| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 10.99 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 26.10.99 |
| PATENT TITLE |
Compositions and methods for cystic fibrosis therapy |
| PATENT ABSTRACT |
Compositions and methods for therapy of cystic fibrosis and other conditions are provided. The compositions comprise one or more flavones and/or isoflavones capable of stimulating chloride transport in epithelial tissues. Therapeutic methods involve the administration (e.g., orally or via inhalation) of such compositions to a patient afflicted with cystic fibrosis and/or another condition responsive to stimulation of chloride transport. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 16.10.97 |
| PATENT REFERENCES CITED |
Brown et al., "Chemical chaperones correct the mutant phenotype of the .DELTA.F508 cystic fibrosis transmembrane conductance regulatory protein," Cell Stress & Chaperones 1(2): 117-125, 1996. Hwang et al., "Genistein potentiates wild-type and .DELTA.F508-CFTR channel activity," American Journal of Physiology273(3, part 1): C988-C-998, 1997. Scott and Cooperstein, "Ascorbic acid stimulates chloride transport in the amphilbian cornea," Investigative Ophtalmology 14(10): 763-766, 1975. Smith, "Treatment of cystic fibrosis based on understanding CFTR," J. Inher. Metab. Dis 18:508-516, 1995. Rubenstein et al., "In Vitro Pharmacologic Restoration of CFTR-mediated Chloride Transport with Sodium 4-Phenylbutyrate in Cystic Fibrosis Epithelial Cells," J. Clin. Invest. 100(10): 2457-2465, 1997. Sheppard et al., "Mutations in CFTR associated with mild-disease-form CI channels with altered pore properties," Nature 362: 160-164, 1993. Anderson, et al., "Generation of cAMP-Activated Chloride Currents by Expression of CFTR," Science 251:679-682, 1991. Knowles et al., "In Vivo Nasal Potential Difference: Techniques and Protocols for Assessing Efficacy of Gene Transfer in Cystic Fibrosis," Human Gene Therapy 6: 445-455, 1995. Riordan et al., "Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNA," Science 245: 1066-1073, 1989. Illek et al., "cAMP-independent activation of CFTR C1 channels by the tyrosine kinase inhibitor genistein," Cell Physiol. 37: C886-C893, 1995. |
| PATENT CLAIMS |
What is claimed is: 1. A method for enhancing chloride transport in epithelial cells, comprising contacting epithelial cells with a compound selected from the group consisting of flavones and isoflavones, wherein the compound is capable of stimulating chloride transport, and wherein the compound is not genistein. 2. A method according to claim 1, wherein the compound is quercetin, apigenin, kaempferol or biochanin A. 3. A method according to claim 1, wherein the epithelial cells are airway epithelial cells. 4. A method according to claim 3, wherein the airway epithelial cells are present in a mammal. 5. A method according to claim 4, wherein the compound is administered orally. 6. A method according to claim 4, wherein the compound is administered by inhalation. 7. A method according to claim 1, wherein the epithelial cells are intestinal cells. 8. A method according to claim 7, wherein the intestinal epithelial cells are present in a mammal. 9. A method according to claim 7, wherein the compound is administered orally. 10. A method according to claim 1, wherein the compound is present within a pharmaceutical composition. 11. A method according to claim 1, wherein the epithelial cells produce a CFTR protein having a deletion at position 508. 12. A method for treating cystic fibrosis in a mammal, comprising administering to a mammal one or more compounds selected from the group consisting of flavones and isoflavones, wherein the compound is capable of stimulating chloride secretion, and wherein the compound is not genistein. 13. A method according to claim 12, wherein the compound is quercetin, apigenin, kaempferol or biochanin A. 14. A method according to claim 12, wherein the compound is administered orally. 15. A method according to claim 12, wherein the compound is administered by inhalation. 16. A method according to claim 12, wherein the compound is present within a pharmaceutical composition. 17. A method for increasing chloride ion conductance in airway epithelial cells of a patient afflicted with cystic fibrosis, wherein the patient's CFTR protein has a deletion at position 508, the method comprising administering to a mammal one or more compounds selected from the group consisting of flavones and isoflavones, wherein the compound is capable of stimulating chloride secretion. 18. A pharmaceutical composition for treatment of cystic fibrosis, comprising one or more flavones or isoflavones capable of stimulating chloride secretion in combination with a pharmaceutically acceptable carrier, where the composition does not comprise genistein as an active ingredient, and wherein the composition further comprises 4-phenylbutyrate. 19. A pharmaceutical composition for treatment of cystic fibrosis, comprising quercetin in combination with a pharmaceutically acceptable carriers and wherein the composition further comprises 4-phenylbutyrate. 20. A pharmaceutical composition for treatment of cystic fibrosis, comprising apigenin in combination with a pharmaceutically acceptable carrier, and wherein the composition further comprises 4-phenylbutyrate. 21. A pharmaceutical composition for treatment of cystic fibrosis, comprising kaempferol in combination with a pharmaceutically acceptable carriers and wherein the composition further comprises 4-phenylbutyrate. 22. A pharmaceutical composition for treatment of cystic fibrosis, comprising biochanin A in combination with a pharmaceutically acceptable carrier, and wherein the composition further comprises 4-phenylbutyrate. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
TECHNICAL FIELD The present invention relates generally to the treatment of cystic fibrosis. The invention is more particularly related to compositions comprising one or more flavones and/or isoflavones, which may be used to activate chloride transport (ie., absorption and/or secretion) in epithelial tissues of the airways, the intestine, the pancreas and other exocrine glands, and for cystic fibrosis therapy. BACKGROUND OF THE INVENTION Cystic fibrosis is a lethal genetic disease afflicting approximately 30,000 individuals in the United States. Approximately 1 in 2500 caucasians is born with the disease, making it the most common lethal, recessively inherited disease in that population. Cystic fibrosis affects the secretory epithelia of a variety of tissues, altering the transport of water, salt and other solutes into and out of the blood stream. In particular, the ability of epithelial cells in the airways, pancreas and other tissues to transport chloride ions, and accompanying sodium and water, is severely reduced in cystic fibrosis patients, resulting in respiratory, pancreatic and intestinal ailments. The principle clinical manifestation of cystic fibrosis is the resulting respiratory disease, characterized by airway obstruction due to the presence of a thick mucus that is difficult to clear from airway surfaces. This thickened airway liquid contributes to recurrent bacterial infections and progressively imparied respiration, eventually resulting in death. In cystic fibrosis, defective chloride transport is generally due to a mutation in a chloride channel known as the cystic fibrosis transmembrane conductance regulator (CFTR; see Riordan et al., Science 245:1066-73, 1989). CFTR is a linear chloride channel found in the plasma membrane of certain epithelial cells, where it regulates the flow of chloride ions in response to phosphorylation by a cyclic AMP-dependent kinase. Many mutations of CFTR have been reported, the most common of which is a deletion of phenylalanine at position 508 (.DELTA.F508-CFTR), which is present in approximately 70% of patients with cystic fibrosis. A glycine to aspartate substitution at position 551 (G55 ID-CFTR) occurs in approximately 1% of cystic fibrosis patients. Current treatments for cystic fibrosis generally focus on controlling infection through antibiotic therapy and promoting mucus clearance by use of postural drainage and chest percussion. However, even with such treatments, frequent hospitalization is often required as the disease progresses. New therapies designed to increase chloride ion conductance in airway epithelial cells have been proposed, but their long term beneficial effects have not been established and such therapies are not presently available to patients. Accordingly, improvements are needed in the treatment of cystic fibrosis. The present invention fulfills this need and further provides other related advantages. SUMMARY OF THE INVENTION Briefly stated, the present invention provides compositions and methods for the therapy of cystic fibrosis. Within one aspect, the present invention provides methods for enhancing chloride transport in epithelial cells, comprising contacting epithelial cells with a compound selected from the group consisting of flavones and isoflavones, wherein the compound is capable of stimulating chloride transport, and wherein the compound is not genistein. Within certain embodiments, the compound is quercetin, apigenin, kaempferol or biochanin A. For enhancing chloride transport in airway epithelial cells of a mammal, compounds may be administered orally or by inhalation. Within other aspects, the present invention provides methods for treating cystic fibrosis in a patient, comprising administering a compound selected from the group consisting of flavones and isoflavones, wherein the compound is capable of stimulating chloride transport, and wherein the compound is not genistein. Within certain embodiments, the compound is quercetin, apigenin, kaempferol or biochanin A. Compounds may be administered orally or by inhalation. Within further related aspects, the present invention provides methods for increasing chloride ion conductance in airway epithelial cells of a patient afflicted with cystic fibrosis, wherein the patient's CFTR protein has a deletion at position 508, the method comprising administering to a mammal one or more compounds selected from the group consisting of flavones and isoflavones, wherein the compound is capable of stimulating chloride secretion. Within further aspects, pharmaceutical compositions for treatment of cystic fibrosis are provided, comprising one or more flavones or isoflavones capable of stimulating chloride transport in combination with a pharmaceutically acceptable carrier. Within certain embodiments, such compositions may comprise quercetin, apigenin, kaempferol and/or biochanin A in combination with a pharmaceutically acceptable carrier. These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a recording of transepithelial short-circuit current (Y axis) as a function of time (X axis), showing the effect of apigenin on the current across a Calu-3 cell monolayer. Measurements were performed in an Ussing chamber, where the basolateral membrane was permeabilized with .alpha.-toxin and a chloride gradient was applied across the apical membrane as a driving force. Tissue was first stimulated with cAMP (100 .mu.M). Apigenin (50 .mu.M) was subsequently added as indicated. The horizontal bar represents 100 seconds, and the vertical bar represents 12 .mu.A/cm.sup.2. FIG. 2 is a recording showing the effect of quercetin on transepithelial short-circuit current across a Calu-3 cell monolayer in an Ussing chamber, where the basolateral membrane was permeabilized with .alpha.-toxin and a chloride gradient was applied across the apical membrane as a driving force. Tissue was first stimulated with cAMP (100 .mu.M). Quercetin (30 .mu.M) was subsequently added as indicated. Bars are 140 seconds (horizontal) and 12 .mu.A/cm.sup.2 (vertical). FIG. 3 is a recording illustrating the dose-dependent stimulation of transepithelial chloride secretion by quercetin (in the amounts indicated) across a primary bovine tracheal epithelium. Amiloride (50 .mu.M) was added to block sodium transport as indicated. The CFTR channel blocker diphenylcarboxylate (DPC, 5 mM) was added as shown. FIG. 4 is a recording showing the effect of biochanin A on transepithelial short-circuit current across a Calu-3 cell monolayer in an Ussing chamber, where the basolateral membrane was permeabilized with .alpha.-toxin and a chloride gradient was applied across the apical membrane as a driving force. The tissue was first stimulated with forskolin (Fsk, 10 .mu.M). Subsequent addition of biochanin A (Bio, 100 and 300 .mu.M) was subsequently added as indicated. FIG. 5 is a cell-attached single channel patch clamp recording from a 3T3 cell expressing .DELTA.F508-CFTR. The cell was treated with 10 .mu.M forskolin as shown. Genistein (50 .mu.M) and apigenin (50 .mu.M), were added where indicated by boxes. The holding potential was 75 mV, and channel openings were upward. FIG. 6 is a whole cell patch clamp recording on an airway epithelial cell homozygous for .DELTA.F508-CFTR. Before the measurement, the cell was incubated for 2 days in 5 mM 4-phenylbutyrate. 30 .mu.M quercetin was added where indicated by the box. Further stimulation by forskolin (10 .mu.M) is also shown. The holding potential was -60 mV. FIG. 7 is a recording illustrating the effect of genistein on G551D-CFTR expressed in a Xenopus oocyte. Current was measured with the two-electrode voltage clamp technique. G551D-CFTR was injected in oocyte. Current was first stimulated with forskolin (10 .mu.M) and isobutylmethylxantine (IBMX; 2 mM). Genistein (50 .mu.M) was added as indicated. The right panel shows current voltage relations recorded after treatment with forskolin and IBMX (F/I) and after treatment with genistein (F/I+Geni). A voltage ramp from -130 mV to +70 mV was applied and current was recorded during the two conditions. FIG. 8 is a recording illustrating the effect of quercetin on nasal potential difference (PD) measurement in a healthy human volunteer. Amiloride (50 .mu.M) was added to block sodium transport as indicated. Conditions were rendered chloride free (Cl free) and chloride secretion was stimulated with isoproterenol (iso; 5 .mu.M). Quercetin (querc; 10 .mu.M) was added as indicated. FIG. 9 is a recording illustrating the effect of apigenin and kaempferol on nasal PD in mice. Chloride secretion was stimulated with isoproterenol (iso; 5 .mu.M), and amiloride (50 .mu.M) was added to block sodium transport as indicated. Under chloride-free conditions (Cl free), apigenin (50 .mu.M, left panel) and kaempferol (kaemp, 50 .mu.M, right panel) were added as indicated. DETAILED DESCRIPTION OF THE INVENTION As noted above, the present invention is generally directed to compositions and methods for the treatment of diseases characterized by defective chloride transport in epithelial tissues, including cystic fibrosis, and diseases with excessive accumulation of mucus, including cystic fibrosis, chronic bronchitis and asthma. It has been found, within the context of the present invention, that certain flavones and isoflavones are capable of stimulating chloride transport in epithelial tissues (e.g., tissues of the airways, intestine, pancreas and other exocrine glands) in a cyclic-AMP independent manner. Such therapeutic compounds may be administered to patients afflicted with cystic fibrosis as described herein. The term "flavones", as used herein refers to a compound based on the core structure of flavone: ##STR1## An "isoflavone" is an isomer of a flavone (i.e., the phenyl moiety at position 2 is moved to position 3), and having the core structure shown below: ##STR2## Many flavones are naturally-occurring compounds, but synthetic flavones and isoflavones are also encompassed by the present invention. A flavone or isoflavone may be modified to comprise any of a variety of functional groups, such as hydroxyl and/or ether groups. Preferred flavones comprise one or more hydroxyl groups, such as the trihydroxyflavone apigenin, the tetrahydroxyflavone kaempferol and the pentahydroxyflavone quercetin. Preferred isoflavones comprise one or more hydroxyl and/or methoxy groups, such as the methoxy, dihydroxy isoflavone biochanin A. Flavones and isoflavones for use within the context of the present invention have the ability to stimulate chloride transport in epithelial tissues. Such transport may result in secretion or absorption of chloride ions. The ability to stimulate chloride transport may be assessed using any of a variety of systems. For example, in vitro assays using a mammalian trachea or a cell line, such as the permanent airway cell line Calu-3 (ATCC Accession Number HTB55) may be employed. Alternatively, the ability to stimulate chloride transport may be evaluated within an in vivo assay employing a mammalian nasal epithelium. In general, the ability to stimulate chloride transport may be assessed by evaluating CFTR-mediated currents across a membrane by employing standard Ussing chamber (see Ussing and Zehrahn, Acta. Physiol Scand. 23:110-127, 1951) or nasal potential difference measurements (see Knowles et al., Hum. Gene Therapy 6:445-455, 1995). Within such assays, a flavone or isoflavone that stimulates a statistically significant increase in chloride transport at a concentration of about 1-300 .mu.M is said to stimulate chloride transport. Within one in vitro assay, the level of chloride transport may be evaluated using mammalian pulmonary cell lines, such as Calu-3 cells, or primary bovine tracheal cultures. In general, such assays employ cell monolayers, which may be prepared by standard cell culture techniques. Within such systems, CFTR-mediated chloride current may be monitored in an Ussing chamber using intact epithelia. Alternatively, chloride transport may be evaluated using epithelial tissue in which the basolateral membrane is permeabilized with Staphylococcus aureus .alpha.-toxin, and in which a chloride gradient is imposed across the apical membrane (see Illek et al., Am. J. Physiol. 270:C265-75, 1996). In either system, chloride transport is evaluated in the presence and absence of a test compound (i.e., a flavone or isoflavone), and those compounds that stimulate chloride transport as described above may be used within the methods provided herein. Within another in vitro assay for evaluating chloride transport, cells are transfected with a chloride channel gene (e.g., CFTR) having a mutation associated with cystic fibrosis. Any CFTR gene that is altered relative to the normal human sequence provided in SEQ ID NO: 1, such that the encoded protein contains a mutation associated with cystic fibrosis, may be employed within such an assay. The most common disease-causing mutation in cystic fibrosis is a deletion of phenylalanine at position 508 in the CFTR protein (.DELTA.F508-CFTR; SEQ ID NO:4). Accordingly, the use of a CFTR gene encoding .DELTA.F508-CFTR is preferred. However, genes encoding other altered CFTR proteins (e.g., G551D-CFTR; SEQ ID NO:6) may also be used. Cells such as NIH 3T3 fibroblasts may be transfected with an altered CTFR gene, such as .DELTA.F508-CFTR, using well known techniques (see Anderson et al., Science 25:679-682, 1991). The effect of a compound on chloride transport in such cells may be evaluated by monitoring CFTR-mediated currents using the patch clamp method (see Hamill et al., Pflugers Arch. 391:85-100, 1981) with and without compound application. Within another in vitro assay, a mutant CFTR may be microinjected into cells such as Xenopus oocytes. Chloride conductance mediated by the CFTR mutant in the presence and absence of a test compound may be monitored with the two electrode voltage clamp method (see Miledi et al., Proc. R. Soc. Lond. Biol. 218:481-484, 1983). Alternatively, such assays may be performed using a mammalian trachea, such as a primary cow tracheal epithelium using the Ussing chamber technique as described above. Such assays are performed in the presence and absence of test compound to identify flavone and isoflavones that stimulate chloride transport. In vivo, chloride secretion may be assessed using measurements of nasal potential differences in a mammal, such as a human or a mouse. Such measurements may be performed on the inferior surface of the inferior turbinate following treatment of the mucosal surface with a test compound during perfusion with the sodium transport blocker amiloride in chloride-free solution. The nasal potential difference is measured as the electrical potential measured on the nasal mucosa with respect to a skin electrode placed on a slightly scratched skin part (see Alton et al., Eur. Respir. J. 3:922-926, 1990) or with respect to a subcutaneous needle (see Knowles et al., Hum. Gene Therapy 6:445-455, 1995). Nasal potential difference is evaluated in the presence and absence of test compound, and those compounds that results in a statistically significant increase in nasal potential difference stimulate chloride transport. As noted above, any flavone or isoflavone that stimulates chloride transport within at least one of the above assays may be used for therapy of cystic fibrosis, other diseases characterized by abnormally high mucus accumulation in the airways or intestinal disorders such as constipation. Preferred therapeutic compounds include quercetin (3,3',4',5,7-pentahydroxyflavone), apigenin (4'5,7-trihydroxyflavone), kaempferol (3,4',5,7-tetrahydroxyflavone) and biochanin A (4'-methoxy-5,7-dihydroxyisoflavone), as depicted below: ##STR3## Other suitable therapeutic compounds may be identified using the representative assays as described herein. Quercetin, apigenin, kaempferol, biochanin A and other flavones and isoflavones may generally be prepared using well known techniques, such as those described by Shakhova et al., Zh. Obshch. Khim. 32:390, 1962; Farooq et al., Arch. Pharm. 292:792, 1959; and Ichikawa et al., Org. Prep. Prog. Int. 14:183, 1981. Alternatively, such compounds may be commercially available (e.g., from Indofine Chemical Co., Inc., Somerville, N.J. or Sigma-Aldrich, St. Louis, Mo.). Further modifications to such compounds may be made using conventional organic chemistry techniques, which are well known to those of ordinary skill in the art. For in vivo use, a therapeutic compound as described herein is generally incorporated into a pharmaceutical composition prior to administration. Within such compositions, one or more therapeutic compounds as described herein are present as active ingredient(s) (ie., are present at levels sufficient to provide a statistically significant effect on nasal potential difference, as measured using a representative assay as provided herein). A pharmaceutical composition comprises one or more such compounds in combination with any pharmaceutically acceptable carrier(s) known to those skilled in the art to be suitable for the particular mode of administration. In addition, other pharmaceutically active ingredients (including other therapeutic agents) may, but need not, be present within the composition. Administration may be achieved by a variety of different routes. One preferred route is oral administration of a composition such as a pill, capsule or suspension. Such compositions may be prepared according to any method known in the art, and may comprise any of a variety of inactive ingredients. Suitable excipients for use within such compositions include inert diluents (which may be solid materials, aqueous solutions and/or oils) such as calcium or sodium carbonate, lactose, calcium or sodium phosphate, water, arachis oil, peanut oil liquid paraffin or olive oil; granulating and disintegrating agents such as maize starch, gelatin or acacia and/or lubricating agents such as magnesium stearate, stearic acid or talc. Other inactive ingredients that may, but need not, be present include one or more suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia), thickeners (e.g., beeswax, paraffin or cetyl alcohol), dispersing or wetting agents, preservatives (e.g., antioxidants such as ascorbic acid), coloring agents, sweetening agents and/or flavoring agents. A pharmaceutical composition may be prepared with carriers that protect active ingredients against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art. Particularly preferred are methods in which the therapeutic compound(s) are directly administered as a pressurized aerosol or nebulized formulation to the patient's lungs via inhalation. Such formulations may contain any of a variety of known aerosol propellants useful for endopulmonary and/or intranasal inhalation administration. In addition, water may be present, with or without any of a variety of cosolvents, surfactants, stabilizers (e.g., antioxidants, chelating agents, inert gases and buffers). For compositions to be administered from multiple dose containers, antimicrobial agents are typically added. Such compositions are also generally filtered and sterilized, and may be lyophilized to provide enhanced stability and to improve solubility. Pharmaceutical compositions are administered in an amount, and with a frequency, that is effective to inhibit or alleviate the symptoms of cystic fibrosis and/or to delay the progression of the disease. The effect of a treatment may be clinically determined by nasal potential difference measurements as described herein. The precise dosage and duration of treatment may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Dosages may also vary with the severity of the disease. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. In general, an oral dose ranges from about 200 mg to about 1000 mg, which may be administered 1 to 3 times per day. Compositions administered as an aerosol are generally designed to provide a final concentration of about 10 to 50 .mu.M at the airway surface, and may be administered 1 to 3 times per day. It will be apparent that, for any particular subject, specific dosage regimens may be adjusted over time according to the individual need. As noted above, a pharmaceutical composition may be administered to a mammal to stimulate chloride transport, and to treat cystic fibrosis. Patients that may benefit from administration of a therapeutic compound as described herein are those afflicted with cystic fibrosis. Such patients may be identified based on standard criteria that are well known in the art, including the presence of abnormally high salt concentrations in the sweat test, the presence of high nasal potentials, or the presence of a cystic fibrosis-associated mutation. Activation of chloride transport may also be beneficial in other diseases that show abnormally high mucus accumulation in the airways, such as asthma and chronic bronchitis. Similarly, intestinal constipation may benefit from activation of chloride transport by a flavone or isoflavone as provided herein. Summary of Sequence Listing SEQ ID NO:1 is a DNA sequence encoding human CFTR. SEQ ID NO:2 is an amino acid sequence of human CFTR. SEQ ID NO:3 is a DNA sequence encoding human CFTR with the .DELTA.F508 mutation. SEQ ID NO:4 is an amino acid sequence of human CFTR with the .DELTA.F508 mutation. SEQ ID NO:5 is a DNA sequence encoding human CFTR with the G551D mutation. SEQ ID NO:6 is an amino acid sequence of human CFTR with the G55ID mutation. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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