DETAILED DESCRIPTION OF THE INVENTION

[0055] As noted above, the present invention is generally directed to compositions and methods for the treatment of diseases characterized by defective chloride transport, particularly with regard to defective cellular chloride transport (e.g., defective export from or import to the cell of chloride, such as chloride anion or in the form of a chloride salt or other chloride-containing compound), for example, in epithelial tissues, including diseases such as cystic fibrosis, asthma, chronic obstructive pulmonary disease, and other inflammatory disorders of the airways, intestinal constipation, pancreatitis, and dry eye syndrome and also including diseases that feature excessive accumulation of mucus, including cystic fibrosis, chronic bronchitis and asthma. Within the context of the present invention, defective chloride transport may comprise a decrease (e.g., in a statistically significant manner) or lack of chloride transport as compared to normal physiological levels with which the skilled artisan will be familiar as a property of a particular cell or tissue type, organ system, or the like, or an increase(e.g., in a statistically significant manner) in chloride transport as compared to such normal levels.

[0056] It has been found, within the context of the present invention, that certain flavones and isoflavones, as well as other polyphenolic compounds, and ascorbate compounds such as ascorbic acid (vitamin C), and derivatives thereof, are capable of stimulating CFTR-mediated chloride transport in epithelial tissues (e.g., tissues of the airways, intestine, pancreas and other exocrine glands) in a cyclic-AMP independent manner. It has further been found, within the context of the present invention, that, under appropriate conditions, such compounds may stimulate chloride transport in certain cells, for example, cells having a mutated CFTR (e.g., ΔF508-CFTR or G551D-CFTR). Such therapeutic compounds may be administered to patients suspected of having or afflicted with a disease characterized by defective chloride transport (e.g., defective cellular chloride transport) such as, cystic fibrosis, asthma, chronic obstructive pulmonary disease, and other inflammatory disorders of the airways, intestinal constipation, pancreatitis, and dry eye sydrome as described herein.

[0057] The term “flavones,” as used herein refers to a compound based on the core structure of flavone: 3

[0058] 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: 4

[0059] Certain flavones and isoflavone have the structure: 5

[0060] wherein carbon atoms at positions 2, 3, 5, 6, 7, 8, 2′, 3′, 4′, 5′ and 6′ are bonded to a moiety independently selected from the group consisting of hydrogen atoms, hydroxyl groups and methoxyl groups, and wherein X is a single bond or a double bond. Stereoisomers and glycoside derivatives of such polyphenolic compounds may also be used within the methods provided herein.

[0061] 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. Genistein is yet another preferred isoflavone for use within the methods provided herein.

[0062] Any flavone or isoflavone, or ascorbate compound (e.g., ascorbic acid, ascorbate, or a derivative thereof), that stimulates (e.g., increases with statistical significance) chloride transport within at least one of the assays described herein may be used for therapy of diseases characterized by defective chloride transport as provided herein including, for example, cystic fibrosis and other diseases characterized by abnormally high mucus accumulation in the airways, asthma, chronic obstructive pulmonary disease, and other inflammatory disorders of the airways, intestinal constipation, pancreatitis, and dry eye syndrome. Preferred therapeutic compounds include flavones and isoflavones that occur naturally in plants and are part of the human diet. Preferred compounds include genistein (4′,5,7-trihydroxyisoflavone), as well as 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: 6

[0063] Other suitable therapeutic compounds may be identified using the representative assays as described herein. Additional representative flavones and isoflavones include flavanone, flavone, dihydroxyflavone, trimethoxy-apigenin, apigenin 7-O-neohesperidoside, fisetin, rutin, daidzein and prunetin. Representative flavones and isoflavones are summarized in Tables I and II. 1

[0064] where=a double bond,—is a single bond, ONeo is Neohesperidoside, ORut is rutinoside, OCH3 is methoxy, OH is hydroxy 2

[0065] where=a double bond,—is a single bond, ONeo is Neohesperidoside, ORut is de, OCH3 is methoxy, OH is hydroxy.

[0066] Genistein, quercetin, apigenin, kaempferol, biochanin A and other s and isoflavones may generally be prepared using well known techniques, 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.

[0067] As noted above, other polyphenolic compounds may be used within the methods provided herein. For example, trihydroxystilbenes such as reservatrol (trans-3,5,4′-trihydroxystilbene) may be employed. Reservatrol is a polyphenolic compound having the following structure: 7

[0068] Other compounds that may be used within the methods provided herein are ascorbate compounds such as ascorbic acid (vitamin C) and derivatives thereof. Accordingly and as provided herein, reference to ascorbate, ascorbic acid or vitamin C contemplates similar uses of other ascorbate derivatives having similar effects on chloride transport (e.g., cellular chloride transport) as those presently disclosed. Vitamin C, is a required micronutrient for humans (Nishikimi, M., et al., (1994) J. Biol. Chem. 269, 13685-13688.) Dietary vitamin C acts as a cofactor for several intracellular enzymes and scavenges free radicals (Rumsey, S. C. et al., (1998) J. Nutr. Biochem. 9, 116-130.). In the lungs and respiratory tract vitamin C mainly functions as an electron donor for reactive oxygen species (Willis, R. J., et al., (1976) Biochim. Biophys. Acta 444, 108-111; Slade, R., et al., (1993) Exp. Lung Res. 19, 469-484; van der Vliet, A., et al., (1999) Am. J. Physiol. 276, L289-L296; Kelly, F. J., et al., (1999) Lancet 354, 482-483). Epidemiological studies suggested a link between high dietary intake of vitamin C and its protective effects against respiratory symptoms (Schwartz, J., et al., (1994) Am. J. Clin. Nutr. 59, 110-114), however an alarming 25% of the U.S. population did not meet the recommended dietary intake levels for vitamin C as published in the Third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) (Food and Nutrition Board Institute of Medicine (2002) in Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids (Natl. Acad. Press, Wash. D.C.), pp. 95-185). Insufficient dietary intake of vitamin C (Kodavanti, et al., (1996) Exp. Lung Res. 22(4), 435-448), environmental pollutants (Kodavanti, et al., Supra; Cross, et al., (1992) FEBS Lett. 298, 269-272) and a number of inflammatory disorders of the airways are known to severely deplete physiological pools of vitamin C in the respiratory tract. For example, low levels of vitamin C were found in patients with bronchial asthma (Kelly, et al., Supra; Menzel, D. B. (1992) Ann. N Y Acad. Sci. 669, 141-155.), cystic fibrosis (Winklhofer-Roob, etal., (1997) Am. J. Clin. Nutr. 65, 1858-66; Brown, et al., (1997) Am. J. Physiol. 273, L782-L788), chronic obstructive pulmonary disease (Calikoglu, et al., (2002) Clin. Chem. Lab. Med. 40, 1028-1031), acute respiratory distress syndrome (Cross, et al., (1990) J. Lab. Clin. Med. 115, 396-404), and smokers (Lykkesfeldt, et al., (2000) Am. J. Clin. Nutr. 71, 530-536) as well as in children exposed to environmental tobacco smoke (Preston, et al., (2003) Am. J. Clin. Nutr. 77, 167-172). In addition to its well known function as an antioxidant in many species including mammalian species, in amphibians a regulatory function of vitamin C on Cl transport across the cornea has been demonstrated (Scott, et al., (1975) Invest. Ophthalmol. 14, 763-6).

[0069] The cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) was cloned in 1989 (Riordan, et al., (1989) Science 245, 1066-73). In the respiratory tract CFTR mediates the transport of Cl⁻ across the epithelial cell apical membrane into the extracellular airway surface liquid (ASL), which transport is regulated to properly adjust ASL salt composition. The dynamics of the ASL affect its physiological function, the most important of which is the removal of inhaled particles and the support of mucociliary clearance (Widdicombe (1995) Am. J. Respir. Crit. Care Med. 151, 2088-2092). CF-like symptoms such as thickened airway secretions are often seen in chronic inflammatory airway diseases without mutations in the CFTR gene, and there is emerging evidence that post-translational damage to CFTR by reactive oxygen and nitrogen species decreases CFTR function (Bebok, et al. (2002) J. Biol. Chem. 277, 43041-43049).

[0070] As described in greater detail below and in the examples, the experiments described herein were designed to investigate the role of ascorbate compounds such as vitamin C in controlling Cl⁻ secretion and to clarify the potential involvement of CFTR in the underlying Cl⁻ conductance.

[0071] Accordingly, other compounds that may be used within the methods provided herein are ascorbic acid and derivatives thereof. Such compounds include L-ascorbic acid (L-xyloascorbic acid), dehydroascorbic acid (L-threo-2,3-Hexodiulosonic acid γ-lactone) and salts of the foregoing acids (e.g., L(+)-Ascorbic acid sodium salt, L(+)-Ascorbic acid iron(II) salt), as well as other ascorbate compounds as provided herein (e.g., L-Ascorbic acid 6-palmitate, L-Ascorbic acid 2-phosphate sesqui-magnesium salt, L-ascorbic acid 2-phosphate trisodium salt, L-Ascorbic acid 2-sulfate dipotassium salt) and other ascorbate compounds known to the art (see for example, J Biol Chem. 1999 Aug. 13; 274(33): 23215-22; Yamamoto, et al., J Nutr Sci Vitaminol (Tokyo). 1992; Spec No: 161-4; Hornig D.

[0072] Ann N Y Acad Sci. 1975 Sep. 30; 258: 103-18), and which have detectable effects on chloride transport (e.g., cellular chloride transport) as herein disclosed. 8

[0073] Within certain preferred embodiments, ascorbic acid or a derivative thereof is used in combination with a polyphenolic compound as described above. Certain representative combinations include ascorbic acid and one or more flavenoids and/or isoflavenoids (such as genistein and ascorbic acid; and kaempferol and ascorbic acid). Ascorbic acid may generally be used to treat or prevent genetic loss of chloride secretory function (e.g., cystic fibrosis), as well as other related loss or reduced chloride secretory function (e.g., intestinal constipation, dry eye syndrome, asthma, and obstructive airway diseases).

[0074] Certain embodiments disclosed herein relate to the observation that the effect of vitamin C on chloride transport depends on the function of the sodium-dependent vitamin C transporters (SVCT) (see in particular Example 13; the sequences for SVCTs are known in the art and are available, for example, via Genbank Accession Numbers SVCT1: NM_—005847 and SVCT2: AY380556). In certain embodiments, such as for the treatment of diarrhea resulting from high dose vitamin C treatment, it may be desirable to block vitamin C transport in intestinal epithelial cells. As such, the present invention contemplates the use of agents, including but not limited to, for example, phloretin and derivatives thereof, that block or otherwise decrease vitamin C transport in intestinal epithelia, in a statistically significant manner, such as through SVCT1 and SVCT2. Accordingly, provided herein are methods for identifying agents that alter chloride transport by contacting a cell (e.g., an epithelial cell) such as a cell that expresses SVCT1 and/or SVCT2, with a test agent and an ascorbate compound (e.g., vitamin C, ascorbic acid, ascorbate salts, dehydroascorbic acid or derivatives thereof) and measuring chloride transport in the cell that has been contacted with the test agent and vitamin C as compared to chloride transport in a cell that has been contacted with vitamin C in the absence of the test agent, for instance, in the presence of a control compound known not to affect chloride transport. In this regard a statistically significant increase or decrease in chloride transport in cells contacted with the test agent and vitamin C as compared to the cells contacted with vitamin C in the absence of the test agent, or in the presence of a control compound, indicates the agent alters chloride transport. Such agents can be identified by measuring chloride transport using any of the assays described herein and can also be identified using assays that screen for inhibition of SVCT1 and SVCT2 (e.g., by measuring vitamin C transport). Such agents can be identified using the assays as described herein, or known in the art (Tsukaguchi, H., Tokui, T., Mackenzie, B., Berger, U. V., Chen, X. Z., Wang, Y., Brubaker, R. F. & Hediger, M. A. (1999) Nature 399, 70-75).

[0075] As used herein, epithelial cells include any cell of epithelioid origin as known to those familiar with the art, and may be present in a biological sample as provided herein, for example a cell derived from a mammalian organ outer layer (e.g., skin, renal cortical layer, etc.) or an organ lining layer (e.g., gastric epithelia, intestinal epithelia, etc.) such as mammalian epithelial cells that are capable of forming one or more specialized structures, including desmosomes, tight junctions, adhesion plaques, distinct apical and basolateral regions, and the like.

[0076] Assays for Evaluating Chloride Transport

[0077] Flavones, isoflavones, ascorbate compounds such as ascorbic acid and derivatives thereof 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, in certain preferred embodiments, at a concentration of about 1-10,000 μM, and in certain other preferred embodiments at a concentration of 0.1-1,000 μM (and all concentrations therebetween) is said to stimulate chloride transport. Generally, with regard to an ascorbate compound such as ascorbic acid or a derivative thereof, stimulation of a statistically significant increase in chloride transport, for example, at a concentration of about 1 to about 300 μM and in certain other preferred embodiments at a concentration of 0.1-1,000 μM (and all concentration therebetween) is said to comprise stimulated chloride transport. In another embodiment, at least about 5% increase of chloride transport is considered beneficial. In certain embodiments, increased chloride transport by at least about 10%-15% may be considered beneficial. In an additional embodiment, increased chloride transport by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, is considered beneficial. In other embodiments, it is considered beneficial if a compound, such as those described herein, provides a stimulatory effect on chloride transport at a level that is about 25%-60% of the level of chloride transport that is stimulated using commonly known CFTR agonists (e.g., forskolin). By way of non-limiting theory and as described herein, and in contrast to the situation that pertains to certain presently known CFTR agonists such as forskolin, a known elevator of intracellular cAMP, according to certain embodiments the invention relates to chloride transport that may be altered (e.g., increased or decreased in a statistically significant manner) under conditions that do not effect an increased level of intracellular cAMP, which may refer to any detectable increases in intracellular cAMP levels that are statistically significant and that are typically greater than 5%, 10%, 20%, 30%, 40%, 50% or more above the intracellular cAMP levels that exist in a particular biological system prior to introduction of the conditions that alter chloride transport.

[0078] Certain embodiments of the invention as disclosed herein relate to detecting chloride transport, and in particular to detecting chloride transport by cells, which may typically pertain to detection of cellular export of intracellular chloride to the extracellular environment. As described herein and known in the art, CFTR represents an important transmembrane channel for chloride transport through the cellular plasma membrane, and in humans CFTR is the major chloride transport channel. For example, certain single amino acid defects in CFTR manifest themselves as lethal mutations in CF, underscoring the importance of CFTR as a primary means for transporting chloride. Accordingly, based upon the present disclosure any of a number of art accepted methodologies may be used for detecting chloride transport such as CFTR-mediated chloride transport. By way of illustration and not limitation, descriptions of several such chloride transport assays, including in vitro and in vivo methodologies, are presented.

[0079] 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 human, rat, mouse and bovine tracheal and alveolar cell cultures, as well as intestinal epithelial cell lines, such as T84, HT-29, Caco-2 and cell lines of other epithelial origin such as MDCK and FRT. 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. Anion efflux assays may be used to detect CFTR-mediated transport (e.g., iodide efflux, see for example RNA. 2003 October; 9(10): 1290-7). Additionally, chloride transport (or generally airway surface liquid composition), can be evaluated using any number of indicators such as described in J Gen Physiol. 2003 November; 122(5): 511-9. Alternatively, chloride transport may be evaluated using epithelial tissue in which the basolateral membrane is permeabilized with anion-selective ionophores such as nystatin, amphotericin B or Staphylococcus aureus α-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, or ascorbate or derivative thereof), and those compounds that stimulate chloride transport as described above may be used within the methods provided herein.

[0080] 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 (ΔF508-CFTR; SEQ ID NO:4). Accordingly, the use of a CFTR gene encoding ΔF508-CFTR is preferred. However, genes encoding other altered CFTR proteins (e.g., G551D-CFTR; containing a glycine to aspartate point mutation at position 551; SEQ ID NO:6) may also be used. Cells such as NIH 3T3 fibroblasts may be transfected with an altered CTFR gene, such as ΔF508-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.

[0081] 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 (e.g., flavone, isoflavone, ascorbate or derivatives thereof) may be monitored with the two electrode voltage clamp method (see Miledi et al., Proc. R. Soc. Lond. Biol. 218:481-484, 1983).

[0082] Alternatively, such assays may be performed using a mammalian trachea, such as a primary human, mouse, sheep, pig or 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 flavones, isoflavones, ascorbate or derivatives thereof that stimulate chloride transport.

[0083] Any of the assays described herein may be performed in the presence of cAMP agonists known in the art and as described herein (see Example 8 and other examples herein).

[0084] Any of the above assays may be performed following pretreatment of the cells with a substance that increases the concentration of CFTR mutants in the plasma membrane. Such substances include chemical chaperones, which support correct trafficking of the mutant CFTR to the membrane, and compounds that increase expression of CFTR in the cell (e.g., transcriptional activators). A “chemical chaperone,” as used herein is any molecule that increases trafficking of proteins to a cell membrane. More specifically, a chemical chaperone within the context of the present invention increases trafficking of a mutant CFTR (e.g., the Δ508-CFTR and/or G551D-CFTR) to the membrane by a statistically significant amount. Chemical chaperones for use herein include, but are not limited to, glycerol, dimethylsulfoxide, trimethylamine N-oxide, taurin, methylamine and deoxyspergualin (see Brown et al., Cell Stress Chaperones 1:117-125, 1996; Jiang et al., Amer J. Physiol.-CellPhysiol . 44:C171-C178, 1998). Compounds that increase expression of CFTR in the cell include 4-phenylbutyrate (Rubenstein et al., J. Clin. Invest. 100:2457-2465, 1997), sodium butyrate (Cheng et al., Am. J. Physiol. 268:L615-624, 1995) and S-Nitrosoglutathione (Zaman, et al., Biochem Biophys Res Commun 284: 65-70, 2001; Snyder, et al., American Journal of Respiratory and Critical Care Medicine 165: 922-6, 2002; Andersson, et al. Biochemical and Biophysical Research Communication 297(3): 552-557, 2002.). Other compounds that increase the level of CFTR in the plasma membrane (by increasing correct trafficking and/or expression of the CFTR) may be readily identified using well known techniques, such as Western Blotting and immunohistochemical techniques, to monitor maturation of CFTR and evaluate effects on levels of plasma membrane CFTR, respectively.

[0085] 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.

[0086] Compounds as provided herein may generally be used to increase chloride transport within any of a variety of CFTR-expressing epithelial cells. CFTR is expressed in may epithelial cells, including intestinal, airway, lung, pancreas, gallbladder, kidney, sweat gland, lacrimal gland, salivary gland, mammary epithelia, epithelia of the male and female reproductive tract, and non epithelial cells including lymphocytes. All such CFTR-expressing organs are subject to stimulation by the compounds provided herein.

[0087] Accordingly, certain embodiments of the invention relate to a method of identifying an agent that stimulates (or impairs, and in either event in a statistically significant manner) chloride transport (e.g., cellular transport of chloride across a plasma membrane), comprising contacting in the absence and presence of a candidate agent an ascorbate compound and a biological sample comprising a cell, under conditions and for a time sufficient to induce chloride transport; and detecting chloride transport, wherein a level of detectable chloride transport that is increased in the presence of the candidate agent relative to the level of detectable chloride transport in the absence of the agent indicates an agent that stimulates chloride transport (and wherein, similarly, a level of detectable chloride transport that is decreased in the presence of the candidate agent relative to the level of detectable chloride transport in the absence of the agent indicates an agent that impairs chloride transport). In certain related further embodiments the ascorbate compound may be vitamin C or a derivative thereof, and in certain other related further embodiments the candidate agent may be a flavonoid. In certain other related embodiments the cell comprises a CFTR and/or a vitamin C TR. In certain preferred embodiments the step of contacting does not increase the intracellular cAMP level.

[0088] Certain chloride transport assays may be adapted to a high throughput screening (HTS) format and thus may be especially suited to automated screening of large numbers of candidate agents for activity in assays of chloride transport. HTS has particular value, for example, in screening synthetic or natural product libraries for active compounds. The methods of the present invention are therefore amenable to automated, cost-effective high throughput drug screening and have immediate application in a broad range of pharmaceutical drug development programs. In a preferred embodiment of the invention, the compounds to be screened are organized in a high throughput screening format such as a 96-well plate format, or other regular two dimensional array, such as a 384-well, 48-well or 24-well plate format or an array of test tubes. For high throughput screening the format is therefore preferably amenable to automation. It is preferred, for example, that an automated apparatus for use according to high throughput screening embodiments of the present invention is under the control of a computer or other programmable controller. The controller can continuously monitor the results of each step of the process, and can automatically alter the testing paradigm in response to those results.

[0089] Typically, and in preferred embodiments such as for high throughput screening, candidate agents are provided as “libraries” or collections of compounds, compositions or molecules. Such molecules typically include compounds known in the art as “small molecules” and having molecular weights less than 10⁵ daltons, preferably less than 10⁴ daltons and still more preferably less than 10³ daltons. For example, members of a library of test compounds can be administered to a plurality of samples in each of a plurality of reaction vessels in a high throughput screening array as provided herein, each containing at least one cell and being present in a form that permits conditions for inducing detectable chloride transport according to principles described herein. Candidate agents further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels. For example, various starting compounds may be prepared employing one or more of solid-phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions. The resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides (see e.g., PCT/US91/08694 and PCT/US91/04666) or other compositions that may include small molecules as provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. Pat. No. 5,798,035, U.S. Pat. No. 5,789,172, U.S. Pat. No. 5,751,629). Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries may be prepared according to established procedures, and tested for effect on chloride transport according to the present disclosure, using a biological sample (e.g., a cell such as an epithelial cell, or other suitable preparation of a cell, tissue, organ, or the like, for instance primary cell cultures, biopsy cells, tissue explants, established cell lines including transformed, immortal or immortalized cells, or other naturally occurring or genetically engineered cells or artificial cells).

[0090] Compositions and Methods of Use

[0091] For in vivo use, a 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) (i.e., 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 physiologically acceptable carrier(s) and/or excipient(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.

[0092] In certain embodiments, an ascorbate compound or a derivative thereof and a flavonoid or isoflavonoid can be combined to additively open CFTR and thereby stimulate chloride transport. Within certain methods provided herein, a flavone, isoflavone, or derivative thereof, ascorbic acid or derivatives thereof, alone or in combination, may be combined with a substance that increases the concentration of CFTR mutants in the plasma membrane of a cell. As noted above, such substances may include a chemical chaperone, which supports correct trafficking of the mutant CFTR to the membrane, and may also include compounds that increase expression of CFTR in the membrane. These substances may be contained within the same pharmaceutical composition or may be administered separately. Preferred chemical chaperones include, for example, glycerol, dimethylsulfoxide, trimethylamine N-oxide, taurin, methylamine and deoxyspergualin, or the like, and compounds that increase expression of CFTR in the membrane include 4-phenylbutyrate and sodium butyrate, or the like. The use of flavonoid and/or isoflavonoid compounds, or ascorbic acid and derivatives thereof, as described herein, in combination with such substances may increase mutant CFTR activity, and ameliorate symptoms of diseases associated with defective chloride transport, such as cystic fibrosis.

[0093] In certain embodiments, the compounds described herein may be used to treat diarrhea, in particular diarrhea associated with vitamin C treatment. By way of non-limiting theory, CFTR blockers have been employed in the treatment of diarrhea (e.g., U.S. Pat. No. 5,234,922) whereas according to the instant disclosure and as described herein, agents that decrease vitamin C transporter activity are for the first time recognized as effecting decreased CFTR-mediated chloride transport. As such, the compositions, such as compositions comprising compounds that block vitamin C transporters (e.g., SVCT1 and/or SVCT2), can be administered alone or in combination with CFTR blockers. Such compositions as well as other compositions described herein may be in combination with any physiologically acceptable carrier(s) and/or excipient(s) known to those skilled in the art to be suitable for the particular mode of administration.

[0094] Administration may be achieved by a variety of different routes. 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.

[0095] Another 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.

[0096] 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.

[0097] In general, the compositions of the present invention may be administered by the topical, transdermal, oral, rectal (e.g., via suppository or enema) or parenteral (e.g., intravenous, subcutaneous or intramuscular) route. In addition, the compositions may be incorporated into biodegradable polymers allowing for sustained release of the composition, the polymers being implanted in the vicinity of where delivery is desired, for example, in the intestinal epithelia. The biodegradable polymers and their use are described, for example, in detail in Brem et al. J. Neurosurg. 74:441-446 (1991).

[0098] Pharmaceutical compositions are administered in an amount, and with a frequency, that is effective to inhibit or alleviate the symptoms of a disease characterized by defective chloride transport, such as cystic fibrosis, asthma, chronic obstructive pulmonary disease, and other inflammatory disorders of the airways, intestinal constipation, pancreatitis, and dry eye syndrome, and/or to delay the progression of the disease. The effect of a treatment may be clinically determined by nasal potential difference measurements, or other measurements as described herein and known in the art. 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 2000 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 μM 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.

[0099] As noted above, a pharmaceutical composition may be administered to a mammal to stimulate chloride transport, and to treat diseases characterized by defective chloride transport, such as cystic fibrosis, asthma, chronic obstructive pulmonary disease, and other inflammatory disorders of the airways, intestinal constipation, pancreatitis, and dry eye syndrome and diseases with excessive accumulation of mucus, including cystic fibrosis, chronic bronchitis and asthma and to prevent local vitamin C deficits in the respiratory tract due to oxidatative stress (ozone, tobacco smoke, air pollution, allergic reactions, and the like). Patients that may benefit from administration of a therapeutic compound as described herein are those afflicted with one or more diseases characterized by defective chloride transport, such as cystic fibrosis, asthma, chronic obstructive pulmonary disease, and other inflammatory disorders of the airways, intestinal constipation, pancreatitis, and dry eye syndrome and diseases with excessive accumulation of mucus, including cystic fibrosis, chronic bronchitis and asthma. 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.

Summary of Sequence Listing

[0100] SEQ ID NO:1 is a DNA sequence encoding human CFTR.

[0101] SEQ ID NO:2 is an amino acid sequence of human CFTR.

[0102] SEQ ID NO:3 is a DNA sequence encoding human CFTR with the ΔF508 mutation.

[0103] SEQ ID NO:4 is an amino acid sequence of human CFTR with the ΔF508 mutation.

[0104] SEQ ID NO:5 is a DNA sequence encoding human CFTR with the G551D mutation.

[0105] SEQ ID NO:6 is an amino acid sequence of human CFTR with the G551D mutation.

[0106] SEQ ID NOs:7-10 are PCR primers.

[0107] SEQ ID NO:11 is the polynucleotide of SVCT2 from human trachea epithelia as set forth in GenBank accession No. AY380556 and is identical to the sequence set forth in SEQ ID NO:12 with the exception of one nucleotide exchange at position 1807 T→C.

[0108] SEQ ID NO:12) is the polynucleotide of SVC2 from human kidney as set forth in GenBank accession No. AJ269478.

[0109] The following Examples are offered by way of illustration and not by way of limitation.

EXAMPLES

Example 1

Stimulation of Chloride Transport by Representative Flavones and Isoflavones in Airway Cells

[0110] This Example illustrates the use of the representative compounds apigenin, quercetin and biochanin A to enhance chloride secretion in Calu-3 human pulmonary cultures or in primary bovine tracheal cultures.

[0111] A Calu-3 cell monolayer was prepared in an Ussing chamber as described by Illek et al., Am. J. Physiol. 270:C265-275, 1996. The basolateral membrane was permeabilized with α-toxin and a chloride gradient was applied across the apical membrane as a driving force (see Illek et al, Am. J. Physiol. 270:C265-C275, 1996). The tissue was first stimulated with cAMP (100 μM), and then with a representative flavone or isoflavone.

[0112] As shown in FIGS. 1 and 2, subsequent addition of apigenin or quercetin further stimulated chloride current. FIG. 1 illustrates the short circuit current across the Calu-3 cell monolayer before and after addition of apigenin (50 μM). FIG. 2 illustrates the effect of quercetin (30 μM) on chloride current across a Calu-3 monolayer. In both cases, the flavone stimulated chloride current beyond the stimulation achieved by cAMP.

[0113] FIG. 3 illustrates the results of a related experiment to evaluate the dose-dependent stimulation of transepithelial chloride secretion by quercetin across a primary bovine tracheal epithelium. The epithelial cells were first treated with amiloride (50 μM), and then with quercetin at the indicated concentrations. The dose-response relation yielded a half maximal stimulation at 12.5 μM. At high concentrations of quercetin, the current was blocked. Current was fully inhibited by the CFTR channel blocker diphenylcarboxylate (DPC, 5 mM).

[0114] To evaluate the effect of biochanin A, a Calu-3 cell monolayer was prepared and permeabilized as described above. The tissue was first stimulated with forskolin (Fsk, 10 μM). The effect of biochanin A (Bio, 100 and 300 μM) on short-circuit current (I_sc) across the Calu-3 monolayer was evaluated in an Ussing chamber. As shown in FIG. 4, biochanin A further stimulated chloride secretion.

Example 2

Activation of Mutant CFTR by Representative Flavones and Isoflavones

[0115] This Example illustrates the use of the representative compounds apigenin, quercetin and genistein to activate ΔF508-CFTR and G551D-CFTR in different cell types.

[0116] A cell-attached single channel patch clamp recording was obtained from a 3T3 cell expressing ΔF508-CFTR as described by Hamill et al., Pflugers Arch. 391:85-100, 1981 and Fischer and Machen, J. Gen. Physiol. 104:541-566, 1994. As shown in FIG. 5, stimulation of the cell with 10 μM forskolin did not activate ΔF508-CFTR channel, but addition of genistein (50 μM) or apigenin (50 μM, where indicated by boxes) induced ΔF508-CFTR channel openings, and removal of these compounds inactivated the channels. The holding potential was 75 mV, and channel openings were upward.

[0117] FIG. 6 presents a whole cell patch clamp recording on an airway epithelial cell homozygous for ΔF508-CFTR (cell type: μME cell, see Jeffersen et al., Am. J. Physiol. 259:L496-L505, 1990). Before the measurement, the cell was incubated for 2 days in 5 mM 4-phenylbutyrate to enhance ΔF508-CFTR expression in the plasma membrane (Rubenstein & Zeitlin, Ped. Pulm. Suppl. 12:234, 1995). Measurements were performed as described by Fischer et al., J. Physiol. Lond. 489:745-754, 1995. Addition of 30 μM quercetin activated chloride current in the whole cell mode, which was further stimulated by forskolin. The holding potential was −60 mV.

[0118] The effect of genistein on chloride current in a Xenopus oocyte expressing G551D-CFTR was measured with the two-electrode voltage clamp technique (see Miledi et al., Proc. R. Soc. Lond. Biol. 218:481-484, 1983). G551D-CFTR (2 ng in 50 nL of water) was injected into the oocyte. Current was first stimulated with forskolin (10 μM) and isobutylmethylxantine (IBMX; 2 mM). Genistein (50 μM) was found to further activate chloride currents. As shown in FIG. 7, genistein increased conductance and shifted reversal potential to the right, which is indicative of a stimulated chloride current.

Example 3

Effect of Representative Flavones on Nasal Potential Difference

[0119] This Example illustrates the in vivo use of quercetin, apigenin and kaempferol to activate the nasal potential difference in humans and mice.

[0120] The effect of quercetin on nasal potential difference (PD) measurement in a healthy human volunteer was measured as described by Knowles et al., Hum. Gene Therapy 6:445-455, 1995. Under conditions where sodium transport was blocked with amiloride (50 μM) and chloride secretion was stimulated under chloride-free conditions with isoproterenol (5 μM), quercetin (10 μM) stimulated nasal PD further (FIG. 8).

[0121] The effect of apigenin and kaempferol on nasal PD in mice was evaluated using a method similar to that employed for measurements in humans, except that a plastic tube of approximately 0.1 mm diameter was used as an exploring nasal electrode. The plastic tube was perfused with test solutions at approximately 10 μL/min. After blocking sodium transport with amiloride (50 μM) and during stimulation of chloride secretion with isoproterenol (iso;5 μM) under chloride-free conditions, apigenin (50 μM, left panel) and kaempferol (kaemp, 50 μM, right panel) further stimulated nasal PD.

[0122] These results show that the representative flavenoids quercetin, apigenin, kaempferol and biochanin A stimulate chloride transport across epithelial tissues derived from the airways in vitro, and across nasal epithelium in vivo. The results also show that the CFTR mutants ΔF508 and G551D can be activated by the representative compounds genistein and apigenin.

Example 4

Effect of Genistein on Chloride Current in Cells Expressing a Mutant CFTR

[0123] This Example illustrates the ability of the representative isoflavone genistein to activate chloride current in cells expressing a mutant CFTR.

[0124] In one experiment, genistein was used in combination with 4-phenylbutyrate. Chloride current was measured in JME cells (human nasal epithelial cell line homozygous for the Δ508 mutation of CFTR; see Jefferson et al., Am. J. Physiol. 259:L496-505, 1990). The recording was performed at 0 mV holding potential with a 17:150 mM chloride gradient from bath to pipette. Under these conditions, the recorded current, shown in FIG. 10, is chloride current (Illek and Fischer, Am. J. Physiol. (Lung Cell. Mol. Physiol.):L902-910, 1998). The bottom trace in FIG. 10 is from an untreated cell. Neither forskolin (10 μM nor genistein (30 μM activated current. The top tracing in FIG. 10 is from a cell that had been incubated in 5 mM 4-phenylbutyrate (4-PB) for two days (Rubenstein et al., J. Clin. Invest. 100:2457-2465, 1997). After 4-PB treatment, chloride current was stimulated by forskolin (by on average 30.3±19.4 pS/pF, n=6), and further activated by genistein (to an average 105±84 pS/pF) in a CF cell with the Δ508-CFTR mutation. These results further demonstrate the ability of a flavenoid compound to optimize chloride currents elicited in CF cells by other means.

[0125] Within another experiment, HeLa cells infected with the G551D-CFTR-containing adenovirus were investigated in the patch clamp mode. Stimulation of the cell with forskolin (10 μM) stimulated only a very small current (FIGS. 11A and 11B). On average, forskolin-stimulated conductance was 9.5±5 pS/pF (n=4). Additional stimulation with genistein (30 μM) stimulated significant chloride currents, which were time- and voltage-independent (FIG. 11B) and well fitted with the Goldman equation (line in FIG. 11B; Illek and Fischer, Am. J. Physiol. (Lung Cell. Mol. Physiol.):L902-910, 1998), which are characteristics of CFTR-mediated currents. Average forskolin+genistein-activated conductance was 120±30 pS/pF (n=4). Current variance to mean current plot (FIG. 11C) were used to calculate the average open probability (P_o shown on top of axis) of the population of channels carrying the total current (as described in Illek and Fischer, Am. J. Physiol. (Lung Cell. Mol. Physiol.):L902-910, 1998). During forskolin stimulation, maximal P_o reached was 0.04 (open circles) and after additional stimulation with genistein P_o reached a maximum of 0.42 in this recording. On average, after forskolin stimulation, P_o=0.05±0.02 and after forskolin+genistein stimulation P_o=0.54±0.12. For comparison, wild type CFTR expressed in HeLa cells and recorded under the same conditions resulted in P_o=0.36±0.05 (n=3) after forskolin stimulation and P_o=0.63±0.16 after forskolin+genistein treatment.

Example 5

Effect of Representative Flavones on Nasal Potential Difference in CF Patients

[0126] This Example illustrates the in vivo use of quercetin and genistein to activate the nasal potential difference in CF patients bearing the G551D mutation.

[0127] Measurements were performed on patients as described by Alton et al., Eur. Respir. J. 3:922-926, 1990; Illek and Fischer, Am. J. Physiol. (Lung Cell. Mol. Physiol.):L902-910, 1998; and Knowles et al., Hum. Gene Therapy 6:445-455, 1995). The results are presented in FIGS. 12A and 12B. FIG. 12A shows a recording from a patient with the genotype G551D/ΔF508. Initial treatment with amiloride and chloride free solution had the purpose to isolate and amplify the chloride selective potentials. Addition of the beta-adrenergic agonist isoproterenol showed no effect, which is typical for CF patients (Knowles et al., Hum. Gene Therapy 6:445-455, 1995). However, addition of genistein hyperpolarized nasal PD. Average responses of nasal PD to genistein and quercetin of four CF patients with the G551D mutation are shown in FIG. 12B (open bars). The filled bars show for comparison the respective responses in healthy subjects. The genotypes of the 4 CF patients were: two G551D/ΔF508, one G551D/G551D and one G551D/unknown. Responses are most likely due to the G551D mutation because the homozygous G551D patient responded not different compared to the heterozygous G551D patients. Genistein and quercetin responses of nasal PD in CF patients were significant (p<0.05).

[0128] These results demonstrate that CFTR mutations are sensitive to flavenoid treatment, and provide additional evidence for therapeutic benefit of such compounds for the treatment of cystic fibrosis.

Example 6

Effect of Additional Representative Polyphenolic Compounds on Epithelial Cell Chloride Currents

[0129] This Example illustrates the effect of further flavenoids and isoflavenoids on chloride currents in airway epithelial cells.

[0130] Airway epithelial cells were prestimulated with 10 μM forskolin. The percent increase in chloride current was then determined following treatment with a series of polyphenolic compounds. FIG. 13A summarizes the stimulatory effect of these compounds. On average, chloride currents were further stimulated by reservatrol (100 μM) to 135%, by flavanone (100 μM) to 140%, by flavone (200 μM) to 128%, by apigenin (20 μM) to 241%, by apigenin 7-O-neohesperidoside (30 μM) to 155%, by kaempferol (20 μM) to 182%, by fisetin (100 μM) to 108%, by quercetin (30 μM) to 169%, by rutin (30 μM) to 149%, by genistein (30 μM) to 229%, by daidzein (50 μM) to 162%, by biochanin A (100 μM) to 139% and by prunetin (100 μM) to 161%.

[0131] The stimulatory effect of 7,4′ Dihydroxyflavone is shown in FIG. 13B. Addition of 7,4′-Dihydroxyflavone to the mucosal perfusion dose-dependently stimulated transepithelial C1 currents in unstimulated Calu-3 monolayers. This experiment was performed using unstimulated tissue.

[0132] The stimulatory effect of trimethoxy-apigenin is shown in FIG. 13C. Addition of trimethoxy-apigenin to the mucosal perfusion dose-dependently stimulated transepithelial C1 currents in unstimulated Calu-3 monolayers. Kinetic analysis is depicted on the right panel and estimated half maximal stimulatory dose was 11.7 μM.

[0133] These results indicate that a variety of polyphenolic compounds stimulate chloride currents in epithelial cells.

Example 7

Effect of Reservatrol on Chloride Currents

[0134] This Example illustrates the stimulatory effect of reservatrol on transepithelial chloride currents.

[0135] Unstimulated Calu-3 monolayers were treated with increasing concentrations of reservatrol. FIG. 14 shows the recording generated following the addition of reservatrol to the mucosal perfusion dose-dependently stimulated transepithelial chloride currents in unstimulated Calu-3 monolayers. For comparison, currents were further stimulated by serosal addition of forskolin. The stimulated chloride current was completely blocked by the Cl channel blocker DPC. These results indicate that reservatrol stimulates transepithelial chloride transport.

Example 8

Effect of Ascorbic Acid and Dehydroascorbic Acid on Chloride Currents

[0136] This Example illustrates the stimulatory effect of ascorbic acid and dehydroascorbic acid on transepithelial chloride current.

[0137] Unstimulated Calu-3 monolayers were stimulated with L-ascorbic acid, as shown in FIG. 15. Addition of L-ascorbic acid to the mucosal or serosal perfusion very effectively stimulated transepithelial chloride secretion in unstimulated Calu-3 monolayers. For comparison, chloride currents were further stimulated by serosal addition of forskolin. In the continued presence of L-ascorbic acid and forskolin, it is remarkable that addition of genistein further stimulated chloride currents. These results indicate that genistein serves as a potent drug that is able to hyperstimulate chloride secretion and thereby maximize chloride transport across epithelia. The stimulated chloride current was completely blocked by the chloride channel blocker DPC.

[0138] The stimulatory effect of L-ascorbic acid is also shown in FIG. 16. Addition of 100 μM L-ascorbic acid to the mucosal or serosal perfusion very effectively stimulated transepithelial chloride currents in unstimulated Calu-3 monolayers. For comparison, ascorbic acid-stimulated chloride currents were stimulated by the cAMP elevating agonist forskolin (20 μM, serosal). Under these stimulated conditions kaempferol further hyperstimulated chloride currents. The stimulated chloride current was completely blocked by the chloride channel blocker DPC (5 mM).

[0139] The stimulatory effect of dehydroascorbic acid is shown in FIG. 18. Addition of dehydroascorbic acid at 10, 100 or 300 μM to the mucosal and serosal perfusion effectively stimulated transepithelial chloride currents in unstimulated Calu-3 monolayers. Stimulated C1 currents returned to baseline after 5-15 min.

Example 9

Effect of Ascorbic Acid on Chloride Currents in vivo

[0140] This Example illustrates the stimulatory effect of ascorbic acid on human nasal potential difference.

[0141] Nasal potential difference measurement was performed on a human volunteer according to a protocol by Knowles et al., Hum. Gene Therapy 6:445-455, 1995. Addition of L-ascorbic acid (100 μM) to the luminal perfusate in the nose (in the presence of amiloride (blocks Na currents) and in chloride-free solution) hyperpolarized nasal potential difference (PD) by 6.3 mV (FIG. 17). Addition of the β-adrenergic agonist isoproterenol further hyperpolarized nasal PD. Stimulation was reversed by washing out drugs with NaCl Ringer solution. These results demonstrate the ability of ascorbic acid to stimulate chloride transport in epithelia in humans.

Example 10

Effect of Genistein on Chloride Currents in Mammary Epithelia

[0142] This Example illustrates the stimulatory effect of genistein in mammary epithelial cells.

[0143] The stimulation of transepithelial short-circuit current (Isc) across 31EG4 mammary epithelial monolayers by addition of 20 μM genistein is shown in FIG. 19. Na currents were blocked by mucosal addition of amiloride (10 mM). Chloride currents were further stimulated by forskolin (20 μM, serosal). Currents were recorded in symmetrical NaCl Ringers solution at 0 mV and pulses were obtained at 2 mV.

[0144] The following Methods were used for the experiments described in Examples 11-16.

[0145] Human airway cell culture. The human submucosal serous gland-like cell line Calu-3, the CF nasal epithelial cell line homozygous for ΔF508 CFTR (CF15) and human tracheal primary cultures (hTE) were cultured as described (Jefferson, D. M., Valentich, J. D., Marini, F. C., Grubman, S. A., lannuzzi, M. C., Dorkin, H. L., Li, M., Klinger, K. W. & Welsh, M. J. (1990) Am. J. Physiol. 259, L496-L505. Sachs, L. A., Finkbeiner, W. E. & Widdicombe, J. H. (2003) In Vitro Cell Dev. Biol. Anim. 39, 56-62.). Wildtype CFTR-corrected CF15 cells were generated via adenovirus-mediated gene transfer. Growth media were nominally free of ascorbic acid (<10 μm, University Pathology Inc., Salt Lake City, Utah). For transepithelial measurements cells were grown on permeable filter inserts and used after 3 to 10 days.

[0146] Patch clamp analysis. Single channel patch clamp studies were performed on Calu-3 cells at 37° C. The outside-out recording mode was established via the whole cell mode as described (Fischer, H. (2001) in Methods in Molecular Medicine, eds. Skatch, W. R. & Walker, J. M. (Humana Press Inc, Totowa), pp. 49-66.). The bath solution contained (in mM) 145 NaCl, 1.7 CaCl₂, 1 MgCl₂, 10 Hepes, 10 glucose,