WO1997043643A1

WO1997043643A1 - Inhibitors of pseudomonas aeruginosa induced mucin production: compositions and treatments for cystic fibrosis patients

Info

Publication number: WO1997043643A1
Application number: PCT/US1997/008310
Authority: WO
Inventors: Jian Dong Li; Carole Basbaum; Young S. Kim; Jim Gum
Original assignee: The Regents Of The University Of California
Priority date: 1996-05-15
Filing date: 1997-05-15
Publication date: 1997-11-20
Also published as: AU3069097A

Abstract

The invention relates to methods for screening for inhibitors of mucomone induced mucin production, methods for inhibiting mucin production, methods for treating cystic fibrosis. Methods for identifying compounds that inhibit mucomone induced mucin production involve contacting cells with a compound in the presence of a mucomone, and detecting a change in mucin production or mucin gene activation of the cells.

Description

INHIBITORS OF PSEUDOMONAS AERUGINOSA INDUCED MUCIN PRODUCTION: COMPOSITIONS AND TREATMENTS FOR CYSTIC

FIBROSIS PATIENTS

ACKNOWLEDGEMENTS

This invention was supported in part by grants from R01-HL 24136, NRHNHLBI and RO1-HLA43762. The U.S. Government may have rights in this invention.

INTRODUCTION

TECHNICAL FIELD

The present invention provides for methods of identifying compounds for treating medical conditions related to the inappropriate production of mucin, as well as compounds and methods for treating such conditions, such as Pseudomonas infections of cystic fibrosis patients.

BACKGROUND

Pseudomonas aeruginosa ("PA") often resides in the respiratory tract of patients with cystic fibrosis ("CF"). It is a virulent pathogen. Once such patients acquire a Pseudomonas aeruginosa infection, the infection is rarely, if ever, eradicated. Infection leads to progressive pulmonary deterioration and ultimately to death. Although the extraordinary predisposition of CF patients to colonization and infection with P. aeruginosa has been recognized for many years, a satisfactory explanation for this phenomenon remains elusive. Cystic fibrosis commonly occurs among Caucasians (approximately 1 in

2,000 newborns). The mode of inheritance is autosomal recessive and about 5% of the normal population carries the defective gene. Patients suffer from airway obstruction due to mucus, which is the leading case of death in CF patients. Unfortunately, current treatments fail to result in the complete eradication or prevention of Pseudomonas aeruginosa infections in cystic fibrosis patients or treatment of the overproduction of mucus. Antimicrobial therapy using antibiotic therapeutic protocols unfortunately have complications. Patients with CF dispose of antimicrobial agents more rapidly than do normal individuals, which results higher doses than those normally recommended. Strains of Pseudomonas aeruginosa also dissociate into multiple serotypic forms, which often have different antimicrobial susceptibility patterns.

Since PA infection is chronic and the infecting strains of Pseudomonas aeruginosa are rarely eradicated, resistance to multiple antimicrobial agents frequently develops to thwart antibiotic therapies. Therapeutic levels of antimicrobial agents in sputum are difficult to achieve because of poor penetration and inactivation. Mucoid exopolysaccharides of mucoid PA strains additionally present a barrier to penetration of some antibiotics. Finally, allergy to certain antibiotics (such as betalactam) precludes antibiotic therapy with some patients.

Anti- inflammatory agents have also been tried in the therapy of Pseudomonas aeruginosa infections in CF patients because it has been postulated that host-mediated inflammation may be responsible for a large part of the pulmonary damage in the CF lung. Efforts have been made to dampen the inflammatory response in the CF lung by use of a systemic steroidal anti- inflammatory therapy, such as prednisone. Unfortunately, prednisone therapy carries substantial risks including growth retardation, glucose intolerance and development of cataracts. Preliminary studies are underway to use non-steroidal anti- inflammatory agents.

Consequently, there is a need to provide therapies for reducing mucus production in CF patients and to reduce the effect of Pseudomonas aeruginosa infections on CF patients.

SUMMARY OF THE INVENTION The present invention now recognizes for the first time an important role of mucomones, such as bacterial mucomones, in mucin production by epithelial cells. Mucomones are molecules that stimulate or induce mucin production by binding to epithelial cells, as described herein.

For example, one embodiment of the invention provides for methods for identifying compounds useful as therapeutics, such as CF therapeutics, by: 1) screening compounds using epithelial cells that express mucins, such as HM3 and NCIH292 cells,

2) determining a change in mucin production in the presence of the test compound compared to the absence of the test compound, and 3) selecting compounds useful for treating medical conditions related to mucin production, such as CF or respiratory microorganism infections. The present invention also recognizes for the first time that induction of mucin in epithelial cells is linked to activation of epithelial cell tyrosine kinases by mucomones, especially bacterial mucomones. Binding of mucomones to epithelial cells causes activation of tyrosine kinases that regulate the induction of mucin production, particularly MUC-2 and MUC-5 production. Mucomone induced mucin production can be blocked by inhibiting binding of mucomones to an epithelial cell, inhibiting tyrosine kinase activity, and inhibiting any crucial step in the signal transduction cascade between binding of a mucomone and mucin gene activation, including inhibiting transcription factors that interact with mucin promoters. Such inhibitors can be used in treatments of medical conditions related to the inappropriate expression of mucin, such as, for example, cystic fibrosis. The invention includes a number of methods and composition related to drug discovery and therapeutic treatments.

Another embodiment of the invention provides for methods for inhibiting the production of mucins by cells, such as CF cells, by applying an effective amount of an inhibitor of mucomone induced mucin production to the cells. Other methods, compounds and compositions are more fully described herein.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 A-H shows an in situ hybridization analysis of MUC-2 mRNA expression in normal and CF human bronchial explants. In situ hybridizations were performed with the HAM1 anti-sense probe recognizing human MUC-2 mRNA. MUC-2 mRNA expression was shown in P. aeruginosa non-treated- and treated- bronchial explant epithelia from non-CF individuals (A and B) and CF individuals (C and D), in P. aeruginosa non-treated- and treated- bronchial explant submucosal glands from non-CF individuals (E and F) and CF individuals (G and H). Arrows in A, B, C and D show airway surface epithelial cells. In these experiments both non- CF and CF bronchial explants were exposed for 6 hours to P. aeruginosa conditioned medium. Similar results were observed in bronchial explants from four CF and four non-CF individuals. L: airway lumen. SG: submucosal glands.

FIG. 2A shows RPA analysis of MUC-2 mRNA expression in human MUC-2- expressing epithelial cell lines. Upregulation of MUC-2 mRNA by P. aeruginosa conditioned medium occurred in NCIH 292, HM3 cells. Cells were treated with P. aeruginosa conditioned medium (PA) or vehicle (CON) for 6 hours prior to cell lysis and RNA extraction. The results are typical of 3 separate experiments.

FIG. 2B shows induction of MUC-2 gene transcription by P. aeruginosa. A 2.8 Kb DNA fragment of the 5 '-flanking region of human MUC-2 gene cloned into a luciferase reporter gene ( p-28641uc) was transfected into NCIH 292, HM3 and CFTE29O cells. Luciferase activity was then assessed in both P aeruginosa treated- and non- treated cells, as described herein. Induction by P. aeruginosa was detected in all cell lines. Transfections were carried out in triplicate, and the results are typical of 5 separate experiments. The luciferase activity was normalized with respect to β- galactosidase activity.

FIG. 3A, B, C, D show identification of DNA cis-elements required for MUC-2 upregulation by P. aeruginosa. Luciferase activity of two deletion mutants of MUC-2 promoter fused to the luciferase reporter gene was assayed in various epithelial cell lines. (A) p-28641uc and p-731uc were transfected into NCIH 292, HM3 and CFTE29O cells. Promoter activity was then assessed in P. aeruginosa-treated- and non-treated- cells. (B) HM3 cells were transfected with p-731uc, p-1141uc, p- 3431uc, p-6211uc, p-13081uc and p-28641uc. (C) HM3 cells were transfected with p-

731uc, p-911uc, p-1021uc, p-1141uc and p-3431uc. (D) HM3 cells were transfected with p-13081uc, p-16271uc and p-28641uc. Luciferase activity was then measured in HM3 cells treated with or without P. aeruginosa conditioned medium.

FIG. 4A and B show the affect of mutant P. aeruginosa conditioned medium on MUC-2 transcription. HM3 cells were transfected with p-28641uc. (A) After 42 hours the cells were exposed to conditioned medium from 4 defined mutants of the PAO strain: PAO1 (wild-type), PAO-R1, PAO-pmm, PAO/NP, AK1152. After 6 hours the cells were harvested for luciferase activity measurement. (B) PA LPS (lipopolysaccharide) at 25 μg/ml induced mucin production in HM3 cells compared to the control. All transfections were carried out in triplicate. Results are typical of 4 separate experiments. Luciferase activity was normalized with respect to β- galactosidase. Controls contain no added mucomone.

FIG. 5 A and B show upregulation of MUC-2 transcriptional activity by various components of PA or bacterial conditioned media. (A) Conditioned media from PAOl and PAO-pmm strains were compared to LPS isolated from the PAO- pmm strain (20 μg/ml). (B) Conditioned media from PAOl strain was compared to LPS isolated from PAO-pmm, E. coli, 055-B5 S.e (Salmonella enteritidis) and K.p.

(Klebsiella pneumonia). FIG. 5C shows upregulation of MUC-2 transcriptional activity using samples normalized for LPS concentration. FIG. 5D shows upregulation of MUC-2 transcriptional activity by LPS compared to Lipid A. Luciferase activity was normalized with respect to β-galactosidase. Controls contain no added mucomone.

FIG. 6A and B show induction of MUC-2 by after various treatments or molecular weight fractionation. (A) Conditioned media from PAOl wildtype strain was subjected to boiling, DNAse or protease treatment. (B) Conditioned media from PAOl wildtype strain was subjected to fractionation on a filtron spin column. FIG. 7A and B show inhibition of P. aeruginosa induced upregulation of

MUC-2 transcriptional activity by tyrosine kinase inhibitors genistein and AG126. (A) HM3 cells were pretreated with genistein (100 μg/ml) for 2 hours and then exposed to P. aeruginosa conditioned medium (PAO) or vehicle (Control) for 6 hours before a reporter gene assay. HM3 cells were transfected with p-28641uc. After 40 hours the cells were pretreated with genistein for 2 hours and then exposed to P. aeruginosa conditioned medium for 6 hours. (B) HM3 cells were pretreated with AG126 (50 μg/ml) for 2 hours and then exposed to P. aeruginosa conditioned medium (PAO) or vehicle (Control) for 6 hours before a reporter gene assay. HM3 cells were transfected with p-28641uc. After 40 hours the cells were pretreated with genistein for 2 hours and then exposed to P. aeruginosa conditioned medium or PA LPS (20 μg/ml) for 6 hours. Luciferase activity were assessed as described herein. FIG 8 shows inhibition of P. aeruginosa induced upregulation of MUC-2 transcriptional activity by PP1.

DESCRIPTION OF SPECIFIC EMBODIMENTS DEFINITIONS

Production, when used in the context of describing a cellular process, typically refers to a cellular process or processes involved in maintaining the steady state level of a molecule, such as a mucin, for example MUC-2 or -5. Consequently, production includes the cellular processes of gene activation or induction, transcription, protein synthesis, protein translation, and secretion. Production also refers to cellular and extracellular process responsible for maintaining steady state levels of a molecule, such degradation pathways and extracellular structural elements that anchor molecules to cells or in an extracellular matrix.

Secretion, when used in the context of describing a cellular process, typically refers to a cellular process or processes of transporting a molecule from inside the cell to an extracellular location.

Protein synthesis, when used in the context of describing a cellular process, typically refers to a cellular process or processes involved in making a molecule, such as a mucin, for example MUC-2 or -5. Protein synthesis may involve transport of the molecule within the cell making the molecule. The term protein synthesis, however, does not include reference to the term secretion described herein.

Mucomones typically refer to molecules that induce mucin production and include proteins, amino acids, simple sugars, complex sugars, LPSs (lipopolysaccharides), glycoproteins and the like. Structurally, LPS consists of a variable polysaccharide domain covalently linked to an invariable, diglucosamine- based, acylated phospholipid, lipid A. Lipid A is a key factor in MUC-2 stimulation and lipid A from diverse bacterial species is potent in this respect. Often mucomone antagonists and LPS antagonists will be glucosamine based precursors and analogs of bacteria LPSs, such as analogs of E. Coli Lipid A, or Lipid X. Diglucosamine-based analogs are also potent LPS inhibitors. Mucomones may interact with epithelial cell receptors either alone or with a mucomone binding protein. Such mucomone binding proteins or analogs thereof can be used to inhibit mucomone induction of mucins. For instance, bacterial permeability-increasing protein (BPI) binds endotoxin and can neutralize LPS.

Mucin or mucins typically refer to the structural components of mucus from epithelial cells that protect tissues, such as the respiratory and reproductive tracts. Typically, mucins form extremely large oligomers through linkage of glycoprotein monomers using disulfide bonds. Usually, such glycoproteins are large > 100,000 daltons or > 250,000 daltons and usually consist of approximately 75% carbohydrate and 25% protein. Mucins include proteins encoded by the MUC genes described herein. Altered mucins also occur in pathological conditions, such as inflammatory diseases.

Lipopolysaccharide ("LPS"), a complex glycolipid, is the major component of the outermost membrane of Gram-negative bacteria. LPSs can provide a potent and pleiotropic stimulus to immune and epithelial cells, both in vivo and in vitro, and has been implicated in the clinical syndrome of gram-negative bacterial septic shock.

INTRODUCTION

Until the present invention it was not recognized that PA could directly induce mucin production in epithelial cells, particularly airway cells of CF patients. It was not established whether mucin production in CF cells was due to a mutation in the CF cells; whether PA infections stimulated a first cell type in airway tissue that would, in turn, induce mucin production in a second cell type in the airway tissue; and whether direct contact of PA cells to airway cells contributed to induction of mucin production.

Before the invention described herein, airway obstruction due to inappropriate mucin production was postulated to be related to changes in salt and water balance across airway that consequently altered mucin hydration in the airway. The increase in mucin synthesis observed in CF tissues compared to normal tissues suggested that CF abnormalities, such as mutation, caused mucin over production. The present invention now recognizes for the first time an important role of mucomones, such as bacterial mucomones, in mucin production by epithelial cells. Mucomones are molecules that stimulate or induce mucin production by binding to epithelial cells, as described herein. In particular, mucomones can be released or secreted by microorganisms, such as PA, to induce mucin production by epithelial cells, which benefits the invading bacteria by providing a substrate for growth, protection and attachment.

In the case of CF, airway cells produce abnormal salt secretions rich in sodium chloride compared to normal airway tissues. The salt rich airway fluids increase susceptibility of the tissue to infection by bacteria, such as PA, due to the reduced ability of endogenous bactericidal agents to ward off sustained infection of the airway tissue at high salt concentrations. Consequently, chronic PA infections develop in CF airway tissues defective in salt transport. PA residing in the airway tissue releases mucomones to induce mucin production of airway epithelial cells, which can potentially occur within minutes to hours. The increased mucin production facilitates the growth and maintenance of PA, permitting virulent, chronic PA infections and allowing PA to reside in the mucus even after the cells have lost their pili. The present invention also recognizes for the first time that induction of mucin in epithelial cells is linked to activation of epithelial cell tyrosine kinases by mucomones, especially bacterial mucomones. Binding of mucomones to epithelial cells causes activation of tyrosine kinases that regulate the induction of mucin production, particularly MUC-2 and MUC-5 production. PA mucomone induced mucin production can be blocked by tyrosine kinase inhibitors and such compounds can be used in treatments of medical conditions related to the inappropriate expression of mucin.

As a non-limiting introduction to the breadth of the invention, the invention provides at least five categories of useful methods, compounds and compositions:

1) methods directed to identifying therapeutic compounds for treating medical conditions related to mucin production, such as CF, bronchitis, asthma and respiratory infections by microorganisms,

2) methods directed to reducing mucin production by epithelial cells, particularly in medical conditions related to the inappropriate production of mucin, 3) methods directed to the treatment of CF using tyrosine kinase inhibitors, 4) compositions related to the 5' flanking region of the MUC-2 gene responsible for MUC-2 induction by mucomones,

5) compositions related to the methods described in 1-3.

For example, one embodiment of the invention provides for methods for identifying compounds useful as therapeutics, such as CF therapeutics, by:

1) screening compounds using epithelial cells that express mucins, such as HM3 and NCIH292 cells,

2) determining a change in mucin production in the presence of the test compound compared to the absence of the test compound, and 3) selecting compounds useful for treating medical conditions related to mucin production, such as CF or respiratory microorganism infections. Another embodiment of the invention provides for methods for inhibiting the production of mucins by cells, such as CF cells, by applying an effective amount of an inhibitor of mucomone induced mucin production to the cells. Such inhibitors include, but are not limited to, inhibitors of mucomone binding to epithelial cells, tyrosine kinase inhibitors, and other inhibitors. Combinations of the above methods, compounds and composition are also contemplated. Other methods, compounds and compositions are more fully described herein.

METHODS FOR SCREENING FOR INHIBITORS OF MUCOMONE INDUCED MUCIN

PRODUCTION

The present invention recognizes that epithelial cells involved in mucin production would be useful components of in vitro or in vivo methods for identifying compounds that are useful as therapeutics, such as compounds that inhibit mucomone induced mucin production. Although not essential for practicing the invention, such identifying methods can involve screening assay systems that permit high throughput automated screening. A method of identifying compounds useful for therapeutics comprises: 1) contacting a compound with cells that induce or upregulate mucin production in the presence of a mucomone, and 2) detecting a change in mucin production or mucin gene activation of the cells.

Such a method includes the use of mucomones that can induce mucin production in the cell type employed in the method. Examples of such mucomones include, but are not limited to, PA conditioned media, E. coli conditioned media, LPS from PA, E. coli and other gram negative bacteria, Lipid A from PA, E. coli and other gram negative bacteria, and other mucomones known in the art and described herein. Such a method can be used to screen any of a number of compounds for the ability to inhibit mucomone induced mucin products, including, but not limited to, tyrosine kinase inhibitors, inhibitors of mucomone binding, and other types of compounds.

A number of methods may be used to detect mucin production or mucin gene activation indirectly or directly. For instance, mucin production can be directly detected by measuring protein synthesis, and mucin gene activation can be directly detected by measuring transcription, and RNA levels using the appropriate labels or probes for such assays. For example, an RNase protection assay (RPA), as described herein and known in the art, can be used to study the effect of a compound on the transcriptional activity of a MUC gene by detecting changes in MUC-1 (Gendler, S. J,

JCB. 1990, 265: 15288-93), MUC-2 (Gum J.R. Jr. et al., JBC. 1994, 269(4): 2440-46, Velcich A. et al., JBC 1997, 272:7968-7976), MUC-3 (Gum J.R. BBRC 1990, 171:407-15, MUC-4 (Aubert J.P. et al. Am J Respir. Cell. Mol. Biol. 1991, 5: 178- 85), MUC-5 (Rose M.C. et al. , JBC, 1989, 264:8193-99), MUC-6 (Toribara, N.W. et al., JCB. 1993, 268:5879-95), MUC-7 (Reddy, M.S. Biochemical J. 1992,

287:639-43) and MUC-8 (Sachdev, G. , Biochemical J. , 1994, 300 (pt 2): 295-8) RNA levels. Mucin probes can be selected from among the known nucleotide sequences for mucin, such as for human mucins and can be used for RPA or traditional hybridization techniques, such as, for example, performing northern blots. Oligonucleotides comprising known mucin nucleotide sequences can also be used to measure RNA levels by quantitative PCR. The methods and sequences of the above- referenced publications are herein incorporated by reference. Direct detection methods may also include antibody assays for mucins to assess regulation at the protein level, such as the use of A10G5 monoclonal antibody to detect MUC-2. It should be remembered that mucin secretion is not a desirable method for detecting mucin production because mucin secretion from the cells, especially mucin secretion within 2-3 hours in response to a mucomone, is not indicative of gene upregulation. Such rapid secretion measurements provide a better measurement of release of stored mucin from the cell and not synthesis of a large molecule comprised both of protein and sugar, which often has a total molecular weight of 1,000,000 daltons.

A reporter gene assay can be used to indirectly determine the amount of mucin gene activation. For example, luciferase or CAT assays can be used in conjunction with a 5' or 3' flanking region or intron region containing mucin transcriptional control elements, such as promoters, response elements or enhancers, that are operably associated with a mucin gene, and are known to participate in mucin gene activation. Mucin transcriptional control elements can be readily employed in a reporter gene construct using common cloning techniques. For example, 2.8 Kb of the 5 '-non-coding region includes the transcriptional response elements for the MUC- 2 gene. These transcriptional response elements are described and characterized herein for the first time in Example 5. A response element is a transcriptional control element that is sufficient to effect mucomone induced upregulation of transcriptional activity of a mucin gene. For example, a MUC-2 response element is a region of the

2.8 Kb noncoding region of the MUC-2 gene that is sufficient to effect upregulation of MUC-2 transcriptional activity in response to mucomone stimulation. Transcriptional activity is upregulated when mucomone stimulation results in at least 2-fold greater transcriptional activity than that observed in unstimulated cells. Reporter gene constructs can be prepared by operatively linking a reporter gene with at least one transcriptional regulatory element of a mucin gene. Such constructs typically include a promoter and a response element. At least one of the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the mucomone receptor, whereby activity of the mucomone and the receptor can be monitored via transcription of the reporter genes.

Reporter gene constructs can be used in a method for identifying inhibitors of MUC-2 upregulation in response to mucomone stimulation. Such constructs can include a reporter gene operably linked to fragments of the 2.8 Kb 5 '-non-coding region of the MUC-2 gene that when stimulated by a mucomone can result in at least a 2-fold increase in MUC-2 transcriptional activity, preferably a 5-fold increase or greater, more preferably a 10-fold increase or greater and most preferably a 50-fold increase or greater. Fragments of the non-coding region yielding increases in activity having a numerical value falling within the ranges described above or in excess of 50- fold are also included among the report constructs of the invention. For example, a fragment of the 5 '-non-coding region of MUC-2 yielding an increase in activity of 3- fold, 4-fold, 6-fold, 7-fold, 8-fold, 9-fold, 11-fold, 20-fold, 30-fold, 40-fold, 50-fold, 90-fold, 200-fold, 2000-fold, etc. , or any fraction thereof is also included in the invention. Examples of preferred 5'-non-coding region fragments include, but are not limited to, a fragment comprising base pairs - 91 to 0, -102 to 0, -114 to 0, -343 to 0, -621 to 0, -1308 to 0, -2864 to 0, -2864 to -73, -2864 to -1308, -2864 to -343, - 2864 to + 14, -1627 to +14, -1308 to + 14, -114 to + 14, -102 to + 14, -91 to + 14, -91 to -73, -102 to -73, -114 to -73, -343 to -73, -1627 to -73, and -1627 to -1308.

Most preferred are -1627 to -1308, -2864 to -1308, -2864 to -73, -114 to -73, -91 to -73, -2864 to + 14, and -1627 to + 14. Such fragments can be operably linked to the MUC-2 promoter or to a heterologous promoter, such as, for example, the thymidine kinase (tk) promoter to render such heterologous promoters responsive to mucomone stimulation.

Constructs may contain additional transcriptional regulatory elements or other sequences, that are not necessarily regulated by the mucomone receptor or mucomone, but are selected for their ability to reduce background level transcription or to amplify the transduced signal and to thereby increase the sensitivity and reliability of the assay.

A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable. The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. Many reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art.

Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282:864-869), luciferase, and other enzyme detection systems, such as beta-galactosidase, firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1 :4154-4158; Baldwin et al. (1984), Biochemistry 23:3663-3667), alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182:231-238, Hall et al. (1983) 7. Mol. Appl. Gen. 2: 101), and GFP (green fluorescent protein).

Any cell, especially an epithelial cell, that produces mucin can be used as a component of the assays described herein. If it is not known whether a cell produces mucin in response to a mucomone, mucomone induced mucin production can be easily determined using the assay described herein. Cells that test positive in such assays can then be used in to identify therapeutic compounds. Often it will be desired to select cells from different epithelial tissues to aid in identifying a tissue specific inhibitor. Although most epithelial cells will have mucomone inducible mucin production, the regulation of mucin production can vary from tissue to tissue as well as depend on the type of mucomone. Thus, if an inhibitor of colon or airway mucin production is desired it will often be preferable to use colon or airway derived mucin producing cells in identifying such an inhibitor. Selection of such cells is not restricted to this manner of selection and it will often be the case that some cell lines will be preferred irrespective of the tissue source, such as HM3 and NCIH292, because the cells respond well to mucomones, are convenient to culture, and produce large reporter gene signals in the presence of a mucomone compared to in the absence of a mucomone, i.e. cells that have a low level of mucin production in the absence of a mucomone. Preferably, NCIH292, HM3, Hela, CFTE29O, NCIH292 and 16Lu cells are used in the identification methods and most preferably HM3 and NCIH292 are used. Cells from a variety of epithelial tissues can be used as well, such as airway secretory cells, ciliated cells, epithelial cells of the respiratory tract, kidney secretory cells and reproductive tract epithelial cells. Once a particular cell type has been selected various direct and indirect detection assays on mucin production can be used, as described herein.

For example, a compound can be tested indirectly for its ability to inhibit mucin production by epithelial cells, such as HM3 or NCIH292 cells, by measuring a change in the production of a reporter gene product, using the 5' flanking region of MUC-2 or a functional fragment thereof operatively linked to reporter gene, such as luciferase, in the presence and absence (control) of the compound being tested. It will be recognized that such controls can be used in any of the assays described herein and that other controls can be readily interchanged to achieve specific detection, such as using cells without functional 5' flanking regions of the mucin gene operatively linked to a functional reporter gene, by not adding a mucomone or by blocking the action of an added inhibitor. Any of a number of compound types can be tested for inhibition of mucomone induced mucin production, including, but not limited to, inhibitors of mucomone binding, tyrosine kinase inhibitors, inhibitors of other components of the signal transduction pathway, inhibitors of transcription factors that interact with mucin promoters, and other compound types. Inhibitors of mucomone binding can include mucomone receptor antagonists such as, for example, LPS antagonists. Inhibitors include small molecule organic compounds, simple or complex sugars, peptides, dominant negative mutants of proteins involved in mucomone binding, signal transduction, and transcription, and other compounds. LPS antagonists can include Lipid A analogs, lipid X analogs and other diglucosamine analogs, and other compound types. Inhibitors of transcription factors include, for example, inhibitors of NF-κB, API, AP2, and C/EBP. For example, CAPE (CALBIOCHEM Catalog No. 211200), an NF-κB inhibitor, can inhibit mucomone induced mucin production. Signal transduction inhibitors include inhibitors of sarc, ras, rafl, mekl/2, erkl/2, and rsk. Inhibitors of sarc that are effective inhibitors of mucomone induced mucin production include PP1 and a dominant negative mutant of sarc. Inhibitors of ras that are effective inhibitors of mucomone induced mucin production include a dominant negative mutant of ras. Inhibitors of mekl/2 that are effective inhibitors of mucomone induced mucin production include a PD98059 and a dominant negative mutant of mekl/2. Inhibitors of erkl/2 that are effective inhibitors of mucomone production include AG126. Inhibitors of rsk that are effective inhibitors of mucomone production include a dominant negative mutant of rsk.

Once it is determined that a compound inhibits mucomone induction of mucin production, useful inhibitors can be selected. Selection criteria are usually based on the extent of modulation produced by the tested compound. Compounds will usually be selected on their ability to inhibit or reduce such cellular processes. Such compounds will be useful for the treatment of medical conditions caused by inappropriate mucin production or secretion. Such compounds will also be useful in treating CF or bronchitis. Typically, compounds that inhibit mucomone induced mucin production by at least 10%, preferably by at least 30% and more preferably by at least 70% compared to control mucomone induced mucin production will be selected as useful compounds. Such percent inhibition criteria can be applied to other measurements in assays described herein, such as detection of reporter gene assays and RPA assays.

More specific selection criteria can be advantageously used to identify compounds that more specifically modulate a cellular process. It will be recognized that the affinity of the compound being tested for its receptor can often dictate the specificity of the compound, such as a compounds with an affinity for a cellular tyrosine kinase, LPS, LPS binding protein, or an LPS receptor. Consequently, it is desirable to select compounds that bind to receptors or modulate function with a high apparent or actual affinity. In the case of tyrosine kinase inhibitors, affinities of 10³ or less are typical under physiological conditions, 10⁵ or less are preferred, and 10"⁶ are more preferred. To achieve such desired results, new compounds or known tyrosine kinase inhibitors or LPS antagonists can be synthesized and screened at predetermined concentrations. Typically, compound concentrations will be 500 micromolar or less, preferably 50 micromolar or less, more preferably 10 micromolar or less, even more preferably 1 micromolar or less and most preferably 0.1 micromolar or less. The percentage inhibition criteria discussed herein can be applied to these concentration selection criteria.

Many different types of tyrosine kinase inhibitors (TKIs) can be tested for inhibitory activity of mucomone induced mucin production. Such TKIs are described herein and known in the art. Tyrosine kinases are typically classified as either receptor tyrosine kinases or non-receptor tyrosine kinases. Some TKI's inhibit both types of tyrosine kinases and some TKI's, such as, for example, pyrazolopyrimidine (PP1) are specific for non-receptor tyrosine kinases. Often, the TKI will either bind to the ATP or tyrosine site on the tyrosine kinase. Many chemical structures that mimic ATP or tyrosine binding to the tyrosine kinase can be used. For molecules that bind to the ATP site (ATP analogs), it is desirable to test compounds that comprise at least an adenosine base or adenosine base derivative. For molecules that bind to the tyrosine site (tyrosine analog), it is desirable to test compounds that comprise at least an substituted benzene ring, such as (HO)n-benzene-X, where X can be a stable moiety of 1 to 30 atoms, usually comprised of N, S, C, O, or H atoms. TKI's that are identified as inhibitors of mucomone induced production can be used for the other methods described herein. Identifying useful compounds for modulating cellular processes, such as mucin production, can involve both in vitro and in vivo screening assays. In vivo assays and measurements described herein can be used to further select for useful compounds. For example, a compound that has been identified in an in vitro assay can be further assayed in vivo, for example, by applying it to an airway of a mammal. Alternatively, the in vivo assay can be used to identify compounds alone or prior to an in vitro assay.

PHARMACEUTICAL COMPOSITIONS

The present invention also includes pharmaceutical compositions that can be used in the methods described herein and known in the art. Typically, the compositions will comprise an inhibitor of mucomone induced mucin production, such as, for example, a tyrosine kinase inhibitor or an LPS antagonist, as the active ingredient. Compositions are often matched with a particular mode of delivery as described herein and known in the art. The precise type and amount of compound for use in the present compositions will vary depending, for example, on the specific drug chosen, the dosage form thereof, i.e. , standard versus sustained release, the condition for which the drug is administered, and the size and kind of the mammal treated. Compounds of the invention, as described herein, can be selected to modulate cellular and physiological processes, such as, for example, to prevent an increase in mucomone induced mucin production or to decrease mucomone induced mucin production. Preferably, such compounds include inhibitors which provide a reduction in or prevention of mucomone induced mucin production when used in an amount sufficient to provide a concentration of 1X10^"3M or less, preferably an amount of about 1X10^"8M to about lXlO^M, and more preferably about 1X10^"7M to about 1X10^"5M in the extracellular fluids or treated tissue of the target epithelium.

A variety of tyrosine kinases can be used for the compositions and methods described herein. For example, different types of TKIs can be used such as oxinole derivatives (such as those disclosed in U.S., 5,488,057 issued January 30, 1996 and WO 95/01349), bicyclic aryls (such as those disclosed in 5,326,905 issued July 5, 1994), heteroaryl (such as those disclosed in U.S. 5,409,930), carbocyclic and heterocyclic aryls, benzylphosphonics (such as those disclosed in U.S. 5,326,905), genisteins, diaminophthalimdies (such as disclosed in U.S. 5,491, 144 issued February

13, 1996) and pyrazolopyrimidines. The compounds and formulae of the patent documents referred to above are herein incoφorated by reference. Specific compounds may also be used, such as those disclosed by Calbiochem's 1995 and 1997 Catalogs as follows with catalog numbers in parentheses: Aminogenistein (155100), Baicalein (196322), Daidzein (251600) Emodin (324694), Erbstatin Analog

(324930), Genistein (345834), Herbimycin A (375670), HNMPA-(AM)s (397100), Lavendustin A (428150), Lavendustin B (428160), Lavendustin C (234460), Lavendustin C Methyl Ester (234455), Psi-tectorigenin (540100), Quercetin (55160), ST 638 (567790), Tyrosine-Specific Protein Kinase inhibitor (657015), Tyrphostin A 1 (658390), Tyrphostin A B (658475), Tyrphostin A 9 (658425), Tyrphostin A 23

(658395), Tyrphostin A 25 (658400), Tyrphostin A 46 (658430), Tyrphostin A 47 (RG 50864) (658405), Tyrphostin A 48 (658448), Tyrphostin A 51 (658410), Tyrphostin A 63 (658450), Tyrphostin AG 126 (658452), Tyrphostin AG 370 (658454), Tyrphostin AG 879 (658480), Tyrphostin AG 1288 (658510), Tyrphostin AG 1295 (658550), Tyrphostin AG 1296 (658551), Tyrphostin AG 1478 (658552),

Tyrphostin B42 (658401), Tyrphostin B 44 (*) (658402), Tyrphostin B 46 (658404), Tyrphostin B 48 (658407), Tyrphostin B 50 (+) (658409), Tyrphostin B56 (658415), RG-13022 (554725), RG-14620 (554740), Bis-Tyrphostin (658418) and PP1 (529572). "Genistein" as used herein refers to the isoflavone compound as described in the Merck Index (7th Edition, 1960, p 474), or a derivative or analogue thereof that functions as a tyrosine kinase inhibitor. The ability of a genistein analogue to inhibit tyrosine kinase, and particularly, to inhibit pp60^rrc, may be readily determined by methods described herein and known to those of skill in the art, for example in Akiyama et al. (1987), herein incorporated by reference. The present inventions thus encompasses the use of any genistein derivative that has a significant (i.e. , consistently above background) inhibitory effect on tyrosine kinases. Also encompassed are genistein-derived compounds that may be formed upon ingestion. For example, genistein glucuronides, as for other glucuronides, are hydrolyzed by β- glucuronidases in the large bowel, and the unconjugated forms reabsorbed. In this environment the genistein compounds may undergo other chemical modifications, such as reduction to isoflavans or B-ring-opened forms, all such compounds are intended to fall within the scope of the present invention. To establish as an effective concentration of genistein concentration at the tissue, a daily intake of at least about 2-200 mg of genistein would be required for a human patient. More preferably, such doses would be between about 5-50 mg, about 10-50 mg, and most preferably, about 20-50 mg per person per day. Compositions for aerosol and enteral, especially oral, and for parenteral administration are especially preferred. The compositions comprise an inhibitor of mucomone induced mucin production alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the inhibitor depends upon the disease to be treated and upon the species, its age, weight and individual condition, and also upon the mode of administration.

Preferred is a pharmaceutical composition suitable for administration to a warm-blooded animal, especially a human, suffering from a medical condition described herein, for example CF, comprising a compound described herein, or a salt thereof when salt-forming groups are present, in an amount effective for the inhibition of the protein kinase, together with at least one pharmaceutically acceptable carrier.

Preferably the compounds of the invention are formulated for pulmonary administration. One such method of administration involves the aerosol ization of a solution containing preferably an aqueous-soluble compound of the invention.

Aerosol compositions can alternatively include the active compound packaged in reverse micelles or liposomes. Pharmaceutical compositions suitable for such a method of administration can additionally include aerosol propellants and a surfactant. Examples of small compounds administered by this method can be found in U.S. Patent Nos. 5,364,615, 5,292,499, and 5,238,683, which are herein incorporated by reference. Both phospholipid and nonconventional liposomes are rapidly becoming accepted as pharmaceutical agents which improve the therapeutic value of a wide variety of compounds (Cancer Res. 43:4730 (1983)) and can be applied to tyrosine kinase inhibitors. Compounds with poor solubility in aqueous systems require formulation by using solubilizing agents such as ionic surfactants, cholates, polyethylene glycol (PEG), ethanol, or other agents which may have undesirable effects when used for inhalation. In addition, a treatment requiring successful delivery into alveoli of the lower pulmonary region may preclude from the formulation the use of certain irritants such as chlorofluorocarbons and should involve a minimum number of required doses. Alternatively, to avoid such limitations, liposomes or hydrophobic particles can be used. An inhalation formulation providing for a sustained release of such a compound using aerosol droplet particles approximately 1-2.1 μ in size, preferably less than 1 μ, would satisfy these special needs.

Small particle aerosol liposomes and liposome-drug combinations for medical use have been previously disclosed (EP 87309854.5), which herein incorporated by reference. Conventional liposomal formulations sometimes have an uncontrollable and fast release rate and frequently have larger particle sizes than are useful for directing a drug to alveoli. However, nonconventional liposomes, which are formed solely by cholesterol and cholesterol derivatives, or alternatively amphipathic lipid components, have been used successfully with controllable sustained release, improved solubility, high encapsulation, absence of need for multiple dosing, and extended stability. Suitable liposomal formulations for sustained release of such compounds include sodium cholesterol sulfate: cholesterol: compound % molar ratios of 55:40:5; 50:40: 10; 53:37:9, and most preferably 50:40: 10 (U.S. Patent No. 5,049,389), which is hereby incorporated by reference.

The pharmaceutical compositions comprise from approximately 5% to approximately 95% active ingredient, dosage forms in single dose form preferably comprising from approximately 20% to approximately 90% active ingredient and dosage forms that are not in single dose form preferably comprising from approximately 5% to approximately 20% active ingredient Unit dose forms, such as dragees, tablets or capsules, comprise from approximately 0.05 g to approximately 1.0 g of active ingredient. The pharmaceutical compositions of this invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilizing processes. For example, pharmaceutical compositions for oral use can be obtained by combining the active ingredient with one or more solid carriers, optionally granulating a resulting mixture, and, if desired, processing the mixture or granules, if appropriate with the addition of additional excipients, to form tablets or dragee cores. Suitable carriers include fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients include flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

Dragee cores can be provided with suitable, optionally enteric, coatings, there being used inter alia concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the production of enteric coatings, solutions of suitable cellulose preparations, such as acetyl-cellulose phthalate or hydroxypropylmethylcellulose phthalate. Colourings or pigments may be added to the tablets or dragee coatings, for example for identification purposes or to indicate different doses of active ingredient.

Orally administrable pharmaceutical compositions also include dry-filled capsules consisting of gelatin, and also soft, scaled capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The dry-filled capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as corn starch, binders and/or gildants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil, liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxethylen-sorbitan fatty acid ester type, may also be added.

Other oral dosage forms are, for example, syrups prepared in customary manner which comprise the active ingredient, for example, in suspended form and in a concentration of about 5% to 20%, preferably about 10%, or in a similar concentration that provides a suitable single dose, for example, when administered in measures of 5 or 10 ml. Also suitable are, for example, powdered or liquid concentrates for the preparation of shakes, for example in milk. Such concentrates may also be packaged in single dose quantifies. Suitable rectally administrable pharmaceutical compositions are, for example, suppositions that consist of a combination of the active ingredient and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.

For parenteral administration compositions include suitable aqueous solutions of an active ingredient in water-soluble form, for example in the form of a water- soluble salt, or aqueous injection suspensions that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers. The active ingredient, optionally together with excipients, can also be in the form of a lyophilisate and can be made into a solution prior to parenteral administration by the addition of suitable solvents. Solutions such as those that are used, for example, for parenteral administration can also be used as infusion solutions.

The invention relates also to a method of treating pathological conditions associated with mucin production and responsive to the inhibition of mucin production. The compounds of this invention can be administered prophylactically or therapeutically, preferably in an amount effective against the said diseases, to a warm-blooded animal, for example a human, requiring such treatment, the compounds preferably being used in the form of pharmaceutical compositions. In the case of an individual having a body weight of about 70 kg the daily dose administered is from approximately 1 mg to approximately 5000 mg, preferably from approximately 20 mg to approximately 500 mg, of a compound of this invention. METHODS FOR TREATING CF AND OTHER MEDICAL CONDITIONS RELATED TO MUCOMONE INDUCED MUCIN PRODUCTION

The present invention recognizes that mucomones can directly induce mucin production by epithelial cells, such as airway cells. Consequently, the methods of the invention are directed to inhibiting inappropriate mucin production using compounds, such as, for example, small molecule organic compounds, simple or complex sugars, peptides, other inhibitors of signal transduction components, transcription factors, and mucomone binding, and other compounds as described herein and are known in the art that have been identified as inhibitors of mucin production. Such cells include airway secretory cells, reproductive tract epithelial cells, ciliated cells, epithelial cells of the respiratory tract and reproductive tract epithelial cells. Delivery of such inhibitors will depend on the area to be treated. For example, treatment of the respiratory tract can be achieved via aerosol or oral application. Intravenous application is a useful mode of delivery and is sometimes preferred in emergency cases.

The invention includes a method of inhibiting mucin production, comprising: applying an effective amount of a compound to target cells that can produce mucin, wherein the compound can, at a concentration of 500 μM or less, inhibit at least 10% of total mucin production from cultured epithelial cells capable of mucomone induced mucin production, preferably HM3 or HCIH292 cells, cultured under conditions conducive to mucin production and in the presence of a mucomone, compared to untreated cells. Preferably, the compound inhibits at least 30% and most preferably at least 70% total mucin production at concentrations of preferably 50 μM or less, more preferably 10 μM or less, even more preferably 1 μM or less, and most preferably 0.1 μM or less.

The method incudes a number of compounds, such as, for example, tyrosine kinase inhibitors (TKI's). Preferred TKI types include oxinole derivatives, bicyclic aryls, heteroaryl, carbocyclic aryls, heterocyclic aryls, benzylphosphonics, diaminophthalimides and pyrazolo-pyrimidines. Preferred specific compounds include quercetin, genistein, AG 126, erbstatin, levendustin, herbimycin, PP1, aeroplysinin, and the like as described above. Coadministration of second compounds is also contemplated, such as an LPS antagonist or antibiotic specific for the bacterial infection of the tissue being administered ihe TKI. Coadministration, as well as administration of the TKI alone, is preferably in the presence of a lung compatible pharmaceutical carrier, when such a tissue has comprised physiological responses due to the inappropriate expression of mucin. TKIs can be administered to the lung as particles with an average particle size 50 microns or less and preferably with an average size of 10 microns or less.

Other embodiments of the invention include methods for treatment of bronchitis, asthma and CF in a mammal, preferably a human. Such treatments can involve systemic administration. Preferably, these methods rely on non-systemic administration of a therapeutically effective amount of active compound to the tissue or cells in need of treatment.

For example, the present invention provides for a method of treating CF in patients comprising administering to a patient in need thereof a therapeutically effective amount of a compound that inhibits mucin production, wherein the compound, at a concentration of 500 micromolar or less, can inhibit at least 10% of total mucin production from cultured airway epithelial cells capable of mucomone induced mucin production, preferably HM3 or HCIH292 cells, cultured under conditions conducive to mucin production and in the presence of a mucomone, compared to untreated cells. Preferably, administration comprises aerosol delivery of the compound. The administered compound can also specifically prevent the binding of a heat resistant, PA derived sugar complex to a CF airway cell. Usually the daily dose is in the range of .1 μg to 500 mg and preferably the effective amount is 100 μg to 20 mg per day for particularly high affinity compounds. Administered compounds are preferably active in an in vitro assay at a concentration of 10 micromolar or less, more preferably at 1 micromolar or less and inhibit at least 10%, preferably 30%, and more preferably 70% of total mucin production from cultured epithelial cells, compared to untreated cells.

Additionally, the present invention also includes cells and tissues generated from the methods of the invention. For example, a cell comprising an epithelial cell and a tyrosine kinase inhibitor described herein. Examples of epithelial cells include a Hela, HM3 and HCIH 292 cells and other cells described herein. Often the cells will be in the presence of a mucomone from bacteria which is attached to the cell and the mucomone will stimulate mucin production in an epithelial cell but does not stimulate mucin production in a fibroblast cell. Other examples include a cell comprising a CF cell and a LPS antagonist.

EXAMPLES

EXAMPLE 1 - MATERIALS AND METHODS.

In situ hybridization analysis can be carried out as described herein and known in the art.

Tissue preparation: The bronchial explants from CF and non-CF individuals were treated with or without P. aeruginosa conditioned medium for 6 hours and were then fixed in 4% paraformaldehyde/ 0.1M phosphate buffer for 4 hours and cryoprotected in 30% sucrose/ 0.1M phosphate buffer overnight at 40 C. The next day, samples were embedded in OCT compound and quickly frozen in liquid nitrogen-cooled Freon-22. The frozen tissue was sectioned (6 mm), placed on Superfrost Plus slides (Fisher Scientific, Pittsburgh, Pa.) and air dried quickly. The sections were stored at -800 C until use.

RNA probes: The HAM1 cDNA contained a tandem repeat unit of the MUC-2 mucin gene, which can be cloned using the sequences described in publication WO 90/12892 and WO 91/08217, which ar incorporated herein by reference. Northern blots confirmed that the HAM1 cDNA recognized mRNA transcripts in the human bronchus, before using the probes in in situ hybridization. [35S] UTP-labeled RNA transcripts were synthesized from the cDNAs in linearized pBluescript plasmid using T7 and T3 polymerases to generate antisense and sense probes at concentrations of 2-5 x 10⁵cpm/ml. The probes of A. Dohrman, et al., Exp. Lung Res. 20: 367 (1994), which is herein incorporated by reference, are examples of the many probes that can be used.

In situ hybridization: Frozen sections of human bronchus were air dried quickly, heated at 55°C for 10 min, fixed with 4% paraformaldehyde in PBS for 10 min, washed with 2 x SSC (0.3 M NaCl/0.03 M sodium citrate, pH 7.0), immersed in 0.1 M triethanolamine HCl (pH 7.5) containing 0.25% acetic anhydride for 10 min, rinsed with 2 x SSC, dehydrated with ethanol and air dried. RNA probe was applied in a hybridization mixture containing deionized formamide (50%), dextran sulfate (10%), tRNA (0.5 mg/ml), Ficoll 400 (0.02 % (W/V)), salmon sperm DNA (1 mg/ml), polyvinylpyrrolidone (0.02% (W/V)), lOmM DTT, 0.3 M NaCl, 0.5 mM EDTA, 10 mM Tris-HCL, and 10 mM NaPO4 (pH 6.8). The mixmre was heated at 70 °C for 15 min and chilled on ice. Fresh DTT was added to achieve a concentration of 20 mM. Then 100 ml of the mixture was applied to each section, and parafilm coverslips applied. Hybridization was carried out in humid chambers overnight at 55 °C. Coverslips were removed in 5 x SSC, 10 mM DTT, at 55 °C. Sections were washed three times in wash buffer (2 x SSC, 1 mM EDTA, 10 mM β- mercaptoethanol (βME)) for 5 min at room temperature. Subsequently, they were treated with 20 mg/ml of RNase A in 500 mM NaCl, 10 mM Tris (pH 8.0) for 30 min at room temperature. This was followed by 2 x 5 min changes of wash buffer and a high stringency wash in 4 L of a wash solution containing 0.1 x SSC, 1 mM EDTA and 10 mM βME (2 h at 55 °C). Slides were then washed 5 min at room temperature in 0.5 x SSC without bME and EDTA. Finally, sections were dehydrated with ethanol and air-dried. The slides were exposed to Ilford K5D emulsion, and stored in the dark at 4°C until developed after 3-10 days exposure. The methods of H.B. Tang et al, Infect. Immun. 63 (1995), which is herein incoφorated by reference, is one of many methods that can be used.

Gene activation can be carried out as described herein and known in the art. MUC-2 transcriptional control elements: The 5 '-flanking region of human

MUC-2 gene was cloned by screening a human placental FIXII genomic library using the 5 '-region of human MUC-2 cDNA as the probe. The 5 '-flanking region was sequenced by dideoxy sequencing. Deletional mutants of the 5'-flanking region DNA was obtained by combining restriction digestion of the upstream region of the gene and PCR amplification. The restriction DNA fragments or PCR-amplified fragments were ligated into a luciferase reporter gene. All junctions and identifications of the DNA sequences in the chimeric constructs were confirmed by DNA sequencing. Transfection was performed by a standard electroporation method, p. aeruginosa conditioned medium was added to the transfected cells 42 hours after transfection. After 6 hours, the cells were harvested for luciferase assay. All transfections were carried out in triplicate. The luciferase activity was normalized by the β-galactosidase activity. Reference to nucleotide numbering. of the human MUC-2 gene is in relation to the transcriptional start site for the MUC-2 gene sequence.

Bacterial strains can be used to make bacterial conditioned media as described herein and known in the art. P. aeruginosa strains: The P. aeruginosa strains used in these studies were grown in M9 medium with aeration at 37 °C to late log phase. All the mutant strains have been described in detail previously in H.B. Tang et al, Infect. Immuno. 63: 1278-1285 (1995) and P. Massion, et al. , J. Cli. Invest. 96, 2204 (1995). In brief, PAOl was wild-type nonmucoid laboratory strain. PAO-pmm, an algC mutant, lacked the phosphomannomutase gene, resulting in a defective LPS core, the lack of

O side chains, and the inability to synthesize alginate. The LasR mutation in PAOR1 affected the production of P. aeruginosa autoinducer, elastase, and alkaline protease. PAO/NP was constructed by allelic exchange in which the wild-type gene pil A was replaced by a homologous piece of DNA containing a tetracycline resistance (TcR) cartridge within the coding sequence. The construction was confirmed by Southern hybridization and the phenotype documented by resistance to the pilus-specific phage lamda F116. AK1152 was nonmotile PAOl mutant generated by chemical mutagenesis.

LPS isolation from bacterial strains can be used to make bacterial LPS as described herein and known in the art.

LPS isolation. LPS can be isolated by a modification of the method of Hitchcock and Brown, J. Bacteriol. 154:269-277 (1983). Strains were harvested is sterile phosphate-buffered saline (PBS) after overnight growth on appropriately supplemented L agar plates. The suspensions were autoclaved and vigorously vortexed, and the cells were pelleted by centrifugation. The supernatants were combined with four volumes of 95% ethanol and precipitated at -20°C overnight, the precipitate were collected by centrifugation and redissolved in sterile PBS. Aliquots of these preparations were digested with RNase and DNase (each at lOOμg/ml) at 37°C overnight and then with pronase (100 μg/ml) for 2 h at 56°C (enzymes were purchased from Boehringer Mannheim Biochemicals Coφoration) and stored at -20°C until analysis. EXAMPLE 2 - PA INDUCES MUCIN PRODUCTION IN AIRWAY CELLS OF CF AND NON- CF PATIENTS.

To investigate whether P. aeruginosa (PA) stimulates or induces mucin synthesis, epithelial cells from airway tissues were contacted with media from PA cells and mucin production measured. In situ hybridization experiments measured mRNA corresponding to MUC-2, a mucin gene expressed in airway epithelial, in both CF and non-CF airway bronchial explants exposed for 6 hours to P. aeruginosa- conditioned or vehicle-containing control medium. In situ hybridization revealed elevated MUC-2 mRNA in the surface epithelium and submucosal glands of P. aeruginosa-treated bronchial explants versus controls (FIG.l, A to H) While occurring in both CF and non-CF airway bronchial explants, the induction was of greater magnitude in the latter. This shows that induction due to endogenous P. aeruginosa exposure, prior to the experiments, was already near maximal in the CF explants.

EXAMPLE 3 - PA INDUCES MUCIN PRODUCTION IN DIFFERENT EPITHELIAL CELLS

AND INDUCTION REQUIRES ONLY ONE CELL TYPE.

To investigate whether PA induced mucin production requires more than one airway cell type, cultures of different epithelial cells were contacted with PA conditioned media and mucin production measured using an RNase protection assay

(RPA) for MUC-2. P. aeruginosa induced MUC-2 production in four MUC-2- expressing cell lines, NCIH 292 (human pulmonary mucoepidermoid carcinoma) cells, Hela (cervix) cells, CFTE290 (airway CF mutant homozygous for ΔF508CFTR) cells and HM3 (human colon carcinoma) cells (FIG. 2 A).

TABLE 1

These findings indicate that P. aeruginosa upregulates MUC-2 mRNA by direct interaction with epithelial cells. That the effect could be obtained in cell free bacterial filtrates (conditioned medium) indicates that the active factors are shed into the bacterial environment and that MUC-2 is induced by these shed molecules and does not require direct contact between bacteria and epithelial cells.

EXAMPLE 4 - PA DERIVED MUCOMONE ACTIVATES MUC-2 TRANSCRIPTION IN

EPITHELIAL CELLS.

To investigate whether PA derived mucomone induces mucin transcription, activation of the 5' region of the mucin gene by PA derived mucomone was tested using epithelial cells transfected with a luciferase reporter gene operatively linked to the 5' flanking region of a mucin gene. MUC-2 promoter activity was measured using luciferase assays in transient expression experiments. The NCIH292, HM3 and CFTE29O (a transformed airway epithelial cell line homozygous for ΔF508 CFTR) cells were transfected with 2.8 kb of the MUC-2 5 '-flanking (non-coding region) region fused to a luciferase reporter gene. Promoter activity increased strikingly in all three cell lines when they were exposed to P. aeruginosa conditioned medium for 6 hours (FIG. 2B) suggesting that P. aeruginosa- induced MUC-2 mRNA levels are controlled at least partly at the level of transcription. EXAMPLE 5 - PA DERIVED MUCOMONE ACTIVATES MUC-2 TRANSCRIPTION AT

TWO DISCRETE 5' FLANKING REGIONS OF THE MUC-2 GENE.

To identify DNA response element(s) critical for this response, NCIH292, HM3 and CFTE29O cells were transfected with serial deletion mutants of the human MUC-2 gene promoter fused to a luciferase reporter gene. Findings in all three cell lines indicated the presence of P. aeruginosa response elements between -2.8kb/-73bp (FIG. 3A). To further define these, we analyzed additional deletion mutants in HM3 cells, the cell line showing the strongest P. aeruginosa response. Plasmids names refer to the length of the 5' flanking region used and its position relative to the transcription start site.

TABLE 2

Transfected cells were treated with either P. aeruginosa culture supernatants or vehicle for 6 hours prior to cell lysis. Luciferase activity was then assessed in P. aeruginosa treated- and non-treated cells. All transfections were carried out in triplicate. Values are the means ±SD; n=4. Luciferase activity was normalized with respect to β -galactosidase activity.

The results shown in FIG. 3B, FIG. 3C and FIG. 3D indicate the presence of (1) a minor inducible response element located in the -91 to -73 region [Jian-Dong, please verify-looks as potent as -114 or -343] of the MUC-2 gene, and (2) the presence of a major inducible response element located in the -1627 to -1308 region of the MUC-2 gene. EXAMPLE 6 - CF CELLS ARE NOT HYPERSENSITIVE TO PA INDUCED MUCIN

PRODUCTION.

To investigate whether CF cells might be hypersensitive to PA conditioned media, responses to PA conditioned media of CF mutant cell lines before and after complementation with wild type CFTR expression plasmid was measured using the luciferase assays described herein. Co-transfection of p-28641uc and pREP4.7kbCFTR into CFTE29O cells did not reduce their responsiveness to P. aeruginosa. Thus, CF airway epithelial cells homozygous for ΔF508 CFTR do not appear to be hypersusceptible to the P. aeruginosa-induced upregulation of MUC-2 transcription.

EXAMPLE 7 - BACTERIA AND PA DERIVED LPS INDUCES EPITHELIAL CELL MUC-2

TRANSCRIPTION.

To investigate the type of mucomones secreted by PA that cause mucin production in epithelial cells, conditioned media from PA mutants and LPS

(lipopolysaccharide) isolated from PA was added to epithelial cultures and mucin production determined. MUC-2 transcription was analyzed using p-28641uc in HM3 cells, conditioned medium from 4 defined mutants of the PAO strain: PAOl (lasR), PAO-pmm (algC), PAO/NP, AK1152. Although most mutant conditioned media were as potent as that of wild-type, conditioned medium from mutant PAO-pmm, an algC mutant which lacked the phosphomannomutase gene, resulting in a defective LPS core, the lack of O side chain, and the inability to synthesize alginate, evoked only 50 % of the wild type response (FIG. 4A). LPS isolated from PA also induced mucin production in epithelial cells. HM3 cells transfected with p-28641uc for 42 hours were incubated in the presence and absence of LPS (20μg/ml; from Sigma Company cell culture graft). After 6 hours the cells were harvested and luciferase activity measured. Transfections were in triplicate. Luciferase activity was normalized with respect to /3-galactosidase activity. LPS induced mucin production compared to the control, as shown in FIG. 4B, indicating that PA LPS is a mucomone. To determine if mucin transcriptional activity can be induced by PA pmm conditioned medium having an incomplete LPS structure, upregulation of MUC-2 transcriptional activity due to LPS isolated from PA pmm mutant strain was compared to PA pmm conditioned medium and PAOl conditioned media. The results in FIG. 5A show that the incomplete LPS complex in PA pmm has an effect similar to PA pmm conditioned medium, indicating the incomplete LPS complex of pmm mutant induce mucin production.

To determine if LPS from other gram-negative bacteria also increased upregulation of MUC-2 transcription, the effects of LPS from 3 other bacteria (E. Coli, Salmonella enteritides, and Klebsiella pneumonia) on human MUC-2 promoter activity was examined. FIG. 5B shows that all the LPSs tested upregulate MUC-2 transcription, indicating that the use of different LPS antagonists for different bacterial LPS can be used to treat mucin oveφroduction induced by not only PA but also other Gram-negative bacteria. When experiments such as those described above are performed normalizing the LPS concentrations of all the conditioned medias, PAO-pmm conditioned media (also referred to as culture supernatent, CS) and purified LPS was as potent as the culture supernatent or purified LPS of wild-type (FIG.5C). Thus, maximal induction of MUC-2, does not appear to require the complete LPS complex or alginate. An experiment showing that lipid A (purified from E. coli) could essentially mimic the LPS effect (FIG. 5D) indicates that lipid A is a key factor in the MUC-2 stimulation and that lipid A from diverse bacterial species is potent in this respect. HM3 cells were transfected with p-28641uc. After 42 hours the cells were exposed to E. coli lipid A (5 μg/ml) (Sigma Chemical Co. St. Louis M) or LPS (5μg/ml) (P. aeruginosa serotype 10). After 6 hours the cells were harvested for luciferase activity measurement. All transfections were carried out in triplicate. Values are the means +SD; n=3. Luciferase activity was normalized with respect o /3-galactosidase. Thus, Lipid A is sufficient to upregulate MUC-2 transcriptional activity to about 60% of the maximal level achieved with either complete LPS or LPS lacking the O side chain. EXAMPLE 8 - PA DERIVED MUCOMONES ARE HEAT, PROTEASE AND DNASE RESISTANT.

To further determine the nature of PA mucomones, PA conditioned media was treated with either heat, proteases or DNAses before contacting epithelial cells with the PA conditioned media. Relative to the control (no treatment) and PAOl wildtype

PA strain, boiling, protease and DNAse treatments all did not decrease the ability of PA conditioned media to induce mucin production HM3 cells transfected with p- 28641uc. Mucin production was measured as MUC-2 gene activation from the increase in reporter gene activity, as shown in FIG. 6A. These results are consistent with a heat stable, non-protein structure and non-DNA structure for PA mucomone(s).

EXAMPLE 9 - PA DERIVED MUCOMONES HAVE DIFFERENT SIZE RANGES.

To determine the molecular weights of the PA mucomones, PA conditioned media was size fractionated and then tested in mucin production assays using HM3 cells transfected with p-28641uc. PA conditioned media was sized fractionated using a spin column for each size range. PA mucomones have molecular weight of 100, 30, 10, 3 and 1 kd, as shown in FIG. 6B. PA mucomones can be further isolated as described herein.

EXAMPLE 10 - TYROSINE KINASE INHIBITOR GENISTEIN COMPLETELY INHIBITS PA

DERIVED MUCOMONE INDUCED MUCIN PRODUCTION.

To investigate whether tyrosine kinase inhibitors can block PA mucomone induced mucin production, tyrosine kinase inhibitors were tested in mucin production assays using HM3 cells transfected with p-28641uc or with RPA analysis. HM3 cells were pretreated with Genistein (100μg/ml=0.37 mM) for 2 hours and then exposed to PA conditioned media for 6 hours before RPA analysis. HM3 cells transfected with p-28641uc for 40 hours were pretreated with genistein for 2 hours and then exposed to PA conditioned media for 6 hours and luciferase activity measured as described herein. Genistein completely abolished P. aeruginosa- induced upregulation of MUC-2 gene transcription as assessed by the induction of steady-state mRNA as assessed by RPA assays or luciferase assays (FIG 7A), indicating that inhibition of tyrosine phosphorylation completely prevents PA conditioned media induction of mucin production, in this case MUC-2 production.

EXAMPLE 11 - TYROSINE KINASE INHIBITOR TYRPHOSTIN AG126 INHIBITS PA DERIVED MUCOMONE INDUCED MUCIN PRODUCTION.

To investigate whether a tyrosine kinase inhibitor specific for LPS-activated kinase, such as pp42 mapk, can block PA mucomone induced mucin production, tyφhostin AG126 was tested in mucin production assays using HM3 cells transfected with p-28641uc. HM3 cells transfected with p-28641uc for 40 hours were pretreated with AG126 (25 μM) for 2 hours and then exposed to PA conditioned media for 6 hours or PA LPS before luciferase activity was measured as described herein.

AG 126 abolished P. aeruginosa- induced upregulation of MUC-2 gene transcription as assessed by luciferase assays (FIG 7B). AG 126 blocked both PA conditioned media and PA LPS induction of mucin production. These results indicate that inhibition of tyrosine phosphorylation completely prevents PA LPS induced mucin production, in this case MUC-2 production.

EXAMPLE 12 - NON-RECEPTOR TYROSINE KINASE INHIBITOR PP1 INHIBITS PA MUCOMONE INDUCED MUCIN PRODUCTION HM3 cells were transfected with p-28641uc. 40 hours after being transfected with p-28541uc, HM3 cells were pretreated with PP1 (5, 14 and 28 μM) (CALBIOCHEM, La Jolla, CA, Catalog No 529572) for 30 min. and then exposed to PAOl culture supernatant (CS) 6 hours before harvesting. Luciferase activity was measured as described above. All transfections were carried out in triplicate. Values are the means ± SD; n=3. Luciferase activity was normalized with respect to β- galactosidase. As shown in FIG. 8, concentrations of 5 μM, 14 μM and 28 μM of

PP1, reduced upregulation of MUC-2 transcriptional activity by 37%, 49% and 65%, respectively, compared to MUC-2 transcriptional activity in the absence of PP1.

Reduction of transcriptional activity was calculated as follows: % Reduction = 100-100 activity of PP1 treated cells activity of untreated cells EXAMPLE 13 - IN VIVO MODEL FOR PA INFECTIONS OF CF PATIENTS.

To investigate mucomone induced mucin production in vivo, several in vivo animal models for cystic fibrosis and lung diseases can be used. For example, the Johansen, H.K. et al. (Am J Respir Crit Care Med. Vol. 152 p. 1337-1346, 1995) rat model of chronic P. aeruginosa pneumonia mimicking cystic fibrosis can be used. Pier, G.B. et al. 's murine model of chronic mucosal colonization by p. aeruginosa (Infect. Immun. 1992, Vol. 60, No. 11, p. 4768-4776) will be used to measured mucomone-induced mucin expression level in airway epithelial cells compared to control animals without infection and animals administered PA LPS to the lung. Mucomone induced mucin expression will then be treated with the tyrosine kinases, as described herein. Effects on the airway mucin expression level in these animals can then be determined.

To assess the treatment in animals with mutant CFTR alleles, animal models for the ΔF508 allele of cystic fibrosis can be used (Doorninck, J.H. et al., EMBO, 1995, 14 (18): 4403-4411; Zeiher, B.G. et al. JCI, 96: p. 2051-2064;

Colledge, W.H. et al. , Nature Genetics, Vol. 10, p. 445; Snouwaert, J.N., et al., Am J. Respir Crit Care Med 1995, Vol. 151, p. 559-564). Typically, mice will first receive 1 mg of streptomycin per ml of drinking water for a minimum of 5 days using an aerosol. Airway mucin expression level is measured as described herein, for example, using in situ hybridization. Measurements will be made in the presence or absence of TKIs and LPS antagonists. Inhibitors can be administered as an oral, aerosol or intravenous dose.

Inhibitors of mucomone-induced mucin production can also be assessed in human bronchial xenografts, as described by Engelhardt, J.F. et al. (JCI, 1992, Vol. 90, p. 2598-2607). Briefly, explants of "open-ended" human bronchial xenografts are made subcutaneously in the flanks of female nu/nu BALB/c mice. Human bronchial xenografts from CF patients can also be used to assess directly the effect of inhibitors on mucin expression in these xenografts using an assay, such as an in situ hybridization assay. Human bronchial xenograft from normal individuals can be exposed to P. aeruginosa to assess mucomone induction and the affect of inhibitors described herein. The inhibitors can be introduced into mice as described herein. PUBLICATIONS

1. U.S. patent 5,436,128, issued July 25, 1995.

2. J.M. Rommens, et al. , Science 245, 1059 (1989).

3. J. Riordan, et al., Science 245, 1066 (1989). 4. P.H. Gilligan, Clin. Microbiol. Rev. 4, 35 (1991).

5. S.E. Gabriel, et al. , Nature 363, 263 (1993).

6. T. Boat, R. Boucher, In " Textbook of respiratory medicine" J. Murray and J. Nadel, 7. W.B. Sauders Co, Philadelphia, 1418 (1994).

8. E.M. Schweibert, et al. , Cell 81, 1063 (1995). 9. M. Yamaya, et al.,Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5),

L491 (1991).

10. R.C. Frates, et al., Pediatr. res. 17, 30 (1983).

11. H.B. Tang, et al., Infect. Immun. 63 (1995)

12. G.B. Pier, et al. , Science 271 , 64 (1995) 13. P. Massion, et al., J. Cli. Invest. 93, 26 (1994).

14. E. DiMango, et al.J. Cli. Invest. 96, 2204 (1995)

15. A. Dohrman, et al., Exp. Lung Res. 20, 367 (1994)

16. M. J. Coyne, et al., J. Bactriol. 176, 3500 (1994).

All publications and patent applications mentioned in this specification are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of identifying compounds useful as therapeutics comprising: contacting epithelial cells with a compound, said cells being being capable of mucomone induced mucin production and said cells being cultured in the presence of mucomone, detecting a change in mucin gene activation in said epithelial cells in the presence of said compound, wherein said detecting is selected from the group consisting of measuring mucin protein levels using a mucin antibody, measuring levels of RNA encoding said mucin, and measuring MUC-2 gene activation with a reporter gene.

2. The method of claim 1, wherein said detecting is selected from the group consisting of measuring levels of RNA encoding said mucin and measuring MUC-2 gene activation with a reporter gene.

3. The method of claim 2, wherein said reporter gene is operably linked to a response element of said MUC-2 gene.

4. The method of claim 2, where said mucomone is LPS from gram negative bacteria.

5. A method of inhibiting of mucin production comprising: applying an effective amount of an inhibitor of mucomone induced mucin production to a target cell, wherein said inhibitor, at a concentration of 500 micromolar or less, inhibits at least 10% of mucin gene activation in epithelial cells capable of said mucomone induced mucin production, cultured under conditions conducive for said mucin production and in the presence of said mucomone, compared to said epithelial cells cultured in the absence of said inhibitor, as determined by measuring levels of RNA encoding said mucin or by measuring MUC-2 gene activation with a reporter gene.

6. The method of claim 5, wherein said inhibitor is a TKI selected from the group consisting of oxinole derivatives, bicyclic aryls, heteroaryl, carbocyclic aryls, heterocyclic aryls, benzylphosphonics, diaminophthalimides and py razolopy rimidines .

7. A method of treating cystic fibrosis comprising: administering to a patient in need thereof a therapeutically effective amount of a compound that inhibits mucomone induced mucin production, wherein said compound at a concentration of 10 micromolar or less inhibits at least 10% of mucin gene activation in epithelial cells capable of said induced mucin production, cultured under conditions conducive for said mucin production and in the presence of a mucomone, compared to said epithelial cells cultured in the absence of said compound, as determined by measuring levels of RNA encoding said mucin or by measuring MUC-2 gene activation with a reporter gene.

8. The method of claim 7, wherein said compound is administered with a lung compatible pharmaceutical carrier.

9. An epithelial cell comprising a tyrosine kinase inhibitor and a bacterial mucomone.

10. A mammalian cell transfected with a reporter gene operably linked to a promoter and a transcriptional response element of a MUC-2 gene, wherein said cell is capable of mucomone induced mucin production.