAGATCCG TTTCTTTCCT CTCCTTCTAC AAAAAATAAA GCAACTTTCC AGAACAATAC CCAGGTGATG ATTTCTCCCC TGCTCCCTCC CTAAGATATT GGCAAGTTTG GAGGGTTCAA GGAGAAACAG AGCATGTAGA GAAGATACCT CTCTCATAAC CATTTGTGAT TTACAAGTCT TACCTGATTC TT
  • CTCCGGGCTC TACCTCACTC TTTTCTCCCA CACCCAGGGG GAAGTGTAGG GTTCTCAGAC TTTAGAATAA AGAGGAATCA CCTGGACAAC TCACCTAAAA TGCACATCTT CAGGTCTCAT ACTCAGAGGC TCTGACTCAA CAGGTCTGGG TGGCGCCCAA GAATTTGGGC TTTAAATGAG TATCTCAGAT GATTCTAATA CAGAATGTGT AAGATGACCA GATCCTATCA CACTTAGATG TATTGGCCTA GGGCCACCTA ACTTGGAGAA AATGTTAGTA AGACCCCGTG GTTGGTGCTC AGCTATAGGT ACCAGAATTT TGATCAAAAT TTACTATCAT TGTGACACTT CTCTTCGGAA CTGGAAGGCC AGAACCCCAC TTGTAAAGTG CTGGGAAAAT ACAAGGAAAA TTTAGGGTGA GTAGCATTTT GAATTCTTAC ACATGGAAAG TAAATGTATA AGAATTCTTAC A
  • CAAATAAGCA GCTTCTCTCA AAATGTTGTA ATTACAAAAA TTCCAAGGCA AATATAATAA ACTCCTTGTC GGTGCTATGT CTAGAAACTT AACAGCCCCA AAGAAAGTCC TGACAAGGCA AAAAATATAT ATATATATAC AAATTGTGGA AGCAGGGTGT TGAAAGAAGA ATAAAGACTA TATAAGGACA AACTGTTTAA AAGGGAGGGT ATCCTTGAAA GCTTGACACT TGACTCTTTT GACGAGGCTG AGGGAAAACA CTCAGTTTCA TAGATTGCTG GTACGGATGT AAAATAGTGA CATCCCTATA GAGAGGAATT TGGCAATATC TAGCAAAAGT GCTTATGCAT TTATTCTTTG ACCTAGTAAT CCCGCTTCTA GGATTAGTGG TGAAGATACA CCTCAACAAT AAAAATATAT ATACATTAGG TTATTAGTTA TGGTTTAATT TTTAATAGCA AAATATTTAA AACAACC
  • AAAAAAAAAA AAATGTTGAG AATAATGGAT TCTAACACTT AAAACAAAAA ATAATCCATA GCCCACAGAA GGGGAAGAGA GGGGGAGCTC TTATTTACAG ATGAATATCA AATAGCAAAG ACAGAAGAAA TGACAGAATT AGAGAAACAT CATTTTGCAA AACACCACTG TAATAATCAA TTCAGGCAAG TATTATTAAT GGATGTATTA CTATTGCGTA AAACCAGTTG GGGAACAGGA TATTCATACA GTCTGAAGGT GTCACCCTAA ACATAACTTA TTACAAGTGG AAAATGGTGC CTTTACAATG AAGAAATCTA GCAGAAACCA TCTTAATCTA GTGATCAAAC TTAGTATCAC CAATAATGGA TCATACTGAG TCATGTGTCT CCTAATATGA TGCACCAGGA AGGATGCAAC GTCATGAACG TTGTATTCTT TTGTATTCAA CAGACCACCC AGGGTAAAGG CAGCTTTCTC
  • GCTCATGCCT GTAATCCCAG CACTTTGGGA GGCTGAGGTG GGGGATCACA AGGTCAGGAG ATCGAGACCA TCCTGGCTAA CACGGTAAAA CCCAGTCTCT GCTTAAAAAA TACAAAAAAT TAGCCAGGCG TGGTGGTGGG TGCCTGTAGT CCCAGATACT TGGGAGGCTG AGGCAGGAGA ATGGCGTGAA CCCAGGAGGT GGAGCTTGCA GCGAGCTGAG ATTGTGCCAC TGCACTCCAG CCTGGGCGAC AGAGCAAGAC TCCATCAAAA AAAAAAACAA AAAAACCATT CTGGGGTCTG GAGAATGGTA GCCCTTACAG CACCACCAGG CAGTGCCCCA GTGGGGACTC TGTGTGGGGG CTCTGACCCC ACATTTCCCT TCTGCACGGC CCTAGTAGAG GTTCTCCATG AGGGTTCTAC CCCTGCAGCA AACTTCTGCC TGGACATCCA GGCATTTCCA TACATCCTCG GA
  • TGTAAGCCAC CAATGCTTGG TCAGGGCTTG AACCCTCTGA AGCAATGGCC TGAGCTGTAC GTTGACACCT TTTAGCCTAG ACATCTAGGA CACAGGGCAC CATGACCCGA AGCTTCATAA AGTGGGAGGG CCTTGGGACT AGCTGAGGAA ACCATTTTTC CATCCTAGGC CTCCAGGCCT GTGATGGGAA GGGCAGCCAT GAAGGTGCCT GACATGCCCT GGAGACGTTT TCCCCATTGT CTTGGTAACT AACATTCAGC TCCGTGTGCA GCACCAACTT ACTTATGCAA ATTTCTGTCA CTGGTTTGAA TTTCTCCCCA GAAAACAGGA TTTTTCTTTT CTATTGCATC ATCATGCTGC AAATTTTCAA ACTTTTATGC TATGCTTCCT GTTGAAGACT TTGCGGCTTA GAAATTTCTT CCCCCAGATA CCCAAAATTA TCTCTCTCAA GTTCAAAGTT CCACAGATAT CTA
  • TTTCCCCAGA ATAGGTCTAT GTTTTGCAAT CTGCTACTCC ATACAGAGAT TTGAGTTCAC TTGGCAATTT AGTGCTGCTT ATATGTGACC AGTTAGTCTG TTTTACTTAT CTATGCCTTA AACATTACTA TACTTACTAA CTCCAAGATG CCTGGTCTCA ACTTGACAAA AATACCCCAA GTTGGGAAAT CCTTATGTGA ATATGTAGAT AGTCACAATT GCTGGTTGAT GATGATCTGT CTTTTCCTGT ATTTGAGAAA ATGGAGATAA AATGGACCAA TCCAAATAAT GGATTAAACA TGGGAATAGG TGAGAGAG AGAGGAATAC ATGGTGGCTC TCAGTGTCTG GCTTAGGCAG TAAACACTTT CGTTAATAAA GACGGAAAAT AAAAAAGGAA TAATTGGTGT CTAGGGGAAA ATAATGAGCT CAAGTTTTAA CACTCTGAGT TCCCGGATGT GAGACATCCA GGCGCATTTA
  • AGCCCCCACC CAAAAAAATC ACTCTGTTCT CTCCCCATTC TTTGATAGGC ATACTTGCTG TTTTCTCACA GCCAAGGTAC AGAGGGGACT TAGAGGAACT AGAACTCTAA TACACTGCTA GCAGGAATGT AAAATGAAGC ATCTACTTCA GAAAACCATT TTATCAGTTT CTAGAAAGTT AAACATAGAC CCACCATGCA GCCCAGCCAC TCTACTCCTA AGTATTTACA CAAGAGAAAT GAAAACGTGT CCCCACACAG TTGTATTTAA AGGTGATGGT TAGCCTTGTG TGTCAACTTG GCTAGGCTAT AATACCCAGT TACTGAATCA AATAGTAATC TAGGTGCATC TGTGAAGGTA TTTTGTAGAT GTGGTTAACA GCTACAATCT GTTGACTTCA AGTAAAGGAG ATTGCTCTTG ATAGTATGGG TGGGCTTCAT CCAATCAATT GAAGGCCTTA AGAGCAAAAAAAGGTTTC CC
  • GCCTAAAGAT TTGCTAGGCA TTATAATCAC ATCAGCTAAT TTCTTAAAAT AAACCTCTTT ATATATATTG ATACAATGAA TGGTTATAGC AGCCTTATTT GTAATAGCCA CAAACTGGAA ACAACCTAAA TGTCCTTCAA TAAGTGAATA CATAAACAAA TTGTGGTATA TCCACAATTT TTACGCAGCA GTAAAAAGGA ATAAATGGTT GAATAAGGAA TAAACACATA ACAAGGATGA ACCTTAAAAC CGTAAGGCTG AATGGAAAAA GTCAGACAAA ACTAATACAT ACTGAATAAT TCCATTTATA TTGAAGTTCT AGAAAATGAG GACTAACCTA TAGTAACAAA AAGCAGAAAAAA ATTTTGCCCA CTGGTGATGG AGGGGGCGCA GGTATTGTAG AGTATCTGAG AAAGGACAAC TGGATAAAAG GGGGCACAAG AAAACTTTTG AGGGTGATTG ATATGTTCAT TATCTTGTGG CAT
  • AGTTTACTGT ATATCTATTA TACTTCAGTA GAGAGGAAGG AAGAAAGTGG GCAGGGTGGG GGAGAGGAAA GGAAACGAGG GAGGAAAGGC CCTAATAGGA AGGATTTTGG AGTTTAGATT TTAAAATGAT AAAGGATGTT TGACACTCTA GGCATATGAC GAATATAGGA TTATGAGTCC ACAAAAACCA CCAGGAAGTC ATGTATGTTT ATACTTTTAA GTGAAGGATC AGTGGATTAT CAACTCCCTA ATGCTTTGCC TCTATGAC TGGCTGCTGT CCTTCTCATC CCAATACTCC TTCCAAAGCC CCTTGCTTAA ATGTAAGCCT TCTTTCCTCC TTTCAACACA TCCTGCATTC CGTGACAAAA TAAGTTTTCC TTAAACAGAA TGTACAGCAT ATTATTTGTA CAATTAAAAAAA TTTTTGGCCA GGTGTGATGA CTCATGCCTG TAATCCCAGC AATT
  • CTACTAAAAA TACAAAAATT AGCTGAGTGT AGTGTGGCAG GTACCTGTAA TCCCAGCTAC TCAGGAAGCT GAGGCAGGAG AATCGCTTGA ACCTGGGAGG TGGAGGTTGC TGTGAGCAGA GATCAGACTA TTGCATTCTA GGCTAGGAGA CAGAGTGAGA CTCGGTCCCC AAAAAAAAAC ACATTTTTTT TTAATGTTTC CTCCTTGCCT GTAGGAAAAA GGCTCTGACT CCTTAGCCTG GGCATCAGAG CTCTATCTAA ATGGACTTTA ACCTGATTTT GTGGCACTAA TTCCATTGCA GTACTTGTCC GCTCACTGGC CTGTGCCTCT CTGCCACTAT TTTTGGAATA ATGTCCTCTCTCTCTCTCCTACA AAGCCTCCCC TGGCTACTTC AGCCCACAGA GATATTTAAC TGCTCTGCAG TTCAGGACAT TCTTCTGACT CT
  • GCGTCTAGAA GCTGTGCCCT GTGGGAAGTG GTGGTGCTTG GTAAGAGATG CCAGGACCAG TGGTACCCAC TGGGAGCACT GCCAATACCC AGCAAGGAGC ATGGGTGCAC AGTAAGGCAT TGCACTGTGA TTCAGCATAA AATAACAATA AGGGAACGTC ACGGAGAAAA GGCCAGACTT CCTTTGTTTA GAATGTGGGA AATGTCTTCT GAAAAATGGT AGTAAAAAAG CATGCTTGGA TGGTCCACTC CAGGCAAAAC TGACTAATCG GGGGTCAGGG ATACAACCCC TGCATCATAT GTTTGTTTCT GTTGGGCTGA CATGAGGTTC ACTGTGACCA CTGTGGTTTA ACCCCATAGT CTCCTGGAAA TACAGCCAGG TCAAGAGAGC TCCACATAAA ACATAATCAA AAAAATAAAC TCAAGTTTCC ACTGATCAGC TTTTCACAAC TCTTATCCTT TCACTAACTT TGGAGC
  • TGTTGAGTTA CTATACAACA CAAGAGTAAA CTTTCTTATA AGTGGTAATT TTTTTTTACA GGAATAATTG AAAATGGAAA TTACCTTCTC TACTCATAGT AAGTACTCAG TGCGTTCTTG ATGGGATGAG AATGTGTTTG AGCTTTAGTG TAAGGCAGAA TTCTGTTTAG TCTGCCAGTA TTGGAGAAAA ATAAAACACA AAGGGACTGA CATGTAGGAA GTGGCACCTG GGAGGGTCTC AATTCTTCCT ATTACAAAAA TGCCCCAGAG AAATAAAAAG CTTGTGTACA TGTTGAGATG GGAGTTCT CTGGCCCCCCCC TCGCAGGATG TGTGACAGTG GGGTGGCTCT CTGCTGCGCC ACCATGAGCT CAAACCCCTC ATAGGAGGGG GAGCACACAG GCAGGAAGGT GCAGGAGCTG GGCGAGCTCT TTGGGCTCTG GCCCCGTGGT ACTGTCTAGA GGTG
  • CAGCACAGCC AAGGCACTTG GGGTTTCATG AGACTAAGTA CATGCAATTC TATTGTAAAG GCTTAAAATA TATACAACTG ACCCTTGAAC AACATGAATT TGAATTGCAT GGTCAGTTAT ACGCAGATTT TCTTCCACCT CTGCCACCCC TGAGACAGTA AGATCAATCA ATCCTCTTCC TCCTACTCCT CAGTCTACTC AAAGATACTT GAAGTCTACT TGAAGATGAC AAGCACAAAG ACATTTATGA TGATCCACTT CCACTTAGTG AATAGTAAAT ATGTTTTCTC TTCCTCCTAA TTTTTTAACA CTTTCTTCTC TCTAGCTTAA TTTATTGTTA AGAATACAAT CTATAATACA TATGACATAC AAAATATGTC TTAGTTGACT GTTTATGTTA TCTGTAAGGC TTCAGGTCAA GAGTATGCTA TTAGTGGTTA AGTTCGAG GAGTCAAAAG GTGTATGTGG ACTTTCAACT GCAG
  • AAATAATAGC AAAACAGAAA TCAAACACTC AAATTTTTGG TCCTTCTGTT TATTTCATTT TGGATACTCA GTGAATGTTA ATTAACCAGG AAACTTAAAA GTTATTTCAA TTATGAACCT CTTCAATCCT TCATCAATTA TTTTGAGTAT TCTGGTCTTA AAAACATCTC TTTCTTCTAC AAACTTCTGA AAGAGATGAA CACCTCCACC TACACCAAAAAA TAATGTGCTT TGCTGGCCAA AAGTACACGT CCATTTTTAC TTAACAGTCT AAGGAAAGTC TGGTGCAAAT TACTATAATA ATCTGGGTTG TAAATGGTTT CTGAGGTGAG AATGAGATCA TATTTTACAA AAAGTTTTTC ACTACTTAGT ACAAGCTTAC AAAACTCAGA CCACTCACCA GAAAAAAATC GGCATTTATA TAGTTGTGTT ACTTTTGGTT TCCTGCATCT TTTCACATCT GGCTCATTTA CATTTTC
  • GCATTTTACG TATTATTTTC CAGGCCTTAC CTGCATGTCT GCATAATCAT AACTGACTAA TTTTGGAACA GCTGGTAATT ATTTGAGCTT TACTGAAATT TTTTCATGAG GCCAATTCTA CCCTACTGAA CTCAAATTTG AGTTAATGAT GACCTCATTT TGATTGCTGC TGTAAAAAAT AAGATTTCGG AAGAGGAATG AATTCTTGTA TTACTGTGGT AGGACTATGG GTTTTTTTTT GTTTGTTTGT TTGTTTTGAG ACGGAGTCTC ACCCTGTCAC CCAGGCTGGA GTGCAGTGGT GCGATCTCAG CTCACAGCAG CCAGGTTCAA GTGATTCTCC TTCCTCAGCC TCCCGAGTAG CTGAGATTAC AGGCACGTGC CACCATGCCC GGCTAATTTT TTGTATCTTT AGTAGAGATG GTTTCACCAT GTTGGCCAGG CTGGTCTCGA ACTCCTGACC TCGTGATCCG CCTGCC
  • CTGAAAAGCA CAAGCATACC TTTTTTGTTT TAAATGGAGG GAACTAAAGA TACTTTGGTG CCAAAATGAA ACATTATTTG TAATTAATCT CTTATTGAAA TGGGTTTCTA ACTTTAGCTT TGAATCGTAA TCTTTCAAAT TTCTTGTACT CATAGTCACT TGATGATTCT CTATCTGAAA TATTTCTTAG AATTTGTTCT TGACCACCAG AAAAAGATTC AACTGTTACA TAGATGAAAA TGGATGTTGA GTGTTAACAG GCCTATGGGA AACAGTATTT TCTTTAGCTA CATTGTATTG TTGACTGTGT TGCTATTCTT ATAATGTTTA GGTCATTTAA ATTGTTAGAA AGATCCAAGT ATTAAGATCT AGGGTGGCTA ACTTTTCACA GACAAAAAGC TTGTAA GGTCATTTAC GGTCATTTAC TATACCCTTA ATTCAGGAAG GTTAGCTTGA ATTGGGTCAA AAGGAAACTG
  • CCCTGTTCTT GGATCTGCCR TGGAGGGATC TGGTGCCTCC AGACATGTGC ACATGARTCC ATATGGAGCT TTTCCTGATG TTCCACTCCA CTTTGTATAG ACATCTGCCC TGACTGAATG TGTTCTGTCA CTCAGCTTTG CTTCCGACAC CTCTGTTTCC TCTTCCCCTT TCTCCTCGTA TGTGTGTTTA CCTAAACTAT ATGCCATAAA CCTCAAGTTA TTCA AAGCTTCTAC CCTAGTCTGG TGCTACACTT ACATTGCTTA CATCCAAGTG TGGTTATTTC TGTGGCTCCT GTTATAACTA TTATAGCACC AGGTCTATGA CCAGGAGAAT TAGACTGGCA TTAAATCAGA ATAAGAGATT TTGCACCTGC AATAGACCTT ATGACACCTA ACCAACCCCA TTATTTACAA TTAAACAGGA ACAGAGGGAA TACTTTATCC AACTCACACA AGCTGT
  • GCCTTAATCA TTATTTTACT GCATGGTAAT TAGGGACAAA TGGTAAATGT TTACATAAAT AATTGTATTT AGTGTTACTT TATAAAATCA AACCAAGATT TTATATTTTT TTCTCCTCTT TGTTAGCTGC CAGTATGCAT AAATGGCATT AAGAATGATA ATATTTCCGG GTTCACTTAA AGCTCATATT ACACATACAC AAAACATGTG TTCCCATCTT TATACAAACT CACACATACA GAGCTACATT AAAAACAACT AATAGGCCAG GCACGGTGGC TCAGACCTGT AATCCCAGCA CTTTGGGAGG ACCAACCTCT
  • AAAGAAGTCA AG AGCTGCCAGC CAGAGAGGGA GTCATTTCAT TGGCGTTTGA GTCAGCAAAG AAGTCAAGAT GGCCAAAGTT CCAGACATGT TTGAAGACCT GAAGAACTGT TACAGTGAAA ATGAAGAAGA CAGTTCCTCC ATTGATCATC TGTCTCTGAA TCAGAAATCC TTCTATCATG TAAGCTATGG CCCACTCCAT GAAGGCTGCA TGGATCAATC TGTGTCTCTG AGTATCTCTG AAACCTCTAA AACATCCAAG CTTACCTTCA AGGAGAGCAT GGTGGTAGTA GCAACCAACG GGAAGGTTCT GAAGAAGAGAGA CGGTTGAGTT TAAGCCAATC CATCACTGAT GATGACCTGG AGGCCATCGC CAATGACTCA GAGGAAGAAA TCATCAAGCC TAGGTCATCA CCTTTTAGCT TCCTGAGCAA TGTGAAATAC AACTTTATGA GGATCATCAA ATACGAATT
  • CTGCCCCAGC CACCCCCGGA CCCGCCGGCG CCCCCGCTGG CGCCCCTC CTCAGCCTGG GGGGGCATCA GGGCCGCCCA CGCCATCCTG GGGGGGCTGC ACCTGACACT TGACTGGGCC GTGAGGGGAC TGCTGCTGCT GAAGACTCGG CTGTGACCCG GGGCCCAAAG CCACCACCGT CCTTCCAAAG CCAGATCTTA TTTATTTATT TATTTCAGTA CTGGGGGCGA AACAGCCAGG TGATCCCCCC GCCATTATCT CCCTAGTT AGAGACAGTC CTTCCGTGAG GCCTGGGGGA CATCTGTGCC TTATTTATAC TTATTTATTT CAGGAGCAGG GGTGGGAGGC AGGTGGACTC CTGGGTCCCC GAGGAGGAGG GGACTGGGGT CCCGGATTCT TGGGTCTCCA AGAAGTCTGT CCACAGACTT CTGCCCTGGC TCTGGAACAT AT
  • TCACAGCTTT TTTCCCTGCC AGAGGGACAA ATTCCCAGCT GACGGGGACC ACAACCTGGA TTCCCTGCCC ACCCTGGCCA TGAGTGCAGG GGCACTGGGA GCTCTACAGG TAAGGGCAAG GGAGTGGGCT GGGGACAAGG TGGGAGGCAG GCAGTGAAGG GGGCGGGGAG GATGAGGGGC ACTGGTCGGG TGTTCTCTGA TGTCCCGGCT CTATCCCCAG CTCCCAGGTG TGCTGACAAG GCTGCGAGCG GACCTACTGT CCTACCTGCG GCACGTGCAG TGGCTGCGCC GGGCAGGTGG CTCTTCCCTG AAGACCCTGG AGCCCGAGCT GGGCACCCTG CAGGCCCGAC TGGACCGGCT GCTGCGCCGG CTGCAGCTCC TGGTATGTCC TGGCCCCAAG ACCTGACACC CCAGACCCCC ACCCCTGGCC CCAAAATCCT GTGGCCTGAG TCCTTGAA
  • CAGCCCTGAC TCCACAGACT TCACCCCCAA CCCCCACACT CAGCTCTGGA AGCCCGTCCT GACTCCAGCC TCCATTTTCG GAACCCCACA GCCTGAAGAG CTCCCGGCCT AAACACTTCA CCCCACGCGC CACAGTCCCC CTGTGAATAT GCAGCCCCGA TTCAGCTGCA GCTCCACAGC ACCCCTGCCC TGCACCCCCG CTGCACCCCC TACCTGTGAC TCACCTCT CCTCTCCCCA CAGATGTCCC GCCTGGCCCT GCCCCAGCCA CCCCCGGACC CGCCGGCGCC CCCGCTGGCG CCCCCCTCCT CAGCCTGGGG GGGCATCAGG GCCGCCCACG CCATCCTGGGGGGGCTGCAC CTGACACTTG ACTGGGCCGT GAGGGGACTG CTGCTGCTGA AGACTCGGCT GTGACCCGGG GCCCAAAGCC ACCACCGTCC TTCCAAAGCC AGATCTTATT TATTTATTTA TTTCAGT
  • CTGATGACTA G ATGAGTGTCA GAAGTGTGAA GGGTGCCTGT TCTGAATCCC AGAGCCTCCT CTCCCTCTGT GAGGCTGGCA GGTGAGGAAG GGTTTAACCT CACTGGAAGG AATCCCTGGA GCTAGCGGCT GCTGAAGGCG TCGAGGTGTG GGGGCACTTG GACAGAACAG TCAGGCAGCC GGGAGCTCTG CCAGCTTTGG TGACCTTGGG CCGGGCTGGG AGCGCTGCGG CGGGAGCCGG AGGACTATGA GCTGCCGCGC GTTGTCCAGA GCCCAGCCCA GCCCTACGCG CGCGGCCCGG AGCTCTGTTC CCTGGAACTT TGGGCACTGC CTCTGGGACC CCTGCCGGCC AGCAGGCAGG ATGGTGCTTG CCTCGTGCCC CTTGGTGCCC GTCTGCTGAT GTGCCCAGCC TGTGCCCGCC ATGCCGCCCT CCATCTCAGC TTTCCAGGCC GCCTACATCG
  • GCTGGCAATT GCTGTGGACC GCTACCTCCG GGTCAAGATC CCTCTCCGGT ACAAGATGGT GGTGACCCCC CGGAGGGCGG CGGTGGCCAT AGCCGGCTGC TGGATCCTCT CCTTCGTGGT GGGACTGACC CCTATGTTTG GCTGGAACAA TCTGAGTGCG GTGGAGCGGG CCTGGGCAGC CAACGGCAGC ATGGGGGAGC CCGTGATCAA GTGCGAGTTC GAGAAGGTCA TCAGCATGGA GTACATGGTC TACTTCAACT TCTTTGTGTGTGTGGGTGCTGCCC CCGCTTCTCC TCATGGTCCT CATCTACCTG GAGGTCTTCT ACCTAATCCG CAAGCAGCTC AACAAGAAGG TGTCGGCCTC CTCCGGCGAC CCGCAGAAGT ACTATGGGAA GGAGCTGAAG ATCGCCAAGT CGCTGGCCCT CATCCTCTTC CTCTTTGCCC TCAGCTGGCT GCCTTTGCAC
  • CATCCTCACC CAGAGCTCCA TCCTGGCCCT GCTGGCAATT GCTGTGGACC GCTACCTCCG GGTCAAGATC CCTCTCCGGT ACAAGATGGT GGTGACCCCC CGGAGGGCGG CGGTGGCCAT AGCCGGCTGC TGGATCCTCT CCTTCGTGGT GGGACTGACC CCTATGTTTG GCTGGAACAA TCTGAGTGCG GTGGAGCGGG CCTGGGCAGC CAACGGCAGC ATGGGGGAGC CCGTGATCAA GTGCGAGTTC GAGAAGGTCA TCAGCATGGA GTACATGGTC TACTTCAACT TCTTTGTGTGTGTGGGTGCTGCCC CCGCTTCTCC TCATGGTCCT CATCTACCTG GAGGTCTTCT ACCTAATCCG CAAGCAGCTC AACAAGAAGG TGTCGGCCTC CTCCGGCGAC CCGCAGAAGT ACTATGGGAA GGAGCTGAAG ATCGCCAAGT CGCTGGCCCT CATCCTCTTC CT

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Pulmonology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Otolaryngology (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne une composition comportant un excipient, des oligonucléotides qui sont antisens aux récepteurs d'adénosine, et contiennent de faibles quantités ou pas d'adénosine (A), et des agents bronchodilateurs. Ces agents et la composition et les formulations de l'invention sont aptes au traitement de la voie respiratoire, des maladies pulmonaires et malignes associées à la bronchoconstriction, de l'inflammation et des allergies de la voie respiratoire, des voies aériennes altérées, y compris la maladie pulmonaire et les maladies dont les effets secondaires affectent les poumons d'un sujet, telles que les allergies, l'asthme, les troubles respiratoires, la rhinite allergique, la douleur, la mucoviscidose, la fibrose pulmonaire, l'infection des voies respiratoires, la broncho-pneumopathie chronique obstructive, et les cancers, entre autres. Les agents et la composition de l'invention peuvent être administrés de manière préventive, prophylactique ou thérapeutique conjointement avec d'autre thérapies, ou peuvent être utilisés en tant que substitut pour des thérapies susceptibles de provoquer des effets secondaires négatifs importants.
PCT/US2002/013143 2001-04-24 2002-04-23 Composition, preparations et trousses pour le traitement de maladie respiratoire et pulmonaire au moyen d'oligonucleotides antisens et d'un agent bronchodilateur WO2002085309A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002305236A AU2002305236A1 (en) 2001-04-24 2002-04-23 Composition, formulations and kits for treatment of respiratory and lung disease with anti-sense oligonucleotides and a bronchodilating agent
US10/627,930 US20040049022A1 (en) 2001-04-24 2003-07-25 Composition & methods for treatment and screening

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28603601P 2001-04-24 2001-04-24
US60/286,036 2001-04-24

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/627,930 Continuation-In-Part US20040049022A1 (en) 2001-04-24 2003-07-25 Composition & methods for treatment and screening

Publications (2)

Publication Number Publication Date
WO2002085309A2 true WO2002085309A2 (fr) 2002-10-31
WO2002085309A9 WO2002085309A9 (fr) 2012-02-16

Family

ID=23096778

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/013143 WO2002085309A2 (fr) 2001-04-24 2002-04-23 Composition, preparations et trousses pour le traitement de maladie respiratoire et pulmonaire au moyen d'oligonucleotides antisens et d'un agent bronchodilateur

Country Status (2)

Country Link
AU (1) AU2002305236A1 (fr)
WO (1) WO2002085309A2 (fr)

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006003492A2 (fr) * 2004-07-02 2006-01-12 Warner-Lambert Company Llc Compositions et methodes de traitement d'infections pathologiques
WO2006094406A1 (fr) * 2005-03-11 2006-09-14 Sarissa Inc. Oligonucleotides antisens cibles sur la region de codage de la thymidylate synthase et utilisations de ceux-ci
EP1758998A2 (fr) * 2004-01-30 2007-03-07 Quark Biotech, Inc. Oligoribonucleotides et procedes d'utilisation de ceux-ci dans le traitement d'etats fibreux et d'autres maladies
US7507810B2 (en) 2005-02-25 2009-03-24 Isis Pharmaceuticals, Inc. Compositions and their uses directed to IL-4R alpha
US7585500B2 (en) 2004-11-17 2009-09-08 Amgen Inc. Fully human monoclonal antibodies to IL-13
US7713521B2 (en) 2005-08-12 2010-05-11 Schering Corporation MCP1 fusions
WO2010120524A2 (fr) 2009-03-31 2010-10-21 Altair Therapeutics, Inc. Procédés de modulation d'une réponse immunitaire à une infection virale
US7893243B2 (en) 2005-04-12 2011-02-22 Intradigm Corporation Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases
US7893244B2 (en) 2005-04-12 2011-02-22 Intradigm Corporation Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases
US20110319475A1 (en) * 2009-02-12 2011-12-29 Opko Curna, Llc Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf
EP2314691A3 (fr) * 2002-11-14 2012-01-18 Dharmacon, Inc. SIRNA fonctionnel et hyperfonctionnel
CN102370620A (zh) * 2010-08-26 2012-03-14 同济大学附属上海市肺科医院 一种肺靶向性的免疫纳米脂质体及其制备方法
US8524217B2 (en) 2010-05-11 2013-09-03 Merck Sharp & Dohme Corp. MCP1-Ig fusion variants
US8546349B2 (en) 2010-07-28 2013-10-01 Thermo Fisher Scientific Biosciences Inc. siRNA targeting VEGFA and methods for treatment in vivo
US8575329B2 (en) 2002-11-14 2013-11-05 Thermo Fisher Scientific Biosciences Inc. siRNA targeting kinase insert domain receptor (KDR)
US20150159162A1 (en) * 2008-12-04 2015-06-11 Curna, Inc. Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene
US20170355990A1 (en) * 2010-06-23 2017-12-14 Curna, Inc. Treatment of sodium channel, voltage-gated, alpha subunit (scna) related diseases by inhibition of natural antisense transcript to scna
WO2019020371A1 (fr) * 2017-07-28 2019-01-31 Universität Zu Lübeck Médicaments antisens contre l'icam-1 canine destinés à être utilisés dans le traitement de l'inflammation de la cavité buccale
US10370657B2 (en) * 2009-06-16 2019-08-06 Curna, Inc. Treatment of Collagen gene related diseases by inhibition of natural antisense transcript to a collagen gene
US10688044B2 (en) 2018-03-19 2020-06-23 Bryn Pharma, LLC Epinephrine spray formulations
US20210252039A1 (en) * 2018-06-14 2021-08-19 Ionis Pharmaceuticals, Inc. Compounds and methods for increasing stmn2 expression
WO2021247770A1 (fr) * 2020-06-03 2021-12-09 Cedars-Sinai Medical Center Méthodes et systèmes de mesure de la récurrence d'une maladie après opération
WO2022122872A1 (fr) * 2020-12-09 2022-06-16 Ucl Business Ltd Agents thérapeutiques pour le traitement des troubles neurodégénératifs
US11578329B2 (en) 2021-04-19 2023-02-14 Novo Nordisk A/S Compositions and methods for inhibiting nuclear receptor subfamily 1 group H member 3 (NR1H3) expression
AU2020204409B2 (en) * 2013-09-05 2023-05-25 Murdoch University Antisense-induced exon2 inclusion in acid alpha-glucosidase

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2314691A3 (fr) * 2002-11-14 2012-01-18 Dharmacon, Inc. SIRNA fonctionnel et hyperfonctionnel
US8575329B2 (en) 2002-11-14 2013-11-05 Thermo Fisher Scientific Biosciences Inc. siRNA targeting kinase insert domain receptor (KDR)
JP2007528216A (ja) * 2004-01-30 2007-10-11 クアーク・ファーマスーティカルス、インコーポレイテッド 線維性状態およびその他の疾患の治療のための、オリゴリボヌクレオチドおよびその使用方法
US7939652B2 (en) 2004-01-30 2011-05-10 Quark Pharmaceuticals Inc. Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
EP1758998A4 (fr) * 2004-01-30 2008-02-20 Quark Pharmaceuticals Inc Oligoribonucleotides et procedes d'utilisation de ceux-ci dans le traitement d'etats fibreux et d'autres maladies
JP4755113B2 (ja) * 2004-01-30 2011-08-24 クアーク・ファーマスーティカルス、インコーポレイテッド 線維性状態およびその他の疾患の治療のための、オリゴリボヌクレオチドおよびその使用方法
EP2330111A3 (fr) * 2004-01-30 2011-08-17 Quark Pharmaceuticals, Inc. Oligoribonucléotides et procédés d'utilisation de ceux-ci dans le traitement d'états fibreux et d'autres maladies
US8198258B2 (en) 2004-01-30 2012-06-12 Quark Pharmaceuticals Inc. Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases
EP1758998A2 (fr) * 2004-01-30 2007-03-07 Quark Biotech, Inc. Oligoribonucleotides et procedes d'utilisation de ceux-ci dans le traitement d'etats fibreux et d'autres maladies
WO2006003492A3 (fr) * 2004-07-02 2006-09-28 Warner Lambert Co Compositions et methodes de traitement d'infections pathologiques
WO2006003492A2 (fr) * 2004-07-02 2006-01-12 Warner-Lambert Company Llc Compositions et methodes de traitement d'infections pathologiques
US7585500B2 (en) 2004-11-17 2009-09-08 Amgen Inc. Fully human monoclonal antibodies to IL-13
US7994302B2 (en) 2004-11-17 2011-08-09 Amgen Inc. Fully human monoclonal antibodies to IL-13
US8153603B2 (en) 2005-02-25 2012-04-10 Isis Pharmaceuticals, Inc. Compositions and their uses directed to IL-4R alpha
EP2316941A2 (fr) 2005-02-25 2011-05-04 Isis Pharmaceuticals, Inc. Compositions et leurs utilisations ciblées sur IL 4R-alpha
US7507810B2 (en) 2005-02-25 2009-03-24 Isis Pharmaceuticals, Inc. Compositions and their uses directed to IL-4R alpha
WO2006094406A1 (fr) * 2005-03-11 2006-09-14 Sarissa Inc. Oligonucleotides antisens cibles sur la region de codage de la thymidylate synthase et utilisations de ceux-ci
US7893244B2 (en) 2005-04-12 2011-02-22 Intradigm Corporation Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases
US7893243B2 (en) 2005-04-12 2011-02-22 Intradigm Corporation Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases
US8282914B2 (en) 2005-08-12 2012-10-09 Merck, Sharp & Dohme Corp. Method for treating atherosclerosis by administering human MCP1 fusions
US7713521B2 (en) 2005-08-12 2010-05-11 Schering Corporation MCP1 fusions
US7972591B2 (en) 2005-08-12 2011-07-05 Schering Corporation Methods for treating rheumatoid arthritis and multiple sclerosis using MCP1 fusions
US11697814B2 (en) 2008-12-04 2023-07-11 Curna, Inc. Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene
US20150159162A1 (en) * 2008-12-04 2015-06-11 Curna, Inc. Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene
US10519448B2 (en) 2009-02-12 2019-12-31 Curna, Inc. Treatment of brain derived neurotrophic factor (BDNF) related diseases by inhibition of natural antisense transcript to BDNF
US9074210B2 (en) * 2009-02-12 2015-07-07 Curna, Inc. Treatment of brain derived neurotrophic factor (BDNF) related diseases by inhibition of natural antisense transcript to BDNF
US20110319475A1 (en) * 2009-02-12 2011-12-29 Opko Curna, Llc Treatment of brain derived neurotrophic factor (bdnf) related diseases by inhibition of natural antisense transcript to bdnf
WO2010120524A2 (fr) 2009-03-31 2010-10-21 Altair Therapeutics, Inc. Procédés de modulation d'une réponse immunitaire à une infection virale
WO2010120511A2 (fr) 2009-03-31 2010-10-21 Altair Therapeutics, Inc. Procédé de traitement de troubles respiratoires
US10370657B2 (en) * 2009-06-16 2019-08-06 Curna, Inc. Treatment of Collagen gene related diseases by inhibition of natural antisense transcript to a collagen gene
US11339394B2 (en) 2009-06-16 2022-05-24 Curna, Inc. Treatment of collagen gene related diseases by inhibition of natural antisense transcript to a collagen gene
US8524217B2 (en) 2010-05-11 2013-09-03 Merck Sharp & Dohme Corp. MCP1-Ig fusion variants
US20170355990A1 (en) * 2010-06-23 2017-12-14 Curna, Inc. Treatment of sodium channel, voltage-gated, alpha subunit (scna) related diseases by inhibition of natural antisense transcript to scna
US9150862B2 (en) 2010-07-28 2015-10-06 Thermo Fisher Scientific Inc. siRNA targeting VEGFA and methods for treatment in vivo
US8546349B2 (en) 2010-07-28 2013-10-01 Thermo Fisher Scientific Biosciences Inc. siRNA targeting VEGFA and methods for treatment in vivo
CN102370620A (zh) * 2010-08-26 2012-03-14 同济大学附属上海市肺科医院 一种肺靶向性的免疫纳米脂质体及其制备方法
AU2020204409B2 (en) * 2013-09-05 2023-05-25 Murdoch University Antisense-induced exon2 inclusion in acid alpha-glucosidase
WO2019020371A1 (fr) * 2017-07-28 2019-01-31 Universität Zu Lübeck Médicaments antisens contre l'icam-1 canine destinés à être utilisés dans le traitement de l'inflammation de la cavité buccale
US11773394B2 (en) 2017-07-28 2023-10-03 Universität Zu Lübeck Antisense drugs against canine ICAM-1 for use in the treatment of inflammation of the buccal cavity
JP2020528443A (ja) * 2017-07-28 2020-09-24 ウニヴェルジテート ツー リューベック 口腔の炎症の治療のために用いられるイヌｉｃａｍ−１に対するアンチセンス薬物
US11008574B2 (en) 2017-07-28 2021-05-18 Universität Zu Lübeck Antisense drugs against canine ICAM-1 for use in the treatment of inflammation of the buccal cavity
JP7153947B2 (ja) 2017-07-28 2022-10-17 ウニヴェルジテート ツー リューベック 口腔の炎症の治療のために用いられるイヌｉｃａｍ－１に対するアンチセンス薬物
US10925841B2 (en) 2018-03-19 2021-02-23 Bryn Pharma, LLC Epinephrine spray formulations
US11000489B2 (en) 2018-03-19 2021-05-11 Bryn Pharma, LLC Epinephrine spray formulations
US11723884B2 (en) 2018-03-19 2023-08-15 Bryn Pharma, LLC Epinephrine spray formulations
US10688044B2 (en) 2018-03-19 2020-06-23 Bryn Pharma, LLC Epinephrine spray formulations
US20210252039A1 (en) * 2018-06-14 2021-08-19 Ionis Pharmaceuticals, Inc. Compounds and methods for increasing stmn2 expression
US11833168B2 (en) * 2018-06-14 2023-12-05 Ionis Pharmaceuticals, Inc. Compounds and methods for increasing STMN2 expression
WO2021247770A1 (fr) * 2020-06-03 2021-12-09 Cedars-Sinai Medical Center Méthodes et systèmes de mesure de la récurrence d'une maladie après opération
WO2022122872A1 (fr) * 2020-12-09 2022-06-16 Ucl Business Ltd Agents thérapeutiques pour le traitement des troubles neurodégénératifs
US11578329B2 (en) 2021-04-19 2023-02-14 Novo Nordisk A/S Compositions and methods for inhibiting nuclear receptor subfamily 1 group H member 3 (NR1H3) expression

Also Published As

Publication number Publication date
AU2002305236A1 (en) 2002-11-05
AU2002305236A8 (en) 2012-03-08
WO2002085309A9 (fr) 2012-02-16

Similar Documents

Publication Publication Date Title
WO2002085308A2 (fr) Compositions, formulations et trousses contenant des oligonucleotides anti-sens et des steroides anti-inflammatoires et/ou un ubiquinone pour le traitement de maladies respiratoires ou pulmonaires
WO2002085309A2 (fr) Composition, preparations et trousses pour le traitement de maladie respiratoire et pulmonaire au moyen d'oligonucleotides antisens et d'un agent bronchodilateur
WO2000062736A2 (fr) Oligonucleotide antisens a faible teneur en adenosine, compositions, kit et procede pour le traitement d'affections des voies aeriennes associees a la bronchoconstriction, a l'inflammation pulmonaire, aux allergies et a la depletion de surfactant
US7034007B1 (en) Low adenosine anti-sense oligonucleotide, compositions, kit & method for treatment of airway disorders associated with bronchoconstriction, lung inflammation, allergy(ies) & surfactant depletion
AU2018236699B2 (en) Methods and compositions for modulating apolipoprotein(a) expression
KR102604132B1 (ko) 상염색체 우성 정신 지체 5 및 드라베 증후군의 치료를 위한 안티센스 올리고머
KR102482890B1 (ko) 아포지질단백질 (a) 발현을 조절하는 조성물 및 방법
AU2018203564A1 (en) Antisense modulation of gccr expression
US6025339A (en) Composition, kit and method for treatment of disorders associated with bronchoconstriction and lung inflammation
CN1317009A (zh) 低腺苷反义寡核苷酸制剂、组合物、试剂盒及处理
AU9395198A (en) Multiple target hybridizing nucleic acids, their preparation, compositions, formulation, kits and applications
WO1998023294A9 (fr) Agent et methode de traitement de maladies et d'etats pathologiques associes a des malaises respiratoires et a une inflammation pulmonaire
CN110951731A (zh) 用于调节c9orf72表达的组合物
JPH11507218A (ja) アンチセンスオリゴヌクレオチドを用いる肺疾患の治療方法
US6825174B2 (en) Composition, formulations & method for prevention & treatment of diseases and conditions associated with bronchoconstriction, allergy(ies) & inflammation
KR102431353B1 (ko) B형 간염 감염을 치료하기 위한 PAPD5 및 PAPD7 mRNA의 감소용 핵산 분자
EP1411967B1 (fr) Peptide bloquant agissant sur la secretion cellulaire inflammatoire
AU5071002A (en) Low adenosine oligonucleotide, composition, kit & methods for obtaining oligonucleotide & for treatment of airway disease(s)
MXPA01000971A (es) Agente composicion, estuche y tratamientos que emplean un oligonucleotido con bajo contenido de adenosina
AU724817B2 (en) Method of treatment for asthma
MXPA00002640A (en) Multiple target hybridizing nucleic acids, their preparation, compositions, formulation, kits and applications
CN109706234A (zh) 解热镇痛类药物个性化基因检测试剂盒
AU2008200379A1 (en) Blocking peptide for inflammatory cell secretion

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP