MXPA97009865A - Method of treatment for a - Google Patents

Abstract

A method for reducing bronchoconstriction in an individual in need of such treatment is described. The method comprises administering to the subject an antisense oligonucleotide molecule directed against the adenosine A1 or A3 receptor in an amount effective to reduce bronchoconstriction. The method is useful to treat patients suffering from asthma. Pharmaceutical formulations are described

Description

ASTHMA TREATMENT METHOD This invention was made with government support under grant RO 1 CA47217-06 of the National Cancer Institute. The Government has certain rights to this invention.

FIELD OF THE INVENTION This application relates to a method for administering antisense oligonucleotides against adenosine A &sub2; | or A3 as for treatment for asthma.

BACKGROUND OF THE INVENTION Asthma is one of the most common conditions in industrialized countries and in the United States it amounts to approximately 1% of all costs for health care. K. Weiss et al. , New England. J. Med. 326, 862-866 (1992). There has been an alarming increase in both the frequency and mortality of asthma during the past decade, Asthma-United States, 1980-1990, MMWR 41, 733-735 (1992), and it is predicted that occupational asthma will be the condition Occupational pulmonary disease in the next decade. M. Chan-Yeung and J. Malo, European Resp. J. 7, 346-371 (1994). Although the increasing mortality of asthma in industrialized countries could be attributed to increased confidence in beta-blockers in the treatment of this condition, the underlying causes of asthma remain poorly understood. J. Gern and R. Lemanske, In Immunology and Allergy Clinics of North America 13, Bush, R.K. ed. W.B. Saunders Company, London, pp. 839-860 (1993). Adenosine can be an important mediator of bronchial asthma. R. Pauwels et al., Clinical & Exp. Allergy 21 Suppl. 1, 48-55 (1991); S. Holgate et al., Annal of the New York Acad. Sci. 629, 227-236 (1991). The potential role of adenosine in asthma in humans is based on the experimental discovery that, in contrast to normal individuals, asthmatic individuals respond to aerosolized adenosine with marked bronchoconstriction. M. Church and S. Holgate, Trends Pharmacol. Sci. 7, 49-50 (1986); M. Cushley et al., Br. J. Clin. Pharmacol. 15, 161-165 (1983). Similarly, the asthmatic rabbits produced using the allergic rabbit to the dust mite of the human model also showed a response to aerosolized adenosine with marked bronchoconstriction, whereas * _ > non-asthmatic rabbits showed no response. S. Ali et al., Agents Action 37, 165-176 (1992). Recent work using this model system has suggested that adenosine-mediated bronchoconstriction and bronchial hypersensitivity in asthma are mediated primarily through the stimulation of adenosine receptors. S. Ali et al., J. Pharmacol. Exp. Ther. 268, 1328-1334 (1994); S. Ali et al., Am. J. Physiol 266, L271-277 (1994). Theophylline, an important drug in the treatment of asthma, is a known adenosine receptor (see M. Cushley et al., Am. Rev. Resp. Dis. 129, 380-384 (1984)) and was found to eliminate the Adenosine-mediated bronchoconstriction in asthmatic rabbits (Ali, et al., supra.). The pretreatment of allergic rabbits with another specific receptor antagonist Ai, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), potently inhibited adenosine-mediated bronchoconstriction and bronchial hypersensitivity in asthmatic rabbits. Id.

The therapeutic potential, however, of the specific receptor antagonists A- | currently available is limited by its toxicity.

H. Klitgaard et al., European J. Pharmacol. 242, 221-228 (1993). Theophylline has been widely used in the treatment of asam, but is associated with frequent significant toxicity resulting from its narrow therapeutic dose regimen. E. Powell et al., Pediatric Emergency Care 9, 129-133 (1993); S. Nasser and P. Rees, Drug Safety 8, 12-18 (1993); P. Epstein, Annals of Interna! Med. 119, 1216-1217 (1993). The availability of an alternative strategy to regulate adenosine-mediated bronchoconstriction would clearly be of therapeutic interest.

BRIEF DESCRIPTION OF THE INVENTION A first aspect of the present invention is a method for reducing adenosine-mediated bronchoconstriction in an individual in need of such treatment. The method comprises administering an adenosine receptor antisense oligonucleotide to the subject's lungs in an amount effective to reduce bronchoconstriction, wherein the adenosine receptor is selected from the group consisting of adenosine A-receptors | and A3 adenosine receptors. A second aspect of the present invention is a method of treating asthma in an individual in need of such treatment. The method comprises administering an adenosine receptor antisense oligonucleotide oligonucleotide to the lungs of the subject in an amount effective to treat asthma, wherein the adenosine receptor is selected from the group consisting of adenosine A <receptors.; | and A3 adenosine receptors. A third aspect of the present invention is a pharmaceutical composition, comprising, together in a pharmaceutically acceptable carrier, an adenosine receptor antisense oligonucleotide wherein the adenosine receptor is selected from the group consisting of adenosine A receptors and receptors. of adenosine A3 in an effective capacity to reduce adenosine-mediated bronchoconstriction. A fourth aspect of the present invention is the use of an adenosine receptor antisense oligonucleotide as presented above for the preparation of a medicament for (a) to reduce adenosine-mediated bronchoconstriction in an individual in need of such treatment, or ( b) treat asthma in an individual who needs such treatment. Antisense oligonucleotides have received considerable theoretical consideration as pharmacological agents potentially useful in human conditions. R. Wagner, Nature 372, 333-335 (1994). However, the practical applications of these molecules in the real models of human suffering have been evasive. An important consideration in the practical application of these molecules is the route of administration. Most experiments using antisense oligonucleotides in vivo have included direct application to limited regions of the brain (see C. Wahlestedt, Trends in Pharmacological Sciences 15, 42-46 (1994); J. Lai et al., Neurireport 5 , 1049-1052 (1994), K. Stafinder et al., Neuron 12, 805-810 (1994), A. Akabayashi et al., Brain Research 21, 55-61 (1 994)), or the spinal fluid / see for example L. Tseng et al. , European J, Pharmacol. 258, R 1 -3 (1994); R. Raffa et al. , European J, Pharmacol. 258, R5-7 (1994); F. Gillardon et al. , European J, Neurosci. 6, 880-884 (1994). Such applications have limited clinical utility due to their invasive nature. The systematic administration of antisense oligonucleotides also poses significant problems with respect to pharmacological application, of which the difficulty in targeting the tissues involved in the disease is not the least. In contrast, the lung is an excellent potential target for the application of antisense oligonucleotide since it can be reached in non-invasive form and in a specific tissue form.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the effects of adenosine receptor antisense oligonucleotide A-j and non-corresponding control antisense oligonucleotide on dynamic bronchial airway submission in a rabbit model. The two stars represent significant difference in p < 0.01, Student's t test. Figure 2 illustrates the specificity of adenosine receptor antisense oligonucleotides A- | as indicated by the adenosine A receptor number - | and A2 present in the airway tissue treated with the adenosine receptor antisense oligonucleotides A < | .

DETAILED DESCRIPTION OF THE INVENTION The nucleotide sequences are presented herein * _ > by a single chain only, in the 5 'to 3' direction, from right to left. Nucleotides and amino acids are presented in the form in the manner recommended by the IU PAC-IU B Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR 1 .822 and the established use . See, for example, Patentln User Manual, 99-102 (Nov. 1990) (Patent and Trademark Office of E. U.A., Office of the Deputy Commissioner for Patents, Washington, D.C. 20231); Patent of E U .A. No. 4, 871, 670 to Hudson et al. in Col. 3, lines 20-43 (the applicants specifically intend that the description thereof and all patent references cited therein are incorporated herein by reference). The method of the present invention can be used to reduce adenosine-mediated bronchoconstriction in the lungs of a subject for any reason, including (but not limited to) asthma. The antisense oligonucleotides or for the A - | and A3 show to be effective in the regulation of A -j or A3 in the cell. A novel aspect of this treatment, compared with traditional treatments for adenosine-mediated bronchoconstriction, is that the administration is directed to the lungs. Additionally, a receptor-like protein is reduced in shortest way, instead of simply interacting with a drug, and the toxicity is reduced. As used herein, the term "treating" or "treating" asthma refers to treatment that decreases the likelihood that the subject to whom said treatment is administered manifests symptoms of bronchoconstriction or asthma. The term "regular" refers to inducing a decrease in production, secretion or availability (and therefore a decrease in concentration) of adenosine receptor Ai or intracellular A3. The present invention relates primarily to the treatment of human subjects, but may also be employed for the treatment of other mammalian subjects, such as dogs and cats for veterinary purposes.

In general, "antisense" refers to the use of small synthetic oligonucleotides resembling single-stranded DNA, to inhibit gene expression by inhibiting target messenger RNA (mRNA) function. Milligan, J.F. Et al, J. Med. Chem. 36 (14), 1923-1937 (1993). Inhibition of the expression of At or A3 adenosine receptor genes is desired in the present invention. Gene expression is inhibited through hybridization to (sense) coding sequences in a specific messenger RNA (mRNA) target via linkage ligation in accordance with the Watson-Crick base pair formation rules. The mechanism of antisense inhibition is that the oligonucleotides applied exogenously decrease the mRNA and protein levels of the target gene or cause changes in the growth or shape characteristics of the cells. ID See also Helene, C. and Toulme, J. Biochim. Biophis. Acta 1049, 99-125 (1990); Cohen, J.S., DE., Oligodeoxinucleotides as Antisense Inhibitors of Gene Expression; CRC Press: Boca RaVón, FL (1987). As used herein, "adenosine receptor antisense oligonucleotide" is defined as a short, synthetic nucleotide sequence that (1) hybridizes to any coding sequence or to an mRNA that is encoded for the adenosine receptor At or the A3 adenosine receptor, according to the hybridization conditions described below, and (2) after the hybridization causes a decrease in the expression of adenosine Ai or A3 receptor genes.

The mRNA sequence of adenosine A receptors < | or A3 it is derived from the DNA base sequence of the gene that expresses either the adenosine A-] receptor or A3. The A-adenosine receptor sequence | human genomic is known and described in the Patent of E.U.A. No. 5,320,963 to G. Stiles et al. The adenosine receptor A- | has been cloned, sequenced and expressed in rats (see F. Zhou et al., Proc. Nati Acad. Sci. E.U.A. 89: 7432 (1992)) and humans (see M.A. Jacobson et al., UK Patent Application No. 9304582. 1 (1993)). Therefore, the antisense oligonucleotides regulate the production of the adenosine receptor A-j or A3. may occur according to normal techniques. One aspect of this invention is an antisense oligonucleotide having a sequence capable of specifically binding to any sequence of a mRNA molecule encoding an A- adenosine receptor. or human A3 adenosine receptor to avoid translation of the mRNA molecule. The antisense oligonucleotide may have a sequence described herein in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. The analogous chemicals of the oligonucleotides (for example, the oligonucleotides in which the phosphodiester linkages have been modified, for example, to the methylphosphonate, the phosphotriester, the phosphorothioate, the phosphorodithioate or the phosphoroamidate, to make the oligonucleotide more stable in vivo ) are also an aspect of the present invention. Phosphodiester bonds as found in nature in oligonucleotides are susceptible to degradation by endogenously occurring cellular nucleases, while many analogue linkages are highly resistant to nuclease degradation. See Milligan et al., And Cohen, J.S., supra. Protection against degradation can be achieved by the use of a "3-end blocked" strategy by which the nuclease-resistant bonds are replaced by phosphodiester bonds at the 3'end of the oligonucleotide. See Tidd, D.M. and Warenius, H.M., Br. J. Cancer 60, 343-350 (1989); Shaw, J.P. et al., Nucleic Acids Res. 19, 747-750 (1991). Phosphoramidates, phosphorothioates and methylphosphonate bonds all function properly in this way. The more extensive modification of the phosphodiester base structure has been shown to impart stability and may allow for improved affinity and increased cell permeation of the oligonucleotides. See Millogan et al., Supra. Many chemical strategies have been employed to replace the entire phosphodiester base structure with novel bonds. Id. ' Analogs of the base structure include phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, boranophosphate, phosphotriester, formacetal, 3'-thioformacetal, 5'-thioformacetal, 5'-thioether, carbonate, 5'-N-carbamate, sulphate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylenemethylimine (MMI) or methyleneoxymethylimino (MOMI). Oligonucleotides modified with phosphorothioate and methylphosphonate are particularly preferred due to their availability through synthesis of tunable oligonucleotide. Id. Where appropriate, the antisense oligonucleotides can be administered in the form of their pharmaceutically acceptable salts. The antisense oligonucleotides can be of any suitable length (for example, from about 10 to 60 nucleotides in length), depending on the particular objective being linked and the mode of delivery thereof. Preferably the antisense oligonucleotide oligonucleotide is directed to a mRNA region containing an ion between intron and exon. When the antisense oligonucleotide oligonucleotide is directed to an intron / exon junction, it may either lie completely on the junction or it may be sufficiently close to the junction to inhibit splicing away from the exon that is intervening during the process of the precursor mRNA to the mature mRNA. (for example, the 3 'or 5' terminus of the antisense oligonucleotide is positioned within, for example, 10, 5, 3 or 2 nucleotides of the intro / exon junction). Also preferred are antisense oligonucleotides that overlap the initiation codon. When the present invention is practiced, the antisense oligonucleotides administered may be related in origin to the spices to which it is administered. When treating humans, the human antisense can be used if desired. Pharmaceutical compositions comprising an antisense oligonucleotide ol as described above are effective for reducing the expression of an adenosine A receptor. or A3 passing through the cell membrane and specifically binding with mRNA encoding an adenosine A receptor - | or A3 in the cell to prevent translation thereof are another aspect of the present invention. Such compositions are provided in a suitable pharmaceutically acceptable vehicle (eg, sterilized pyrogen-free saline solution). The antisense pligonucleotides can be formulated with a hydrophobic vehicle to pass through a cell membrane (eg, in a liposome transported in a pharmaceutically acceptable vehicle). The oligonucleotides can also be coupled to a substance that inactivates the mRNA, such as a ribosome. Such oligonucleotides can be administered to a subject to inhibit the activation of adenosine A receptors. or A3, whose subject needs such treatment for any of the reasons discussed herein. In addition, the pharmaceutical formulation may also contain chimeric molecules comprising antisense oligonucleotides attached to molecules that are known to be internalized by the cells. These oligonucleotide conjugates use uptake pathways to increase cellular concentrations of oligonucleotides. Examples of macromolecules used in this form include transferrin, asialoglycoprotein (linked to the oligonucleotides through polylysine) and streptavidin. In the pharmaceutical formulation the antisense compound can be contained within a lipid particle or vehicle, such as a liposome or microcrystal. The particles may be of any suitable structure, such as unilamellar or plurimelar, provided that the antisense oligonucleotide is contained therein. Positively charged lipids such as N- [1- (2,3-dioleoloxy) propyl] -N, N, N-trimethylammoniomethylsulfate, or "DOTAP", are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, for example, Patents of the U.S.A. Nos. 4,880,635 to Janoff et al., 4,906,477 to Kurono et al., 4,911,928 to Wallach; 4,917,951 to Wallach; 4,920,016 to Alien et al .; 4, 921,757 to Wheatley et al .; etc. The active composition can be administered to individuals by any means that transports the antisense nucleotide composition to the lung. The antisense compounds described herein may be administered to the lungs of a patient by any suitable means, but are preferably administered by generating an aerosol composed of respirable particles, the respirable particles composed of the antisense compound, of which particles the individual inhales. The respirable particles can be liquid or solid. The particles may optionally contain other therapeutic ingredients. The composite particles of the antisense compound for practicing the present invention should include particles of respirable size: ie particles of a size small enough to pass through the mouth and the larynx after inhalation and of the bronchi and alveoli of the lungs. In general, particles that vary in size from about .5 to 10 microns are respirable. Particles of non-respirable size that are included in the aerosol tend to deposit in the throat and be swallowed, and the amount of non-respirable particles in the aerosol is preferably reduced to the minimum. For nasal administration, a particle size on the scale of 10-500 microns is preferred to ensure retention in the nasal cavity. Liquid pharmaceutical compositions of active compound for producing an aerosol can be prepared by combining the antisense compound with a suitable vehicle, such as sterilized pyrogen-free water. Other therapeutic compounds may optionally be included. The solid particle compositions containing respirable dry particles of the micronized antisense compound can be prepared by grinding the dry antisense compound with a mortar and pestle and then passing the microchipped composition through a 400 mesh screen to break or separate the large agglomerates. A solid particle composition composed of the antisense compound may optionally contain a dispersant which serves to facilitate the formation of an aerosol. A suitable dispersant is lactose, which can be mixed with the antisense compound in any suitable ratio (eg, a weight ratio of 1 to 1). Again, other therapeutic compounds may also be included. The dose of the antisense compound administered will depend on the condition being treated, the condition of the individual, the particular formulation, the route of administration, the administration time to the subject, etc. In general, intracellular concentrations of the oligonucleotide from 0.05 to 50 microns or more particularly 0.2 to 5 microns are desired. For administration to a subject such as a human, a dose of .01, .1 or 1 mg / kg to 50, 100 or 150 mg / kg or more is typically employed. Depending on the solubility of the particular formulation of the active compound administered, the daily dose may be divided among one or more dose unit administrations. The administration of the antisense compounds can be effected therapeutically (ie, as a rescue treatment) or prophylactically. The aerosols of liquid particles comprising the antisense compound can be produced by any suitable means, such as a nebulizer. See, for example, the Patent of E. U .A. No.4, 501, 729. Nebulizers are commercially available devices that transform solutions or suspensions of active reagent into a therapeutic aerosol mist either by accelerating the compressed gas, typically air or oxygen, through a narrow venturi orifice. ultrasonic agitation medium. Formulations suitable for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w / w of the formulation, but preferably less than 20% w / w. The carrier is typically aqueous or a dilute aqueous alcohol solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the preparation is not prepared sterilized, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, pH regulating agents and surfactants. The aerosols of solid particles comprising the active ingredient can likewise be produced with any aerosol generator of medicament in solid particles. Aerosol generators for administering solid particle drugs to an individual produce solid particles that are respirable, as explained above, and generate an aerosol volume containing a predetermined metered dose of a medicament at a regimen suitable for administration in humans. An illustrative type of aerosol generator in solid particles is an insufflator. Formulations suitable for administration by insufflation include finely ground powders which can be supplied by means of an insufflator or taken into the nasal cavity in the form of an aspiration. In the insufflated? ' the powder (eg, a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either perforated or opened in situ and the powder is supplied by air drawn through the device after inhalation or by means of a manually operated pump. The powder employed in the insufflator consists either solely of the active ingredient or of a powder mixture comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically comprises from 0.1 to 100 w / w of the formulation. A second illustrative type of aerosol generator comprises a metered dose inhaler. The metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension formulation or solution of the active ingredient in a liquid made propellant. During use these devices discharge the formulation through a valve adapted to supply a measured volume, typically from 10 to 150 meters, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane and mixtures thereof. The formulation may additionally contain one or more cosolvents, for example, ethanol, surfactants, such as oleic acid or -. sorbitan trioleate, antioxidants and suitable flavoring agents. The aerosol, whether formed of liquid or solid particles, can be produced by the aerosol generator at a rate of about 10 to 150 liters per minute, more preferably about 30 to 50 liters per minute, and more preferably around 60 liters per minute. Aerosols that contain large amounts of medication can be administered more quickly.

The following examples are given to illustrate the present invention and should not be considered as limiting thereof. In these examples, μM means micromolar, ml means milliliters, μm means micrometers, mm denotes my limeters, cm means centimeters, ° C means degrees centigrade, μg means micrograms, mg means milligrams, g means grams, kg means kilograms, M means molar and h means hours.

EXAMPLE 1 Design and synthesis of antisense oligonucleotides The design of antisense oligonucleotides against adenosine A receptors - | and A3 may require the solution of the complex secondary structure of the mRNA of the A- receptor. object and the AR Nm of the receiver A3 object. After generating this structure, antisense nucleotides are designed whose target regions MRNA that can be constructed to confer activity and functional stability to the mRNA and that can optimally overlap the initiation codon. Other object sites are easily usable. As a demonstration of the specificity of the antisense effect, other nucleotides not fully complementary to the target mRNA, but containing identical nucleotide compositions on a w / w basis, are included as controls in the antisense experiments. The secondary structure of the A R N m receptor A -j of the subject adenosine was analyzed and used as described above to designate a phosphorothioate antisense oligonucleotide. The antisense oligonucleotide that was synthesized was designated HAdAIAS and had the following sequence: 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ ID NO: 1) As a control, a phosphorothioate antisense nucleotide that does not correspond designated HAdAIMM was synthesized with the following sequence: 5'-GTA GCA GGC GGG GGG GGC-3 '(SEQ ID NO: 2) Each oligonucleotide had identical base content and general sequence estrctura. Homology investigations in GENBANK (publication 85.0) and EMBL (publication 40.0) indicated that the antisense oligonucleotide was specific for the A- receptor genes. of rabbit and human adenosine and that the control that no corresponding was not a candidate for hybridization with any known gene sequence. The secondary structure of the mRNA of the A- receptor | of adenosine The object was analyzed and used as described above to designate two phosphorothioate antisense oligonucleotides. The first antisense oligonucleotide (HAdA3AS1) synthesized had the following sequence: 5'-GTT GTT GGG CAT CTT GCC-3 '(SEQ ID NO: 3) As a control, an irrelevant phosphorothioate antisense oligonucleotide (HAdA3MM) was synthesized, having the following sequence: 5'-GTA CTT GCG GAT CTA GGC-3 '(SEQ ID NO: 4) A second phosphorothioate antisense oligonucleotide that does not correspond (HAdA3AS2) was also designed and synthesized, having the following sequence : 5'-GTG GGC CTA GCT CTC GCC-3 '(SEQ ID NO: 5) Its oligonucleotide control (HAdA3MM2) had the following sequence: 5'-GTC GGG GTA CCT GTC GGC-3' (SEQ ID NO: 6) Phosphorothioate oligonucleotides were synthesized on an Oligonucleotide Synthesizer Model 396 from Applied Biosystems and purified using N ENSORB chromatography (DuPont, MD).

MPLO AXIS 2 Adenosine A-j receptor antisense oligonucleotide oligonucleotide test in vitro The antisense oligonucleotide against the adenosine A ih umano receptor (SEQ ID NO: 1) described above was tested for its effectiveness in an in vitro model using lung adenocarcinoma HTB-54 cells.LBB lung adenocarcinoma cells -54 showed to express the adenosine receptor A -j using standard norther blotting procedures and receptor probes designed and synthesized in the laboratory.The lung adenocarcinoma cells HTB-54 (106/100 mm tissue culture dish) were exposed to 5.0 microns of HAdA I AS or HAdAI MM for 24 hours with a fresh change of media and oligonucleotides after 12 hours of incubation.

After the 24 hour exposure to the oligonucleotides, the cells were harvested and their AR N was extracted by normal procedures. A 21-mer probe corresponding to the region of target Nm by the antisense (and therefore having the same sequence as the antisense, but without being subjected to phosphorothioate) was synthesized and used for northern blots of a probe. RNA prepared from HAdA I AS treated, HAdAI MM treated and untreated HTB-54 cells. These blots clearly showed that HAdA I AS, but not HAdA I MM effectively reduced the A R N m of human adenosine receptor in < fifty% . This result showed that HAdA I AS is a good candidate for an anti asthma drug since it evacuates the intracellular mRNA for the A - receptor. of adenosine, which is involved in asthma.

EXAMPLE 3 Efficacy of adenosine A ^ receptor adenosine receptor oligonucleotides in vivo A fortuitous homology between the human and rabbit DNA sequences within the A- gene | of adenosine that overlaps the initiation codon allowed the use of the phosphorothioate antisense oligonucleotides initially designed for use against the human A adenosine receptor A in a rabbit model.

Newborn White New Zealand free Pasteurella rabbits were immunized intraperitoneally within 24 hours after birth with extract (Berkeley Biologicals, Berkeley, CA) of house dust mites (D. farinae) units / ml of 312 antigen mixed with Caolín to 1 0%. Immunizations were repeated weekly during the first month and then every two weeks for the next two months. At the age of 3-4 months, eight sensitized rabbits were anesthetized and relaxed with a mixture of 44mg / kg of ketamine hydrochloride and 0.4mg / kg of acepromazine maleate intramurally administered. The rabbits were left on their backs in a comfortable position on a small molded animal shelter board and were intubated with a 4.0mm intratracheal tube (Mallinkrodt, Inc., Glens Falls, NY). A 2.4 mm external diameter polyethylene catheter with a fixed latex balloon was passed into the esophagus and kept at the same distance (approximately 16 cm) from the mouth throughout the experiments. The intratracheal tube was fixed to a warm Fleisc pneumotachograph (size 00; DOM Medical, Richmond, VA), and flow was measured using a Validyne differential pressure transducer (Model DP-45161927; Validyne Engineering Corp., Northridge, CA) driven by a Gould vehicle amplifier (Model 1 1 -41 13; Gould Electronic, Cleveland, OH). The esophageal balloon was attached to one side of the differential pressure transducer, and the outward flow of the intratracheal tube was connected to the opposite side of the pressure transducer to allow recording of the transpulmonary pressure. The flow was integrated to give a continuous tidal volume and the measurements of total lung resistance (LR) and dynamic submission (Cdyn) were calculated at cervolumometric and flow points, respectively, using an automated respiratory analyzer (Model 6). Buxco, Sharon, CT). The animals were chosen at day 3 and pretreatment values were obtained for PC50 on day 1 for aerolized adenosine. Antisense (HAdA I AS) or non-corresponding control oligonucleotides (HAdA I MM) were dissolved in sterile physiological saline at a concentration of 5000 ug (5 mg) per 1.0 ml. The animals were subsequently administered the antisense or non-corresponding oligonucleotide aerosolized through the intratracheal tube (approximately 5000 micrograms in a volume of 1.0 ml), twice daily for two days. The aerosols of either saline, adenosine, or antisense or non-corresponding oligonucleotides were generated by an ultrasonic solubilizer (DeVilbiss, Somerset, PA), producing aerosol droplets 80% of which were less than 5%. micrometers in diameter. In the first end of the experiment, four randomly selected allergic rabbits were given antisense oligonucleotide and at four the non-corresponding control oligonucleotide. On the morning of the third day, PC50 values were obtained (the concentration of adenosine aerosolized in mg / ml required to reduce the dynamic submission of the bronchial airway 50% from the baseline value) and were compared with the PC50 values obtained for these animals prior to exposure to the oligonucleotide. After an interval of one week, the animals were crossed, those previously administered with the non-corresponding control oligonucleotide were now administered the antisense oligonucleotide and those previously treated with the antisense oligonucleotide were now admired. the ol igonucleotide control not corresponding. The treatment methods and measurements were identical to those used at the first end of the experiment. It should be noted that in six of the eight animals treated with antisense oligonucleotide, adenosine-mediated bronconstriction could not be obtained until the solubility limit of adenosine, 20 mg / ml. For the purpose of calculations, PC50 values for these animals were set at 20 mg / ml. The values given therefore represent a minimum figure for the effectiveness of antisense. The real effectiveness was superior. The results of this experiment are illustrated both in Figure 1 and Table 1.

TABLE 1 Effects of adenosine receptor A i oligonucleotide on the PC50 values in asthmatic rabbits Non-corresponding control Oligonucleotide antisense receptor A < | Pre Post Pre Post oligonucleotide oligonucleotide oligonucleotide oligonucleotide 3. 56+ 1.02 5.16+ 1.93 2.36+ 0.68 > 19.5+ 0.34 ** The results are presented as the mean N = 8) + SEM. The significance was determined by repeated measurements of variation analysis (ANOVA) and Tukey's protected t-test. significantly different from all other groups, P < 0.01.

In both ends of the experiment, the animals that received antisense oligonucleotide showed an increase in the order of magnitude in the dose of aerosolized adenosine required to reduce the dynamic submission of the lung by 50%. No effect of the non-corresponding control oligonucleotide on PC50 values was observed. No toxicity was observed in any animal that received either the antisense or control inhaled oligonucleotide. The results clearly show that the lung has an exceptional potential as a target for therapeutic intervention based on antisense oligonucleotide in pulmonary disorders. They also show, in a model system that closely resembles asthma in humans, that the regulation of the receptor A- | from Adenosine largely eliminates adenosine-mediated bronchoconstriction in the asthmatic airways. Bronchial hypersensitivity in the allergic rabbit model of human asthma is an excellent end point for antisense intervention since the tissues involved in this response are close to the point of contact with aerosolized oligonucleotides and the model closely stimulates a significant human condition .

EXAMPLE 4 Specificity of adenosine receptor antisense oligonucleotide A-j At the conclusion of the crossover experiment of Example 3, the airway smooth muscle of rabbits was analyzed quantitatively for receptor number A- | of adenosine as a control for the specificity of the antisense oligonucleotide were also qualified A2 adenosine receptors which should not be affected. Airway smooth muscle was cut from each rabbit and a membrane fraction was prepared according to the methods described (J. Kleinstein and H. Gloss an, Naunyn-Schmiedeberg's Arch. Pharmacol 305, 191-200 (1978) with light modifications The crude plasma membrane preparations were stored 170 ° C until the time of the test. The protein content was determined by the method of Bradford (M. Bradford, Anal. Biochem. 72, 240-254 (1976)). The frozen plasma membranes were melted at room temperature and incubated with 0.2U / ml amide adenosine for 30 minutes at 37 ° C to remove the endogenous adenosine. The binding of [3H] DPCPX (specific receptor A- |) or [3H] CGX (specific receptor) was measured as described above.

A2). S. Ali et al., J. Pharmacol. Exp. Ther. 268, 1328-1334 (1994); S. Ali et al., Am. J. Physiol.266, L271-277 (1994). As illustrated in both Figure 2 and Table 2, animals treated with adenosine receptor antisense oligonucleotide A- | in the crossed experiment they had a decrease of almost 75% in the number of receptor A- | compared to the controls, as tested for specific binding of the DPCPX specific antagonist A- |. There was no change in the receiver number A2, as tested for specific ligation of 2- [p- (2-carboxyethyl) -phenethylamino] -5 '- (N-ethylcarboxamido) adenosine specific A2 receptor agonist (CGS-21680.

TABLE 2 Specificity of the action of the oligonucleotide of receptor A- | of adenosi na Control oligonucleotide Non-corresponding antisense oligonucleotide of receptor A - | The results are presented as the mean N = 8) + _ SEM. The meaning was determined by repeated measurement of variation measurements (A NOVA) and Tukey's protected t-test. "Significantly different from all, the other groups, P <0.01.

The above examples are illustrative of the present invention and should not be considered as limiting thereof. The definition is defined by the following claims with equivalents of the claims to be included therein.

SEQUENCE LIST (1. GENERAL INFORMATION: (i) APPLICANT:, Nyce. Jonathan W. (ii) TITLE OF THE INVENTION: METHOD FOR TREATMENT OF ASTHMA (iii) SEQUENCE NUMBER: 6 (iv) POSTAL ADDRESS: (A) RECIPIENT: Kenneth D. Sibley (B) STREET: Post Office Drawer 34009 (C) CITY: Charlotte (D) STATE- North Carolina (E) COUNTRY: E.U.A. (F) POSTAL CODE: 28234 (v) COMPUTER READING FORM: (A) TYPE OF MEDIA: diskette (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln elease No.1.0 Version No .1.30 (vi) CURRENT APPLICATION DATE: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (viii) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: Sibley, Kenneth D. (B) REGISTRATION NUMBER: 31,665 (C) REFERENCE NUMBER / CEDULA: 5128-29 (ix) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (919) 881-3140 (B) TELEFAX: (919) 881-3175 (C) TELEX: 575102 (2) INFORMATION-FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 GATGGA GGGC GGCA TGGCGG G (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTAGCAGGCG GGGATGGGGG C (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GTTGTTGGGC ATCTTGCC (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GTACTTGCGG ATCTAGGC (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) DE-MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GTGGGCCTAG CTCTCGCC (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRING: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTCGGGGTAC CTGTCGGC

Claims (34)

1. CLAIMS 1 . A method for reducing adenosine-mediated bronchoconstriction in a subject in need of such treatment, characterized in that it comprises administering an adenosine receptor antisense oligonucleotide to the lungs of the subject in an amount effective to reduce bronchoconstriction, such adenosine receptor selected from the group which consists of adenosine A receptors < | and A3 adenosine receptors.
2. 2. A method according to claim 1, further characterized in that said adenosine receptor is the adenosine A receptor. .
3. 3. A method according to claim 1, further characterized in that said adenosine receptor is the A3 adenosine receptor.
4. 4. A method according to claim 1, further characterized in that said antisense oligonucleotide comprises nucleotides in which at least one phosphodiester bond is replaced with a bond selected from the group consisting of methyl phosphonate bonds, phosphotriester bonds, phosphorothiato bonds, phosphorodithioate bonds and phosphoroamidate bonds.
5. 5. A method according to claim 1, further characterized in that said antisense oligonucleotide has the sequence given herein as SEQ I D NO: 1.
6. 6. A method according to claim 1, further characterized in that said antisense oligonucleotide has the sequence given herein as SEQ ID NO: 3.
7. 7. A method according to claim 1, further characterized in that said antisense oligonucleotide has the sequence given herein as SEQ ID NO: 5.
8. 8. A method according to claim 1, further characterized in that said antisense oligonucleotide is delivered by administering an aerosol of respirable particles containing said antisense oligonucleotide to the lungs of such a subject.
9. 9. A method according to claim 8, further characterized in that said particles are selected from the group consisting of solid particles and liquid particles.
10. 10. A method according to claim 9, further characterized in that said aerosol is composed of particles having a particle size within the regime of about 0.5 to 10 microns.
11. 11. A method according to claim 8, further characterized in that said particles are liposomes containing said antisense oligonucleotide.
12. 12. A method according to claim 8, further characterized in that said antisense oligonucleotide is administered in an amount sufficient to reach intracellular concentrations of said antisense oligonucleotide in the subject of about 0.1 to 10 microns.
13. 13. A method of treating asthma in a subject in need of such treatment, characterized in that it comprises administering an adenosine receptor antisense oligonucleotide to said subject in an amount effective to treat asthma, such adenosine receptor selected from the group consisting of of adenosine A ^ and adenosine A3 receptors.
14. 14. A method according to claim 13, further characterized in that said adenosine receptor is the adenosine receptor A -j.
15. 15. A method according to claim 13, further characterized in that said adenosine receptor is the A3 adenosine receptor.
16. 16. A method according to claim 13, characterized ad * emás because such an antisense oligonucleotide oligonucleotide comprises nucleotides in which at least one phosphodiester bond is replaced with a bond selected from the group consisting of methylphosphonate bonds, phosphotriester bonds, phosphorothiato bonds, phosphorodithioate bonds and phosphoroamidate bonds.
17. 17. A method according to claim 13, further characterized in that said antisense oligonucleotide has the sequence given herein as SEQ I D NO: 1.
18. 18. A method according to claim 13, further characterized in that said antisense oligonucleotide oligonucleotide has the sequence given herein as SEQ ID NO: 3.
19. 19. A method according to claim 13, further characterized in that said antisense oligonucleotide. has the sequence given in the present as S EQ ID NO: 5.
20. 20. A method according to claim J 3, further characterized in that said antisense oligonucleotide oligonucleotide is delivered by administering an aerosol of respirable particles containing said antisense oligonucleotide to the lungs of such a subject. twenty-one .
21. A method according to claim 20, further characterized in that said particles are selected from the group consisting of solid particles and liquid particles.
22. 22. A method according to claim 21, further characterized in that said aerosol is composed of particles having a particle size within the range of about 0.5 to 10 microns.
23. 23. A method according to claim 20, further characterized in that said particles are liposomes containing said antisense oligonucleotide.
24. 24. A method according to claim 13, further characterized in that said antisense oligonucleotide is administered in an amount sufficient to reach intracellular concentrations of said antisense oligonucleotide in the subject of about 0.1 to 10 microns.
25. 25. A pharmaceutical composition characterized by comprising together in a pharmaceutically acceptable carrier: an adenosine receptor antisense oligonucleotide; such adenosine receptor selected from the group consisting of adenosine A-receptors | and A3 adenosine receptors; in an effective amount to reduce adenosine-mediated bronchoconstriction.
26. 26. A method according to claim 25, further characterized in that said adenosine receptor is the adenosine receptor A- | .
27. 27. A method according to claim 25, further characterized in that said adenosine receptor is the A3 adenosine receptor.
28. 28. A method according to claim 25, characterized in that said antisense oligonucleotide comprises nucleotides in which at least one phosphodiester bond is replaced with a bond selected from the group consisting of methylphosphonate bonds, phosphotriester bonds, linkages of phosphorothiate, phosphorodithioate bonds and phosphoroamidate bonds.
29. 29. A method according to claim 25, further characterized in that said antisense oligonucleotide has the sequence given herein as SEQ ID NO: 1.
30. 30. A method according to claim 25, further characterized in that said antisense oligonucleotide has the sequence given herein as SEQ I D NO: 3.
31. 31 A method according to claim 25, further characterized in that said antisense oligonucleotide has the sequence given herein as S EQ I D NO: 5.
32. 32. A pharmaceutical composition according to claim 25, characterized in that it comprises such vehicle are selected from the group consisting of solid vehicles and liquid vehicles.
33. 33. A pharmaceutical composition according to claim 25 further characterized in that it comprises a liposome, such a liposome containing said antisense oligonucleotide.
34. 34. A pharmaceutical composition according to claim 25 further characterized in that said antisense oligonucleotide is conjugated to a molecule capable of cellular uptake. SUMMARY A method for reducing bronchoconstriction in an individual in need of such treatment is described. The method comprises administering to the subject an antisense oligonucleotide molecule directed against the adenosine receptor A- | or A3 in an amount effective to reduce bronchoconstriction. The method is useful to treat patients suffering from asthma. Pharmaceutical formulations are described.