BG105158A - Prevention of the formation of mucus coating on the respiratory tracts by the administration of egf-r antagonist - Google Patents

Abstract

Hypersecretion of mucus in the lungs is inhibited by the administration of an epidermal; growth factor receptor (EGF-R) antagonist. The EGF-R antagonist may be in the form of a small organic molecule, an antibody, or portion of an antibody that binds to and blocks the EGF receptor. The EGF-R receptor antagonist is preferably administered by injection in an amount sufficient to inhibit formation of goblet cells in pulmonary airways. The degranulation of goblet cells that result in airway mucus production is thereby inhibited. Assays for screening candidate agents that inhibit goblet cell proliferation are also provides. 27 claims

Description

TECHNICAL FIELD

In general, the invention relates to the field of pulmonary treatment. More specifically, the invention relates to suppression of mucus over-secretion in the lungs and airways by administering an antagonist against EGF-R. In addition, the present invention also relates to methods for the development and evaluation of proposed agents that are capable of suppressing the secretion of mucus in the lungs.

BACKGROUND OF THE INVENTION

In the respiratory tract from the respiratory system, the secretory system serves as the primary protective mechanism for the removal of inhalable particles or infectious agents outside the respiratory tract into the lungs. In addition, the substances present in the respiratory fluid are used to limit the toxicity of the particles and to deactivate infectious agents. The physical mechanism of coughing serves to expel secretion from the airways (see, e.g., Foundations of Respiratory Care, Piesrson and Kacmarek, eds (1992) Churchill Livingstone Inc. New York; Harrison's Principles of Intematics Medicine Fauci et al. , eds (1997) 14th Edition, McGraw Hill, New York, New York).

The secretory system consists of twisted epithelial cells, goblet epithelial cells, and serum and mucus secreting cells located in the submucosal glands. The twisted bundle (cilia) is surrounded by a single aqueous layer (periciliary fluid), which is secreted in the passage (lumen) of the respiratory canal by the active transfer of chloride and the passive movement of water through the epithelium. The bundle comes into contact with the mucus floating on this water layer and, in a unidirectional rotational motion, promotes the movement of the mucus towards the pharynx (see Pierson and Kacmarek, supra and Fauci, et al., Supra). The mucus is produced by epithelial goblet cells and submucosal cells and is secreted in the airway after degranulation.

While mucus usually helps to clear away inhaled particles or infectious agents, the excess secretion of mucus in the respiratory tract can cause them to progressively block. In peripheral roads, coughing is not effective for clearing secretions. Moreover, because of their small size, small airways containing many tapestry cells are highly susceptible to mucus obstruction. Excessive airway secretion affects a significant number of people; this is evident in various pulmonary diseases such as chronic bronchitis, acute asthma, cystic fibrosis and bronchiectasis.

Mucosal secretion is a major symptom in patients with chronic pulmonary obstruction (COPD) and determines the condition (eg, chronic cough and sputum). This condition alone affects 14 million Americans and can cause progressive disability and death. Asthma has been estimated to affect at least 4% of the US population and causes at least 2,000 deaths per year (Pierson and Kucmarek, supra). During an acute asthmatic attack, the bronchial walls swell, increase the volume of secretions and contract the smooth bronchial muscle, narrowing the airway. As a result of over-secretion in acute asthma, a large-scale mucus blockage can be a major cause of morbidity and mortality.

Excess secretion is also involved in dietary fibrosis, which is one of the most common fatal genetic diseases in the world. Cystic fibrosis is a recurrent autosomal disease,

which causes the mucous cell in the airway to become insensitive to the activation of cyclic AMP-dependent protein kinase in the membrane of chloride ion channels (Pierson and Kacmarek, supra and Fauci, et al., supra). The ensuing electrolyte imbalance reduces the level of hydration of the mucus in the respiratory tract, resulting in high density mucus in the lungs of the individual and they are affected by dietary fibrosis. Excess secretion clogs the body's air ducts with dietary fibrosis, which subsequently impairs lung function.

Other diseases of over-secretion include chronic pulmonary obstruction (COPD). Stress from oxidizing agents has an important role in the pathogenesis of COPD. Cigarette smoke, which produces oxygen-free radicals, is highly associated with pathogenesis. Neutrophils are common at the site of inflammation in COPD, and it is interesting to note that oxygen-free radicals are released by the neutrophils during activation.

Mechanical intubation is often required to provide involuntary inhalation of patients with various pulmonary diseases. For this purpose, a tube is inserted through the oropharynx and inserted into the trachea. To prevent air leakage around the endotracheal

a tube, a balloon inflates around the tube in the lower trachea, which can rub the epithelium and cause metaplasia of the goblet cells. Injury to the epithelium leads to recovery processes that can lead to copious secretions. Such prolonged incubation of the trachea may lead to adverse effects in patients due to over-secretion.

High secretion in the lungs of patients with pulmonary disease with secretion decreases secretion clearance. Pathological agents, such as bacteria, e.g. Pseudomonas aeruginosa, often create secretions colonies, causing frequent pulmonary infections.

Classical forms of treatment for individuals with respiratory tract secretion include antibiotic treatment, bronchodilators (such as methylxanthines, sympathomimetics with strong β2 adrenergic stimulating properties, anticholinergics), use of systemic or inhaled corticosteroids, top secretion by administration oral to expectorants, such as goifenesin, and aerosol delivery of "mycolytic" agents such as water, hypertonic saline (see Harrison's supra). One new therapy for cystic fibrosis is the administration of DNAse by targeting DNA rich mucus or phlegm (Shak, et al., (1990) Proc. Natl. Acad. (USA) 87: 9188-9192; Hubbard, RC et. al., (1991) N. Engl. J. Med. 326: 812). In addition, breast physical therapy consisting of percussion, vibration and drainage is also used to help clear mucus with high density. A lung transplant may be one extreme option for patients with severe pulmonary disease. Therefore, more effective or alternative treatment that addresses mucus secretions is needed. In particular, there is a need for a specific form of therapy that will reduce airway secretion.

References

The use of EGF inhibitors to stop the growth of cancer cells has been reviewed by Levitski (1994) Eur J. Biochem. 226 (1): 1-13; Powis (1994) Pharmac. Ther. 62: 57-95; Kondapaka and Reddy (1996) Mol. Cell. Endocrine. 117: 53-58.

SUMMARY OF THE INVENTION

Overexpression of mucus in the respiratory tract is an unfavorable symptom in a number of different lung diseases. Secretion is the result of goblet cell degranulation, the development of which is supported by stimulation of the epidermal growth factor receptor (EGF-R). The present invention treats pulmonary over-secretion by administering therapeutic amounts of EGF antagonists, preferably kinase inhibitors. The antagonists may be in the form of small molecules, antibodies, or antibody portions that bind to EGF or its receptor. In another aspect of the invention, in vitro or in vivo methods are provided that predict the therapeutic potential of the agents being proposed to suppress excess secretion.

It is a primary object of the invention to provide a method of treating diseases involving over secretion in the lungs.

It is another object of the invention to provide compositions which are useful in the treatment of diseases involving over secretion in the lungs.

It is also another object of the invention to provide an in vitro assay for the evaluation of the proposed agents that suppress overexpression, this method comprising the steps of (I) contacting an in vitro model of rapid development of goblet cells with EGF or its functional equivalent; (ii) subsequently contacting an in vitro model with one agent available; and (iii) assessing the rapid growth of the goblet cells, whereby suppressing this increase is indicative of the therapeutic potential of the agent proposed.

It is another object of the invention to provide an in vivo assay for the evaluation of proposed agents that suppress mucus over-secretion, the method comprising (i) creating an animal model of pulmonary over-secretion disease by inducing EGF-R, e.g. with tumor necrosis factor alpha (TNF-α); (ii) stimulating the induced EGF-R with its ligand, i. transformation into alpha growth factor (TNF-α) or EGF to produce mucin-producing goblet cells; (iii) treatment with the agent proposed; and (iv) assessing the growth of the goblet cells or the secretion of mucus, the inhibition of goblet cell growth or secretion being indicative of the therapeutic potential of the proposed agent.

It is another object of the invention to provide in vitro and in vivo studies for the evaluation of EGF-R antagonists that suppress mucus overexpression.

An advantage of the invention is that it provides a means of preventing excessive mucus formation in the airways.

One feature of the invention is that a range of different types of antagonists can be used to block the effects of EGF and / or TGF-a and their interaction with EGF-R.

One aspect of the invention is compositions of EGF antagonists for reducing the secretion of airways in a mammalian patient, preferably a human patient.

Another aspect of the invention is one method for the pulmonary administration of EGF antagonists to reduce mucus secretion in the mammalian airways, preferably a human patient.

Another aspect of the invention is to provide a method of treating a range of various diseases that have as a symptom excessive formation of mucus secretions in the respiratory tract. These diseases include, but are not limited to, chronic bronchitis, acute asthma, dietary fibrosis, bronchiectasis, chronic obstructive pulmonary diseases, over-secretion resulting from epithelial disorders such as allergic stimuli or mechanical damage, and over-secretion through the nose.

These and other objects, advantages and features of the invention will be elucidated by those skilled in the art after learning the details of the in vitro and in vivo treatment methods and methods, which are fully described below.

EXPLANATION OF THE FIGURES Attached

Figure 1A is a western print of EGF-R in NCLH292 and in A431 cells. Figure 1B, immunocytochemical analysis with antiEGF-R antibody in cultures of NCL-H292 cells. Figure 1C Northern analysis of EGF-R in NCL-H292 cells.

Figure 2. Alcium blue / PAS staining of NCL-H292 cells for identification of mucin glycoproteins.

Figure 3. Northern analysis for expression of the MUC5 gene in HCLH292 cells.

Figure 4A and 4B. Immunohistochemical analysis of EGF-R with an anti-EGF-R antibody in pathogen-free rats. Figure 4A, TNFα-treated rats. Figure 4B, rats with ovalbumin sensitivity.

Figure 5 is a graph showing the effect of EGF-R tyrosine kinase inhibitor (BIBX1522) on goblet cell production (expressed as% of stained area in the airway epithelium occupied by Alpine blue / PAS-positive stained cells).

EXAMPLES FOR THE IMPLEMENTATION OF THE INVENTION

Methods and compositions are provided for the treatment of mucosal secretion in the respiratory tract by administering therapeutic amounts of EGF antagonists, preferably kinase inhibitors. The antagonists may be in the form of small molecules, antibodies, or a portion of antibodies that bind EGF or its receptor. In diseases with excessive secretion of the respiratory tract, such as chronic bronchitis, bronchiectasis, cystic fibrosis, acute asthma, COPD, etc. increases the synthesis of mucin in the respiratory tract and excess mucus secretion occurs. The secretion of mucus causes obstruction in the respiratory tract, an effect that causes death in these diseases.

Several causes of airway damage and inflammation are shown here to stimulate the expression of the epidermal growth factor receptor in airway epithelial cells. Following EGF-R induction, subsequent stimulation of EGF-R by both ligand-dependent and ligand-independent mechanisms leads to the production of mucin both in gene expression and in protein levels. Selected EGF-R tyrosine kinase inhibitors have been demonstrated to block this expression of mucin in gene and protein.

It is suggested, without limiting the invention, that an evolutionary sequence of goblet cell production may be based on the expression of EGR-R. TNFα stimulation stimulates intense staining of unpelleted secreted cells; their subsequent activation by EGF-R ligands induces increasing staining for mucosal glycoconjugates in the cytoplasm, with the cells becoming pre-goblet and then goblet cells. The data suggest that EGF-R activation exhibits selective cell differentiation but no increase. Apparently, tablet cells are derived from non-granular secreted cells that express EGF-R and are stimulated by EGF-R ligands to produce mucin.

In addition to cytokine stimulation, EGR-F can be stimulated by other signaling mediators. For example, prolonged smoking is associated with progressive pathological changes in the peripheral airways, including tablet cell hyperplasia. Inflammatory cytokine and cigarette smoke-activated neutrophils have been shown to induce mucin synthesis in human bronchial epithelial cells by ligand-independent activation of EGF-R, involving recruited neutrophils and cigarette smoke as regulators of epithelial cell differentiation induction of mucin-producing cells in the respiratory tract. Neutrophils activated by a variety of stimuli, including IL-8, N-formyl-methionyl-leucyl-phenylanine, TNF-α, cigarette smoke or H ₂ O, regulate the expression of mucin in epithelial cells whose synthesis is inhibited by EGF-R inhibitors . The neutrophils are also able to produce EGR-F ligands, EGF and TGFα. In addition, epithelial cells are sources of EGF-R ligands.

Mechanical injury to the epithelium of the respiratory tract can also cause over-secretion and cause mucosal obstruction. EGF-R tyrosine kinase inhibitors serve to prevent mucosal over-secretion after tracheal intubation.

Epithelial damage is often found in the study of patients, even with mild asthma, and the damage is increasingly associated with worsening clinical symptoms. Epithelial damage resulting from an allergic reaction triggers EGF-R activation, resulting in super-normal production of goblet cells. EGF-R is involved in epithelial damage, such as "airway remodeling," which occurs in asthma, wound repair, and closure. Mechanical damage to the epithelium and injury to the epithelium in asthma may involve a similar EGF-R growth sequence that results in an abnormal increase in epithelial secretion cells.

Overexpression is also an important demonstration of inflammatory diseases of the nose. When goblet cells in the nose are "irritated" by inducing goblet cell degranulates using a neutrophil-dependent mechanism, the expression of EGF-R and mucin is highly regulated. These events are related to re-granulation of goblet cells. When inflammation, such as stimulation of neutrophil infiltrate, causes goblet cell breakdown and mucin secretion, EGF-R overregulation and activation again deliver mucin to the airway epithelium.

Before describing the present treatment methods and compositions, it should be understood that this invention is not limited to specific methods and compositions as described, since, of course, they can be modified. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not considered to be limiting, since the scope of the present invention will be limited to the appended claims only.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly interpreted by anyone with normal experience in the practice to which this invention belongs. Although similar or equivalent methods and materials to those described herein may be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to indicate and describe the methods and / or materials in connection with the publications cited.

The publications reviewed here are provided solely for the purpose of disclosure prior to the filing date of this application. Nothing here can be interpreted as assuming that the present invention is not entitled to make such publication at an earlier date by virtue of a prior invention. Further, publication dates may differ from actual publication dates, which may need to be independently verified.

DEFINITIONS

By "epidermal growth factor" or "EGF" is meant one protein or part thereof having biological activity characterized by the mitogenic activity of epithelial cells ((eg Cohen (1986) Biosciences Reports 6 (12): 1027; Aaronson , SA, Growth Factors and Cancer, Science <1991) 254: 1146-1153). The human epidermal growth factor, for example as described by Urdea et al., (1983) Proc. Nat. Acad, Sei. 80: 7461-7465.

The mitogenic activity of EGF on goblet cells is of particular interest for the purposes of the present invention. This definition also seeks to include proteins and parts thereof that are functional equivalent to EGF with respect to the biological reaction caused by EGF.

By "epidermal growth factor receptor" or "EGFR" is meant one protein or a portion thereof capable of binding an EGF protein or part thereof. An example is the human epidermal growth factor receptor (see Ulrich et al., (1984) Nature 309: 418-425; Gene Bank Assay No. NM 005228). Preferably, the binding of the EGF ligand activates EGF-R (resulting in activation of intracellular mitogenic signaling, autophosphorylation of EGF-R). Anyone experienced in the art will appreciate that other ligands, in addition to EGF, can bind to and activate EGF-R. Examples of such ligands include, but are not limited to: TGF-α, betacellulin, amphiregulin, heparin-binding EGF (ΗΒ-EGF), and nuregulin (also known as hergulin) (Strawn and Shawver (1998) Exp.-Opin, Invest Drugs 7 (4) 553753, and "The Protein Kinase Facts Book; Tyrosine Kinasis Protein" (1995) Hardie, et al., (Eds., Academic Press, NY, NY).

By "EGF-R antagonist" is meant any agent that is able to directly or indirectly suppress the effect of EGF-R, in particular the effect of EGF-R on goblet cell growth or the secretion of goblet cell mucus. EGF-R can be triggered by ligand-dependent and ligand-independent mechanisms, resulting in autophosphorylation or trans-phosphorylation, respectively. Interesting EGF-R antagonists can suppress one or both of the above mechanisms, for example, binding of THF-α to EGFR results in a ligand-dependent phosphorylation that can be blocked by an antibody that binds EGF-R by this way prevents EGF from interacting with a ligand that would activate the EGF receptor. Examples of such antibodies are described by Golds ein et al., (1995) Clin. Cancer really. 1: 1311-1318; Lorimer et. al., (1995) Clin. Cancer Res. 1: 859-864; Schmidt and Weis (1996) Br. J. Cancer 74:85 3-862. Small molecule tyrosine kinase inhibitors are also effective as EGF-R antagonists.

Alternatively, compounds such as free oxygen radicals have been shown to stimulate EGF-R transphosphorylation, producing ligand-independent receptor activation. Other means of activating EGF-R by transphosphorylation include ultraviolet and osmotic stress, stimulation of G-prototype coupled with endothelin-1, lysophosphatidic acid and thrombin, receptor of ml muscarinic acetylcholine, and human growth hormone. The antagonists of this ligand-independent mechanism include antioxidants such as uperm oxide dismutase, DMSO, DMTU, ascorbic acid and the like.

An EGF-R antagonist may be an antibody that binds to a factor that stimulates the production of EGF or EGF-R, thus suppressing EGF help for growth of goblet cells (i.e., a developmental inhibitor of phosphorylation affecting EGF-R). For example, a fusion protein of T 3FaPseudomonas exotoxin 40, as described by Arteaga et al., (1995) Cancer Res. 54: 4703-4709.

In one preferred embodiment, the EGF-R antagonist is an inhibitor of tyrosine kinase activity, in particular small molecule inhibitors having a selective effect on EGF R, compared to other therosin kinases - the preferred small molecules block the natural receptor of EGF- R is preferably in a mammal and have a molecular weight of less than 1 kD.

EGF and EGF-R inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as quinazolines such as PD 153035, 4- (3-chloroanilino) quinazoline, or CP-358,774, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, 59 pyrrolopyrimidines, pyrrolopyridines , CGP 60261 and CGP 62706, and pyrazolopyrimidines (Shawn and Shawver, supra.), 4- (phenylamino) -7H-pno [2,3-d] pyrimidines (Traxler et al., (1996) J. Med. Chem 39 : 2285-2292), curcumin (diferuloyl methane) (Lexamin arayana, et al., (1995), Carcinogen 16: 1741-1745) 4-5-bi (4fluoroanilino) phthalimide (Buchdunger et al., (1995) Clin. Cancer Res 1: 813-821; Dinney et al., (1997) Clin. Cancer Res. 3: 161-168); tyrphostins containing nitrothiophene moieties (Brunton et al., (1996) Anti Cancer Drug Design 11: 265-295); the protein kinase inhibitor ZD-1839 (AstraZeneca); CP-358774 (Pfizer, Inc.); PD-0183805 (Warner-Lambert); or as described in International Patent Application W099 / 09016 (American Cyanamid; WO98 / 43960 (American Cyanamid); WO97 / 38983 (Warener Labert); WO99 / 06378 (Warner Lambert); WO99 / 06396 (Warner Lambert); WO96 / 30347 (Pfizer, Inc.); WO96 / 33977 (Zeneca) and WO96 / 33980) Zeneca; all are included here for reference; or antisense molecules.

By "suppression" is meant to neutralize, attenuate or prevent the growth of goblet cells, degranulation of goblet cells or the over-secretion of goblet cell mucus.

The terms "treatment", "cure" and the like are used herein in a general sense, to achieve the desired pharmacological and / or other physiological effect. The effect may be prophylactic, in the sense of complete or partial prevention of disease or symptom, and / or may be therapeutic, in the sense of partial or complete cure of the disease and / or deleterious effect contributing to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, especially a human, and includes:

(a) preventing the onset of a disease or symptom in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it;

(c) suppression of the disease or symptom, i. stopping their development; or (c) alleviating the disease or symptom, i. causing regression in the disease or symptom. The invention is directed to the treatment of patients with pulmonary or respiratory diseases, and in particular to the treatment of patients with mucus over-secretion, i. preventing, suppressing or alleviating mucus over-secretion. In the context of treating symptoms, the invention is directed to reducing mucus or sputum in the respiratory tract, suppressing infection by pathological organisms, relieving cough, and preventing hypoxia due to obstruction of the respiratory tract.

More specifically, "treatment" is intended to mean providing a therapeutically traceable and beneficial effect in a patient suffering from pulmonary disease including mucus over-secretion.

More specifically, "treatment" should mean preventing, alleviating and / or suppressing mucus over-secretion with a composition selected from the group of EGF and EGF-R antagonists such as antibodies, protein tyrosine kinase inhibitors, and antisense molecules and the like. . Alternative treatment may include preventing the expression of EGF-R in the airways by blocking the passage in the early phase. For example, reagents that block the binding of TNFα to its receptors may prevent the growth of EGF-R.

Treatment includes the prevention or suppression of infections by pathological agents caused by and / or associated with mucus over-secretion.

An "antibody" means a protein immunoglobulin that is able to bind an antigen. An antibody as used herein should include fragments of an antibody, e.g. F (ab ') 2, Fab', Fab, which are able to bind the antigen or fragment of the antigen to be considered. Preferably, binding of an antibody to the α and γ gene inhibits the activity of EGF and EGF-R.

The term "humanized antibody" is used here to sleep complete antibody molecules, e.g. consisting of two complete light chains and two complete heavy chains, as well as antibodies, having only fragments of antibody e.g. Fab, Fab ', F (ab') 2 and Fv, wherein the CDRs are derived from a non-human source and the remainder of the Ig molecule or fragment is derived from a human antibody, preferably derived from a nucleic acid sequence encoding a human antibody.

The terms "human antibody" and "humanized antibody" are used herein to describe an antibody in which all parts of the molecule, as well as fragments of the antibody, are described above in the definition of the term "humanized antibody".

The term "chimeric antibody" encompasses humanized antibodies. Chimeric antibodies have at least one portion of a heavy or light amino acid chain derived from a first mammalian species and another portion of a heavy or light amino acid chain derived from a second, different mammalian species. Preferably, the variable zone is derived from a non-human mammal species and the permanent zone is derived from a human species. Specifically, the chimeric antibody is preferably derived from one nucleotide sequence from a non-human mammal encoding a variable region and one nucleotide sequence from a human object encoding the constant portion of an antibody.

By "specific binding" is meant a strong craving and / or strong affinity for binding an antibody to a specific polypeptide. An antibody binding to its epitope in one specific polypeptide is stronger than binding to the same antibody to another epitope, especially one that may be present in molecules bound to or in the same sample as the specific polypeptide under consideration. Antibodies that bind specifically to one polypeptide under consideration may be able to bind other polypeptides to one weak but noticeable level, e.g. 10% or less of the binding demonstrated to the polypeptide in question. Such a weak binding, or basic binding, is well distinguished from the specific antibody bound to the compound or polypeptide under consideration, e.g. using appropriate controls.

By "distinct labeled body", "distinct labeled anti-EGF" or distinct labeled anti-EGF fragment "is meant an antibody (or antibody fragment that retains the binding specificity) that has a recognizable label attached. This label

is normally attached by chemical conjugation, where the label is a polypeptide, optionally it can be attached by genetic engineering technology. Methods for producing distinct labeled proteins are well known in the art. Distinctive labels can be selected from one of many such labels known in the art, but usually these are radioisotopes, fluorophores, enzymes, such as horseradish peroxidase or other parts or compounds that either emit a distinct signal (such as radioactivity, fluorescence, color), or emit a distinct signal after being labeled with a substrate. Various distinct label / substrate pairs (such as peroxidase / diaminobenzidine, avidin / streptavidin, luciferase / luziferin), as well as antibody labeling methods and methods for using labeled antibodies

for the detection of an antigen are well known in the art (e.g., see Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

THERAPEUTIC METHODS

The present invention provides a method of treating pulmonary over-secretion by administering therapeutic amounts of EGF-R antagonists. Any disease, and in particular a pulmonary disease characterized by excessive secretion of the mucus or the accumulation of pathological levels of the mucus, can be treated by the methods described herein. Examples of pulmonary over-secretion diseases that can be treated by this method include, but are not limited to, chronic diseases of the lung, such as chronic bronchitis, inflammatory diseases such as asthma, bronchiectasis, pulmonary fibrosis, COPD diseases, through the nose, such as nasal allergies and other over-secreted diseases. Genetic diseases such as cystic fibrosis, Cartagena syndrome, alpha-Gantitrypsin deficiency, congenital cystic fibrosis with accumulation of mucus in the respiratory tract are also considered.

Antagonists that directly target EGF EGF-R are preferred. In addition, anyone with knowledge and experience in the field will appreciate that any factor or cell that is involved in the biological sequence that promotes EGF-R goblet cell growth may be a target for TGFα antagonist inhibition. Without being bound theoretically, a sequence (cascade) begins during an inflammation reaction when cells, such as fat or neutrophils, release TNFα, which then stimulates the emergence of EGF-R. Stimulation of EGF-R, e.g. EGF ligand, in turn, triggers the growth of goblet cells. Thus, all cells or factors involved in the cascade, as well as in the THFα pathway, can be targets of antagonist activity.

EGF-R antagonists administered by the therapeutic method may take a variety of forms. By way of example, the EGF-R antagonist may be in the form of a small molecule (such as an antisense oligonucleotide, a tyrosine kinase inhibitor, etc.) antibodies or a portion of antibodies that bind to EGF, TGFα or EGF-R.

SMALL MOLECULES AS EGF-R ANTAGONISTS

Tyrosine kinase inhibitors that act on the EGF receptor and are selective for EGF-R are known in the art and can be used in the proposed methods. Examples which may include BIBX 1522 Boehringer Ingelheim, Inc., Ingelheim, Germany) are described above; CGP59326B (Novartis Corporation, Basel, Switzerland); 4-aminoquinazoline EGF-R inhibitors (described in U.S. Patent 5,760,041); substituted sterol compounds, which may also be naphthalene, indane or one benzoxazine; including nitrile and malononitrile compounds (described in U.S. Patent 5,217,999) the inhibitors disclosed in U.S. Patent 5,773,476; potato carboxypeptidase (PCI) inhibitor, one 39-amino acid protease inhibitor with three disulfide bridges. (Blanco-Aparicio et al., (1998) J. Biol Chem 273 (200: 12370-12377); Bombesin antagonist RC-3095 (Szepeshazi et al., (1997) Proc Natl Acad Sci USA 94: 10913-10918) and Other tyrosine kinase inhibitors include quinazolines, such as D153035, 4- (3-chloroanilino) quinazoline, or CP-358,774, pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261, and CGP 6270wn, and CGP 6270wn, and CGP 6270wn, supra), 4- (phenylamino) -7H-pyrrolo [2,3-0] pyrimidines (Traxler et al. (1996) J. Med. Chem. 39: 2285-2292) curcumin (Korutla et al. (1994) Biochim Biophys Acta 1224: 587: 600); (Laxmin arayana (1995);

Preferred tyrosine kinase inhibitors have a selective effect on the EGF receptor, with the EGF-R receptor being more inhibited by other cell surface receptors having tyrosine kinase activity. This selectivity is enhanced by the delivery methods of formulations and drugs, i. when the inhibitor is preferably administered to the inflamed airways, and the like.

Typical dosages for systemic administration are in the range of 0.1 µg to 100 milligrams per kg bodyweight of the subject for single administration. Specialists can immediately appreciate that dose levels can change as a function of the specific compound, the severity of symptoms, and the susceptibility of the subject to side effects. Some of the specific compounds are stronger than others. Preferred dosages for a compound can be readily determined by one skilled in the art. One preferred means is to measure the physiological strength of a compound, for example, with the in vitro and in vivo assays described herein.

ANTIBODIES AS EGF-R ANTAGONISTS

Antibodies such as AGF-R antagonists are of particular interest (e.g., Viloria, et al., American Journal of Pathology 151: 1523). EGF and EGF-R antibodies are obtained by immunization with a xenogeneic immunocompetent mammalian host, including mice, ungulates, lagomorphs, sheep, pigs, cattle and the like. with EGF or EGF-R, or parts thereof. As an immunogen, human EGF or EGF-R, or portions thereof, are preferably used. Choosing a particular receiver is basically a matter of convenience. Immunizations are performed in accordance with conventional techniques, the immunogen being injectable subcutaneously, intramuscularly, intramuscularly, intravenously, and the like. in the animal host. Normally, about 1 mg / kg to about 10 mg / kg of EGF or EGF-R is administered intramuscularly as an immunogen daily. Injections can be with or without an adjuvant, such as a complete or incomplete adjuvant of Freund, spec, alum, etc. Upon completion of the immunization program, an antiserum may be harvested in accordance with conventional methods of producing a polyclonal antiserum specific for EGF or EGF-R.

Both the monoclonal and polyclonal antibodies are obtained from the immunized animal, preferably monoclonal antibodies. Polyclonal antisera can be collected from serum, by conventional methods, from animals after completion of the immunization program. For the production of monoclonal antibodies, lymphocytes from the spleen, drainage of the lymph node, etc. are collected from a suitable lymphoid tissue and synthesized with a synthesizer, usually a myeloma line, to produce a hybridoma that secretes a specific monoclonal antibody. In accordance with conventional methods, a review of the clones of the antigen specificity hybridomas of interest is performed.

Of particular interest are antibodies, preferably monoclonal antibodies, that bind to EGF or EGF-R so as to suppress EGF-binding to EGF-R, i.e. an antibody that specifically binds to the extracellular region of EGF-R, thereby preventing EGF binding. Such antibodies can be obtained by the conventional methodology described above or by other known methods in the art. Examples of antibodies that would function as EGF antagonists include, but are not limited to: neutralizing anti-EGF-R monoclonal antibody C225 (Kawamoto et al., (1993) Proc. Nat'l. Acad Sci. (USA) 80 : 1337-1341; Petit et al., (1997) J. Path. 151: 1523-153, produced by ImClone Systems Nwe York, NY) and the anti-EGF-R monoclonal antibody EMD55900 (also called Mab 425), (Merck, Darmstadt , Germany).

The proposed antibodies can be obtained as a single strand instead of a normal multimeric structure. Single chain antibodies are described in Jost et al., (1994) J.B.C. 269: 26267-73, and others. The DNA sequences encoding the heavy chain variable region and light chain variable region are linked to a single spacer encoding at least 4 amino acids of small, neutral amino acids, including glycine and / or series. The protein encoded by this synthesis allows the construction of a variable region that preserves the specificity and affinity of the original antibody.

Methods for humanizing antibodies are known in the art. The humanized antibody may be an animal product having genes from the constant region of transgenic human immunoglobulin (see, for example, International Patent Applications WO 90/10077 and WO 90/04036). Alternatively, such an antibody can be achieved by genetic engineering of recombinant DNA technologies, to replace CHI, CH2, CH3, joining regions and / or framework sludge with a corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for the production of chimeric immunoglobulin genes is also known in the art (Liu et al. (1987) PNAS 84: 3489 and (1987) J. Immunol. 139: 3521. The mRNA is isolated from a hybridoma or other antibody producing cell using specific primers (US Patent Nos. 4,683,195 and 4,683,202) Alternatively, a library can be made that is screened to isolate the sequence of interest. connects to human sequences from a constant region The human region C genes are located in known clones. Then, the chimeric, humanized antibody is expressed in terms of the human immunogenic antibody, which is expressed in Rabat et al. (1991) Sequences of Proteins of Immunological Interest, NIH publication No. 91-3242. conventional methods.

Fragments of an antibody such as Fv, F (ab ') 2 and Fab can be obtained by cleaving the intact protein, e.g. with protease or chemical cleavage. Alternatively, a truncated gene is constructed, for example, a chimeric gene encoding part of a fragment of F (ab ') 2 would comprise DNA sequences encoding a CH1 region and a binding region of a single H chain, followed by a translation stop codon to extract cut off molecule.

In an individual suffering from a mucous-secretion disease, initially an amount of an EGF-R antagonist may be administered in the range of about 20 mg to about 400 mg per kg of patient weight, twice daily, e.g. by inhalation.

ANTISENS MOLECULES AS EGF-R ANTAGONISTS

In another embodiment of the invention, the therapeutic agents provided are antisense molecules specific for human sequences encoding EGF or EGF-R. The therapeutic agent introduced may be antisense oligonucleotides, in particular synthetic oligonucleotides having chemical modifications from natural nucleic acids, or nucleic acid constructs that exhibit such antisense molecules as RNA. The antisense sequence is complementary to the mRNA of attacked EGF or EGF-R genes and inhibits the expression of attacked genius products (see, for example, Nyce et al. (1997) Nature 385: 720). Antisense molecules suppress gene expression by reducing the amount of mRNA available for translation by activating PHKse H or a spatial obstacle. One or a combination of several antisense molecules may be introduced, and the combination may include numerous different sequences of a single attacked gene or sequences that complement several different genes.

One preferred attack gene is EGF or EGF-R. Access to the gene sequence can be obtained through publicly available basins (human epidermal growth factor, access to Genabank No. K01166; human epidermal growth factor receptor precursor mRNA, access to Genabank No. X00588). Typically, the antisense sequence would have the same ancestral origin as the animal host.

Antisense molecules can be obtained by expressing all or part of the attacked gene sequence in a suitable vector, the vector being introduced and represented in the attacked cells. The transcription start is oriented in such a way that the antisense strand is obtained as a single RNA molecule. Antisense RNA hybridizes with the endogenous sensitive (sense) bundle of mRNA, blocking the expression of the attacked gene. A natural transcription start region or an exogenous natural start transcription region may be used. The promoter can be introduced by recombinant methods in vitro or as a result of homologous integration of the sequence into the chromosome. Many, strong, muscle-accelerating accelerators are known in the art, including β-actin accelerator, early and slow-acting SV40 accelerators, human cytomegalovirus accelerator, retroviral LTRs, etc.

Transcription vectors typically have convenient restriction sites located close to the accelerator sequence to aid in nucleic acid sequence alignment. Transcription cassettes can be prepared by including one transcription start region, the gene or fragment thereof, and one transcription termination region. Transcription cassettes can be introduced into a plurality of vectors, such as a plasmid, a retrovirus, or a lintivirus; and the like, the vectors being able to remain in the cell in a transient or stable state, typically for at least one day, more often for at least several days.

Alternatively, in a preferred embodiment of the invention, the antisense molecule is a synthetic oligonucleotide. The antisense nucleotides are generally from about 7 to 500, usually from 12 to 50 nucleotides, more often from about 20 to 35 nucleotides, the length being governed by the inhibition efficiency, specificity, including the lack of cross-reactivity and the like. Short oligonucleotides, 7 to 8 bases in length, have been found to be potent and selective inhibitors of gene expression (see Wagner et al. (1996) Nature Biotrchnology 14: 840-844).

One specific region (s) from the endogenous gpRNA strand-sensitive strand is selected to complement the antisense sequence. The 5 'sRNA region has been shown to be particularly susceptible to antisense inhibition. Recent facts, however, indicate that analysis of the secondary structure of the sRNA may play an important role in the accessibility of countries to suppression. Selection of a specific sequence of oligonucleotides may employ an empirical method in which several proposed sequences are tested to suppress the expression of the attacked gene in an in vitro or animal model. One combination of sequences may also be used, with several regions of the mRNA sequence selected for antisense supplementation.

Antisense oligonucleotides can be chemically synthesized by methods known in the art (see Wagner et al. (1993) supra, and

Milligan et al., Supra.). Preferred oligonucleotides are chemically modified by a natural phosphodiester structure in order to increase their intracellular resistance and binding affinity. Several such modifications have been described in the literature that alter the chemistry of the skeletons of oligonucleotides, sugars or heterocyclic bases.

The oligonucleotides may further comprise an attacking portion that enhances the uptake of the molecule by the cells. The attacking portion specifically binds the molecules, e.g. an antibody or portion thereof that recognizes molecules present on the surface of lung epithelial cells, in particular, epithelial cells containing EGF-R.

Double specific antibodies, chimeric antibodies and single chain antibodies are known in the art. Properly prepared non-human antibodies can be humanized in various ways. The linkage between the oligonucleotide and the attacked portion can be accomplished by any conventional method, for example by a disulfide, amide or thioether linkage, depending on the chemistry of the oligonucleotide skeleton. Preferably, the linkage is cleaved within the cell to release an oligonucleotide.

The oligonucleotides can be conjugated to hydrophobic precipitates, e.g. cholesterol to protect the nuclei and improve the transfer through the membranes of cells. Alternatively, conjugation to poly-L-lysine or other polyamines may also enhance cell delivery. Another modification can be made by adding one accommodating component, such as acridine, which is able to fit into the attacked mRNA and stabilize the resulting hybrid. Antisense oligonucleotides can be combined with an enzyme (s) that degrades antisense-TPPK complexes in the cell, e.g. RNase-H. Similarly, any protein or enzyme that may advantageously degrade or secrete antisense-mRNA duplexes may also be useful.

As an alternative to antisense inhibitors, catalytic nucleic acid compounds may be used to suppress gene expression, e.g. ribozymes, antisense conjugates, etc. Ribozymes can be synthesized in vitro and introduced into a patient, or can be encoded into a single vector of expression from which the ribisim is synthesized in the attacked cell (for example, see International Patent Application WO / 9523225, and Beigelman et al. (1995) Nucl. Acids Res 23: 4432-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Antisense conjugates of a single metal complex, e.g. terfyridylCi (I) which are capable of promoting the hydrolysis of mRNA are described in Bashkin et al. (1995) Appl Biochem Biotechnol 54: 43-56

PHARMACEUTICAL RECIPES

EGF-R antagonists can be obtained in solution or in any other pharmaceutically suitable form of administration, such as a liposomal suspension. Suitable antibodies or other forms of antiEGF are prescribed for administration in a conventional manner for administration of such materials. Typical formulations are listed in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. The route of administration is selected on the basis of the compound to be administered, the patient's condition and the disease to be treated. When there is excess mucus secretion, the compound may be administered by different routes depending on the severity of the disease, e.g. emergencies may require in vitro administration; acute but non-life-threatening conditions may be treated orally while chronic treatment may be conducted with an aerosol.

For therapeutic use in diseases of the nose and respiratory tract, local delivery is preferred. Aerosol inhalation provides a high level of drug concentrations compared to systemic administration concentrations. Alternatively, the EGF antagonist may be administered by injection, including intramuscularly, intravenously (IV), with subcutaneous or abdominal injections, preferably IV and local injections. Other forms of administration may be used, provided that agents are available that allow the EGF-R antagonist to enter the systemic circulation, such as muscle and subcutaneous formulations, which may be administered as capsules, stickers or through the nose. Any suitable formulation that affects the transfer of the EGF-R antagonist to the bloodstream or locally into the lungs can be used properly.

For injection, generally suitable formulations include aqueous solutions or suspensions using saline, Hank's solution, or other buffers, optionally containing stabilizing agents or other small components. Liposomal preparations or other forms of microemulsions may be used. The EGF-R antagonist can also be delivered in lyophilized form and converted for administration. Muscle and subcutaneous injections generally include agents that aid the passage of the muscle or skin barrier, such as bile fluid, salts, fusic acid, and their analogs, various disinfectants, and the like. Oral administration is also possible provided that appropriate enteric coatings are prescribed to enable EGF-R antagonists to survive in the digestive tract.

The nature of the formulation will depend to some extent on the nature of the EGF-R antagonist selected. A suitable formulation is prepared using known techniques and principles for formulations that are well known to those skilled in the art. The percentage of EGF-R antagonists contained in a given pharmaceutical composition will also depend on the nature of the formulation; the percentage of an EGF-R antagonist that is an antibody will typically vary over a wide range from about 1% by weight to about 85% by weight.

Many delivery methods are known in practice to enhance the uptake of nucleic acids from cells. Useful delivery systems include Sendai virus-liposomal delivery systems (Rapaport and Shai (1991) J. Biol. Chem. 269: 15124-15131), cationic liposomes, polymeric feed gels or matrices, porous balloon catheters (such as disclosed by Shy et al. (1994) Circulation 90: 955-951; and Shi et al. (1994) Gene Therapy 1: 408-414). retrovirus expression vectors and the like.

The use of liposomes as a delivery vehicle is an interesting method of administration of EGF-R antagonists. The liposomes fuse with the cells in the attacked side and deliver the lumen content intracellularly. Liposomes remain in contact with cells for a time sufficient to fuse, using various means to maintain contact, such as isolation, binding agents and the like. Liposomes can be made with purified proteins or peptides that mediate membrane penetration, such as Sendai virus or influenza virus, and the like. Lipids can be any useful combination of known liposomes forming lipids, including cationic lipids such as phosphatidylcholine. The residual lipid normally represents neutral lipids such as cholesterol, phosphatidyl serine, phosphatidyl glycerol and the like. For the preparation of liposomes, the procedure described by Kato et al. (1991) J. Biol. Chem. 266: 3361.

In one preferred embodiment, the EGF-R antagonist is encapsulated in sterile stabilized "covert" liposomes, i. E. fixed liposomes. When such liposomes are injected i.v., they remain in the bloodstream for long periods. The post-capillary nodes in the veins of the veins open when the airways are inflamed and allow fluid to be collected and molecules left, e.g. adjunct, kininogen, to enter the tissues, initiating inflammatory cascades. Such inflammation allows the liposomes and their contents to be selectively deposited in the inflamed tissue (Zhang et al. (1998) Pharm Res 15: 455-460).

EGF-R antagonists may be administered to the affected patient via a single pharmaceutical respiratory delivery system. The compositions may be formulated in a form suitable for administration by inhalation. The pharmaceutical delivery system is one that is suitable for respiratory therapy by administering EGF-R antagonists to the mucosal lines of the bronchi. The present invention can use a system that depends on the force of compressed gas to push EGF-R antagonists out of a container. An aerosol or pressurized kit may be used for this purpose.

As used herein, the term "aerosol" is used in its conventional sense as it relates to the transport of very fine liquid or solid particles of a pressurized moving gas to the site of therapeutic administration. When a pharmaceutical aerosol is administered in this invention, it contains a therapeutically active compound that can be dissolved, suspended or emulsified in a mixture of liquid carrier and vehicle. The aerosol can be in the form of a solution, suspension, emulsion, powder or semi-solid preparation. The aerosols included in the present invention are intended for administration as fine, particulate matter or as liquid mist through the patient's respiratory tract. Can be used

different types of moving media known to those skilled in the art. Examples of suitable propellants include, but are not limited to: hydrocarbons or other suitable gases. In the case of pressurized aerosol, the unit dose can be determined by indicating the feed rate from the measured quantity.

The present invention can also be implemented with a nebulizer, which is a device for creating very fine particles of liquid, predominantly of the same shape, in gas. Preferably, a liquid containing EGF-R antagonists is sprayed. Small droplets can be carried by a gas stream through the nebulizer outlet pipe. The resulting mist penetrates the patient's respiratory tract.

A powder composition comprising EGF-R antagonists or analogs thereof, with or without a lubricant, carrier or vehicle, may be administered to a mammal in need of therapy. This embodiment of the invention can be achieved with a conventional device for the administration of a powder pharmaceutical composition by inhalation. For example, a powder mixture of the composition and a suitable base powder such as lactose or starch can be presented in a single dose, such as a capsule or cartridge, e.g. gelatin or blister packs from which the powder can be introduced using an inhaler.

A combination of therapies may be used to treat pulmonary diseases with over secretion. In particular, EGF-R antagonists may be combined with conventional treatment to relieve excessive secretion, such as bronchodilators, expectorants, mucolytic agents, and the like, to aid mucosal cleansing.

Depending on the condition of the patient, it may be preferable to administer the composition of the present invention by injection (e.g., intravenously) or by inhalation. Patients with a large amount of mucus in their lungs generally cannot initially

I are treated with inhalation. This is due to the fact that the lungs of the patient are significantly obstructed, so inhaling aerosol compositions in the lungs may not be particularly effective. However, after injection or, alternatively, maintenance for a prolonged period or in cases where the patient's lungs are not severely obstructed, administration by inhalation is preferred. This is preferable because smaller doses can be targeted locally to specific cells that are most in need of treatment. By filing

at smaller doses, all adverse side effects are eliminated or significantly reduced. By delivering directly to the cells most in need of treatment, the effect of treatment is achieved more quickly.

There are several different types of inhalation methodologies that may be applied in connection with the present invention. The antagonists of the present invention can be formulated basically into three different types of inhalation compositions. First, anatagonists of the invention can be combined with low boiling point media. Such formulations are generally administered by conventional dosimetric inhalers (MDI). In doing so, conventional MDIs can be modified to increase the ability to obtain repeatable dosing using technology that measures the patient's respiratory volume and flow rate, as discussed in U.S. Pat. Nos. 5,404,871 and 5,542,410.

Alternatively, the antagonists of the present invention may be incorporated into aqueous or ethanol solutions and administered with conventional nebulizers. Preferably, such formulations are aerosolized using devices and systems as disclosed in U.S. Pat. Nos. 5,497,763; 5,544,646; No. 5,718,222 and 5,660,166.

Finally, the antagonist compositions of the present invention can be formulated as dry powder formulations. Such formulations may be administered by the ordinary inhalation of the dry powder after the formation of the aerosol mist of the powder. A technology for this is described in U.S. Patent 5,775,320, issued July 7, 1998, and U.S. Patent 5,740,794, issued April 21, 1998.

Considering all the patents cited above, the Applicants note that these patents cite other publications in the field of pulmonary drug administration and such publications should be considered for the specific methodology, devices and formulations that may be used in connection with administration of antagonists of the present invention. Further, each of these patents is incorporated herein for the purpose of referring to their completeness for the purposes of the disclosed formulations, devices, packaging, or methodology for filing formulations with antagonists of the present invention.

VERIFICATION RESEARCH

Available drugs

Verification studies may be used to determine the proposed bioactive agents as EGF antagonists. Of particular interest are screening studies on agents that have low toxicity to human cells. For this purpose, a wide range of studies can be used, including in vitro labeled protein-binding assays, electrophoretic mobility shift assays, protein binding immunoassays, and the like. Purified EGF or EGF-R protein can also be used to determine the three-dimensional crystal structure that can be used to model molecular interaction, transfer function, and the like.

The term &quot; agent &quot;, as used herein, describes each molecule, e.g. protein or pharmaceutical agent capable of altering or inhibiting the physiological function of EGF or EGF-R. Generally, a plurality of assay mixtures are operated in parallel at different concentrations of the agent to produce a different reaction to different concentrations. Usually, one of these concentrations serves as a negative control, ie. at zero concentration or below detection level.

Applicant agents cover numerous chemical classes, although typically, they are organic molecules, preferably organic compounds with a molecular weight of over 50 and less than about 2500 daltons. Applicant agents include functional groups required for structural interaction with proteins, in particular hydrogen binders, and typically include at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The agents involved often comprise cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted by one or more of the above functional groups. Participating agents can also be detected among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, their structural analogs or combinations.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, there are numerous agents for the arbitrary or direct synthesis of a wide variety of organic compounds and biomolecules, including the expression of arbitrary oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds, in the form of bacterial, fungal, plant and animal extracts, are available or produced. In addition, natural or synthetic

manufactured libraries and compounds can be easily modified by conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Known pharmacological agents may be subjected to targeted or arbitrary chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to obtain structural analogues.

When the screening test is a binding assay, one or more molecules may be coupled to a single label, the label being able to directly or indirectly give a distinct signal. Various labels include radioisotopes, fluorescents, chemiluminescents, enzymes, specific binding molecules, particles such as magnetic particles and the like. Specific binding molecules include couples such as biotin and streptavidin, digoxin and antidigoxin and the like. In specific binding participants, the adjuvant is normally labeled with a single molecule that allows tracing in accordance with a known procedure.

Various other reagents may be included in the screening studies. These include reagents such as salts, neutral proteins, e.g. albumin, disinfectants, etc., which are used to help optimally bind protein to protein and / or reduce uncharacteristic or basic interactions. Reagents that improve the effectiveness of the assay may be used, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like. The mixture of components is added in a row that provides the necessary binding. Incubation is carried out at a suitable temperature, typically between 4 and 40 ° C. The incubation periods are selected for optimum activity, but can also be optimized to facilitate quick review. Usually 0.1 to 1 hour is sufficient.

The compounds of the desired pharmaceutical activity may be administered with a physiologically acceptable carrier in a patient for the treatment of over-secretion disease with the formulations described herein. Depending on the nature of the administration, the compounds can be formulated in various ways that are described herein. The concentration of the therapeutically active compound in the formulation may vary from about 0.1-100% by weight.

Dosing mode

The appropriate dosage level will vary depending on a number of factors including the subject to be treated, the nature of the over-secretion state to be treated and its severity, the nature of the EGF-R antagonist to be administered as the active ingredient, the formulation the practitioner's introduction, formulation, and judgment. For example, when antibodies are introduced by themselves, such as anti-EGF or EGF-R into a composition for injection, the dosages are in the range of 20 mg / kg to about 40 mg / kg as a single dose. Re-entry after a period of several days or intravenous administration for a long period may be required. Under chronic conditions, administration may be continued for longer periods if necessary.

The efficacy of the dosing regimen is determined by evaluating the patient's improved lung function. This evaluation may include measurement of viscoelasticity of the sputum, improvements in lung function, including improvements in the compulsively investigated volume of sputum, and maximum exhalation flow. The aforementioned therapeutic regimen may be administered in conjunction with concomitant therapies, such as antibiotics, DNAase I, or other current therapies for the treatment of pulmonary over-secretion. If antibiotics are introduced as part of the patient's treatment, a quantitative evaluation of the bacteria may be included after therapy to evaluate the efficacy of the treatment by reduced bacterial growth, decreased mucosal or sputum viscosity, and increased mucosal or lung clearance. confused.

Lung function evaluation tests as well as diagnostic tests for the clinical development of pulmonary disease with hyper secretion are known to those skilled in the art. Standard lung function tests include airway resistance (AR); forced vital capacity (FVC); forced exhalation volume for 1 sec (FEV (l)); forced average exhalation volume; and Peak Exhalation Flow (PEFR). Other functional pulmonary tests include blood gas analysis; drug reaction; irritation and exercise testing; measuring the strength of the respiratory muscles; optical fiber respiratory examination; and the like. Some basic procedures for studying the properties of mucus include rheology, e.g. using a magnetic micrometer, adhesiveness to characterize the forces of attraction between the adhesive surface and one adhesive system by measuring the contact angle between the mucus drop and the surface. The transfer of mucus from cilia (fibers on the cell surface) can be investigated using conventional technologies as well as by direct measurement, e.g. in situ cleansing of the mucus. The transseptic potential difference, as a net result of the activity of the ion-transport system of the pulmonary epithelium, can be measured using suitable electrodes. Quantitative morphology methods can be used to characterize the condition of the epithelial surface.

The treated patient may be a primate, such as a human being, or any other animal demonstrating the symptoms described. As the method of the invention is particularly suited for the treatment of human patients, it is clear that the invention is also applicable in veterinary practice.

In vitro examination assay

In another embodiment of this invention, in vitro studies are used to evaluate the therapeutic potential of the agents proposed to suppress goblet cell growth, i. the extent to which such agents are active as an EGF antagonist. Generally, such studies cover the following steps: (i) contact of an in vitro model of a goblet cell growth with EGF or its functional equivalent; (ii) subsequent contact of the in vitro model with the agent proposed; (iii) estimation of the goblet cell growth, suppressing the goblet cell growth is indicative of the therapeutic potential of the proposed agent.

The assay is preferably performed with two controls, with the second group of cells not coming in contact with the compound and the third group being in contact with EGF but not with the proposed agent. Then, comparisons were made to determine the extent of the effect of EGF and the proposed agent on the cells.

Any in vitro model of goblet cell growth can be used. As an example, rat tracheal cells can be isolated and cultured as described in Guzman et al. (1995) 217: 412–419. Briefly, rat tracheal cells were placed on semipermeable collagen gel coated membranes, initially cultured submerged in medium and subsequently maintained in contact with air / liquid. In vitro cell samples include primary human bronchial cells (obtained from Clonetics, San Diego); NC1-H292 cells (ATCC CRL-1848); and A431 cells (ATCC CRL-1555).

In vitro culture comes in contact with EGF and the proposed agent. The proposed agent may contact the culture before, simultaneously or subsequently with the addition of EGF, depending on the evaluation endpoint and the nature of the proposed agent. Cultured cells are evaluated for suppression of goblet cell growth, which is related to the control means.

Different molecular or biochemical markers can be used to estimate goblet cell growth. Patterns of molecular and biochemical markers that may be used include, but are not limited to: gene expression or protein expression characteristic of goblet cells. Some mucin genes, such as MUC5W (Desseyn et al. (1997) J. Biol. Chem. 272: 3168-3178), are expressed in the respiratory tract and have a gene product highly expressed in the mucus. The expression of mucin genes provides a suitable marker for determining mucus production.

The expression of the mucin gene can be evaluated by conventional technology such as Northern blot analysis, polymerase chain reaction (PCR), mucin gene expression assay, or in situ analysis. Alternatively, mucin proteins are evaluated by conventional methodology, such as Western blot analysis, ELISA, immunochemistry, and the like, using markedly labeled antibodies. Morphological criteria may also be used to determine the presence or absence of goblet cells in culture; as alpine blue / PAS mucin staining (Lou et al. (1998) Am. J. Respir. Crit. Care Med. 157: 1927-1934). Mucin antibodies can be tested by ELISA studies. Because stimulation of EGF-R by a single ligand, such as EGF, TGF-α induces kinase phosphorylation at a specific EGF receptor and goblet cells are produced, EGF-R phosphorylation can be measured as a reflection of goblet cell induction (Donato et al. (1984) 7 Biol. Chem. 264: 20474-20481).

The decrease in molecular or biochemical markers associated with goblet cell growth is indicative of the therapeutic potential of the antagonist.

In vivo models

In another embodiment of the invention, animal models in vivo are used to evaluate the therapeutic potential of proposed agents to suppress goblet cell growth. Typically, the study involves the following steps: (i) creating an animal model of over-secreted lung disease by inducing the expression of EGF-R; (ii) stimulating induced EGF-R to produce mucin-producing goblet cells; (iii) treatment with the agent proposed; and (iv) assessment of goblet cell growth or mucus secretion, while suppression of goblet cell growth or mucus secretion is indicative of the therapeutic potential of the proposed agent.

Any in vivo model of pulmonary disease with top secretion may be used. By way of example, a model of asthma in mice is proposed herein, as described by Temann et al. (1997) Am. J. Respir. Cell. Biol. 16: 471-478. Alternatively, one rat model may be used as described by Takeyama et al. (1998) Am. J. Physiol. Examples of other animal models that may be used include, but are not limited to: Guinea pigs (a species that substantially expresses goblet cells) and rats.

Pulmonary or tracheal tissue from animal models can be evaluated with the same molecular and biochemical markers as described for the in vitro model. Decreasing tablet cell growth is indicative of the therapeutic potential of the EGF-R antagonist.

To present practitioners with normal knowledge, a full disclosure and description of how to operate and use

the present invention, the following examples are presented, which are not intended to limit the scope of what the inventors consider their invention, nor are they intended to represent that the experiments given below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the figures used (such as quantities, temperatures, etc.), but some experimental errors and deviations must also be taken into account. Unless otherwise stated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is close to or equal to atmospheric.

THE EXPERIMENTAL PART

EXAMPLE 1

EGF - SYSTEM REGULATES MUCIN C PRODUCTION

RESPIRATORY TRACT

Goblet cell hyperplasia occurs in various diseases of the respiratory tract with excessive secretion, but since the hidden mechanisms are not known, there is no effective therapy. In healthy respiratory tracts, goblet cells are few, but over-secretion airway diseases exhibit goblet cell hyperplasia. A human bronchial cell line (NC1-H292) was tested. These cells substantially express EGF-R; the expression of EGF-R genes was further stimulated by tumor necrosis factor alpha (TNF-α). EGF-R ligands increase the synthesis of mucin, this effect being enhanced by co-incubation with

Of TNF.

Pathogen-free epithelial cells of the rat expressed little EGF-R protein, but intratracheal dropwise TNF-α (200 ng) induction induced EGF-R in basal, preorbital and goblet cells, but not in cells with cilia; TNFα, EGF or TGFα alone do not induce goblet cell production. In addition, dropwise administration of TNFα followed by EGF-R ligands led to an increase in the number of goblet and pre-goblet cells and a shock increase in Alcian blue / PAS positive staining (reflecting mucosal glycoconjugates) and expression of the MUC5 gene. In sensitized rats, ovalbumin produced goblet cells and EGF-R expression in the airway epithelium. In NCL-H292 cells in TNFα-stimulated rats, followed by EGF-R ligands, and in a rat asthma model, pre-treatment with an EGF-R tyrosine kinase inhibitor (BIBX1522) prevents the production of goblet cells in the airways. These findings determine the role of EGF-R cascade inhibitors in over-secreted respiratory diseases.

© METHODS

RESEARCH IN VITRO

Cell culture. Human lung mucoepidermoid carcinoma cell line, NCL-H292 cells, germinated in RPMI 1640 medium containing 10% bovine embryo serum, penicillin (100 U / ml), streptomycin (100 μτ / ml) at 37 ° C in a water jacket incubator and 5% CO ₂ . In the liquid state, cells were incubated with EGF (recombinant human EGF, 25 ng / ml, Genzyme, Cambridge, MA), TGFα (recombined human TGFα, 20 ng / ml, Genzyme), EGF (25 ng / ml), plus TNFα ( 20 ng / ml) or TGFα (25 ng / ml) plus TNFα (20 ng / ml) for 12 h, 24 h or 48 h. In EGF-R tyrosine kinase inhibitor BIBX1522 (10 cells / ml generously provided by Boehringer Ingelheim Inc. Germany), cells were pretreated with BIBX 1522 for 30 min before growth factors were added. After incubation, cells grown in T-75 flask were used for complete RNA extraction or protein extraction to visually represent the mucin, 8-chamber slides were used for Alpine blue / PAS staining.

Western coloring. Cells grown in T-75 flasks were lysed (disintegrated) and scraped with PBS containing 1% Triton X, 1% sodium dioxycholate and PMSF (10 mg / ml). Total protein was evaluated with a BCA protein assay reagent (Pierce, Rockford, IL). Cell lysates were digested in Tricin sample buffer and 2% βΜΕ at 95 ° C. Proteins were separated from SDS-PAGE in 8% acrylamide gels. The resulting gels were equilibrated in the transfer buffer: 25 µM Tri-HCl, 192 mM glycine, 20% (vol / vol) methanol, pH 8.3. The proteins are then transferred by electrophoresis to nitrocellulose membranes. The membranes were incubated for 1 hour in 5% skim milk in PBS containing 0.05% Tween 20. The membranes were then incubated with mouse monoclonal anti-EGF-R antibody (1: 100) overnight at 4 ° C. The bound antibody is displayed visually according to standard protocols for the complex method for avidin-biotin-alkaline phosphatase (ABC kit, Vector Laboratories). A431 cell lysates (20) are used for positive control of EGF-R.

Immunocytochemical localization of EGF-R in NCI-H292 cells. Cells grown on 8-chamber slides were fixed with 4% paraformaldehyde for 1 h. To determine the presence of EGF-R, PBS containing 0.05% Tween 20.2% normal goat serum and 2 mM livamisole was used as antibody diluent. Parts were incubated with mouse monoclonal antibody to EGF-R (1: 250) overnight at 4 ° C, then washed three times with PBS to remove excess primary antibody. The cells were then incubated with biotinylated anti-mouse equine immunoglobulin (Vector Laboratories, Burliname, CA) at a dilution of 1: 200 for 1 h at room temperature. The bound antibody is displayed visually according to standard protocols for a complex method for avidin-biotin-alkaline phosphatase (ABC kit, Vector Laboratories, Burlingame, CA).

Samples. EGF-R mRNA expression was determined using a linearized pTRI-EGF-R human assay template (Ambion, Austin, TX). The sample contains one 360 bp fragment of cDNA from a human EGF-R gene, whose range is 12-14. The expression of the MUC5 gene was determined using a MUC5AC probe containing a single fragment of 298 bp cDNA from the human MUC5 AC gene (generously provided by Dr. Carol Basbaum).

Northern Coloring. RNA was fully extruded from NC1-H294 cells grown in T-75 tissue culture flask using a Tri-reagent (Molecular Research Ctr, Cincinnati, OH) for each condition. Total RNA was electrophoresed on a 1% agarose / formaldehyde gel and transferred to a nylon membrane (Amsterdam, Arlington Heights, IL) by capillary penetration. Samples were labeled with ³² P using a Randon Primed DNA labeling kit (Boehringer Mannheim Corp., Indianapolis, IN). The blots were pre-hybridized at 24 ° C for 4 h, then hybridized at 42 ° C for 16 h with a ³² P-labeled DNA-specific sample. The hybridization solution contains 250 mM tri-HCl (pH 7.5), 5% SDS, 1% BSA, 1% polyvinyl-pyrrolidone, 1% Ficoll, and 0.5% sodium pyrophosphate. After hybridization, the membranes were washed twice with 2 x

SSC for 30 min at room temperature followed by two washes in 2 x SSC with 0.1% SDS for 30 min at 50 ° C and rinsing in 0.1 x SSC with 0.1%

SDS. The membranes are exposed to X-ray film.

RESEARCH IN VIVO

The animal experiment protocol was approved by the Animal Research Committee, University of California, San Francisco. Fisher's characteristic pathogen-free F344 male rats weighing 230-250 g (Simonsen Laboratories, Gilroy, CA) were kept in a room at controlled temperature (21 ° C), and free access to standard laboratory food and water.

Healthy rats. Rats were anesthetized with methohexital sodium (Brevital sodium, 50 mg / kg, i.p .; Eli Lilly & Co, Indianapolis, IN), allowing them to breathe easily. To determine whether THFα overexpresses EGF-R in the airways, THFα (200 ng, 100 µl) is implanted in the trachea, animals being euthanized after 24 h. To determine whether EGF or TGFα induce goblet cells in the epithelium of the respiratory tract, EGF (600 ng, 100 µl) or TGFa (synthetic rat TGFa, 250 ng, 100 µl; Sigma, St Louis, MI) was implanted into the trachea alone or 24 h after TNFα (200 ng administration). 100 µl), and the animals were euthanized after 48 h. In each study, sterile PBS (100 µl) was introduced to control the trachea. To confirm the emergence of mucin production upon EGF-R activation, the effect of an EGF-R tyrosine kinase inhibitor, BIBX 1522, was investigated (the dose determined from studies using the inhibitor to prevent cancer growth). . Rats were pre-treated with BIBX 1522 (3, 10 or 30 mg / kg),

hours before and 24 hours after TGFa implementation. The trachea and lungs were removed for study 48 hours after TGFa administration,

Sensitized rats

Sensitization. Rats were sensitized on days 0 and 10 with abdominal injections of ovalbumin (10 mg, grade V; Sigma, St Louis, MO), in complex with 100 mg of aluminum hydroxide in 0.5 ml of sterile saline. The rats were allowed to rest for 10 days. On day 20, ovalbumin was administered directly to the trachea; the animals were irritated with a three-fold injection (day 20, 22 and 24) into the trachea of 100 µl of 0.1% ovalbumin in brine. Rats were euthanized either on day 20 without irritation or 48 h after the third irritation (day 26). This procedure induces metaplasia of goblet cells. To block goblet cell hyperplasia, sensitized rats were pretreated with an EGF-R tyrosine kinase inhibitor, BIBX1522. In the days of ovalbumin irritation (days 20, 22 and 24), the sensitized rats were pre-treated with BIBX1522 (10 mg / kg, 1 h before irritation), then BIBX 1522 was also implanted in the trachea together with ovalbumin (BIBX 1522, 10 ′ ⁵ M, 100 μΐ). BIBX 1522 was also injected every 24 hours, until the day before the rats were euthanized. The trachea is then removed 48 hours after the third irritation.

Fabric preparation. At pre-selected intervals during anesthesia, systemic circulation was perfused with 1% paraformaldehyde in PBS treated with DEPC at a pressure of 120 mmHg. The trachea was then removed and placed in 4% paraformaldehyde for 24 h. After fixation, the trachea and lungs were immersed in JB-4 plus monomeric solution A for cell analysis, or in OSC. compound (Sekura Finetek U.S.A., Inc., Torrance, CA) for immunohistochemistry and in situ hybridization. The submerged tissues were cut into cross sections (4 mm thick) and placed on slides.

Cell analysis. The total number of epithelial cells is counted, counting the nuclei of epithelial cells over 2 mm from the base plate (lamina) with one objective lens for immersion in oil (magnification x 1000). The linear length of the reference plane below each analyzed area of the epithelium is determined by tracing the contour of the digitized image of the reference plane. After the introduction of a stimulator that "develops" the shape of the goblet cells, these cells have granules positive for Alcian blue / PAS, but the size of the granules is small and the number of cytoplasmic granules is low. We call these "developing" goblet cells "pre-goblet cells," a stage before the cells turn into mature goblet cells. The goblet cells are tall, cuboid, cup-shaped, to column-shaped, with a large amount of granules stained in Alcian blue / PAS filling most of the cytoplasm between the nuclei and the surface of the lamina. Preglobular cells were defined as cells with smaller mucus-coated areas (<1/3 height in epithelium from basement membrane to lumen surface) or partially and slightly stained in Alcian blue / PAS small granules. Cilia cells are recognized along the borders by cilia, a weakly stained cytoplasm and a large circular nucleus. Cells with non-granular sectors are columnar in shape and are fed from the lumen to the base lamina. The cytoplasm is slightly pink in color, with several small PAS-positive and Alcian blue-negative granules observed. Base cells are small and flat with large nuclei located just above the base lamina but do not reach the lumen of the airway.

Quantitative evaluation of goblet cell production. The production of goblet cells is determined by the bulk density of Alcian blue / PAS mucosal matter on the mucosal surface of the epithelium using a semi-automatic imaging system described elsewhere (Weber et al. (1984) Science 224: 294-297). Areas stained in Alcian blue / PASpositive and the entire epithelial area were measured, with the data expressed as a percentage of the total area stained in Alcian blue / PAS. The analysis was performed with the NIH Image Programe Public Access Program (developed by the US National Institutes of Health, available online from anonymous FTP from zippy.nimh.gov or on a floppy disk by the National Technical Information Service, Springfield, VA, part number PB95 -500195GEI).

Immunohistochemical localization of EGF-R in rat epithelium. The localization of EGF-R was examined by immunohistochemical staining of an antibody to EGF-R (Calbiochem, San Diego, CA) in frozen rat tracheal sections. After perfusion with 1% paraformaldehyde in PBS, the tissues were placed in 4% paraformaldehyde in PBS for 1 h, then transferred to 30% sucrose overnight for cryoprotection. The tracheas are immersed in the OST. compound (Sakura Finetek U.S.A. Inc., Torrance, CA) and frozen. Frozen sections (5 µm) were cut and plated on glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA). Immunoblotting was performed in a manner similar to in vitro studies.

Sample preparation. The rat MUC5 cDNA was generously provided by Dr. Carol Basbaum. One fragment of 320 bp cDNA from rat MUC5 was subcloned into the Xba / hindIII side of a transcription vector, pBluescript-SK (-) (Stratagene, La Jolla, CA). In order to prepare RNA samples for in situ hybridization, this recombined plasmid containing the rat MUC5 cDNA fragment was linearized and transcribed in vitro with T7 or T3 polymerase to obtain respectively antisense or sensory samples. On-site hybridization samples are generated in the presence of [ ³⁵ S] UTP. After transcription, the DNA plate was ground with DNase, and the labeled radioisotope RNA was purified through a Sephadex G-25 Quick Spin TM column (Boehringer Mannheim, Indianapolis, INO and precipitated in ethanol / ammonia / acetate solution. Before use, RNA samples were digested. washed with 70% ethanol and diluted in 10 mM DTT.

On-site hybridization. Frozen sections (5 µm) were cut and placed on positively charged glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA). For hybridization with antisense and sensory samples, sections cut in close proximity are used. Other sections are used for Alcian blue / PAS staining. Individual samples were re-fixed in 4% paraformaldehyde, rehydrated in 0.5% x SSC, and acetylated in triethanolamine with acetic anhydride. Hybridization was performed with 2500-3000 cpm / μΐ of antisense or sense sample in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris, 5 mM EDTA, 1 x Denhard solution, 20mM dithiothreitol, 10% dextran sulfate, 0.5 mg / ml. yeast tPHK and 0.5 mg / ml salmon sperm DNA at 55 ° C overnight. Post-hybridization treatment consisted of washing 2 x SSC, 1 mM EDTA, 10 mM β-mercaptoethanol at room temperature, incubation with RNase solution (20 mg / ml) for 30 min at room temperature, another washing in 0.1 x SSC, 1mM EDTA, 10 mM β mercaptoethanol at 55 ° C for 2 h, then in 0.5 x SSC at room temperature. The samples were dehydrated, air-dried, and coated with a Kodak NBT (Eastman Kodak,

Rochester, NY) for autoradiography. After exposure at 7 to 21 d at 4 ° C, the slides were developed, fixed, and stained with hematoxylin (21).

Statistics. All data are presented as mean +/- SEM. To determine statistically significant differences between groups, one-sided mean deviation analysis is used. The Scheffe F-test is used to correct multiple comparisons when determining statistical significance in the deviation analysis. A significant probability of less than 0.05 was assumed for the null hypothesis when a statistically significant difference is indicated.

RESULTS

TNFα stimulates the production of EGF-R in NCI-H292 cells. First, it is determined whether NC1-H292 cells express organically EGF-R. Western immunoblot analysis showed the presence of EGF-R protein in liquid (confluent) cultures of NC1-H292 cells (Figure 1A, right). Cells were examined after becoming liquid (cofluent). The lysates were electrophoresed on 8% acrylamide gels and stained with anti-EGF-R antibody. The molecular weights of the marker proteins are indicated on the right. A positive control for EGF-R was the A431 cell protein (Figure 1A, left), which organically expresses EGF-R (Weber et al., Supra). Immunocytochemical studies with anti-EGF-R antibody revealed positive staining, most impressive when dividing cells (Fig. 1B, Immunocytochemical analysis with anti-EGF-R antibody in NH1-H292 cell cultures). In cofluence, positive staining is observed, most strongly in dividing cells (arrows, right). In the absence of a primary antibody, no staining was observed (left). Northern coloring indicates that

THFα (20 ng / ml) upregulated the expression of the EGF-R gene, an effect that appeared after 12 h and increased after 24 h (Fig. 1C, Northern analysis of EGF-R in NC1-H292 cells). The assay was performed with all RNA extruded from cofluent cultures incubated with TNFα (20 ng / μπ) for 12 or 24 h. The RNA was electrophoresed on formaldehydegarose gel and transferred to a nylon membrane and hybridized with P labeled EGF- R cDNA sample. After hybridization, the membrane was washed and autoradiographed.

EGF-R ligands stimulate the expression of mucosal glycoconjugates and the expression of the MUC5 gene in NCI-H292 cells. EGF-R is organically expressed in NC1-H292 cells, so the ability of EGF-R ligands (EGF, TGFa) to induce the production of mucosal glycoconjugates (Figure 2, upper column, staining in Alcian blue / PAS of NC1- cells) was evaluated. H292 for mucin glycoprotein recognition) Upper column = incubation of cells without inhibitor, lower column = incubation in the presence of EGF-R tyrosine kinase inhibitor BIBX 1522 (10 µg / ml). When cells were incubated alone (under control), some PAS-positive staining (arrows, top column) was observed; incubation with THFa alone (20 ng / ml) did not affect staining; incubation with EGF; (25 ng / ml) or with THFα (25 ng / ml) increased PASpositive staining (arrows); incubation with THFα and TGFα significantly increases staining (arrow).

Some control cells show staining; incubation with TNFα alone (20 ng / ml) did not affect staining; incubation with EGF or TNFα increases the staining much more than if both ligands act alone. Thus, EGF-R ligands induce mucosal glycoconjugates in NC1-H292 cells.

To study the expression of the MUC5 gene, Northern staining was performed (Figure 3). The whole RNA (10 µg) was extracted from the cells, electrophoresed on formaldehyde / agarose gel, transferred to a nylon membrane, and hybridized with a MUC5 cDNA labeled sample. After hybridization, the membrane was washed and autoradiographed. Cultures were obtained alone in medium (C), EGF or TGFα (25 ng / ml), TNFα (20 ng / ml), or a combination of TNFα and EGF or TGFα for 12 h (upper column) or 24 h (lower) column) upon expression of the MUC5 gene. Cultures were also obtained with TNFα and EGF- or TGFα after pre-incubation with the EGF-R tyrosine kinase inhibitor (BIBX 1522; 10 µg / ml; lower column); the inhibitor prevents the expression of the MUC5 gene.

Cells NC1-H292 show some expression in a control state (Figure 3, lower left column); when cells are incubated with EGF or TGFα, the expression of the MUC5 gene differs very little after 12 h but is clearly expressed after 24 h. TNFα alone does not affect the expression of the MUC5 gene but when TNFα is added to cells incubated with EGF -R ligands, the expression of the MUC5 gene increased markedly, above the level induced by EGF-R kigand alone (Figure 3).

An EGF-R tyrosine kinase inhibitor (BIBX1522) prevents the expression of mucosal glycoconjugates and the MUC5 gene in NCI-H292 cells. To test the hypothesis that activation of EGF-R receptors induces expression of the MUC5 gene, cells with EGF-R tyrosine kinase inhibitor BIBX1522 were incubated. When NC1-H292 cells were pretreated with BIBX1522 (10 µg / ml), PAS-positive staining was inhibited in the control state, and the increased staining that appeared with EGF-R ligands was markedly suppressed (Figure 1, lower column) . In a Northern assay, the expression of the MUC5 gene, which was markedly increased by the combination of TNFα and EGF or with TGFa, was completely suppressed by pre-incubation with BIBX 1522 (Figure

3, lower column). These results interfere with the activation of EGF-R in the incubation of the mucin gene and mucosal glycoproteins in NC1H292 cells.

TNFα stimulates the production of EGF-R in rats. Pathogen-free rats (which organically have several goblet cells in the airway epithelium) have been studied, starting with the role of TNFα. In the control condition, the tracheal epithelium contains several EGF-R positive cells (Figure 4A, left). In addition, intrinsic tracheal incorporation of TNFα (200 g) induced EGF-R protein in different cell types from the tracheal epithelium (Figure 4A, right). EGF-R-positive staining was observed in goblet cells (G), pre-goblet cells (PG), non-granular secretion cells (S) and base cells (Ba) m, but not in cells with cilia. Thus, TNFα induces the production of EGF-R protein.

Role of EGF-R ligands in the production of mucosal glycoconjugates and expression of the MUC5 gene in rats. In control, the tracheal epithelium contains few goblet and pre-goblet cells. Upon incorporation into the trachea of EGF-R ligands, EGF (600 ng; not shown) or TGFα (250 ng; Table 1) alone had no effect on the production in the epithelium of mucosal glycoconjugates. Moreover, when TNFα was first administered, followed for 24 h by EGF or TGFα (Table 1), the animals were euthanized 48 h later, the staining with Alcian blue / PAS increased markedly, with the number of goblet and pre-goblet cells increased markedly without changing the total number of cells or the number of cells with cilia (Table 1). In situ hybridization of the MUC5 gene showed no expression in the controlled animals. When

TNFα followed by EGF or TGFα is introduced into the trachea, the expression of MUC5 in the epithelium is visible. Thus, induction of EGF-R alone or stimulation with EGF-R ligands alone is insufficient to induce metaplasia of goblet cells or production of mucous glycoconjugates. In addition, upon induction of EGF-R by TNFα, the introduction of EGF-R ligands stimulates noticeable metaplasia of goblet cells.

Table 1 Cell analysis in the tracheal epithelium

This sensitization Cell type control TGFa TNFα / TGFα i.p. only i.p. + и.т. Tapestry 2.8 ± 0.7 5.8 ± 1.2 28.8 ± 3.4 * 5.4 ± 1.5 38.2 ± 6.3 * Pre-tapestry 7.8 ± 1.3 12.8 ± 1.6 44.8 ± 3.6 * 13.8 ± 1.4 36.0 ± 6.3 * Secretarial 82.0 ± 2.0 72.2 ± 4.0 40.8 ± 2.4 * 67.6 ± 7.0 49.8 ± 4.2 Scylla 49.6 ± 2.0 54.6 ± 2.3 53.2 ± 1.8 56.4 ± 3.8 52.4 ± 7.1 Basic 57.8 ± 2.6 56.8 ± 2.3 43.0 ± 3.5 60.2 ± 3.4 59.8 ± 2.9 Undefined 1.4 ± 0.5 2.0 ± 0.4 0.8 ± 0.4 1.4 ± 0.2 2.6 ± 0.5 Total 201.4 ± 2.2 204.2 ± 3.3 211.4 ± 4.8 204.8 ± 6.6 238.8 ± 4.4 * % colored area AB / PAS 2.4 ± 0.8 6.8 ± 1.9 35.8 ± 4.2 * 7.8 ± 2.9 38.7 ± 6.2 *

Table 1. Effect of mediators and sensitization of ovalbumin on tracheal epithelial cells in rats. Cells were analyzed as described in Methods; five rats in a group. Characterization is assisted by staining in Alpine Blue (AB) / PAS (which stains mucosal glycoconjugates). In addition to cell counting, the total epithelial area occupied by AB / PAS staining is calculated as a percentage. Control airways and pathways stimulated with TGFα (250 ng) alone contain few goblet and pre-goblet cells; there is some AB / PAS staining. TNF? (200 ng) followed by TGF? Resulted in an increase in the number of tablet and pre-pellet cells and an increase in the area occupied by cells stained with AB / PAS. Sensitization of rats with ovalbumin (OVA) done abdominally (ip) has no effect on cell distribution or on AB / PAS staining, but when OVA is given ip, followed by insertion into the trachea (it) of OVA, a shocking increase in tablets is detected and pre-goblet cells and the percentage area of AB / PAS staining.

Ovalbumin sensitization in rats induces the production of EGF-R and goblet cells. As it was declared that death from acute asthma was due to mucosal obstruction of the airways, a model of asthma was produced in pathogen-free rats. Injection of ovalbumin (10 mg, ip) on day 0 and day 10 did not stimulate goblet cell hyperplasia (Table 1). However, when this is followed by three times the introduction of ovalbumin into the trachea (it) (0.1% in 100 µl) on days 20, 22 and 24, with the animals being euthanized on day 26, the number of goblet and pre-goblet cells noticeably growing; the number of cells with cilia and base cells remained unchanged (Table 1, right side). Immunohistochemical studies with anti-EGF-R antibody showed no staining in the control trachea. Animals sensitized as i.p. both i.t., show EGF-R staining (Figure 4B, left), selectively in cells that are positively stained with AB / PAS (Figure 4B, right). After triple administration to the trachea of ovalbumin (0.1%, 100ml), EGF-R immunoreactivity is strongly expressed in goblet and pre-goblet cells (bottom, left), the same cells that stain positively with AB / PAS (bottom in right). Thus, an ovalbumin model of asthma shows an increase in goblet cells in cells that produce EGF-R.

An EGF-R tyrosine kinase inhibitor (BIBX1522) prevents the production of goblet cells induced by TNFα and EGF-R ligands and by sensitization with ovalbumin in rats. Because BIBX 1522 prevents the production of mucin in cultured cells, the effect of this inhibitor was tested in pathogen-free rats. AB / PAS staining, which increased upon incorporation into the trachea of TNFα, followed by EGF-R ligand TGFa, was suppressed in a dose-dependent manner with pre-treatment with BIBX 1522 (3-30 mg / ml, ip; Figure 5A). TNFα tracheal introduction (to induce EGF-R), followed by EGF-R ligand TGFa, leading to shock metaplasia of goblet cells.

In ovalbumin-sensitized rats, one pre-treatment with BIBX1522 (10 mg / ml, ip) completely suppressed goblet cell production (estimated by AB / PAS staining; Figure 5B). Animals given i.p. ovalbumin alone, exhibit little AB / PAS-positive staining in the bronchial epithelium. Animals that were first sensitized with OVA i.p., followed by a threefold introduction (i.t) into the OVA trachea showed a marked increase in AB / PAS positive staining.

These studies indicate that when EGF-R is stimulated by EGFR ligands, the production of goblet cells, in vitro and in vivo, is induced, an effect due to EGF-R and which is blocked by an EGF-R tyrosine kinase inhibitor . In an ovalbumin model of asthma, the inhibitor is also effective in preventing the production of goblet cells.

In addition to describing the mechanism of goblet cell induction, the present results provide a sequence for the development of goblet cell production based on the expression of EGF-R. TNFα stimulation induces intense staining of unpelleted secretion cells; their subsequent activation by EGF-R ligands induces progressive staining for mucosal glycoconjugates in the cytoplasm, and the cells turn into "pre-goblet" and then "goblet" cells. TNFα incorporation followed by EGF-R ligands induces goblet cell production without altering the total number of epithelial cells, suggesting that activation of EGF-R induces selective cell differentiation (rather than growth). The findings suggest that goblet cells are derived from unpelleted secretory cells that express EGF-R and are stimulated by EGF-R ligands to produce mucin.

Important findings have been found in patients who have died of acute asthma, goblet cell hyperplasia and mucosal obstruction. In a murine model of asthma, respiratory sensitization occurs after re-implantation of ovalbumin, resulting in noticeable goblet cell hyperplasia in the respiratory tract. We show that EGF-R, which does not appear in the controlled airway epithelium, is manifested in sensitized animals. The stained cells are pre-goblet and goblet, suggesting that EGF-R is involved in the production of goblet cells. Pre-treatment with an EGF-R inhibitor terrosine kinase receptor (BIBX 1522) prevents the production of goblet cells in the airways, confirming the role of EGF-R activation in goblet cell production in experimentally developed asthma.

The present results include the EGF-R pathway in goblet cell hyperplasia. Past studies have shown that various stimulants such as ozone, sulfur dioxide, viruses, lipopolysaccharides, an activating factor, and interlukin-4 overregulate mucin expression and secretion. The present invention provides a mechanism for evaluating the relationship of these inflammatory stimulators to the EGF-R system.

Asthma serves as an example of a therapeutic strategy of the invention: A normal human airway epithelium has a ratio of 3-10 cells with cilia to each goblet cell. In asthma, the number of goblet cells may be equal to or greater than cells with cilia; in patients who died in status asthmaticus, there was a 30-fold increase in the percentage area occupied by goblet cells, compared to the number in patients who died from non-asthmatic respiratory disease. Suppression of goblet cell production should eliminate this source of over-secretion. Because the life cycle of goblet cells is unknown, the timing of resolving goblet cell hyperplasia with treatment cannot be predicted with accuracy. In the absence of further allergen exposure, goblet cell hyperplasia in pre-sensitized mice resolves within 50 days, in conjunction with other manifestations of allergic inflammation. Suppression of EGFR activation can suppress goblet cell hyperplasia much faster, depending on the life span of the goblet cells. Recently, inhibitors of ATP-competitive tyrosine kinase with high selectivity have been disclosed. EGF-R tyrosine kinase inhibitors have been evaluated for the treatment of complications associated with the expression of EGF-R.

Excess secretion is a major manifestation of many chronic inflammatory diseases of the respiratory tract. Currently, there is no effective therapy to alleviate the symptoms and stop the development of these diseases. The present disclosures provide one mechanism and strategy for therapy: by inhibiting the activation of EGF-R, the production of goblet cells is prevented. Inhibitors of EGF-R activation are offered as therapy for respiratory tract disease with over-secretion.

Example 2

Role of oxidative stress in the production of goblet cells

In humans, long-term smoking is suspected to be associated with developing pathological changes in the peripheral airways, including goblet cell hyperplasia. Similarly, experimental models of smoking in animals have been shown to cause goblet cell hyperplasia in the airways. However, the mechanism by which cigarette smoke can induce mucin synthesis is unknown. The following data demonstrate that inflammatory cytokine-activated neutrophils and cigarette smoke induce mucin MUC5AC synthesis in human bronchial epithelial cells through ligand-independent activation of EGF-R. These results include recruited neutrophils and cigarette smoke as regulators of epithelial cell differentiation, which may achieve above normal induction of mucin-producing cells in the airways.

Methods

Isolation of neutrophils. Human neutrophils are purified from peripheral blood obtained from healthy human donors. Isolation of neutrophils is performed using standard Ficoll-Hypaque gradient separation techniques, dextran precipitation, and electrocyte hypotonolysis. Cells are> 95% viable proven with trypan blue paint off. To prevent endotoxin contamination, all solutions were passed through a 0.1 µm filter.

Cell culture. NC1-H292 cells, a human cell line from lung mucoepidermoid carcinoma, sprouted in RPMI 1640 medium containing 10% fetal bovine serum, penicillin (100 U / ml), streptomycin (100 μτ / ml) and Hepes (25 mM) at 37 ° C in a 5% CO ₂ humidified water jacket. 6-well culture plates or 8-chamber slides are used for cell cultivation. In the cofluent state, cells were incubated for 1 h only with neutrophils (10 ⁶ cells / ml), with TNFα only (recombinant human TNFα, 20 ng / ml, Genzyme, Cambridge, MA), with IL-8 only (human recombined) IL-8, ΙΟ ' ⁸ M, Genzyme), only with fMLP (10' ⁸ M, Sigma, St. Louis, MO), TNFα plus neutrophils, IL-8 plus neutrophils, fMLP plus neutrophils, hydrogen peroxide (H ₂ O ₂ , 200 μΜ), a solution of cigarette smoke or TNFα (recombinantTORa, 0.1-25 ng / ml, Calbiochem, San Diego, CA). The cells are then washed and incubated in fresh medium only. Experiments were terminated at predetermined time periods (for mRNA, 6 h and 12 h; for protein, 24 h). As controls, cells were incubated only in medium for the same periods of time. In other neutrophil studies, TNFα was selected as a stimulant because it has the highest potential effect on MUC5AC synthesis. NH1-H292 cells were incubated for 1 hour with neutrophils, which were incubated with TNFα (20 ng / ml) for 1 hour and then washed with sterile PBS to avoid contamination with the supernatant (floating composition above). solution), (i.e. molecules released from neutrophils), or NC1-H292 cells are incubated only in the supernatant. In suppression studies with EGF-R tyrosine kinase inhibitors, NC1-H292 cells were pretreated with BIBX1522 (10 µg / ml, generously provided by Boehringer Ingelheim Inc., Ingelheim, Germany) or tirfostin AG 1478 (10 μΜ, Calbiochem). 30 min before the stimulator is added. The effects of a selective tyrosine kinase inhibitor of growth factor derived from platelets (tyrphostin A1, 100 μΜ, Calbiochem) were also investigated. In inhibition studies with blocking antibodies to EGF-R ligands, the supernatants were pretreated with anti61

TNF? Antibody (Calbiochem) or anti-EGF antibody for 30 min, and then added to NC1-H292 cells. The role of oxygen-free radicals was investigated using the addition of oxygen-free radical DMSO (1% Sigma); 1,3-dimethyl-2-thiourea (DMTU, 50 mM, Sigma) or superoxide dismutase (SOD, 300 units / ml, Sigma).

Preparation of a solution of cigarette smoke. Research cigarettes (code2Rl, produced for the University of Kentucky Tobacco and Health Research Foundation) were used for the study. A cigarette smoke solution was prepared as described previously (Dusser et al. (1989) J. Clin. Invest. 84: 900-906). For a short time, cigarette smoke was withdrawn into a polypropylene syringe (35 ml) at one blow / min flow rate and slowly bubbled into 20 ml of RPMI1640 containing 50 mM Hepes buffer. The smoke solution was then titrated to pH 7.4 and used immediately after preparation.

Illustration of mucosal glycoconjugates and MUC5AC protein in NCI-H292 cells. At the end of the experiments, cells grown in 8-chamber slides were fixed in 4% paraformaldehyde for 1 h and then stained with Alcian blue / periodic acid-Schiff (PAS) to illustrate mucosal glycoconjugates or used for immunocytochemistry of MUC5AC. For immunocytochemistry of MUC5AC, PBS containing 0.05% Tween 20, 2% normal goat serum and Levamisol (2 mM) was used as the antibody diluent. Cells were incubated with mAb from mice to MUC5AC (clone 45 Ml, 1: 200, Neo Markers, Fremont, CA) for 1 h at room temperature, then washed 3 times with PBS to remove excess primary antibody. The cells were then incubated with biotinylated equine anti-mouse IgG (Vector Laboratories Inc., Burlingame, CA) at a dilution of 1: 200 for 1 hour at room temperature. The bound antibody is illustrated according to a standard protocol for a complex method for avidin-biotin-alkaline phosphatase.

In situ hybridization for the human MUC5AC gene. One 298 bp cDNA fragment of human MUC5AC was inserted into a TA clone vector (Invitrogen, San Diego, CA). The preparation of RNA samples and in situ hybridization were performed as described above.

Immunoassay of the MUC5AC protein. The MUC5AC protein was measured as described above. Briefly, cell lysates were prepared with PBS at multiple dilutions, and 50 µl of each sample were incubated with biotin carbonate buffer (50 µl) at 40 ° C in 96-well plates (Maxisorp Nunc, Fisher Scientific, Santa Clara, CA ) until they are dried. The plates were washed three times with PBS and blocked with 2% BSA (fraction V, Sigma) for 1 h at room temperature. The plates were again washed three times with PBS, then incubated with 50 µl of mouse monoclonal MUC5AC antibody (1: 100) diluted with PBS containing 0.05% Tween 20. After 1 h, the wells were washed three times with PBS and 100 µl of horseradish peroxidase anti-mouse IgG conjugate (1:10 000, Sigma) was sprayed into each well. After 1 h, the plates were washed three times with PBS. A TMV peroxidase solution (Kirkegaard & Perry Laboratories, Gaitherburg, MD) developed a color reaction which was quenched with 2N H2SO4. Absorption was read at 450 nm.

Quantitative analysis of TGFα protein. TGFα protein was measured using a commercially available ELISA kit (Sigma) following the manufacturer's instructions. The supernatant (surface-separated composition) was recovered after incubation of the neutrophils with TNFα (20 ng / ml) for 1 h, mixed with PBS lysis buffer containing 1% Triton X-100,

1% sodium deoxycholate and several protease inhibitors, (Complete

Mini, Boehringer Mannheim, Germany), then used to measure TGFα.

Immunostaining for EGF-R protein and immunostaining for tyrosine phosphorylation. The cells were left under serum starvation for 24 h, then stimulated with TGFα, H ₂ O ₂ or supernatant of activated neutrophils for 15 min. After stimulation, the cells were lysed and incubated for 30 min in an orbital shaker at 4 h. ° C. To remove the insoluble material, the cell lysates were centrifuged at 14,000 rpm. for 5 min at 4 ° C. Aliquots of supernatants containing equal amounts of protein were immunoprecipitated with an anti-EGF receptor antibody (polyclon, Ab4, Calbiochem) and 20 µl of protein A-agarose (Santa Cruz) for 2 h at 4 ° C. The precipitates were washed three times with 0.5 ml of lysis buffer, suspended in SDS sample buffer, and refluxed for 5 min. The proteins were separated from SDS-PAGE in 8% acrylamide gel. The resulting gel was equilibrated in one transition buffer: 25 mM Tris-HCl, 192 mM glycine, 20% (vol / vol) methanol, pH 8.3. Subsequently, the proteins were transferred by electrophoresis to nitrocellulose membranes (0.22 pm) blocked with 5% skim milk in PBS containing 0.05% Tween 20 overnight and then incubated with a monoclonal anti-phosphotyrosine antibody (1: 100 , Santa Cruz) for 1 h. The bound antibody was visualized according to a standard protocol for the avidin-biotin-alkaline phosphatase complex method (ABC kit, Vector Laboratories).

Statistics. All data are expressed as mean +/- SEM. A one-way analysis of the variable is used to determine statistically significant differences between the groups. A Scheffe F test is used to adjust the comparison of sets when determining statistical significance in the variable analysis.

A probability of less than 0.05 was assumed for the null hypothesis as an indicator of a statistically significant difference.

Results

Activated neutrophils induce the synthesis of MUC5AC mucin. When neutrophils with a stimulator that activates neutrophils (IL-8, fMLP, TNFα) are incubated with NC1-H292 cells for 1 h, the synthesis of MUC5AC protein is significantly increased within 24 h while unstimulated neutrophils (10 ⁶ / ml ), IL-8 alone or fMLP alone did not demonstrate an effect on MUC5AC synthesis; incubation with TNFα alone causes a small, insignificant increase in MUC5AC synthesis. When the neutrophils were pre-incubated for 1 hour with TNFα, after which the neutrophils and their supernatant were separated, subsequent incubation of the supernatant for 1 hour with NC1-H292 cells upregulated the expression of the MUC5AC gene within 12 hours and stimulated AB staining. / PAS and with one antibody to MUC5AC protein for 24 h; resting NC1-H292 cells showed a small expression of the MUC5AC gene and a small, spotty staining of AB / PAS and MUC5AC protein. The synthesis of MUC5AC protein induced by the supernatant was significantly increased compared to the control; neutrophils separated from the supernatant after incubation have no effect. It is concluded that activated neutrophils rapidly secrete an active product that induces MUC5AC synthesis.

EGF-R tyrosine kinase inhibitors prevent MUC5AC synthesis induced by a supernatant of activated neutrophils. As EGF-R ligands are known to induce MUC5AC synthesis in HCl-H292 cells by activating EGF-R tyrosine kinase, the role of EGF-R activation in MUC5AC synthesis induced by a supernatant of activated neutrophils is examined. Pre-treatment of NCI-H292 cells with selective EGF-R tyrosine kinase inhibitors (BIBX1522, AG1478) prevents the synthesis of MUC5AC protein, which is usually induced by the supernatant of activated neutrophils. A selective, platelet-derived, kinase growth factor receptor (AG1295) inhibitor and a negative control for tyrphostins (A1) did not work. These results include the activation of EGF-R tyrosine kinase in the synthesis of MUC5AC induced by the supernatant of activated neutrophils.

Role of EGF-R ligands secreted in the supernatant of activated neutrophils in MUC5AC synthesis. To determine whether activation of EGF-R tyrosine kinase is dependent on EGF-R ligands (EGF TGFα), a supernatant of activated neutrophils is pre-incubated with neutralizing antibodies in EGF-R ligands. Pretreatment of the supernatant with anti-TGFα antibody or with anti-EGF antibody does not suppress MUC5AC synthesis induced by the supernatant of activated neutrophils. Moreover, TGFα is not detected in the supernatant. Thus, EGF-R tyrosine phosphorylation induced by the supernatant of activated neutrophils is induced by a mechanism that is independent of EGF-R ligands, EGF and TGFα.

Cigarette smoke and oxygen-free radicals induce MUC5AC synthesis. Cigarette smoke and the oxygen free radicals, Ngo ₂ upregulate the expression of MUC5AC gene within 12 h., And TGFa. Similarly, all stimulants increase the synthesis of MUC5AC protein and the production of mucosal glycoconjugate within 24 hours - effects that manifest themselves in a dose-independent manner. The maximum synthesis of MUC5AC in response to H2O2 is significantly less than the reaction against TGFα. Pretreatment with AG1478 prevents the increase in synthesis of MUC5AC protein induced by all stimulators, indicating that the stimulants induce mucin synthesis under the influence of EGFR tyrosine kinase. Syntheses of MUC5AC caused by a supernatant of activated neutrophils, cigarette smoke and H2O2 are significantly inhibited by free radical pretreatment (DMSO and DMTU) and SOD, but the synthesis of MUC5AC protein by TGFα is not affected by DMSO or SOD.

Induction of tyrosine phosphorylation of EGF-R supernatant by activated neutrophils and H ₂ O ₂ . The distribution of EGF-R protein in serum starvation control conditions and in all stimulated conditions (supernatant of activated neutrophils, cigarette smoke, H ₂ O ₂ and TGFα) is similar. Complete phosphorylation of protein tyrosine occurs within 15 min after the addition of activated neutrophils, cigarette smoke, H2O2 or TGFα; no effect was demonstrated for control in serum starvation. Total protein tyrosine phosphorylation induced by TGFα is greater than the effect on the supernatant of activated neutrophils, cigarette smoke or H ₂ O ₂ . To determine whether EGF-R is phosphorylated, immunoprecipitation is performed with anti-EGF-R antibody: Supernatant of activated neutrophils, soluble products of cigarette smoke and H ₂ O ₂ , all induce EGF-R specific tyrosine phosphorylation within 15 min, an effect similar to that induced by TNFα. Pretreatment of NC1-H292 cells with AG1478 suppresses EGFR tyrosine phosphorylation with all stimuli. The supernatant inhibited by DMSO-, cigarette smoke-, and H2O2- induced EGF-R tyrosine phosphorylation, s << -X ί, but DMSO had no effect on TGFα-induced EGF-R tyrosine phosphorylation.

The above results indicate that neutrophils induce the synthesis of MUC5AC mucin in NC1-H292 cells when activated by IL-8, fMLP, or TNFα. Moreover, the supernatant collected 1 h after incubation of the neutrophils with TNFα induces the synthesis of MUC5AC, an effect that is suppressed by selective inhibitors of EGF-R tyrosine kinase. Suppression of EGF-R completely blocks the synthesis of MUC5AC induced by the supernatant of activated neutrophils; no effect are: one tyrosine kinase inhibitor without EGF-R, one selective platelet-derived kinase inhibitor acting on the growth factor receptor (AG1295) and one negative control for typhostins (A1), such as EGF-R tyrosine phosphorylation is outlined as the signaling direction for MUC5AC synthesis induced by a supernatant of activated neutrophils.

For further analysis of the mechanism by which supernatants from activated neutrophils indicate EGF-R tyrosine phosphorylation, both ligand-dependent and ligand-independent EGF-R pathways were examined. First, we measured TGFα in the supernatant of activated neutrophils, finding that the supernatant did not contain measurable amounts of TGFα. Previous publications have shown that neutrophils contain only low concentrations (2.5 pg / 10 ⁶ cells) of TGFα. The effect of the supernatant of activated neutrophils on the synthesis of MUC5AC has a force equal to the effect of 1 ng of TGFα, which is 400 times greater than the amount of TGFα detected in neutrophils. Second, we performed blocking studies with neutralizing antibodies to EGF-R ligands: Pretreatment with neutralizing antibodies to EGF and TGFα could not suppress MUC5AC synthesis induced by the supernatant of activated neutrophils. These results suggest that the synthesis of MUC5AC induced by the supernatant of neutrophils is not due to the secretion of EGF-R ligands (TGFα and EGF) by neutrophils. Further, we investigated the ligand-independent pathway: Since it is known that oxygen-free radicals are released during neutrophil activation, it is known that they cause transient activation of EGF-R tyrosine kinase in different cells, our hypothesis is that the release of oxygen-free radicals from activated neutrophils induces EGF-R tyrosine phosphorylation and, as a result, synthesis of MUC5AC in NC1H292 cells. Free radicals (DMSO and DMTU) and SOD suppress the synthesis of MUC5AC from the supernatant of activated neutrophils. TNF has been reported to cause oxidation boom in neutrophils in suspension, with a maximum response within 1 hour; the present results demonstrate a similar time for expression.

In the present study, exogenous H2O2, a major product that is released by neutrophils during an oxidative boom, induces MUC5AC synthesis in NC1-H292 cells. Having the maximum reaction to H2O2 in the synthesis of MUC5 AC is only half the reaction to TGFα. A significant discovery of the present invention is the fact that cigarette smoke alone induces the synthesis of MUC5AC. This suggests that cigarette smoke can cause synthesis of MUC5AC in vivo, both on direct stimulation and on indirect stimulation induced by neutrophil recruitment. The specific molecules in the cigarette smoke causing MUC5AC synthesis are not yet clear. Cigarette smoke has been shown to contain many products (eg nicotine, tar, acrolein and oxidants). In our experiments, DMSO and SOD partially suppressed cigarette smoke induced MUC5AC synthesis. Thus, oxidant stress may be one mechanism for producing this reaction.

The fact that cigarette smoke induced MUC5AC synthesis is completely blocked by EGF-R tyrosine kinase inhibitors indicates that EGF-R activation plays a major role in cigarette smoke induced MUC5AC synthesis.

In respiratory diseases, neutrophilic inflammation of the respiratory tract is a common feature, with neutrophils being recruited and activated by cytokines and cigarette smoke. Current studies show that recruited neutrophils and cigarette smoke also act as regulators of epithelial cell differentiation, resulting in the induction of airway-producing cells. Most importantly, suppression of EGF-R activation will be useful as a treatment for over-secretion of respiratory diseases.

Example 3

Injury of the epithelium in the airways causes metaplasia of goblet cells

As a hypothesis, it is claimed that agarose plugs implanted in the airways are chronically housed in the bronchi without interfering with them, and that the presence of the plugs will cause inflammation, which results in goblet cell mataplasia. Agarose plugs have been shown to induce noticeable local production of goblet cells, as shown by AB / PAS-positive staining and expression of the mucin MUC5AC gene associated with local recruitment of inflammatory cells. The results engage the activation of EGF-R in goblet cell metaplasia induced by blockage.

Methods

Animals. The animal experiment protocol was approved by the Animal Research Committee of the University of California, San Francisco. Specific pathogen-free male R344 rats (weighing 230 to 250 g, Simonsen Lab. Gilroy, CA) were used.

Rats were housed in BioClean cells, free of pathogens, with environmental control through a laminar flow suction umbrella; animals have free access to sterile food and water.

Medicines. Medicines from the following sources were used: cyclophosphamide (Sigma, St Louis, MO), methohexital sodium (Brevital, Jones Medical Industries, Inc., St. Louis, MO), pentobarbital sodium (Nembutal, Abbott Lab., North Chicago, IL) ; BIBX 1522, a selective EGF-R tyrosine kinase inhibitor (generously provided by Boehringer Ingelheim, Inc., Ingelheim, Germany), was dissolved in the following solution: 2 ml polyethylene glycol 400 (Sigma, St. Louis, MO), 1 ml 0.1 N HCl, and 3 ml of a 2% solution of mannitol in water (pH 7.0). NPC 15669 (leukocyte motility inhibitor), is generously provided by Scios Nova, Inc., Mountain View, CA.

Agarose plugs. Agarose plugs (0.7-0.8 mm in diameter) were made from 4% agarose type II EEE medium (Sigma, St. Louis, MO) in sterile phosphate buffered saline (PBS). To illustrate agarose plugs in tissue, a 3% suspension of Monastral Blue B (Sigma, St. Louis, MO) was added after the agarose had melted at 50 ° C.

Test report. We have studied pathogens-free rats as they have few goblet cells in the airways. Animals were anesthetized with sodium methohexital (Brevital, 25 mg / kg, i.p.). The trachea is aseptically sectioned into the narrow section along the axis, with agarose plugs introduced into the bronchus through a 20 Angiocath (Becton Dikinson, Sandy, UT) caliber connected to a polyethylene pipe (PE 90, 0.86 mm inner diameter and 1.27 outer diameter). mm, Clay Adams, Parsippany, NY), sewn into the severed trachea. The polyethylene tube folds under

30 ° angle to allow for optional insertion into the correct bronchus. After implantation, the incision is closed with a seam.

To evaluate the role of EGF-R on goblet cell metaplasia induced by agarose plugs, the animals were treated with BIBX 1522 (80 mg / kg, ip) 1 h before the agarose plugs were repeated, repeated daily (40 mg / kg, ip, recommended). Animals were euthanized 24, 48, or 72 hours after agarose plugs were introduced.

In order to evaluate the role of TNFα in metaplasia of goblet cells induced by agarose plugs, animals were treated with antibody neutralizing TNFα (Genzyme, Boston, MA). The first treatment (100 µl in 2.0 ml saline, i.p.) is given 1 hour before the introduction of agarose plugs, such as injections i.p. are repeated daily. In addition, the TNFα neutralizing antibody was infused (10 µl / h) through an osmotic mini-pump (2ML1, Alza Corp., Polo Alto, CA) implanted subcutaneously.

In order to study the effect of neutrophils on goblet cell metaplasia induced by agarose plugs, rats were pre-treated with cyclophosphamide (a bone marrow leukocyte inhibitor) or a combination of cyclophosphamide and NPC 15669. Cyclophosphamide (100 mg / ml, ip) was given 5 days before the agarose plugs were introduced, and a second injection of cyclophosphamide (50 mg / kg, ip) was given 1 day before the agarose plugs were introduced. In NPC 15669 studies, the drug (10 mg / kg, i.p.) was injected 1 hour before the agarose plugs were introduced and then daily for three days.

All drugs (BIBX 1522, TNFα neutralizing antibody, cyclophosphamide, and NPC 15669) were given i.p. 1 hour before the introduction of agarose plugs, repeating the doses daily for 3 days.

Preparation of fabrics. At various times, after the introduction of agarose plugs, the rats were anaesthetized with pentobarbital sodium (65 mg / kg, i.p.), perfused at 120 mm g into the bloodstream. v. 1% Parafolmaldehyde in PBS-treated with diethyl pyrocarbonate. The right lung is removed and the right soft part of the tail is used for histology. In frozen sections, the tissues were removed and placed in 4% paraformaldehyde for 1 h, then introduced into 30% sucrose overnight for cryogenic protection. The fabrics are applied in an OSC compound. (Sekura Finitek U.S.A., Inc. Torrance, CA). For sections of methacrylate, tissues were placed in 4% paraformaldehyde for 24 h, then dehydrated with calibrated concentrations of ethanol and applied to methacrylate JB-4 (Polysciences, Inc., Warrington, PA). Tissue sections (4 µm thick) were stained with AB / PAS and discolored with hematoxylin.

Morphometric analysis of the bronchial epithelium. The percentage of mucosal glycoconjugate area in the epithelium stained with AB / PAS was determined using an image analysis system according to methods previously published. The area of epithelium and AB / PAS-stained mucous conjugates in the epithelium are delineated by hand and analyzed using an NIH imaging program (developed at the US National Institutes of Health, available online by anonymous FTP or flops disc from the National Technical Information Service, Springfield, Virginia; part number PB95-5001195GEI). Data are expressed as a percentage of the total epithelium area occupied by AB / PAS staining. To determine, semiquantitatively, mucus secretion, determine the percentage of epithelial surface length occupied by AB / PAS-positive staining by calculating the length staining positively as a ratio to the total length. The percentage of the exposed epithelium is determined by calculating the ratio of the length of the exposed epithelium to the total length of the epithelium.

Cell type recognition in methacrylate sections and cell analysis. The total number of epithelial cells was determined by counting the epithelial cell nuclei on 2 mm from the base lamina with an objective oil immersion lens (xlOOO magnification). The linear length of the base lamina under each epithelial region analyzed is determined by tracing the contour of the digitized image of the base lamina. Epithelial cells are recognized as described previously. Briefly, base cells are recognized as small, large-nucleus flat cells located just above the base lamina but not reaching the airway lumen. The cytoplasm stains dark with no AV or PAS-positive granules present. Cells with cilia are recognized by their ciliary borders, slightly colored cytoplasm and large, circular nuclei. Non-granular secretion cells are column-shaped and extend from the bronchial lumen to the base lamina. After the introduction of agarose plugs into the trachea, “developing” goblet cells (pre-goblet cells) are formed. These cells exhibit AB / PAS-positive staining, the granules are small and the cells are not packed with granules; they contain small mucosa colored areas (<1/3 the height of the epithelium from the basement membrane to the surface of the lumen) or scattered and slightly AB / PAS colored small granules. Indeterminate cells are formulated as cellular profiles that lack cytoplasmic features for proper categorization.

Immunohistochemical localization of EGF-R. The presence of EGF-R was determined by immunohistochemical localization using a mouse monoclonal antibody to EGF-R (Calbiochem, San Diego, CA). The previously prepared 4 pm frozen sections were subsequently fixed with 4% paraformaldehyde treated with 0.3% H2O2 / methanol. The tissues were incubated with an antibody for EGF-R (diluted 1: 250). Biotinylated equine anti-mouse IgG (1: 200; Vector Lab., Burlingame, CA) followed by streptavidin peroxidase was used to illustrate antigen-antibody complexes stained with 3.3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO). complex (ABC kit, Vector Lab., Burlingame, CA). Negative control slides were incubated in the primary or secondary antibody omitted and replaced with PBS.

On-site hybridization. From a plasmid containing the 320 bp cDNA fragment of rat MUC5AC of (kindly proposed by Dr. Carol Blausbaum) are created ^[35 8] -nadpisani riboprobes. The sections were hybridized with [ ³⁵ S] -scripted RNA samples (2500-3000 cpm / ml hybridization buffer) and washed under strictly defined conditions, including treatment with PHase A. After autoradiography for 7-21 days, the photographic emulsion and slides developed are stained with hematoxylin.

Enumeration of neutrophils in the airway epithelium. Assessment of the entry of neutrophils into the bronchi is done by staining the neutrophils with 3-3'-diaminobenzidine tetrahydrochloride, counting the number of neutrophils in the lumen of the airway and in the epithelium; results are shown as the number of stained cells per mm length of the base lamina.

Bronchoalveolar lavage (BAL). To evaluate the individual cell counts in each group of animals, five lungs were lavaged with 3 ml aliquots of sterile PBS in the lungs, pooled, and volume was measured. Cells in BAL were harvested by rotating the lavage fluid at 1000 rpm for 10 min. Then, 10 ml of cell suspension was counted with a hematocytometer to determine the number of cells in the BAL fluid. The number of individual cells was determined in the preparation of cytobibes stained with Diff-Quik (American Scientific Products, McGaw Park, IL). The individual cell counts were obtained by sampling at least 200 cells on each slide of cytoblasts.

Statistical analysis. Data are expressed as mean +/- SE. For statistical analysis, as appropriate, two-way or one-way variable analysis (ANOVA) is used, followed by Student t test if necessary. A probability of less than 0.05 is considered as a statistically significant difference.

Results

Influence on airway epithelial structure: Gaplet cell metaplasia. To determine whether agarose plugs affect the structure of the airway epithelium, agarose plugs were implanted in the right bronchus of 5 pathogens without pathogens. In control animals, the bronchial epithelium contains several goblet cells. In addition, after local deployment of agarose plugs, AB / PAS staining showed a time-dependent increase in the area of goblet cells, this was detected, at the earliest after 24 h and reaching a maximum of 72 h after deployment. At 24 h, agarose plugs cause a significant increase in the number of pre-goblet and goblet cells, and at 48 h, more mature goblet cells are detected (Table 1). At 72 h, agarose cells caused an increase in the number of goblet cells (P <0.01); the number of basal and cilia cells did not change (P> 0.05). The total number of epithelial cells per mm of base lamina, 72 hours after implantation, was slight but not significantly increased (P> 0.05, Table 1); epithelium height (measured from baseline membrane to epithelial lumen surface) increased from 16.0 +/- 1.2 μm in control airways to 38.1 + / 9.1 pm at 72 h after insertion of plugs (i = 5, P < 0.01).

In the airway lumen of control animals, there is no AB / PAS staining. In addition, near the agarose plugs, a positive coloration is observed in the lumen, indicating that it appears

secretion of mucous glycoconjugates. In agarose plug airways, staining increases with time. The percentage of total epithelial length occupied by AB / PAS-positive staining in the airways adjacent to the plugs increased from 0.1 +/- 0.1% in control animals to 4.7 +/- 1.4%, 13.3 +/- 0.7%, and to 19.1 +/- 0.7% at 24 h and 72 h (n = 5). Furthermore, the agarose plugs epithelial the obstructed bronchial epithelium by 13.5 +/- 2.3%, and 5.1 +/- 1.5% of the total area at 24, 48, and 72 h, respectively (n = 5).

Effect of agarose plugs on the expression of the mucin gene. In control rats, no detectable signal was obtained with a MUC5AC antisense sample in the bronchi (n = 4 per group). In bronchial tubes where agarose plugs are implanted, there is a signal for MUC5AC that increases depending on the time from 24 h to 72 h (n = 4). No signals (such as smooth muscle, connective tissue) were found from other cell types. Sections examined with sense sample for MUC5AC show no expression.

Effect of agarose plugs on the expression of EGF-R in the airway epithelium. In control animals immuno-stained with an antibody to EGF-R, diffuse staining in the epithelium was demonstrated. In addition, after the introduction of agarose plugs, the epithelium adjacent to the agarose plugs shows EGF-R-positive AB / PAS staining. The staining form for EGF-R goes in parallel with the staining for MUC5AC and AB / PAS. Pre-goblet cells and unpelleted secretion cells are immune to EGF-R. Cells with cilia do not show immunoreactivity. In airways not obstructed by agarose plugs, the epithelium exhibited little staining for EGF-R, which was similar to staining in control animals.

Effect of EGF-R tyrosine kinase inhibitor on metaplasia of goblet cells and on expression of the mucin gene. In the present studies, the incorporation of agarose plugs results in the expression of EGF-R in the cells, which produces mucin. EGF-R is a representative of the class of tyrosine kinase receptors. Thus, when EGF-R ligands

(EGF or TGFα) bind to EGF-R, specific EGF-R tyrosine kinase is activated. Therefore, to test the hypothesis that activation of EGF-R induces expression of the MUC5AC gene and mucosal glycoconjugates, after the introduction of agarose plugs, an EGF-R tyrosine kinase inhibitor (BIBX 1522) is injected abdominally in rats. BIBX 1522 markedly inhibited the agarose-induced AB / PAS stained epithelial area at 24, 48, and 72 h. It also suppressed completely, the expression of the MUC5AC gene, 72 h after plug implementation.

Effect of TGFα-neutralizing antibody on metaplasia of goblet cells and expression of EGF-R. Our hypothesis is that during inflammation caused by agarose plugs, THFa is released. For this reason, we investigated the effect of pre-treatment of rats with antibody neutralizing THFα on metaplasia of goblet cells induced by agarose plugs; In animals pretreated with antibody neutralizing THFα (η = 5), agarose plugs no longer stimulate protein expression of THFα or the production of AB / PAS-stained (goblet) cells.

Recruitment of inflammatory cells from agarose plugs. Agarose plugs have been observed to cause damage to the epithelium and penetration of inflammatory cells. With THFα and EGF-R ligands, they can produce different inflammatory cells. EGF-R and its ligands are involved in the EGF-R cascade leading to metaplasia of goblet cells. We evaluated the roles of leukocytes and macrophages in the effects induced by agarose plugs in two ways. First, we examined cells in bronchoalveolar lavages: In control rats, macrophages are the predominant recovered cells (u = 5; Fig. 4, Control). After the introduction of agarose plugs, the number of macrophages increased (P <0.05), with a significant number of neutrophils appearing in the lavage fluid (P <0.01). The number of lymphocytes did not change.

Infiltrated cells in tissue sections were also evaluated: airways without agarose plugs contained few neutrophils, and airways containing plugs demonstrated the presence of neutrophils, both in the epithelium and in the lumen. The number of neutrophils in the airway was 0.2 +/- 0.2, 42.4 +/- 7.1, 40.7 +/- 7.7 and 20.1 +/- 7.2 / mm from the base lamina in the control airways, respectively at 24, 48, and 72 h after implementation of plugs (P <0.05, n = 5). In addition, the number of neutrophils in the airway epithelium was 1.3 +/- 0.4, 15.6 +/- 2.6, 14.9 +/- 1.4, and 14.8 +/- 2.6 / mm from the base lamina at control and 24, 48, and 72 h, respectively. after plug implementation (P <0.01, n = 5).

Effect of cyclophosphamide on neutrophil recruitment, metaplasia of goblet cells and protein expression of EGF-R. In cyclophosphamide-treated rats, blood neutrophils were depleted (number of neutrophils in venous blood after cyclophosphamide, 1.8 +/- 0.5%, n = 5), suppressing BAL recruitment of taprope-induced neutrophils. The number of neutrophils in the airways (2.6 +/- 0.3 / mm from the base lamina) and in the epithelium significantly decreased (0.8 +/- 0.2 / mm) after 24 h. Cyclophosphamide also inhibited goblet cell metaplasia induced by agarose plugs and expression. of EGF-R protein. When lumedin, NPC 15669, is added to cyclophosphamide, the inhibition of goblet cell metaplasia induced by agarose plugs is similar to the effect of cyclophosphamide alone. These results include the involvement of neutrophils in cellular metaplasia induced by tap.

Discussion

In the present study, we investigated the effect of implantation of agarose plugs on metaplasia of goblet cells in the airways of pathogen-free rats that have very few goblet cells in the control phase. Epithelial cells in bronchus of control animals and bronchial tubes without agarose plugs (control lungs) were stained equally negatively with AB / PAS. The introduction of agarose plugs results in a pronounced, time-dependent increase of goblet cells in the area of epithelial goblet cells, adjacent to the implanted plugs, this is detected within 24 h and is most pronounced around 72 h. implementation. The airways adjacent to the blocked airways are also positively stained with AB / PAS. The total number of cells, the number of base cells and cells with cilia did not change, but the number of goblet cells increased and the number of unpelleted secretion cells decreased with time after the introduction of agarose plugs (Table 2). These results suggest that goblet cell metaplasia results from the conversion of unpelleted secretory cells into goblet cells.

Table 2

The effect of agarose plugs on the distribution of bronchial epithelial cells in pathogen-free rats *

Cell type Control 24 hours 48 hours 72 hours Tapestry oo ± oo 13.115.6 25.7115.0 51.519.0 Pre-tapestry 0.010.0 32.812.9 25.7115.0 51.519.0 Secretarial 43.513.0 24.413.3 18.413.7 8.912.3 Scylla 98.514.0 83.817.9 81.615.0 84.013.9 Basic 18.415.7 10.610.8 11.61 1.7 11.012.3 Undefined + 1.310.5 1.410.8 1.110.1 0.610.4 Total 16117.2 166.116.1 175.516.2 180.917.5

Cells were analyzed as described in Methods: n = 5 for each group. Characterization is aided by AB / PAS staining (which stains mucosal glycoconjugates). Control airways contain several pre-goblet and goblet cells. Following the introduction of agarose plugs, there was a time-dependent (24, 48 72 h) increase in the number of preglobular and goblet cells, and a decrease in the number of unpelleted secreted cells compared to control animals.

* Data are mean +/- SE, cell count / mm base lamina.

+ P <0.05 compared to control & P <0.01 compared to control !! Cells lack sufficient categorization of cytoplasmic features.

Goblet cells in the airways have been reported to express the MUC5AC gene. In the present studies, control bronchial tubes do not express the MUC5 AC gene, with the airways obstructed by plugs or adjacent to plugs that positively stain with AB / PAS express

MUC5AC gene, suggesting that the MUC5AC gene is involved in the production of mucus induced by agarose plug. These results indicate that agarose plugs induce the expression of mucin genes and the production of mucosal glycoconjugates in selected cells in the respiratory tract.

pathways of rats.

The mechanism of goblet cell metaplasia induced by agarose plugs is investigated. EGF-R is not normally expressed in the airway epithelium of non-pathogenic rats but is induced by THFα. In the presence of EGF-R in the epithelium, the incorporation of EGF-R ligands (EGFR or TGFα) results in an increase in mucin gene and protein expression. A selective inhibitor of EGF-R tyrosine kinase (BIBX 1522) completely suppresses these reactions by engaging EGF-R signaling in goblet cell metaplasia. The effect of BIBX 1522 on goblet cell metaplasia induced by agarose plug was determined; Suppression of mucosal glycoconjugate production and expression of a MUC5AC gene induced by agarose plugs. These results engage an EGF-R cascade in goblet cell metaplasia induced by agarose plugs.

The mechanisms by which the EGF-R cascade induces metaplasia of goblet cells with agarose plugs have been investigated. First, we examined the expression of EGF-R protein in the bronchial epithelium. Control airways stain equally negatively for EGF-R, but airways containing agarose plugs show, selectively, time-dependent, positive staining for EGF-R. Positively stained cells include unpelleted secretion, pre-goblet and goblet cells. Thus, agarose plugs induce the expression of EGF-R protein. Rats previously pretreated with an antibody to neutralize THFa do not develop aplasia of goblet cells induced by agarose plugs, and THFa is involved in the expression of EGF-R induced by agarose plugs.

Cyclophosphamide is a drug that selectively inhibits leukocyte production, prevents the recruitment of neutrophils into the lavage fluid from the airways and into the airway epithelium, following the introduction of agarose plugs, and prevents metaplasia of goblet cells induced by agarose plugs. Macrophages also increase after the introduction of agarose plugs, but cyclophosphamide does not inhibit macrophage recruitment. These results engage the neutrophils in goblet cell metaplasia induced by agarose plugs. The fact that cyclophosphamide also reduces the expression of EGF-R protein after agarose plugs suggests that the neutrophils contribute, at least in part, to the expression of EGF-R in this inflammatory state.

The neutrophils are also able to produce EGF-R ligands, EGF and TGFα. In addition, epithelial cells are derived from EGF-R ligands and a shocking epithelium is found adjacent to the agarose plugs. Thus, the epithelium can be an important potential source of THFα and EGF-R ligands.

It is logical to assume that an effective agarose plug stimulator is related to the movement of the plugs during respiration without subsequent abrasion of the epithelium. Mechanical injury to the airway epithelium has been reported to cause over-secretion. These previous studies give credence to the hypothesis that mechanical trauma to the airway epithelium leads to over-secretion. Orotracheal intubation has been reported to result in abundant mucus secretion in horses. Chronic intubation in patients may cause mucosal over-secretion and may cause mucosal obstruction. EGF-R tyrosine kinase inhibitors may serve to prevent mucosal over-secretion after tracheal intubation.

Epithelial damage is often reported as a common finding in patient studies, even with mild asthma, with the damage increasingly associated with worsening clinical symptoms. Damage to the epithelium caused by an allergic reaction can induce activation of EGF-R, resulting in over-normal production of goblet cells. The data presented above engage the activation of EGF-R in a different reaction, which ® typically involves metaplasia of goblet cells. Mechanical epithelial damage and epithelial injury in asthma may involve a similar (EGF-R) cascade, resulting in a normal increase in epithelial secretion cells. This provides a mechanism for over-secretion that occurs in fatal cases of acute asthma.

Example 4

Reverse granulation of goblet cells by EGF-R receptors u Degranulation of goblet cells in rat nasal respiratory epithelium is induced by inhalation through the nose of fMLP. 4 hours after inhalation through the nose of fMLP (10 ' ⁷ M), significant degranulation was induced in the nasal septal epithelium. The reverse granulation of goblet cells occurs 48 hours after inhalation. In the control condition, MUC5AC protein is expressed in goblet cells, but EGF-R protein is not expressed. Like EGF-R, the MUC5AC mucin gene and protein were absent in the control epithelium but manifested significantly 48 h after inhalation. Pre-treatment with an EGF-R tyrosine kinase inhibitor, BIBX1522, inhibited the expression of the mucin MUC5AC gene and protein after fMLP-induced goblet cell degranulation. These results indicate that EGF-R expression and activation are involved in the reverse granulation of goblet cells in rat epithelium.

METHODS

Animals. The animal experiment protocol was approved by the Animal Research Committee of the University of California, San Francisco. Specific pathogen-free male rats F344 (weighing 200 to 230 g Simonsen Lab, Gilroy, CA) were used. The animals are housed in BioClean cells, free of pathogens, with laminar air exchange umbrellas, the animals have free access to sterile water and food.

Preparation of nasal tissue. At various times, after inhalation, rats were anesthetized with pentobarbital sodium (65 mg / kg, i.p.). The heart of the animal was discovered by inserting a blunt-pointed needle from the apex of the left cavity of the ascending aorta, and the blood circulation was perfused with 1% paraformaldehyde. A cut in the left atrium provides an entrance for the fixative. The eyes, lower jaws, skin and muscles are separated, and the head is immersed in a large volume of the same fixative for 24 hours. After fixation, the head is decalcified with Surgipath (Decalcifier II, Surgical Medical Industries, Inc., Richmond, IL) for 4 -5 days and rinsed in phosphate-buffered saline. The nasal cavity is cut transversely at the level of the acute papilla from the nasal palate (palate). The front block of tissue was immersed in glycol methacrylate (JB 4 Plus, Polysciences, Inc., Warrington, PA) or in OCT (Sekura Finetek, U.S.A., Inc., Torrance, CA) in frozen sections. From the freshest surface of the blocks submerged in glycol methacrylate, sections of 5 µm thickness are cut and stained with Alcian blue (pH 2.5) / periodic acid Schiff (AB / PAS) to give acidic and neutral glycoconjugates, or 3.3 '-Diaminobenzidine (Sigma chemical, St. Louis, MO) to illustrate leukocytes that migrated into the epithelium. From the freshest surface of the frozen, submerged blocks, sections of 5 µm thickness are cut and stained with AB / PAS or used for immuno-staining of EGF-R and MUC5AC.

Enumeration of neutrophils in the nasal epithelium. The neutrophils are counted in the high power fields of the epithelial layer stained with 3.3'diaminobenzidine at magnification x 400. The number of neutrophils in the nasal calyx (from the main membrane to the tips of the cells) is determined by counting the nuclear profiles per unit length of the base laminate.

Quantification of degranulation and reverse granulation of goblet cells. To determine the degranulation and backward granulation of goblet cells, we measured the bulk density of the mucosal staining of AB / PAS on the mucosal surface of the epithelium using a semi-automatic imaging system according to a previously published method. We examined the stained slides using an Axioplan microscope (Zeiss. Inc.) that was connected to a video camera control (DXC7550MD; Sony Corp, of America, Park Ridge, NJ). Images of the nasal epithelium were recorded in high power fields with phase contrast lens at x400 using an IMAXX video system (PDI, Redmond, WA). Intracellular mucin in superficial epithelial secretion cells appears as oval-shaped, violet granules of varying size. We measured the area stained AB / PAS positive and the entire epithelial area, with the data shown as a percentage of the AB / PAS stained area relative to the total area. The analysis was performed on a Macintosh 9500/120 computer (Apple Computer, Inc., Cupertino, CA) using a NIH Image public access program.

Immunolocalization of EGF-R and MEJC5AC protein. Frozen sections of nasal tissue fixed in paraformaldehyde were treated with 3% H ₂ O ₂ / methanol to endogenous peroxide block and incubated with mouse monoclonal antibody to EGF-R (Calbiochem, San Diego, CA) MUC5AC (NeoMarkers Inc., Fremont , CA) for 1 hour in a diluted 1: 100 solution. Immunoreactive EGF-R or MUC5AC was visualized with the Vectastain Elite ABC kit (Vector Lab., Inc., Burlingame, CA) using chromogen using 3,3-diaminobenzidine tetrahydrochloride. Controls include replacement of primary or secondary antibody with PBS.

Methods. We examined rats without pathogens that normally have many goblet cells in the nasal cup (septal) epithelium. To determine the effect of fMLP, in aerosol form, on goblet cell degranulation and on the migration of neutrophils into the nasal mucous epithelium, animals were anesthetized with sodium pentobarbital (60 mg / kg, ip), and administered to the nose with aerosol in 5 min. N-formyl-methionylsilyl-phenalalanine (fMLP; 10 ' ⁵ M, Sigma, St. Louis, MO) in saline without pyrogens. Aerosol exposure was performed by blowing the animals with an ultrasonic nebulizer (PulmoSonic, DeVibliss Co., Somerset, PA), which creates an aerosol mist at a flow rate of 0.3 ml / min. Similarly, only saline spray aerosol was administered to the control animals via the nose.

To investigate the reverse granulation of nasal goblet cells, after inhalation of fMLP aerosol, rats were euthanized 48 h after administration of fMLP through the nose.

To evaluate the effect of EGF-R tyrosine kinase activation on the reverse granulation of goblet cells, animals were pretreated i.p. with one EGF-R tyrosine kinase inhibitor (BIBX 1522, 15 mg / kg, generously provided by Boehringer Ingelheim

Inc., Ingelheim, Germany) 30 min before inhaling fMLP, repeated twice daily.

Statistics. All data are presented as mean +/- SEM. A one-way ANOVA or Student 1 test is applied to each experimental group. A probability less than 0.05 is considered a statistically significant difference.

Results

Effect of JMLP goblet cell degranulation on the structure of the nasal epithelium. In the control condition, the nasal calyx epithelium contains a considerable area of AB / PAS stained goblet cells, but the luminal surface remains unstained. Immunoblotting for mucin MUC5AC protein corresponds to the area of AB / PAS staining, but in situ hybridization demonstrates little or no expression of the MUC5AC gene. Immunohistochemical staining for EGF-R protein was negative. These results indicate that the rat nasal epithelium contained intact goblet cells containing MUC5AC protein in the absence of mucin gene expression. The absence of luminous staining suggests that mucin degradation (secretion) is not present.

The hypothesis is that unstimulated nasal epithelium from rats contains "stable" non-degranular goblet cells containing mucin proteins. Neutrophil chemoattractants (i.e., fMLP) have been shown to recruit neutrophils into the airway epithelium, where they cause goblet cell degranulation by an elastase-dependent process. To check the effect of goblet cell degranulation, an aerosol from a neutrophil chemoattract, fMLP (10 ' ⁷ M) was introduced into the nose for 5 minutes. In rats euthanized 4 h after fMLP, the area stained with AB / PAS and the area of MIC5AS-positive immunostaining decreased significantly, with neutrophil recruitment occurring in the nasal epithelium. AB / PAS staining is expressed in the surface of the nasal airway lumen, confirming that goblet cell degranulation has occurred. Moreover, at 4 h after fMLP, the expression of the MUC5AC gene did not change.

In euthanasia-treated rats 48 h after fMLP, immunohistochemical staining with EGF-R antibody stained positive for EGF-R in pre-goblet and goblet cells, the area of AB / PAS- and MIC5AS-immunopositive staining returned to the level, available in the control condition indicating that reverse granulation of nasal goblet cells has occurred. At this time, neutrophil recruitment was no longer observed; The expression of the MUC5AC gene is visible in the area occupied by goblet cells, indicating that the reverse granulation of goblet cells is associated with an increase in the expression of the mucin gene.

Role of EGF-R tyrosine kinase phosphorylation in reverse goblet cell granulation. Previous studies in rats have reported that activation of EGF receptors (EGF-R) results in expression of the mucin gene and protein. To test the hypothesis that EGF-R activation plays a role in the reverse granulation of nasal goblet cells from rats after fMLP, rats were pretreated (n = 5) with a selective EGF-R tyrosine kinase inhibitor, BIBX 1522; the aerosol form of fMLP induces neutrophil recruitment into the nasal epithelium and goblet cell degranulation, but 48 hours later, the areas of AB / PAS staining and MIC5AS-immunostaining remain reduced. These results commit the activation of EGF-R in the synthesis of mucin in nasal cells after degranulation by fMLP.

In the present study, we examined the regulation of mucin production in the rat nasal epithelium. The control epithelium contains a considerable number of goblet cells, with MUC5AC protein present in these cells. However, no expression of the MUC5AC gene was observed. The expression of MUC5AC mucin has been reported to occur in other airway epithelial cells through the expression of EGF-R and their activation. In nasal epithelial cells from control rats, we did not find expression of the EGF-R gene or protein. As there is no lumen staining for AB / PAS or MUC5AC protein, this suggests that no significant cell degranulation (secretion) occurs. In "stable" goblet cells, EGF-R can be upregulated, preventing further mucin synthesis. Therefore, we "induced" nasal goblet cells by inducing goblet cell degranulation and examined the subsequent changes in the structure of the epithelium in the airways.

Neutrophil chemoattractants induce in the respiratory tract of guinea pig and human goblet cell degranulation, which is dependent on neutrophils and is assisted by neutrophil elastase, with close contact between the neutrophils and the tablet cells. To induce degranulation in normal goblet cells present in the nasal epithelium, fMLP is inhalated through the nose. fMLP recruits neutrophils into the nasal epithelium, followed by tablet cell degranulation; The AB / PAS area is noticeably diminished.

Furthermore, we studied the subsequent events in the nasal epithelium after fMLP-induced goblet cell degranulation. At 4 h after fMLP, when maximal nasal goblet cell degranulation manifested, EGF-R and MUC5AC expression were still missing. In addition, 48 h after fMLP, EGF-R was strongly expressed in pre-goblet and goblet cells. The expression of the MUC5AC gene is now strongly expressed in the epithelium, with these events being associated with reverse granulation of goblet cells (increasing AB / PAS and MUC5AC staining). In fact, 48 hours after fMLP, reverse granulation occurs to the point where the area of

the expression of goblet cells is similar to that in the control state.

These findings suggest that goblet cell degranulation leads to the expression and activation of EGF-R, thereby inducing the expression of goblet cells.

MUC5AC.

To test the role of EGF-R tyrosine kinase activation in the reverse granulation of goblet cells, we pre-treated animals with a selective EGF-R tyrosine kinase inhibitor, BIBX1522. In animals pretreated with BIBX 1522, fMLP still causes goblet cell degranulation. However, pretreatment with BIBX 1522 prevents the reverse granulation of goblet cells and their expression of MUC5AC protein. These results commit the activation of EGF-R in the re-growth of mucins after goblet cell degranulation. In pathogens-free rats, goblet cells are "inactive" (ie, not degranulate) and EGF-R is upregulated. When inflammation (eg stimulation of neutrophil infiltration) causes goblet cell degranulation and mucin secretion, overregulation and activation of EGF-R re-supplies mucin to the airway epithelium. The present findings suggest that selective EGF-R tyrosine kinase inhibitors may be useful in preventing overexpression in nasal diseases.

As the present invention has been described with reference to specific embodiments thereof, it should be made clear to those having the necessary experience and knowledge in the field that various changes can be made and equivalents offered without being separated from them. the actual spirit and scope of the invention. In addition, many modifications can be made to adapt to the particular case, material, composition, process, step or steps of the process, in the spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims (27)

A method for treating mucous secretion in the lung, comprising: administering a therapeutically effective amount of an epidermal growth factor receptor antagonist (EGFR) to a patient suffering from mucous secretion in the respiratory tract. The method of claim 1, wherein said EGF-R antagonist is an EGF-R selective kinase inhibitor. The method of claim 2, wherein said antagonist is BIBX 1522. The method of claim 1, wherein the antagonist is an antibody. The method of claim 4, wherein the antibody is a monoclonal antibody that specifically binds the epidermal growth factor (EGF). The method of claim 4, wherein the antibody is a monoclonal antibody that specifically binds to the epidermal growth factor receptor (EGF-R). The method of claim 1, wherein the antagonist is an antisense molecule characteristic of a sequence encoding a protein selected from the group consisting of EGF and EGF-R. The method of claim 1, wherein the antagonist inhibits EGF-R transphosphorylation. The method of claim 8, wherein said antagonist is an anti-oxidant. The method of claim 1, wherein the antagonist is administered by injection. The method of claim 10, wherein the antagonist is administered intravenously with a carrier in the form of a normal saline solution. The method of claim 1, wherein the antagonist is administered by inhalation. The method of claim 1, wherein the antagonist is introduced by liposome delivery. The method of claim 13, wherein said liposome is spatially stabilized and administered intravenously. 15. An in vitro method for screening the agents proposed, comprising: (i) contact of an in vitro model for the growth (proliferation) of goblet cells with EGF or its functional equivalent; (ii) subsequent contact of the in vitro model with a proposed agent; and (iii) estimation of goblet cell growth; as a decrease in goblet cell growth is an indication of the therapeutic potential of the agent. IMIMn » The in vitro model of claim 14, wherein the in vitro models are lung epithelial cells. 17. An in vivo method for screening agents, including: (i) creation of an animal model of pulmonary over-secretion with the involvement of EGF-R; (ii) stimulating the induced EGF-R with a ligand to produce mucin-producing goblet cells; (iii) treatment with the agent proposed; and (iv) assessment of goblet cell growth or mucus secretion; such as inhibition of goblet cell growth or mucus secretion is indicative of the therapeutic potential of the proposed agent. The in vivo model of claim 17, wherein the animal used in the in vivo model is selected from the group consisting of a mouse, rat, rabbit or guinea pig. The in vivo model of claim 17, wherein the in vivo model is an asthmatic model. 20. Pharmaceutical formulation for the treatment of mucous secretion in the respiratory tract, including: a therapeutically effective amount of an epidermal growth factor receptor (EGF-R) receptor antagonist is a dose sufficient to reduce mucus excess secretion in the airways; and a therapeutically acceptable carrier. The formulation of claim 20, wherein said EGF-R antagonist is a kinase inhibitor selective for EGF-R. The formulation of claim 20, wherein said EGF-R antagonist inhibits EGF-R transphosphorylation. The formulation of claim 20, wherein said antagonist is an anti-oxidant. The formulation of claim 20, wherein the antagonist is an antibody. The formulation of claim 24, wherein the antibody is a monoclonal antibody that specifically binds epidermal growth factor (EGF). The formulation of claim 24, wherein the antibody is a monoclonal antibody that specifically binds to the epidermal growth factor receptor (EGF-R). The formulation of claim 20, wherein the antagonist is an antisense molecule characteristic of a sequence encoding a protein selected from the group consisting of EGF and EGF-R.