EP1827479A1

EP1827479A1 - Methods and agents useful in treating conditions characterized by mucus hyperproduction/ hypersecretion

Info

Publication number: EP1827479A1
Application number: EP05816228A
Authority: EP
Inventors: Lutz Zeitlmann; Johannes Grosse; Klaus Dembowsky; Andreas Popp
Original assignee: Ingenium Pharmaceuticals GmbH
Current assignee: Ingenium Pharmaceuticals GmbH
Priority date: 2004-12-09
Filing date: 2005-12-08
Publication date: 2007-09-05
Also published as: WO2006061414A1

Abstract

The invention relates to compounds capable of inhibiting the interaction of Agr2 protein with mucins, pharmaceutical compositions comprising such compounds and methods of preventing, treating, or ameliorating medical conditions associated with mucus hyperproduction/hypersecretion comprising administering to a mammal a pharmaceutical composition comprising a compound capable of inhibiting Agr2/mucin interaction.

Description

Methods and Agents Useful in Treating Conditions Characterized by Mucus Hyperproduction/Hypersecretion

Background of the Invention

The epithelial mucosal layer is a physical and chemical harrier important in protecting the animal body from dryness, harmful exogenous substances and pathogens. Mucus forms a gel layer covering the epithelial surface, acting as a semi-permeable barrier between the epithelium and the exterior environment. Mucus serves many functions, including protection against shear stress and chemical damage, and, especially in the respiratory tree, trapping and elimination of particulate matter and microorganisms. The mucus layer on top of the intestinal epithelium is the barrier between the host's internal milieu and gut bacteria.

Secretion of mucus occurs by exocytosis of secretory granules (Verdugo, 1991). Constitutive or basal secretion occurs at low levels and is essentially unregulated and continuous. Stimulated secretion corresponds to regulated exocytosis of granules in response to extracellular stimuli such as hormones, neuropeptides and inflammatory mediators (Jackson, 2001; Laboisse et al., 1996). This pathway provides the ability to dramatically increase mucus secretion.

Mucins are high molecular mass, highly glycosylated macromolecules that are the major components of mucus secretions. Many epithelial cells express mucins. For example, mucins are secreted from the apical surface of specialized columnar epithelial cells referred to as goblet cells (Verdugo, 1990; WO

2004/056858).

Mucins have the ability to hydrate and form a viscous gel, producing a protective scaffold overlaying epithelial surfaces. Mucins consist of a polypeptide core (apomucin) covered almost entirely by O-linked carbohydrate chains, which may constitute up to 80% of the total molecular weight. All known mucin genes are characterized by tandem and irregular repeat sequences rich in codons for threonine and serine, e.g., TT and SS, the potential sites of attachment of oligosaccharide chains. After translation, mucin proteins are secreted into the endoplasmatic reticulum (ER). The ER serves as cellular compartment for further posttranslational modification, e.g., protein folding. The O-glycosylation by glycosyltransferase occurs in the Golgi and requires elongated, non three-dimensional forms of mucin molecules. Such non three-dimensional conformation of mucins may be facilitated by chaperon protein activity.

At least 13 different mucins are known which are subdivided into secreted and membrane-bound mucins (see for Review in Dekker et al., 2002 and Moniaux et al., 2001). Mucin2, mucin5ac, mucin5b and mucinό, all of them exhibiting tissue- and cell specific expression, belong to the class of secreted mucins. Mucin2 is mainly expressed in intestinal and in colonic goblet cells. In its most common allelic form the mucin2 protein is more than 5,100 amino acids long. The mucin2 product is polymerized end to end through disulfide bridges to form large secreted polymeric gel-forming mucins (Allen et al., 1998). Mucin5ac is primarily expressed in tracheobronchial goblet cells and in gastric surface epithelial cells. Mucin5b is expressed in tracheobronchial, salivary and esophageal mucous glands, pancreatobiliary and endocervical epithelial cells. Mucin5b is composed of 14.9% protein, 78.1% carbohydrate, and 7% sulfate. Mucinό is expressed in gastric and duodenal mucous glands and in pancreatobiliary and endocervical epithelial cells. Secreted mucin gene expression patterns in mouse are very similar to those described in humans (Audie at al., 1993, Vandenhaute et al., 1997, Bartmann et al., 1998).

WO 2004/056858 discloses reduced amounts of mucin2 mRNA in colon of mice carrying a point mutation in the Agr2 gene (see Example 24 therein). The mutated Agr2 protein carries a charged glutamic acid (E) in position 137 instead of a non-polar valin (V) in the wild type (non mutated) protein. Mutated mice display a reduction in pre-mucin storing granules in the goblet cells, a reduced mucus secretion, and secondary inflammatory infiltrations in the intestinal mucosal epithelium and submucosa. Within a broad spectrum of different murine and human tissue mRNAs analyzed, Agr2 transcripts are detected in mucus secreting tissues, including tissues of the digestive/gastrointestinal tract, in particular salivary gland, esophagus, stomach, small intestine, large intestine, rectum; and tissue of the respiratory tract, in particular nose epithelium, trachea and lung (see Figures 6 to 8 therein). In one Example, Agr2 protein expression is detected in colon goblet cells of wild type mice (see Figure 10 therein). The Agr2 protein is disclosed to be involved in goblet cell differentiation, particularly terminal differentiation and/or goblet cell mucus production or secretion and/or mucus composition.

Mucus hyperproduction/hypersecretion is observed in diseases such as asthma, allergy, COPD and cystic fibrosis. Disease-associated cytokines, bacterial products, proteinases or oxidants are inducers of goblet cell hyperplasia. Goblet cell hyperplasia and associated mucus hypersecretion are clinical and pathophysiological features of asthma and COPD. Especially in asthma, airway mucus hypersecretion contributes to morbidity and mortality.

Known therapeutic targets linked to the pathophysiology of airway mucus hyperproduction/hypersecretion are epidermal growth factor receptor (EGFR) tyrosine kinase, calcium-activated chloride channels (CLCA), and the apoptosis inhibitor Bcl-2. Clinical trials with blockers of these targets are ongoing.

Other strategies of modulating mucus production have been proposed, e.g., by LTB4 antagonists (WO 02/55065), EGF receptor antagonists (WO 02/05842), polycationic peptides (US 6,245,320), KGF (WO 94/23032) (Farrell et al., 2002) and KGF-2 (WO 99/41282).

There is a need for novel methods and agents useful for the treatment of respiratory diseases associated with mucus hyperproduction/hypersecretion, such as asthma, allergy, COPD and cystic fibrosis. Summary of the Invention

The invention described herein demonstrates for the first time that

Agr2 protein interacts directly with mucins, the major components of mucus. The invention offers novel opportunities for the treatment of diseases, where a reduction of mucus hyperproduction/hypersecretion is desirable, particularly respiratory diseases, by interfering with the Agr2/mucin interaction.

Accordingly, in a first aspect the invention relates to a compound capable of inhibiting the interaction of Agr2 protein with a mucin protein. Pharmaceutical compositions comprising such a compound and a pharmaceutically acceptable carrier likewise represent an aspect of the invention.

In another embodiment, the invention relates to a method of preventing, treating, or ameliorating a medical condition (e.g., a medical condition affecting the respiratory system) associated with mucus hyperproduction/hypersecretion in a mammal, e.g., in a human subject, for example asthma, allergic reactions of the respiratory system, COPD (chronic obstructive pulmonary disease), and cystic fibrosis. The method comprises administering to said mammal or said human subject a pharmaceutical composition comprising the compound of the invention capable of inhibiting Agr2/mucin interaction.

Also encompassed by the present invention is the use of the compounds of the invention for the preparation of a pharmaceutical composition for preventing, treating, or ameliorating any of the above-mentioned medical conditions.

In yet another embodiment, the invention relates to a method of identifying a compound capable of interfering with the Agr2/mucin interaction, and thus, identifying compounds useful for preventing, treating, or ameliorating any of the above-mentioned medical conditions. Said methods comprise assaying the ability of a test compound to inhibit the binding of Agr2 protein to mucin protein. Description of the Figures

Figure 1 shows Agr2 protein co-localization with protein disulphide isomerase

(PDI) in the endoplasmatic reticulum (ER) of CaCO2 cells. PDI is a known marker for ER localization. CaCo2 cells, which endogenously express Agr2 and PDI, were stained with anti Agr2 antiserum plus a fluorescent labeled second antibody, or with anti-PDI antibody plus a fluorescent labeled second antibody, respectively. Cellular localization of Agr2 (see Figure IB) and PDI (see Figure IA) was performed with fluorescence microscopic techniques.

Figure 2 shows results of a yeast two-hybrid experiment, indicating protein- protein interaction between murine Agr2 and murine mucin2 (Agr2 x mucin2 fragment), as indicated in the Figure. Agr2 protein was expressed fused to the DNA binding domain of Gal4, whereas murine mucin2 was expressed fused to the Gal4 transcriptional activator domain. Binding of the two fusions products was monitored based on the transcriptional activation of the reporter gene His3. Only yeast cells carrying the fusion products of Agr2 and mucin2 in a protein- protein interaction state are capable of growing on a yeast medium lacking histidine (interaction). The strength of protein-protein interaction correlates with the size of yeast colonies grown and is indicated as score value (+). Positive and negative controls provided by the kit were included in the assay.

Figure 3 shows data of a tracheal ovalbumin challenge assay applied to wild type (WT) mice and Agr2 mutant (Agr2-/-) mice. Tracheal ovalbumin challenge induces goblet cell differentiation from tracheal epithelial cells and glycoprotein synthesis in the goblet cells of wt mice, as seen in Figure 3B. Such mice are well known as a murine asthma model. Control mice do not display such phenotypic alterations in the trachea after saline instillation, as shown in Figure 3A. In Agr2-/- mice, lacking normal Agr2 function, differentiated goblet cell are not visable and no synthesized glycoproteins are detectable after ovalbumin instillation, as shown in Figure 3C. These data provide evidence for a function of Agr2 in goblet cell differentiation and glycoprotein synthesis.

Detailed Description of the Invention

The compounds described and claimed herein are useful in the prevention, treatment, or amelioration of a medical condition, particularly a medical condition affecting the respiratory system, associated with mucus hyperproduction/hypersecretion in a mammal, e.g., in a human subject. These compounds are capable of inhibiting the interaction of Agr2 protein with, or binding of Agr2 protein to mucins, hi this regard, the Agr2 protein is preferably human Agr2, e.g., human Agr2 protein having a primary amino acid sequence corresponding to the amino acid sequence depicted in SEQ ID NO:1.

On the mucin side, the compounds of the invention preferably interfere with the interaction of a human mucin with the Agr2 protein. Particularly preferred compounds inhibit the interaction of mucin2 with Agr2 protein, particularly mucin2 having an amino acid sequence which comprises or consists of the amino acid sequence of SEQ ID NO:2.

Other preferred compounds of the invention inhibit the interaction of mucin5ac and/or mucin5b with the Agr2 protein, particularly mucin5ac having an amino acid sequence which comprises or consists of the amino acid sequence of SEQ ID NO:3, and particularly mucin5b having an amino acid sequence which comprises or consists of the amino acid sequence of SEQ ID NO:4. A compound of the invention may, for example, be a peptide or polypeptide. In a preferred embodiment, the peptide or polypeptide has an amino acid sequence of from 2 to 138 amino acids. Other preferred peptides or polypeptides have an amino acid sequence of from 32 to 136, or from 2 to 16 amino acids. The inventors of the present application have surprisingly found that the Agr2 protein directly interacts with mucins, which appears to be an important aspect of Agr2's physiological role in mucus secreting tissue. Mucins are proteins rich in serine and/or threonine residues. Consistent with that, the inventors have found that Agr2 also binds to other serine and/or threonine rich amino acid sequence motifs. Without intending to be bound by this theory, it is believed that Agr2 acts in a manner similar to a chaperon protein by binding to unfolded parts of mucin proteins, especially including serine and threonine rich amino acid sequences, and thus protecting mucins from protein folding and posttranslational modifications at serine and threonine residues. Loss of this Agr2 function would lead to a reduction in, or abolishment of the expression of functional mucin proteins in the corresponding tissue. Thus, medical conditions characterized by mucus hypersecretion or hyperproduction, e.g., those affecting the respiratory system such as asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis would benefit from inhibitory compounds, such as inhibitory peptides or polypeptides, that interfere with, or inhibit, the binding of Agr2 to mucin.

Accordingly, preferred inhibitory polypeptides of the invention are characterized by a serine and/or threonine rich amino acid sequence. The term "serine and/or threonine rich amino acid sequence" as used herein refers to an amino acid sequence that contains more than 5% serine and/or 5% threonine amino acids compared to the total number of amino acids, more preferably more than 8% serine and/or 8% threonine amino acids compared to the total number amino acids. Other preferred inhibitory polypeptides or peptides of the invention contain amino acid sequences that have at least 12, 15, 20, 25, 30, 35 or even 40% serine amino acids and/or at least 12, 15, 20, 25, 30, 35 or even 40% threonine amino acids compared to the total number of amino acids. The above property of having a serine and/or threonine rich amino acid sequence may be reflected by the presence of one or more uninterrupted repeats of at least two, at least three, or at least four or even more serine or threonine residues. Peptides or polypeptides displaying both serine and threonine repeats of the above kind in their sequence are likewise contemplated by the invention, and represent another preferred embodiment. An example of a preferred peptide of the invention is provided below by SEQ ID NO:5. Other preferred compounds are mucin protein fragments, e.g., fragments of human mucin2, mucin5ac or mucin5b. Preferred embodiments of these mucin fragments have an amino acid sequence of from 2 to 138 amino acids. Other preferred fragments have an amino acid sequence of from 32 to 136, or from 2 to 16 amino acids.

Another preferred inhibitory (polypeptide of the invention is one consisting of, or comprising amino acids 1 to 77 of the N-terminal part of human mucin2, particularly of human mucin2 according to SEQ ID NO:6. Also suitable is a (polypeptide consisting of, or comprising amino acids 1 to 77 of the N-terminal part of mouse mucin2, particularly of mouse mucin2 according to SEQ ID NO:7

The peptides or polypeptides of the invention, including the fragments described and claimed herein, may be fused to other proteins or protein fragments to form fusion proteins. Suitable fusion partners in this regard are, for example, transcriptional activator domains, such as a Gal4 transcriptional activator domain. Other suitable fusion partners, particularly in connection with pharmaceutical uses, are Ig domains.

From the above it will be understood that other compounds that interfere with, or inhibit, the binding of Agr2 to a mucin are likewise useful and contemplated in the context of the present invention. Such compounds may be, for example, small molecules having a molecular weight of no more than 2000 Dalton, or no more than 1500 Dalton. Smaller molecules having a molecular weight of no more than 1000, 500, 400, 300 or even 200 Dalton which display the above property of interfering with, or inhibiting the binding of Agr2 to a mucin are likewise suitable in the context of the present invention. Other preferred compounds that interfere with, or inhibit, the binding of Agr2 to a mucin are peptidomimetics, e.g., peptidomimetics of the molecular weights mentioned above, particularly if they mimick the physico-chemical properties, the inhibitory properties regarding Agr2/mucin interaction and/or the Agr2 binding properties (see below) of the peptides or polypeptides described and claimed herein.

The compounds described and claimed herein preferably exert their inhibitory activity by directly binding to the Agr2 protein, although it is also conceivable that they bind to the mucin, or to an already formed Agr2/mucin complex. The binding affinity regarding the binding to Agr2 is preferably similar to the binding affinity, which the mucin in question has towards the Agr2 protein. Preferably, the binding affinity of the compound to Agr2 is even higher than that of the mucin in question.

Preferred compounds of the invention bind to Agr2 with a dissociation constant (Kd) equal to or lower than 1000 μmol, preferably equal to or lower than 100 μmol, or equal to or lower than a value in the range from 10 μmol to 10 nmol. The binding affinities and dissociation constants of the binding of the compounds of the invention to Agr2 may be readily determined by methods well known in the art. A preferred method in this regard is a biochemical in vitro assay, such as an ALPHAscreen assay (Becton Dickinson, USA). This and other known methods may also be used to determine the relative binding affinity of a given compound of the invention to the Agr2 protein compared to that of the mucin in question.

Pharmaceutical compositions comprising the compounds of the invention together with a pharmaceutically acceptable carrier are another aspect of the invention. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described, e.g., in REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed.), Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ, U.S.A.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

For instance, for oral administration in the form of a tablet or capsule (e.g., a gelatin capsule), the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like. Diluents, include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.

Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers, hi addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

The compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions.

Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension. Additionally, solid forms suitable for dissolving in liquid prior to injection can be formulated. Injectable compositions are preferably aqueous isotonic solutions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.

The compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions.

Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained- released system, which assures that a constant level of dosage is maintained, according to US Pat. No. 3,710,795, incorporated herein by reference.

Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.

For solid compositions, excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used. The active compound defined above, may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier. In some embodiments, suppositories are advantageously prepared from fatty emulsions or suspensions.

The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in US Pat. No. 5,262,564. In other embodiments, large unilamellar vesicles (LUVs) are composed of a mixture of cationic and anionic lipids (see, e.g., Hafez IM, Ansell S, Cullis PR, 2000). The compounds of the present invention are preferably administered intratracheal, e.g., by inhalation. Particularly preferred for intratracheal delivery are aerosols containing the compounds of the present invention together with suitable additives known by those skilled in the art. Alternatively, the compounds of the present invention may be administered to the target cells or tissues in intranasal form, e.g., via aerosols or via topical use of suitable intranasal vehicles.

Liposomal formulations containing the compounds of the present invention are particularly preferred for intratracheal delivery. Such formulations may conveniently be formulated into aerosol formulations suitable for inhalation or intranasal delivery (see, for example, WO 99/34837). In addition, formulations containing large unilamellar vesicles (LUVs) composed of a mixture of cationic and anionic lipids are likewise particularly suitable for these applications. Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross- linked or amphipathic block copolymers of hydrogels.

If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.

Another aspect of the present invention is a method of preventing, treating, or ameliorating a medical condition as defined above in a human subject, said method comprising administering to said human subject a pharmaceutical composition comprising a compound as described and claimed herein. Also encompassed by the present invention is the use of the compounds described and claimed herein for the preparation of a pharmaceutical composition for preventing, treating, or ameliorating a medical condition as defined above.

Preferred target cells for the above method or use are cells of the respiratory tract of the human subject to be treated, which particularly includes goblet cells and/or other mucus secreting cells, such as submucosal cells of the bronchi, nose, larynx, pharynx, and salivary glands of the tongue.

The dosage regimen to be employed in connection with the pharmaceutical compositions, methods and uses of the invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the compound required to prevent, ameliorate, or treat the condition.

It will be understood that all pharmaceutical compositions and formulations described herein may principally be used in the therapeutic methods and methods described and claimed herein. Particularly preferred formulations for delivery of the compounds of the present invention to the target cells of a human subject to be treated include the liposome or unilamellar vesicle formulations as described herein above. In a preferred embodiment of this aspect of the invention, the compounds of the invention are administered intratracheally, intranasally or transmucosally, e.g., by inhalation. Other suitable formulations and modes of administration will be apparent to the skilled person based on the teaching disclosed in the present invention.

Compounds useful in the methods and uses described and claimed herein may be readily identified by screening methods which are per se known in the art. Accordingly, another aspect of the invention are methods of identifying compounds capable of interfering with the Agr2/mucin interaction, said methods comprising assaying the ability of a test compound to inhibit the binding of Agr2 protein to a mucin protein. This assaying may be done, e.g., in the context of a cell- based interaction or binding assay, e.g., by expressing both the Agr2 protein and the mucin, or suitable fragments thereof that interact with each other, optionally fused to another protein, in said cell and allowing them to interact intracellulary. This interaction is preferably monitored with the help of a certain read-out, e.g., the activity of a selectable marker or a reporter gene the expression of which is linked to the interaction of Agr2 and the mucin. If the interaction is affected by the presence of a test compound capable of inhibiting said interaction, this will be reflected by a change in the read-out, e.g., when compared to a control without the test compound. An example of a suitable intracellular interaction or binding assay is the yeast two-hybrid (Y2H) system, which is also commercially available, e.g., from Clontech, USA (Matchmaker™ GaW Two-Hybrid System 3). In such a system, the Agr2 protein, or a suitable fragment thereof, will be expressed fused to the DNA binding domain of Gal4, whereas the mucin protein, or a suitable fragment thereof, is expressed fused to the GaW transcriptional activator domain. Binding of the two fusion constructs via Agr2/mucin interaction is then monitored based on the transcriptional activation of a selectable marker or reporter gene, e.g., His3. A test compound that is taken up by the yeast cells may be assayed in this system for its ability to interfere with the Agr2/mucin interaction.

Alternatively, in case the test compound is a peptide or polypeptide, it may also be expressed in the yeast cells carrying the above two-hybrid system, and the ability of the compound to interfere with the Agr2/mucin interaction is then monitored. This could be done, e.g., by the combined use of the Matchmaker™ two- hybrid system (cat. no. 630303, Becton Dickinson) and a pBridge vector (cat. no. 630404, Becton Dickinson). In one embodiment, the Agr2 protein part is again expressed as DNA-binding fusion protein, and the mucin protein part is expressed as transcription activator domain fusion protein. A test peptide or polypeptide is then co- expressed, for example, from the pBridge vector. If it is capable of inhibiting the Agr2/mucin interaction, the test peptide or polypeptide will inhibit yeast growth under selective medium (e.g., when using his3 as selectable marker in combination with medium lacking histidine) or, if the read-out is activity of a reporter gene, inhibit expression of, e.g., a lacZ reporter gene, the enzymatic activity of which is then monitored.

Another suitable approach to arrive at peptides or polypeptides of the invention is to screen peptide libraries containing random peptides or polypeptides cloned into an expression vector library (for example, Clontech' s commercially available Matchmaker™ Random Library) that expresses the peptides or polypeptides fused to the Gal4 DNA binding domain in yeast cells transformed with these vectors. The Agr2 protein, or a suitable fragment thereof, in turn is again expressed as a fusion with the DNA binding domain of Gal4 in these cells. This system allows in a straightforward manner to assay the ability of the (polypeptides fused to the GaW activator domain to bind to Agr2 protein by monitoring again the readout, e.g., the activity of a selectable marker or a reporter gene, such as His3 or lacZ. Peptides or polypeptides thus identified are candidates to interfere with, or inhibit the Agr2/mucin interaction, which may be further verified in the Y2H assay described above, or by other assays known per se in the art, e.g., a cell-free in vitro binding assay of the kind described below, e.g., a homogeneous binding assay.

An assay likewise suitable for monitoring the interaction of Agr2 protein and mucin protein, and thus the capability of a test compound (such as a peptide or polypeptide, but also a small molecule or a peptidomimetic) to interfere with or inhibit this interaction, is a homogeneous binding assay. Suitable assays of this kind measure, for example, the proximity of the two interaction partners, Agr2 and mucin, by monitoring a read-out that is triggered by proximity of the two proteins. The capability of a test compound to interfere with or inhibit this interaction, and thus, the proximity of the two proteins, may then be readily determined by monitoring said read-out.

An example of such an assay, which readily allows the determination of Agr2/mucin binding or the capability of a test compound to inhibit the Agr2/mucin interaction, is the Amplified Luminescent Proximity Homogeneous Assay (ALPHA; Perkin Elmer). The ALPHA screening technology is a highly sensitive, chemiluminescence assay technology that allows the screening of a large range of biological interactions and activities and is also suitable for high-throughput screening (see, for example, Wilson et al., J. Biomol. Screen. 2003, Oct. 8 (5): 522- 32; Warner et al., Curr. Med. Chem. 2004 Mar. 11 (6): 721-30; or Glickman et al., J. Biomol. Screen. 2002 Feb. 7 (1): 3-10). This screening format offers very good sensitivity and specificity and generates fewer false-negative hits compared to fluorescence polarization. Briefly, donor and acceptor beads are used which are coated with a layer of hydrogel providing functional groups for bioconjugation. Close proximity of the beads is measurable as fluorescence signal, generated by label- transfer: upon laser excitation at 680nm a photosensitizer in the donor bead converts ambient oxygen to a more excited singlet state. The singlet state oxygen molecules diffuse across to react with a chemiluminescer in the acceptor bead that further activates fluorophores contained within the same bead. The fluorophores subsequently emit at 520-620nm. Accordingly, in one example of a method according to the invention,

Agr2 protein is immobilized to ALPHA screen donor beads using, e.g., purified or recombinant Agr2 protein, or a suitable Agr2 protein fragment. The protein may be directly coated or covalently linked onto the beads or coupled to the beads, e.g., via an Agr2 specific antibody. Likewise, a mucin protein, e.g., purified or recombinantly produced, or a suitable mucin fragment having Agr2 binding activity, or an Agr2 binding peptide, e.g., a peptide comprising or consisting of the sequence shown as SEQ ID NO:5, is immobilized onto ALPHA screen acceptor beads. In the absence of a specific molecular interaction due to the presence of an Agr2/mucin binding inhibitor, the singlet state oxygen molecules produced by the donor bead go undetected due to the insufficient proximity of the acceptor bead. Consequently, the fluorescence measured (the label-transfer signal) will be reduced in the presence of a candidate inhibitor compound tested, if that compound is capable of inhibiting the Agr2/mucin interaction.

The assays described above as well as other assays that will be apparent to those skilled in the art in view of the teaching of the present invention are suitable for identifying compounds that inhibit the interaction of Agr2 protein with mucins. In order to further confirm the usefulness of such compounds in the prevention, treatment, or amelioration of medical conditions associated with mucus hyperproduction or hypersecretion, the compounds thus identified may be additionally assayed for their safety and/or efficacy prior to, or after being formulated into pharmaceutical compositions. Such assaying may be done in animal tests, and optionally also in clinical trials on human subjects. Examples

Example 1 : Co-Localization of Agr2 and PDI in the Endoplasmatic Reticulum of

CaCo2 Cells

Agr2 protein co-localizes with protein disulphide isomerase (PDI) in the endoplasmatic reticulum (ER) of CaCO2 cells. PDI is a known marker for ER localization. CaCo2 cells, which endogenously express Agr2 and PDI, were grown in Dulbecco's modified eagle medium (DMEM) + 10% fetal calf serum (FCS) on glass slides. Cells were fixed and permeabilized by treatment with methanol/aceton for 20min at -20°C. Blocking was performed with 2% bovine serum albumine (BSA)/PBS for 30 min at room temperature. Grown cells were split for parallel assays of protein detection: PDI protein was detected by immuno-histochemistry, incubating cells for 1 hour at room temperature with a mouse anti-PDI antibody (cat. no. P71720, Becton Dickinson, USA), followed by a 30 min incubation with a second antibody, goat anti-mouse Alexa (cat. no. Al 1017, Molecular Probes, USA). Agr2 protein was detected by incubating cells for 1 hour with a rabbit anti Agr2 antiserum, followed by a 30 min incubation with a second antibody, goat anti mouse Alexa (cat. no. A21069, Molecular Probes, USA). Nucleus staining was performed with Hoechst33342 (cat. no. H3570, Molecular Probes, USA). Fluorescence staining was monitored with fluorescence microscopic technique, showing co-localization of Agr2 (Agr2, Figure IB) and PDI (PDI (ER marker), Figure IA) in the endoplasmatic reticulum of CaCo2 cells.

Example 2: Yeast Two-Hybrid Assay

A yeast-two hybrid experiment was performed, using a Matchmaker

Gal4 Two-Hybrid-System 3 (cat. no. PT3247-1, Clontech, USA). Data show protein- protein interaction between murine Agr2 and murine mucin2 (Agr2 x mucin2 fragment), as depicted in Figure 2. Agr2 protein was expressed fused to the DNA binding domain of Gal4, whereas murine mucin2 was expressed fused to the Gal4 transcriptional activator domain. Binding of the two fusions products was monitored based on the transcriptional activation of the reporter gene His3. Only yeast cells carrying the fusion products of Agr2 and mucin2 in a protein-protein interaction state are capable of growing on a yeast medium lacking histidine (interaction). The strength of protein-protein interaction correlates with the size of yeast colonies grown and is indicated as score value (+). Positive and negative controls were included.

In detail, a nucleotide sequence encoding amino acid positions 21 to 175 of murine Agr2 (SEQ ID NO:8), and a nucleotide sequence including positions 248 to 478 of the murine mucin2 mRNA, encoding 77 amino acids of murine mucin2 (see SEQ ID NO:7), were cloned into provided vectors, as described above and according to the instructions in the user manual. Transformation was done into yeast strain AH 109, according to the user manual. Fresh transformed yeast cells were plated on medium lacking histidine. Yeast transformed with Agr2 and mucin2 expression vectors grew at medium lacking histidine, indicating Agr2/mucin2 protein-protein interaction. Positive and negative controls were provided within the kit.

Example 3: Tracheal Ovalbumin Challenge in Wild Type and in Agr2-/- Mice

A tracheal ovalbumin challenge assay was performed using wild type (WT) mice and Agr2 mutant (Agr2-/-) mice. Tracheal ovalbumin challenge, performed by intratracheal ovalbumine instillation over a period of 21 days, induces goblet cell differentiation from tracheal epithelial cells and glycoprotein synthesis in the goblet cells of wt mice, as seen in Figure 3B. Such mice are well known as murine asthma model. Control mice do not display such phenotypic alterations in the trachea after 21 days of saline instillation, as shown in Figure 3 A. In Agr2-/- mice, lacking normal Agr2 function, neither differentiated goblet cell are visable nor synthesized glycoproteins are detectable after 21 days of ovalbumin instillation, as shown in Figure 3C. These data provide evidence for Agr2 function in goblet cell differentiation and glycoprotein synthesis. Mucins, in particular mucin5ac, are major components of such glycoproteins. Ovalbumin instillation (125μgram ovalbumine; 50μl final volume) was performed for 5 times at anesthetized mice within a period of 21 days. Mice were sacrificed at day 22 and histologically analyzed. Thin sections of trachea from treated and untreated mice were stained with rhodamine labeled wheat germ agglutinin (WGA) (cat. no. RL- 1022, Vector Laboratories, USA), binding to glycoproteins, including mucin5ac and mucin5b.

Cited Literature

Allen et al., (1998) Allen, A.; Hutton, D. A.; Pearson, J. P.: The MUC2 gene product: a human intestinal mucin. Int. J. Biochem. Cell Biol. 30: 797- 801, 1998.

Audie et al., (1993): Expression of Human Genes in Respiratory, Digestive, and Reproductive Tracts Ascertained by In Situ Hybridization, J. Histochem. Cytochem 41(10):1479-85.

Bartmann et al., (1998) The MUC6 secretory mucin gene is expressed in a wide varitey of epithelial tissues. J. Pathol. 1998 Dec. 186(4): 398-405.

Dekker,J., Rossen,J.W., Buller,H.A., and Einerhand,A-W. (2002). The MUC family: an obituary. Trends Biochem. Sci. 27, 126-131.

Farrell,C.L., Rex,K.L., Chen,J.N., Bready,J.V., DiPalma,C.R., Kaufman,S.A., Rattan,A., Scully,S., and Lacey,D.L. (2002). The effects of keratinocyte growth factor in preclinical models of mucositis. Cell Prolif. 35 Suppl 1:78-85., 78-85. Jackson,A.D. (2001). Airway goblet-cell mucus secretion. Trends Pharmacol. Sci. 22, 39-45.

Laboisse,C, Jarry,A., Branka,J.E., Merlin,D., Bou-Hanna,C, and Vallette,G. (1996). Recent aspects of the regulation of intestinal mucus secretion. Proc. Nutr. Soc. 55, 259-264.

Moniaux et al., (2001) Mucin (MUC) Gene Expression in Human Pancreatic Adenocarcinoma and Chronic Pancreatitis: A Potential Role of MUC4 as a Tumor Marker of Diagnostic Significance. Clin. Cancer Res. 7(12):4033-40

Vandenhaute et al., (1997) Mucin gene expression in biliary epithelial cells. J. Hepatology 27(6): 1057-66.

Verdugo,P. (1990). Goblet cells secretion and mucogenesis. Annu.

Rev. Physiol 52:157-76., 157-176.

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Voynow,J. (2002). What does mucin have to do with lung disease? Paediatr. Respir. Rev. 3, 98.

Sequence Listing

SEQ ID NO:1; amino acid sequence of human Agr2

MEKIPVSAFLLLVALSYTLARDTTVKPGAKKDTKDSRPKLPQTLSRGWGDQLIWTQT YEEALYKSKTSNKPLMIIHHLDECPHSQALKKVFAENKEIQKLAEQFVLLNLVYETT DKHLSPDGQYVPRIMFVDPSLTVRADITGRYSNRLYAYEPADTALLLDNMKKALKLL KTEL SEQ ID NO:2; amino acid sequence of human mucin2; NP_002448

MGLPLARLAAVCLALSLAGGSELQTEGRTRYHGRNVCSTWGNFHYKTFDGDVFRFPG LCDYNFASDCRGSYKEFAVHLKRGPGQAEAPAGVESILLTIKDDTIYLTRHLAVLNG AVVSTPHYSPGLLIEKSDAYTKVYSRAGLTLMWNREDALMLELDTKFRNHTCGLCGD YNGLQSYSEFLSDGVLFSPLEFGNMQKINQPDVVCEDPEEEVAPASCSEHRAECERL LTAEAFADCQDLVPLEPYLRACQQDRCRCPGGDTCVCSTVAEFSRQCSHAGGRPGNW RTATLCPKTCPGNLVYLESGSPCMDTCSHLEVSSLCEEHRMDGCFCPEGTVYDDIGD SGCVPVSQCHCRLHGHLYTPGQEITNDCEQCVCNAGRWVCKDLPCPGTCALEGGSHI TTFDGKTYTFHGDCYYVLAKGDHNDSYALLGELAPCGSTDKQTCLKTVVLLADKKKN AVVFKSDGSVLLNQLQVNLPHVTASFSVFRPSSYHIMVSMAIGVRLQVQLAPVMQLF VTLDQASQGQVQGLCGNFNGLEGDDFKTASGLVEATGAGFANTWKAQSTCHDKLDWL DDPCSLNIESANYAEHWCSLLKKTETPFGRCHSAVDPAEYYKRCKYDTCNCQNNEDC LCAALSSYARACTAKGVMLWGWREHVCNKDVGSCPNSQVFLYNLTTCQQTCRSLSEA DSHCLEGFAPVDGCGCPDHTFLDEKGRCVPLAKCSCYHRGLYLEAGDVVVRQEERCV CRDGRLHCRQIRLIGQSCTAPKIHMDCSNLTALATSKPRALSCQTLAAGYYHTECVS GCVCPDGLMDDGRGGCVVEKECPCVHNNDLYSSGAKIKVDCNTCTCKRGRWVCTQAV CHGTCSIYGSGHYITFDGKYYDFDGHCSYVAVQDYCGQNSSLGSFSIITENVPCGTT GVTCSKAIKIFMGRTELKLEDKHRVVIQRDEGHHVAYTTREVGQYLVVESSTGIIVI WDKRTTVFIKLAPSYKGTVCGLCGNFDHRSNNDFTTRDHMVVSSELDFGNSWKEAPT CPDVSTNPEPCSLNPHRRSWAEKQCSILKSSVFSICHSKVDPKPFYEACVHDSCSCD TGGDCECFCSAVASYAQECTKEGACVFWRTPDLCPIFCDYYNPPHECEWHYEPCGNR SFETCRTINGIHSNISVSYLEGCYPRCPKDRPIYEEDLKKCVTADKCGCYVEDTHYP PGASVPTEETCKSCVCTNSSQVVCRPEEGKILNQTQDGAFCYWEICGPNGTVEKHFN ICSITTRPSTLTTFTTITLPTTPTSFTTTTTTTTPTSSTVLSTTPKLCCLWSDWINE DHPSSGSDDGDREPFDGVCGAPEDIECRSVKDPHLSLEQHGQKVQCDVSVGFICKNE DQFGNGPFGLCYDYKIRVNCCWPMDKCITTPSPPTTTPSPPPTTTTTLPPTTTPSPP TTTTTTPPPTTTPSPPITTTTTPLPTTTPSPPISTTTTPPPTTTPSPPTTTPSPPTT TPSPPTTTTTTPPPTTTPSPPMTTPITPPASTTTLPPTTTPSPPTTTTTTPPPTTTP SPPTTTPITPPTSTTTLPPTTTPSPPPTTTTTPPPTTTPSPPTTTTPSPPTITTTTP PPTTTPSPPTTTTTTPPPTTTPSPPTTTPITPPTSTTTLPPTTTPSPPPTTTTTPPP TTTPSPPTTTTPSPPITTTTTPPPTTTPSSPITTTPSPPTTTMTTPSPTTTPSSPIT TTTTPSSTTTPSPPPTTMTTPSPTTTPSPPTTTMTTLPPTTTSSPLTTTPLPPSITP PTFSPFSTTTPTTPCVPLCNWTGWLDSGKPNFHKPGGDTELIGDVCGPGWAANISCR ATMYPDVPIGQLGQTVVCDVSVGLICKNEDQKPGGVIPMAFCLNYEINVQCCECVTQ PTTMTTTTTENPTPPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTG

TQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITT TTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPT GTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPIT TTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTP TGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPI TTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPT PTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTP ITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTP TPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTT PITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPT PTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTT TPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTP TPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPT TTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVT PTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTP TTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTV TPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQT PTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTT VTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQ TPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTT TVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGT QTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTT TTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTG TQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITT TTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPT GTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPIT TTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTP TGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPI TTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPT PTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTP ITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTP TPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTT PITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPT PTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTT TPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTP TPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPT TTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVT PTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTP TTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTV TPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTGPPTHTSTAPIAELTTSNPPPES STPQTSRSTSSPLTESTTLLSTLPPAIEMTSTAPPSTPTAPTTTSGGHTLSPPPSTT TSPPGTPTRGTTTGSSSAPTPSTVQTTTTSAWTPTPTPLSTPSIIRTTGLRPYPSSV LICCVLNDTYYAPGEEVYNGTYGDTCYFVNCSLSCTLEFYNWSCPSTPSPTPTPSKS TPTPSKPSSTPSKPTPGTKPPECPDFDPPRQENETWWLCDCFMATCKYNNTVEIVKV ECEPPPMPTCSNGLQPVRVEDPDGCCWHWECDCYCTGWGDPHYVTFDGLYYSYQGNC TYVLVEEISPSVDNFGVYIDNYHCDPNDKVSCPRTLIVRHETQEVLIKTVHMMPMQV QVQVNRQAVALPYKKYGLEVYQSGINYVVDIPELGVLVSYNGLSFSVRLPYHRFGNN TKGQCGTCTNTTSDDCILPSGEIVSNCEAAADQWLVNDPSKPHCPHSSSTTKRPAVT VPGGGKTTPHKDCTPSPLCQLIKDSLFAQCHALVPPQHYYDACVFDSCFMPGSSLEC ASLQAYAALCAQQNICLDWRNHTHGACLVECPSHREYQACGPAEEPTCKSSSSQQNN TVLVEGCFCPEGTMNYAPGFDVCVKTCGCVGPDNVPREFGEHFEFDCKNCVCLEGGS GIICQPKRCSQKPVTHCVEDGTYLATEVNPADTCCNITVCKCNTSLCKEKPSVCPLG FEVKSKMVPGRCCPFYWCESKGVCVHGNAEYQPGSPVYSSKCQDCVCTDKVDNNTLL NVIACTHVPCNTSCSPGFELMEAPGECCKKCEQTHCIIKRPDNQHVILKPGDFKSDP KNNCTFFSCVKIHNQLISSVSNITCPNFDASICIPGSITFMPNGCCKTCTPRNETRV PCSTVPVTTEVSYAGCTKTVLMNHCSGSCGTFVMYSAKAQALDHSCSCCKEEKTSQR EVVLSCPNGGSLTHTYTHIESCQCQDTVCGLPTGTSRRARRSPRHLGSG SEQ ID NO:3; amino acid sequence of human mucin5ac; P98088 (only C-terminal and central aa sequences)

TTSTTSASTTSTISPLTTSTTSAPITSMPSGPGTTPSPVPTTSTTSAPTTSTTSGPG TTPSPVPTTSTTSAPTTSTTSASTASTTSGPGTTPSPVPTTSTTSAPTTSTTSASTA STTSGPGTSLSPVPTTSTTSAPTTSTTSGPGTTPSPVPTTSTTSAPTTSTTSGPGTT PSPVPTTSTTPVSKTSTSHLSVSKTTHSQPVTSDCHPLCAWTKWFDVDFPSPGPHGG DKETYNNIIRSGEKICRRPEEITRLQCRAESHPEVNIEHLGQVVQCSREEGLVCRNQ DQQGPFKMCLNYEVRVLCCETPRGCPVTSVTPYGTSPTNALYPSLSTSMVSASVAST SVASSSVASSSVAYSTQTCFCNVADRLYPAGSTIYRHRDLAGHCYYALCSQDCQVVR GVDSDCPSTTLPPAPATSPSISTSEPVTELGCPNAVPPRKKGETWATPNCSEATCEG NNVISLSPRTCPRVEKPTCANGYPAVKVADQDGCCHHYQCQCVCSGWGDPHYITFDG TYYTFLDNCTYVLVQQIVPVYGHFRVLVDNYFCGAEDGLSCPRSIILEYHQDRVVLT RKPVHGVMTNEIIFNNKVVSPGFRKNGIVVSRIGVKMYATIPELGVQVMFSGLIFSV EVPFSKFANNTEGQCGTCTNDRKDECRTPRGTVVASCSEMSGLWNVSIPDQPACHRP HPTPTTVGPTTVGSTTVGPTTVGSTTVGPTTPPAPCLPSPICHLILSKVFEPCHTVI PPLLFYEGCVFDRCHMTDLDVVCSSLELYAALCASHDICIDWRGRTGHMCPFTCPAD KVYQPCGPSNPSYCYGNDSASLGALREAGPITEGCFCPEGMTLFSTSAQVCVPTGCP RCLGPHGEPVKVGHTVGMDCQECTCEAATWTLTCRPKLCPLPPACPLPGFVPVPAAP QAGQCCPQYSCACNTSRCPAPVGCPEGARAIPTYQEGACCPVQNCSWTVCSINGTLY QPGAVVSSSLCETCRCELPGGPPSDAFVVSCETQICNTHCPVGFEYQEQSGQCCGTC VQVACVTNTSKSPAHLFYPGETWSDAGNHCVTHQCEKHQDGLVVVTTKKACPPLSCS LDEARMSKDGCCRFCPLPPPPYQNQSTCAVYHRSLIIQQQGCSSSEPVRLAYCRGNC GDSSSMYSLEGNTVEHRCQCCQELRTSLRNVTLHCTDGSSRAFSYTEVEECGCMGRR CPAPGDTQHSEEAEPEPSQEAESGSWERGVPVSPMH

SEQ ID NO:4; predicted amino acid sequence of human mucin5b; XP 039877

MGAPSACRTLVLALAAMLVVPQAETQGPVEPSWENAGHTMDGGAPTSSPTRRVSFVP PVTVFPSLSRKQMLPLPAGKGVFASPKGGGPDLGVQLPPALNPAHNGRVCSTWGDFH YKTFDGDVFRFPGLCNYVFSEHCRAAYEDFNVQLRRGLVGSRPVVTRVVIKAQGLVL EASNGSVLINGQREELPYSRTGLLVEQSGDYIKVSIRLVLTFLWNGEDSALLELDPK YANQTCGLCGDFNGLPAFNEFYAHSECHLDARLTPLQFGNLQKLDGPTEQCPDPLPL PAGNCTDEEGICHRTLLGPAFAECHALVDSTAYLAACAQDLCRCPTCPCATFVEYSR QCAHAGGQPRNWRCPELCPRTCPLNMQHQECGSPCTDTCSNPQRAQLCEDHCVDGCF CPPGTVLDDITHSGCLPLGQCPCTHGGRTYSPGTSFNTTCSSCTCSGGLWQCQDLPC PGTCSVQGGAHISTYDEKLYDLHGDCSYVLSKKCADSSFTVLAELRKCGLTDNENCL KAVTLSLDGGDTAIRVQADGGVFLNSIYTQLPLSAANITLFTPSSFFIVVQTGLGLQ LLVQLVPLMQVFVRLDPAHQGQMCGLCGNFNQNQADDFTALSGVVEATGAAFANTWK AQAACANARNSFEDPCSLSVENENYARHWCSRLTDPNSAFSRCHSIINPKPFHSNCM FDTCNCERSEDCLCAALSSYVHACAAKGVQLSDWRDGVCTKYMQNCPKSQRYAYVVD ACQPTCRGLSEADVTCSVSFVPVDGCTCPAGTFLNDAGACVPAQECPCYAHGTVLAP GEVVHDEGAVCSCTGGKLSCLGASLQKSTGCAAPMVYLDCSNSSAGTPGAECLRSCH TLDVGCFSTHCVSGCVCPPGLVSDGSGGCIAEEDCPCVHNEATYKPGETIRVDCNTC TCRNRRWECSHRLCLGTCVAYGDGHFITFDGDRYSFEGSCEYILAQDYCGDNTTHGT FRIVTENIPCGTTGTTCSKAIKLFVESYELILQEGTFKAVARGPGGDPPYKIRYMGI FLVIETHGMAVSWDRKTSVFIRLHQDYKGRVCGLCGNFDDNAINDFATRSRSVVGDA LEFGNSWKLSPSCPDALAPKDPCTANPFRKSWAQKQCSILHGPTFAACRSQVDSTKY YEACVNDACACDSGGDCECFCTAVAAYAQACHDAGLCVSWRTPDTCPLFCDFYNPHG GCEWHYQPCGAPCLKTCRNPSGHCLVDLPGLEGCYPKCPPSQPFFNEDQMKCVAQCG CYDKDGNYYDVGARVPTAENCQSCNCTPSGIQCAHSLEACTCTYEDRTYSYQDVIYN TTDGLGACLIAICGSNGTIIRKAVACPGTPATTPFTFTTAWVPHSTTSPALPVSTVC VREVCRWSSWYNGHRPEPGLGGGDFETFENLRQRGYQVCPVLADIECRAAQLPDMPL EELGQQVDCDRMRGLMCANSQQSPPLCHDYELRVLCCEYVPCGPSPAPGTSPQPSLS ASTEPAVPTPTQTTATEKTTLWVTPSIRSTAALTSQTGSSSGPVTVTPSAPGTTTCQ PRCQWTEWFDEDYPKSEQLGGDVESYDKIRAAGGHLCQQPKDIECQAESFPNWTLAQ VGQKVHCDVHFGLVCRNWEQEGVFKMCYNYRIRVLCCSDDHCRGRATTPPPTTELET ATTTTTQALFSTPQPTSSPGLTRAPPASTTAVPTLSEGLTSPRYTSTLGVAGGDMET FENIRAAGGKMCWAPKSIECRAENYPEYPSHQLHGHALLNSGDDLDPHKADHNSHYD CVHWIHGHPDLHPENSSPSQRTTHTPPVPNTMATTHGRSLPPSSPHTVRTAWTSATS GILGTTHITEPSTVTSHTLAATTGTTTPGHTTATSRTTATATPSKTRTSTLLPSSPT SAPITTVVTMGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLE CRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPS TPATSSTAMPSSTPGTTWILTELTTTATTTESTGSTATPSSTPGTTWILTEPSTTAT VTVPTGSTATASSTQATAGTPHPRDGTHASSVDQHNHHTHNQRFHGTTHTPPVPNTT ATTHGRSLSPSSPHTVRTAWTSATSGTLGTTHITEPSTGTSHTPAATTGTTTPGHTR ATSRTTATATPSKTRTSTLLPSSPTSAPITTVVTMGCEPQCAWSEWLDYSYPMPGPS GGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNRE QVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTEQTTAATTTAT TGSTAIPSSTPGTAPPPKVLTSTATTPTATSSKATSSSSPRTATTLPVLTSTATKST ATSFTPIPSFTLGTTGTLPEQTTTPMATMSTIHPSSTPETTHTSTVLTTKATTTRAT SSMSTPSSTPGTTWILTELTTAATTTAATGPTATPSSTPGTTWILTEPSTTATVTVP TGSTATASSTRATAGTLKVLTSTATTPTVISSRATPSSRTTHTPPVPNTTATTHGRS LPPSSPHTVRTAWTSATSGILGTTHITEPSTVTSHTPAATTSTTQHSTPALSSPHPS SRTTESPPSPGTTTPGHTRGTSRTTATATPSKTRTSTLLPSSPTSAPITTVVTTGCE PQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRE LGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSST PGTTWILTKLTTTATTTESTGSTATPSSTPGTTWILTEPSTTATVTVPTGSTATASS TQATAGTPHPRDGTHASSVDQHNHHTHNQWLHGTTHTPPVPNTTATTHGRSLSPSSP HTVRTAWTSATSGTLGTTHITEPSTGTSHTPAATTGTTTPGHTTATSRTTATATPSK TRTSTLLPSSPTSAPITTVVTTGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAA GGAVCEQPLGLECRAQAQPGVPLGELGQVVECSLDFGLVCRNREQVGKFKMCFNYEI RVFCCNYGHCPSTPATSSTAMPSSTPGTTWILTELTTTATTTASTGSTATPSSTPGT APPPKVLTSPATTPTATSSKATSSSSPRTATTLPVLTSTATKSTATSVTPIPSSTLG TTGTLPEQTTTPVATMSTIHPSSTPETTHTSTVLTTKATTTRATSSTSTPSSTPGTT WILTELTTAATTTAATGPTATPSSTPGTTWILTELTTTATTTASTGSTATPSSTPGT TWILTEPSTTATVTVPTGSTATASSTQATAGTPHSKDCNHPSSADKHSHKIHSYQLY THPLLHPVDHVDRTTWILTEPSTIATVMVPTGSTATASSTLGTAHTPKVVTTMATMP TATASTVPSSSTVGTTRTPAVLPSSLPTFSVSTVSSSVLTTLRPTGFPSSHFSTPCF CRAFGQFFSPGEVIYNKTDRAGCHFYAVCNQHCDIDRFQGACPTSPPPVSSAPLSSP SPAPGCDNAIPLRQVNETWTLENCTVARCVGDNRVVLLDPKPVANVTCVNKHLPIKV SDPSQPCDFHYECECICSMWGGSHYSTFDGTSYTFRGNCTYVLMREIHARFGNLSLY LDNHYCTASATAAAARCPRALSIHYKSMDIVLTVTMVHGKEEGLILFDQIPVSSGFS KNGVLVSVLGTTTMRVDIPALGVSVTFNGQVFQARLPYSLFHNNTEGQCGTCTNNQR DDCLQRDGTTAASCKDMAKTWLVPDSRKDGCWAPTGTPPTASPAAPVSSTPTPTPCP PQPLCDLMLSQVFAECHNLVPPGPFFNACISDHCRGRLEVPCQSLEAYAELCRARGV CSDWRGATGGLCDLTCPPTKVYKPCGPIQPATCNSRNQSPQLEGMAEGCFCPEDQIL FNAHMGICVQACPCVGPDGFPKFPGERWVSNCQSCVCDEGSVSVQCKPLPCDAQGQP PPCNRPGFVTVTRPRAENPCCPETVCVCNTTTCPQSLPVCPPGQESICTQEEGDCCP TFRCRPQLCSYNGTFYGVGATFPGALPCHMCTCLSGDTQDPTVQCQEDACNNTTCPQ GFEYKRVAGQCCGECVQTACLTPDGQPVQLNETWVNSHVDNCTVYLCEAEGGVHLLT PQPASCPDVSSCRGSLRKTGCCYSCEEDSCQVRINTTILWHQGCETEVNITFCEGSC PGASKYSAEAQAMQHQCTCCQERRVHEETVPLHCPNGSAILHTYTHVDECGCTPFCV PAPMAPPHTRGFPAQEATAV

SEQIDNO:5

QETRGGGWTLSSSETRRNSYCTTACTTVTIHCTLHPGSPGLQEFDIKLIDTVDLEGG PGTQFAL

SEQ ID NO:6; amino acids 1 to 77 of human mucin2

MGLPLARLAAVCLALSLAGGSELQTEGRTRYHGRNVCSTWGNFHYKTFDGDVFRFPG LCDYNFASDCRGSYKEFAVH

SEQ ID NO:7; 77 amino acids fragment of murine mucin2, derived from murine mucin2 mRNA sequence, Genbank Ace. No. BC034197, nucleotide positions 248 to 478

MPTSSKTTTGPTSPTTRPPSTSTPTSFTVPTETTTQTRPLSTTPTTLETTRTSSWGT FSSTSPITSPSTVWTHTETQ

SEQ ID NO:8; 155 amino acids ofmurine Agr2, amino acids 21-175 ofGenbank Acc.No.NP_035913

KDTTVKSGAKKDPKDSRPKLPQTLSRGWGDQLIWTQTYEEALYRSKTSNRPLMVIHH LDECPHSQALKKVFAEHKEIQKLAEQFVLLNLVYETTDKHLSPDGQYVPRIVFVDPS LTVRADITGRYSNRLYAYEPSDTALLYDNMKKALKLLKTEL

Claims

1. A compound capable of inhibiting the interaction of Agr2 protein with a mucin protein.

2. The compound of claim 1, wherein said Agr2 protein is human Agr2 according to SEQ ID NO: 1 , and wherein the mucin protein is a human mucin.

3. The compound of claim 1 or 2, wherein said mucin is mucin2, preferably mucin2 having an amino acid sequence which comprises or consists of the amino acid sequence of SEQ ID NO.2.

4. The compound of claim 1 or 2, wherein said mucin is mucinSac, preferably mucin5ac having an amino acid sequence which comprises or consists of the amino acid sequence of SEQ ID NO:3.

5. The compound of claim 1 or 2, wherein said mucin is mucin5b, preferably mucin5b having an amino acid sequence which comprises or consists of the amino acid sequence of SEQ ID NO:4.

6. The compound of any of claims 1-5, wherein the compound is a peptide or a polypeptide.

7. The compound of claim 6, wherein the peptide or polypeptide has an amino acid sequence comprising from 2 to 138 amino acids, preferably from 32 to 136 amino acids, also preferably from 2 to 16 amino acids.

8. The compound of claim 6 or 7, wherein the peptide or polypeptide is characterized by a serine and/or threonine rich amino acid sequence.

9. The compound of claim 8, wherein the sequence comprises one or more uninterrupted repeats of at least two, at least three, or at least four serine or threonine residues.

10. The compound of claim 6, wherein the compound has or comprises the amino acid sequence of SEQ ID NO:5.

11. The compound of claim 6, wherein the compound is a fragment of mucin protein, for example a fragment of human mucin2, mucin5ac or mucin5b, preferably a fragment of human mucin2, mucin5ac or mucin5b as defined in any of claims 3-5, said fragment optionally being fused to another protein.

12. The compound of claim 6 or 11, wherein the sequence of the compound comprises or consists of amino acids 1 to 77 of the N-terminal part of human mucin2 according to SEQ ID NO:6, or the 77 amino acid sequence of murine mucin2 according to SEQ ID NO: 7.

13. The compound of any of claims 1-5, wherein the compound is a small molecule, preferably a small molecule having a molecular weight of no more than 2000 Dalton, or no more than 1500, 1000, 500, 400, 300 or 200 Dalton.

14. The compound of any of claims 1-5 or 13, wherein the compound is a peptidomimetic.

15. The compound of any of claims 1-14, wherein the compound binds to said Agr2 protein.

16. The compound of claim 15, wherein the binding to the Agr2 protein occurs with a binding affinity that is similar to, or higher than the binding affinity of said mucin to said Agr2 protein.

17. The compound of claim 15, wherein the binding of said compound to said Agr2 protein is characterized by a dissociation constant (Kd) equal to or lower than 1000 μmol, preferably equal to or lower than 100 μmol, or equal to or lower than a value in the range from 10 μmol to 10 nmol.

18. A pharmaceutical composition comprising a compound according to any of claims 1-17 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein the composition is in the form of a liposome delivery system.

20. The pharmaceutical composition of claim 19, wherein the liposomes are selected from small unilamellar vesicles, large unilamellar vesicles, preferably large unilamellar vesicles composed of a mixture of cationic and anionic lipids, and multilamellar vesicles.

21. The pharmaceutical composition according to any one of claims 18 to 20, wherein the composition is suitable for delivery as an aerosol.

22. A method of preventing, treating, or ameliorating a medical condition associated with mucus hypeφroduction/hypersecretion in a mammal, particularly in a human subject, said method comprising administering to said mammal or said human subject a pharmaceutical composition comprising a compound according to any of claims 1-17, or a pharmaceutical composition according to any of claims 18-21.

23. The method of claim 22, wherein the medical condition is one affecting the respiratory system, preferably a medical condition selected from the group consisting of asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis.

24. The compound according to any of claims 1-17 for use in therapy.

25. The compound of claim 24 for the use specified therein, wherein the compound is for the prevention, treatment, or amelioration of a medical condition associated with mucus hyperproduction/hypersecretion.

26. The compound of claim 24 or 25 for the use specified therein, wherein the medical condition is one affecting the respiratory system, preferably a medical condition selected from the group consisting of asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis.

27. Use of the compound according to any of claims 1-17 for the preparation of a pharmaceutical composition for preventing, treating, or ameliorating a medical condition associated with mucus hyperproduction/hypersecretion.

28. The use of claim 27, wherein the medical condition is one affecting the respiratory system, preferably a medical condition selected from the group consisting of asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis.

29. A method for identifying a compound capable of interfering with the Agr2/mucin interaction, said method comprising assaying the ability of a test compound to inhibit the binding of Agr2 protein to mucin protein.

30. The method of claim 29, wherein the Agr2 protein is human Agr2 protein according to SEQ ID NO:1, and wherein the mucin protein is a human mucin.

31. The method of claim 29 or 30, wherein said mucin is selected from the group consisting of mucin2, mucin5ac and mucin5b, preferably mucin2, mucin5ac and mucin5b as defined in claims 3, 4, and 5, respectively.

32. The method of any of claims 29-31 , wherein the test compound is a peptide or polypeptide as defined in any of claims 6-12, a small molecule as defined in claim 13, or a peptidomimetic as defined in claim 14.

33. The method of any of claims 29-32, wherein the ability of said test compound to inhibit the binding of Agr2 protein to mucin protein is tested in a cell-based interaction assay.

34. The method according to any of claims 29-32, wherein the ability of said test compound to inhibit the binding of Agr2 to mucin protein is tested in a cell free binding assay, e.g., a homogeneous binding assay.

35. A method of preparing a pharmaceutical composition useful in the treatment of a medical condition associated with mucus hyperproduction/hypersecretion, preferably a medical condition affecting the respiratory system, said method comprising

(a) by the method of any of claims 29-34 identifying a compound capable of inhibiting the binding of Agr2 protein to mucin protein; and

(b) formulating the compound thus identified into a pharmaceutical composition.

36. A method of preparing a pharmaceutical composition useful in the treatment of a medical condition selected from the group consisting of asthma, allergic reactions of the respiratory system, COPD, and cystic fibrosis, said method comprising

(b) formulating the compound thus identified into a pharmaceutical composition.

37. The method of claim 35 or 36, further comprising analyzing the safety and efficacy of said compound and/or pharmaceutical composition in animal tests, and optionally also in clinical trials on human subjects.

38. The method of claim 22 or 23, the compound of any of claims 24-27 for the use specified therein, or the use of claim 28 or 29, wherein the compound or the pharmaceutical composition is administered intratracheally, intranasally, or transmucosally.