WO2010137012A1

WO2010137012A1 - Peptide therapy for amphiregulin mediated diseases

Info

Publication number: WO2010137012A1
Application number: PCT/IL2010/000411
Authority: WO
Inventors: Ronit Sagi-Eisenberg; Dana Baram; Ornit Dekel; Yaara Gorzalczany
Original assignee: Ramot At Tel-Aviv University Ltd.
Priority date: 2009-05-25
Filing date: 2010-05-25
Publication date: 2010-12-02

Abstract

The invention is directed to the treatment of amphiregulin-mediated diseases or disorders, specifically to the use of cell permeable peptides, for the treatment of ampheiregulin-mediated diseases including diseases associated with mucus secretion, chronic inflammation and certain types of cancer.

Description

PEPTIDE THERAPY FOR AMPHIREGULIN MEDIATED DISEASES

FIELD OF THE INVENTION

The invention is directed to methods and pharmaceutical compositions for the treatment of amphiregulin-mediated diseases and disorders. In particular, the present invention is directed to the use of cell permeable peptides for the treatment of amphiregulin-mediated diseases including diseases associated with mucus secretion, chronic inflammation and certain types of cancer.

BACKGROUND OF THE INVENTION

Mast cells have long been recognized as key mediators of allergic disorders. However, rapidly accumulating data indicate their major contribution to the progression of chronic inflammatory diseases. Indeed, upon activation by a wide spectrum of stimuli, mast cells express, synthesize and release a variety of inflammatory mediators. Gene array analysis demonstrated the existence of a cluster of remodeling-related genes in mast cells, which are induced by the aggregation of the high-affinity IgE receptor, FcεRI, but are resistant to dexamethasone (Okumura et al., 2005). These genes included amphiregulin (AREG), a polypeptide growth factor that belongs to the epidermal growth factor (EGF) family. AREG is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR mediated by tumor necrosis factor alpha (TNF-α) converting enzyme, also known as TACE or ADAM 17, yields a transmembrane- cytoplasmic fragment (AR-CTF), as well as a soluble ligand.

AREG displays bifunctional properties in that it promotes the growth of fibroblasts, tumor cells, and cultured human epidermal keratinocytes but it was also reported to inhibit the growth of certain normal and neoplastic cell lines. AREG has also been implicated in playing an important role in inflammatory and repair processes, such as cutaneous wound repair and psoriasis. Upregulation of AREG expression was observed in mast cells of asthmatic patients, but not in normal control subjects. Furthermore, upregulation of AREG in mast cells significantly correlated with the extent of goblet cell hyperplasia in the mucosa of patients with bronchial asthma (Wang et al., 2005). Indeed, AREG upregulates mucin gene expression in airway epithelial cells (Okumura et al., 2005). AREG was also found to promote proliferation of airway smooth muscle cells, thus implicating a role in airway remodeling typical to COPD (Shim JY et al., 2008). AREG is also upregulated in polymorphonuclear leukocytes of cystic fibrosis (CF) patients. Moreover, the presence of AREG in the sputum of CF patients was demonstrated thus indicating a role of AREG in cystic fibrosis (Adib-Conquy et al., 2008). AREG binds exclusively to the EGF receptor (EGFR or ErbBl), which has been shown to be a major contributor to cancer development and progression. A role for AREG has been demonstrated in lung cancer and in particular in non-small cell lung carcinoma (NSCLC) (Busser et al., 2008). In addition, AREG is involved in mediating diseases caused by fibrosis, e.g., chronic obstructive pulmonary disease, pulmonary fibrosis, and hepatitis induced fibrosis. AREG promotes the proliferation of primary human lung fibroblasts and AREG treated primary human lung fibroblasts show an increase in the expression of c-fos, a proto-oncogene that facilitates or is required for the proliferation of a wide variety of cells.

A method for treating mammalian diseases mediated by amphiregulin released from mast cells is disclosed in International Patent Application No. WO 06/004593. The method of WO 06/004593 comprises administering an anti-amphiregulin antibody to a mammal useful in preventing or treating allergic diseases, asthma or fibrosis.

International Patent Application No. 2004/068931 provides anti-amphiregulin antibodies for the treatment of patients diagnosed with psoriasis, cancer and/or other proliferative conditions, including benign proliferative conditions. Antibodies against amphiregulin are also disclosed in U.S. Patent No. 5,830,995 and U.S. Patent No. 6,204,359.

U.S. Patent Application Publication No. US 2009/0292007 relates to inhibition of TACE or amphiregulin for the modulation of EGF receptor signal trans-activation. International Patent Application No. WO 09/100445 discloses compositions and methods which modulate G-protein signaling for the treatment of asthma. In particular, WO 09/100445 provides compositions and methods for delivering Gβγ inhibitors into the airway of patients with asthma, particularly, those patients experiencing symptoms associated with β2-adrenergic receptor desensitization. The '445 publication further discloses the inclusion of a membrane permeant peptide sequence from the signal sequence of Kaposi fibroblast growth factor to the Gβγ blocking peptide. While the '445 publication demonstrates a cell-permeable Gβγ blocking peptide effective in treating asthma, cell- permeable Gαi3 and Gαi2 blocking peptides were not effective for such treatment.

Adenosine has long been implicated in a variety of inflammatory processes. Adenosine is released from all cells whenever ATP is degraded, in conditions of increased energy consumption such as stress or hypoxia. Specifically, at sites of inflammation and injury, adenosine levels are markedly elevated. Human mast cells express three members of the adenosine receptor family: The A2a, A2b and A3 (Feoktistov et al., 2003). The A2a and A2b receptors are coupled to Gs, while the A3 R interacts with Ptx sensitive G proteins (Fredholm et al., 2001; Linden, 2001). US Patent No. 6,825,174 discloses compositions and formulations of antisense oligonucleotides exhibiting adenosine receptor inhibitory activity, for the treatment of diseases associated with bronchoconstriction, allergies and inflammation of the lungs.

International Patent Application Publication No. WO 00/78346, by an inventor of the present invention, discloses novel complex molecules useful as anti-allergic agents. The molecules of WO 00/78346 include peptidic or peptidomimetic molecules, having a first segment competent for cell penetration and a second segment able to reduce or abolish mast cell degranulation, particularly histamine secretion from mast cells. Among the peptides disclosed is a novel peptide designated Peptide 2

(AA V ALLP AVLLALLAPKNNLKECGL Y, SEQ ID NO: 10) comprising the C-terminal sequence of Gαi₃ as a second segment (underlined), which inhibited histamine release from activated mast cells.

Additionally, International Patent Application Publication No. WO 02/050097, by an inventor of the present invention, discloses novel anti-allergic complex molecules comprising a first segment competent for cell penetration and a second segment which is able to reduce or abolish mast cell degranulation and prevent late phase inflammatory responses induced by protein kinase activation, and particularly mitogen-activated protein kinase activation. According to WO 02/050097 the first segment is connected to the second segment via a linker or a direct bond that creates a conformational constraint by forming a bend or a turn. Moreover, the second segment having anti-allergic activity is preferably a peptide having a cyclic conformation. The contents of WO 00/78346 and WO 02/050097 are incorporated herein as if set forth in their entirety.

While the complex molecules disclosed in WO 00/78346 and WO 02/050097 were shown to be useful in the treatment of allergies, there is no teaching or suggestion of the use of the molecules for any non-allergic disease or amphiregulin-mediated disorder.

Mucus hypersecretion is a key pathological feature in obstructive inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis and chronic bronchitis, as well as in lung cancer. In normal conditions, airway mucus serves as a protective barrier between the outer environment and the respiratory tract epithelium, withholding pathogens and particles from the respiratory tract. This and other functions, such as hydrating and lubricating, are carried out primarily by the mucus macromolecules, the mucin glycoproteins, produced by and secreted from goblet cells residing in airway epithelium. However, obstruction of the airways by hypersecretion of the same macromolecular complexes is now recognized as a major cause for severe decline in pulmonary function and mortality.

Excess airway mucus contributes to cystic fibrosis (CF) morbidity by increasing the frequency and severity of pulmonary infections. Submucosal gland hypertrophy and airway surface goblet cell metaplasia are also prominent features of COPD, the fourth leading cause of death in the United States and Europe. Histopathological findings from surgical specimens clearly show that increased goblet cell numbers and increased MUC5AC and MUC5B production and secretion are found in the lumen of small airways in COPD patients. Thus, taken together, these results indicate that mucus secretion may be a significant enough factor to result in obstruction of small airways. Therefore, mucus secretion is of considerable impact on disease pathogenesis and prognosis (Evans and Koo, 2008).

Upregulation of mucin gene expression and mucus hypersecretion are also seen in several forms of lung cancer. In particular, mucous metaplasia is a frequent marker in epithelial non-small cell lung carcinomas and is prominent in adenocarcinomas and mucinous bronchoalveolar carcinomas (Evans and Koo, 2008). Membrane associated mucin and mucin-like genes are also altered in lung cancer. These processes include upregulation of Mucl and Muc4, whose membrane bound forms interact with and affect EGF receptor signaling (Hattrup and Gendler, 2008). Indeed, overexpression of MUCl was recorded in more than 90% of breast carcinomas. A high appearance frequency was also demonstrated in other types of cancer, including ovarian, lung, colon, and pancreatic carcinomas. Moreover, the level of MUCl expression correlates with the tumor stage and poor prognosis. The MUCl that is overexpressed in tumors displays a distinct pattern of subcellular localization and O-glycosylation.

There are no direct medications to reduce mucus hypersecretion. Current therapies such as glucocorticoids aim at reducing the inflammatory condition that provokes the overexpression process rather than blocking the production or secretion of the mucin glycoproteins. However, while glucocorticoids demonstrate efficacy in asthma patients, glucocorticoid treatment is largely ineffective in relation to overproduction of sputum and lung tissue remodeling in asthma and is ineffective in CF and COPD.

There remains an unmet medical need for providing additional therapies for amphiregulin-mediated diseases and disorders. In particular, there is an unmet medical need for providing therapies for diseases associated with chronic inflammation and mucus hypersecretion as well as fibrosis and cancer.

SUMMARY OF THE INVENTION

The invention is directed to compositions and methods using peptide therapy for the treatment of amphiregulin (AREG) mediated diseases and disorders. Specifically, the invention provides therapeutic methods utilizing synthetic cell permeable peptides comprising the C-terminal sequence of the alpha subunit of a G protein, fused to a cell importation sequence. The methods are particularly useful for the treatment of AREG- mediated lung diseases associated with chronic inflammation and mucus hypersecretion, including chronic obstructive pulmonary disease (COPD), goblet cell hyperplasia non- allergic asthma and lung cancer. The methods of the present invention are also useful for the treatment of fibrosis and cancer involving amphiregulin as an autocrine activator.

As exemplified hereinbelow, human mast cells (HMC-I) are activated by membranes of stimulated T cells. The invention is based in part, on the discovery that said activated mast cells endogenously generate adenosine which binds the adenosine A3 receptor (A3 R), resulting in the activation of the trimeric G-protein Gi₃. Surprisingly, this process was found to result in up-regulation of expression and release of amphiregulin (AREG), the major cause of mucus overproduction in the lung.

It is now disclosed for the first time that a 26-mer peptide designated herein below ALLl having the amino acid sequence AAV ALLP AVLL ALL APKNNLKECGLY (SEQ ID NO: 10), comprising the C-terminal 10 amino acids of Gαi₃ fused to the Kaposi fibroblast growth factor signal peptide, unexpectedly inhibited the elevated expression and secretion of AREG.

The invention is also based in part on the discovery that supernatants derived from activated, but not resting mast cells, stimulate the proliferation and survival of H 1299 cells, a human non-small cell lung carcinoma (NSCLC) cell line. Surprisingly, pre-incubation of the activated mast cells with ALLl, remarkably reduced NSCLC proliferation. Moreover, direct exposure of NSCLC cells to ALLl, in the absence of mast cell derived supernatants, results in inhibition of NSCLC proliferation.

Thus, the present invention provides therapeutic methods utilizing synthetic cell permeable peptides comprising the C-terminal sequence of the alpha subunit of a G protein fused to a cell importation sequence, particularly useful for the treatment of AREG- mediated diseases. Therefore, the cell permeable peptides are advantageously useful for the treatment of diseases and disorders associated with chronic inflammation and mucus hypersecretion, such as lung diseases. In addition, the cell permeable peptides are particularly useful for the treatment of cancer, particularly of epithelial cell carcinomas. International Patent Application Publication No. WO 2006/046250 and WO

2002/050097, both by an inventor of the present invention, disclose said peptides for the specific treatment of allergies. Unexpectedly, said peptides were found to have a therapeutic effect on AREG-mediated disorders. It is to be explicitly appreciated that the known peptides are claimed for the novel uses as disclosed herein. It is to be understood that the patient populations that are amenable to treatment according to the present invention are not those subjects having allergic inflammatory reactions that were disclosed to be treatable with the known peptides in WO 2006/046250 and WO 2002/050097.

Without wishing to be bound by any theory or mechanism of action it is now disclosed for the first time that diseases and disorders regulated by amphiregulin, including those that are associated with mucus secretion are amenable to treatment with the peptides previously known to prevent histamine secretion and mast cell degranulation. As exemplified hereinbelow the peptides of the invention block upregulation and secretion of amphiregulin. Thus the peptides of the invention are suitable for treatment of amphiregulin-mediated diseases and disorders and particularly phenomena related to fibrosis and mucus hyper-secretion (e.g., CF, COPD and non-allergic asthma). Unexpectedly, cancers (e.g. epithelial cell carcinomas) can also be ameliorated by the treatments of the invention.

According to one aspect, the present invention provides a method for the treatment or prophylaxis of a AREG-mediated disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a therapeutic composition comprising a peptide or peptidomimetic molecule having at least a first segment competent for importation of the peptide or peptidomimetic molecule into cells, and a second segment derived from the C-terminal sequence of a Ga protein, and a pharmaceutical acceptable carrier, wherein the second segment is capable of inhibiting AREG upregulation and/or secretion, and wherein the first segment is joined to the second segment through a linker. According certain embodiments, the first segment is a peptide.

According to other embodiments, the linker joining the first segment to the second segment is a covalent bond. According to particular embodiments, the covalent bond is a peptide bond.

According to certain embodiments, the linker must be of such a nature as to create a conformational constraint at or near the junction between the first segment and the second segment. Preferably the linker must prevent the first segment from being contiguous to the second segment in a linear or an extended conformation. According to one embodiment, the linker will create a bend between the two segments. According to another embodiment, the linker will create a turn at, or near, the junction between the two segments. According to certain currently most preferred embodiments the conformational constraint is selected from the group consisting of, a proline or proline mimetic, an N-alkylated amino acid, a double bond or triple bond or any other moiety which introduces a rigid bend in the peptide backbone.

In addition to Proline, specific examples of moieties which induce suitable conformations include but are not limited to N-methyl amino acids such as sarcosine; hydroxy proline instead of proline; anthranilic acid (2-amino benzoic acid); and 7- azabicyloheptane carboxylic acid.

According to certain embodiments, the second segment is sufficient for ameliorating the symptoms of said AREG-mediated disease or disorder. According to particular embodiments, the second segment is selected from the group consisting of a peptide, a peptidomimetic, or a polypeptide. According to preferred embodiments, the second segment is a peptide, having a cyclic conformation stabilized by bonds selected from the group consisting of hydrogen bonds, ionic bonds and covalent bonds.

Preferably, the second segment has an amino acid sequence selected from the group consisting of: a decapeptide derived from Gαi₃ having the sequence KNNLKECGLY (SEQ ID NO: 1); a decapeptide derived from Gαi₂ having the sequence KNNLKDCGLF (SEQ ID NO: 2); a decapeptide derived from Gαt having the sequence KENLKDCGLF (SEQ ID NO: 3); Cyclic Gαi₃ KNNLKECGLY

I ε-NH (SEQ ID NO: 4); KNNLKECGL-para-amino-F (SEQ ID NO:5);

KQNLKECGLY (SEQ ID NO:6);

KSNLKECGLY (SEQ ID NO:7);

KNNLKEVGLY (SEQ ID NO:8); and

KENLKECGLY (SEQ ID NO:9). Within the scope of the present invention are included all active analogues, homologues and derivatives of these sequences, including but not limited to cyclic derivatives.

Preferably the importation competent segment of the molecule is a peptide taken from a signal peptide sequence. Useful examples thereof include the signal peptide sequence of the Kaposi fibroblast growth factor or a human integrin β3.

According to preferred embodiments, the complex molecule is a peptide having an amino acid sequence selected from the group consisting of:

AAVALLPAVLLALLAPKNNLKECGLY (SEQ ID NO: 10);

AAVALLPAVLLALLAPKNNLKDCGLF (SEQ ID NO: 11); AAVALLPAVLLALLAPKENLKDCGLF (SEQ ID NO: 12);

AAVALLPAVLLALLAPKQNLKECGLY (SEQ ID NO: 13);

AAVALLPAVLLALLAPKNNLKEVGLY (SEQ ID NO: 14);

Succinyl-AAVALLPAVLLALLA-Sar-KNNLKECGLY (SEQ ID NO: 15);

Succinyl- VTVLALG ALAGVGVGPKNNLKECGLY (SEQ ID NO: 16); Succinyl- AAV ALLP AVLLALLAPKSNLKECGLY (SEQ ID NO: 17);

Succinyl-AA V ALLP AVLLALLAPKENLKECGLY (SEQ ID NO: 18);

Succinyl- AAV ALLP AVLLALLAPKANLKECGLY (SEQ ID NO: 19); Succinyl-AAVALLPAVLLALLAPKNNLKECGL-para-amino-F (SEQ ID NO: 20) ; Succinyl-AAV ALLP AVLLALLAPKQNLKECGLY (SEQ ID NO: 21); and Succinyl-AAV ALLP AVLLALLAPKNNLKEVGLY (SEQ ID NO: 22).

Within the scope of the present invention are included all active analogues, homologues and derivatives of these sequences, including but not limited to cyclic derivatives. In particular, active analogs are intended to include esters, such as but not limited to succinylated derivatives.

According to certain embodiments, the AREG-mediated disease or disorder is a chronic non-allergic lung disease. According to particular embodiments, the non-allergic lung disease is selected from the group consisting of chronic obstructive pulmonary disease

(COPD), goblet cell hyperplasia, chronic bronchitis, non-allergic asthma and cystic fibrosis.

Each possibility represents a separate embodiment of the present invention.

According to other embodiments the AREG-mediated disease or disorder is cancer. According to one embodiment, the cancer is an epithelial cell cancer. According to some embodiments, the cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, colon cancer, brain cancer, head and neck cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, rectal cancer, colorectal cancer, genital-urinary cancer, bladder cancer and glioblastoma. Each possibility represents a separate embodiment of the present invention.

According to one embodiment the cancer is lung cancer. According to particular embodiments the lung cancer is selected from the group consisting of small cell lung carcinoma, lung adenocarcinoma, squamous cell lung carcinoma and non-small cell lung carcinoma. Preferably, the lung cancer is non-small cell lung carcinoma. Each possibility represents a separate embodiment of the present invention.

According to other embodiments the AREG-mediated disorder is a fibrotic disease.

According to particular embodiments, the fibrotic disease is selected from the group consisting of fibrosis and remodeling of lung tissue in chronic obstructive pulmonary disease, fibrosis and remodeling of lung tissue in chronic bronchitis, fibrosis and remodeling of lung tissue in emphysema, lung fibrosis and pulmonary diseases with a fibrotic component, fibrosis and remodeling in asthma, fibrosis in rheumatoid arthritis, virally induced hepatic cirrhosis, radiation-induced fibrosis, post angioplasty restenosis, chronic glomerulonephritis, renal fibrosis in patients receiving cyclosporine and renal fibrosis due to high blood pressure, diseases of the skin with a fibrotic component, and excessive scarring. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the fibrotic disease is an organ fibrosis selected from the group consisting of: liver fibrosis (i.e., hepatic fibrosis), lung fibrosis, kidney fibrosis and skin fibrosis. Each possibility represents a separate embodiment of the present invention.

According to other embodiments the AREG-mediated disorder is psoriasis. According to certain embodiments, the AREG-mediated disease or disorder is other than psychogenic or allergic asthma. According to another embodiment, the AREG- mediated disease or disorder is other than asthma. According to another embodiment, the AREG-mediated disease or disorder is other than allergy.

According to another aspect, the present invention provides a pharmaceutical composition comprising a peptide or peptidomimetic molecule having at least a first segment competent for importation of the peptide or peptidomimetic molecule into cells, and a second segment derived from the C-terminal sequence of a Ga protein, and a pharmaceutically acceptable carrier, wherein the second segment is capable of inhibiting AREG upregulation and/or secretion, wherein the first segment is joined to the second segment through a linker, for the treatment or prophylaxis of a AREG-mediated disorder in a subject.

According to yet another aspect, the present invention provides use of a peptide or peptidomimetic molecule having at least a first segment competent for importation of said molecule into cells, and a second segment derived from the C-terminal sequence of a Ga protein, wherein the second segment is capable of inhibiting AREG upregulation and/or secretion, wherein the first segment is joined to the second segment through a linker, for the preparation of a medicament useful in the treatment or prophylaxis of an AREG-mediated disorder in a subject.

The pharmaceutical compositions and methods of the present invention can be used in combination therapy with standard medicaments for the diseases listed herein above.

According to one embodiment, the method of the present invention further comprises administration of at least one anti- inflammatory agent for the treatment of a lung disease or disorder. According to certain embodiments the anti- inflammatory agent is selected from the group consisting of: corticosteroids, sodium cromolyn, IgE inhibitors, phosphodiesterase inhibitors, methylxanthines, beta-adrenergic agents, and leukotriene modifiers. According to one particular embodiment, the method of the present invention further provides administration of corticosteroids.

Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that human mast cells (HMC-I) are activated by T cell membrane. HMC-I cells were incubated with increasing concentrations of membranes isolated from PMA-activated Jurkat T cells (FIG. IA), or resting (r-Tm) and activated (T*m) Jurkat T cells (FIG. IB). Supernatants were collected for measurement of β-hexoseaminidase (β- Hex) release. HMC-I cells were incubated for the indicated time periods with T*m. Cell activation was measured by Western blotting using anti phospho-tyrosine (FIG. 1C), anti phospho-ERKl/2 (FIG. ID) and anti tubulin antibodies, as indicated.

Figure 2 indicates that mast cell activation by T cell membrane is mediated by Gi3 and adenosine. HMC-I cells were preincubated with 200 μM ALLl (FIG. 2A), increasing concentrations of ALLl (FIG. 2C) or 1.5 U/ml adenosine deaminase (ADA; FIG. 2 E). Cells were then stimulated with activated T membranes (T*m). HMC-I activation was measured by Western blotting using anti phospho-tyrosine and anti tubulin (FIG. 2A), anti pohspho-ERKl/2 and anti ERK2 (FIG. 2C) or anti phospho-MEKl/2 or anti MEK1/2(FIG. 2E). The intensities of the bands were quantified and the average relative (phosphorylated/total) pixel densities were calculated and plotted (FIGs. 2B, D and F, respectively). Figure 3 shows that human mast cells, activated by Cl-IBMECA, an adenosine A3 receptor agonist, are inhibited by ALLl. HMC-I cells activation was measured by Western blotting using anti-phospho-MEKl/2 and anti-MEKl/2 (FIG. 3A) or anti-phospho-ERKl/2 and anti-ERK2 (FIG. 3C). The intensities of the bands were quantified and the average relative (phosphorylated/total) pixel densities were calculated and plotted (FIGs. 3B and D, respectively).

Figure 4 shows that AREG and additional growth factors expression is induced by Cl-IBMECA and inhibited by ALLl .

Figure 5 indicates that AREG is induced by Cl-IBMECA and activated T cell membranes.

Figure 6 shows that AREG secretion is partially inhibited by Gi3 or A3R inhibition.

Figure 7 shows the enhanced proliferation of human non small cell lung carcinoma cell line (H 1299 NSCLC) incubated with supernatants of activated mast cells.

Figure 8 shows that H 1299 NSCLC cell proliferation is inhibited upon incubation with either supernatant of ALLl -treated mast cells or direct exposure of ALLl to NSCLC cells.

Figure 9 shows that elevation of cytosolic Ca²⁺ facilitates Cl-IBMECA-induced AREG secretion.

Figure 10 indicat es that Dexamethasone (DEX) enhances Cl -IBMECA-induced AREG secretion.

Figure 11 indicates that elevation of cytosolic levels of cAMP facilitates Cl-IBMECA-induced AREG secretion.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to compositions and methods using peptide therapy for the treatment of AREG-mediated diseases and disorders. Specifically, the invention provides therapeutic methods utilizing synthetic cell permeable peptides comprising the C- terminal sequence of the alpha subunit of a G protein capable of inhibiting AREG upregulation and/or secretion, fused to a cell importation sequence. The methods are particularly useful for the treatment of non- allergic lung diseases associated with chronic inflammation and mucus hypersecretion, including chronic obstructive pulmonary disease

(COPD), goblet cell hyperplasia and non-allergic asthma. The methods are also useful for the treatment of fibrosis and cancer.

Amphiregulin (AREG), a member of the epidermal growth factor family, is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR mediated by TNF-α converting enzyme, TACE-ADAM 17, yields a transmembrane-cytoplasmic fragment (AR-CTF), as well as a soluble ligand. The amino acid sequence of amphiregulin is set forth in SEQ ID NO: 23 (GenBank Accession No. AAA51781.1). As exemplified herein below, a cell permeable peptide termed ALLl, having the amino acid sequence Ala-Ala- Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala- Pro-Lys-Asn-Asn-Leu-Lys-Glu-Cys-Gly-Leu-Tyr as set forth in SEQ ID NO: 10, comprising the C-terminal 10 amino acids of Gαi₃ fused to the Kaposi fibroblast growth factor signal peptide, unexpectedly and specifically inhibited the elevated expression and secretion of AREG, the major cause of mucus overproduction in the lung.

Antiinflammatories are commercially available and are currently prescribed for the treatment of disorders associated with chronic inflammation, particularly lung disorders. The most common antiinflammatories, corticosteroids, have considerable side effects but are commonly prescribed nevertheless. As described herein below, corticosteroids, and particularly dexamethasone, enhances AREG secretion from activated mast cell. Thus, the molecules of the present invention are advantageously effective in decreasing mucus hypersecretion in patients treated with corticosteroids.

As described herein below, supernatants derived from activated, but not resting mast cells, stimulate the proliferation and survival of H 1299 cells, NSCLC cell line. Surprisingly however, supernatants derived from activated mast cells which were pre- incubated with ALLl, remarkably reduced NSCLC proliferation. Moreover, direct exposure of ALLl to NSCLC cells unexpectedly resulted in inhibition of NSCLC cells proliferation.

Thus, the present invention provides a method for the treatment or prophylaxis of AREG-mediated disorder in a subject, comprising administering to said subject a therapeutically amount of a complex molecule having at least a first segment competent for importation of said molecule into cells, and a second segment sufficient for ameliorating the symptoms of said AREG-mediated disorder, said first segment being joined to said second segment through a linker. According to certain embodiments, the complex molecule is a peptide or peptidomimetic.

According to some embodiments, the therapeutic composition consists of the peptide or peptidomimetic molecule having at least a first segment competent for importation of the peptide or peptidomimetic molecule into cells, and a second segment derived from the C-terminal sequence of a Ga protein, and a pharmaceutical acceptable carrier.

According to one embodiment, the first segment is a peptide. According to another embodiment, the first segment is a peptidomimetic.

According to another preferred embodiment, the first segment is a signal peptide. A signal peptide is a peptide which is capable of penetrating through the cell membrane, to permit the exportation and/or importation of proteins or peptides. As used herein, suitable signal peptides are those which are competent for the importation of proteins, peptides or other molecules into the cell. According to certain embodiments, the signal peptide of the present invention is competent for the importation of peptides into mast cells, preferably human mast cells. According to other embodiments, said cells are cancer cells, preferably lung cancer cells (e.g. NSCLC cells). According to other embodiments, the signal peptide is competent for the importation of peptides into cells in need of AREG expression and/or secretion regulation. Such signal peptides generally feature approximately 10-50 amino acids, of which the majority are typically hydrophobic, such that these peptides have a hydrophobic, lipid-soluble portion. Preferably, signal peptides are also selected according to the type of cell into which the complex is to be imported, such that signal peptides produced by a particular cell type, or which are derived from peptides and/or proteins produced by that cell type, can be used to import the complex into cells of that type. Examples of such signal peptides are described above and are also disclosed in U.S. Pat. No. 5,807,746, incorporated by reference as if fully set forth herein for the teachings regarding signal peptides.

Additional exemplary peptides are disclosed, for instance, in Foerg and Merkle, Journal of Pharmaceutical Science, 97: 144-162, 2008, incorporated by reference as if fully set forth herein for the teachings regarding signal peptides. Exemplary signal peptides include the HIV tat peptide, nontoxic membrane translocation peptide from protamine (Park et al. FASEB J. 19(l l):1555-7, 2005), oligoarginine (R_n), and the antimicrobial peptide Buforin 2. Additional exemplary peptides are disclosed in U.S. Patent No. 6,841,535 which relates to peptide-mediated transfection agents (e.g. CHARIOT® delivery reagent).

Non limiting examples of signal peptides include: GRKKRRQRRRPPQQ as set forth in SEQ ID NO: 24;

RQIKIWFQNRRMKWKK as set forth in SEQ ID NO: 25;

RVIRVWFQNKRCKDKK as set forth in SEQ ID NO: 26;

GWTLNSAGYLLGKINLKALAALAKKIL as set forth in SEQ ID NO: 27; GALFLGFLGAAGSTMGAWSQPKKKRKV as set forth in SEQ ID NO: 28;

GALFLAFLAAALSLMGLWSQPKKKRKV as set forth in SEQ ID NO: 29;

KETWWETWWTEWSQPKKKRKV as set forth in SEQ ID NO: 30;

TRSSRAGLQWPVGRVHRLLRK as set forth in SEQ ID NO: 31;

PRPLPFPRPG as set forth in SEQ ID NO: 32; RGGRLSYSRRRFSTSTGR as set forth in SEQ ID NO: 33;

LLIILRRRIRKQAHAHSK as set forth in SEQ ID NO: 34;

DAATATRGRSAASRPTE as set forth in SEQ ID NO: 35;

RPRAPARSASRPRRPVE as set forth in SEQ ID NO: 36;

LGTYTQDFNKFHTFPQTAIGVGAP as set forth in SEQ ID NO: 37; AFGVGPDEVKRKKKP as set forth in SEQ ID NO: 38; and

VRLPPPVRLPPPVRLPPP as set forth in SEQ ID NO: 39.

According to certain embodiments, the signal peptide of the present invention is selected form the group consisting of SEQ ID NO: 24 to SEQ ID NO: 39. Each possibility represents a separate embodiment of the present invention. The second segment is a molecule which has a therapeutic effect for the treatment of amphiregulin-mediated disorders. According to one embodiment, the second segment is a peptide. According to one embodiment, the second segment is a peptidomimetic. According to another embodiment, the second segment is a polypeptide.

Preferably the second segment is derived from the C terminal sequence of Ga protein. According to one embodiment, the second segment is a decapeptide. According to another embodiment, the molecule is a decapeptide derived from the C terminal sequence of Ga protein. According to another embodiment, the Ga protein is Gai₃ (KNNLKECGLY;

SEQ ID NO: 1). According to another embodiment, the Ga protein is Gai₂

(KNNLKDCGLF; SEQ ID NO: 2). According to another embodiment, the Ga protein is Gat (KENLKDCGLF; SEQ ID NO: 3). According to another embodiment, the second segment is selected from the group consisting of a peptidomimetic, a polypeptide, or a protein. Each possibility represents a separate embodiment of the present invention. According to certain embodiments of the present invention the second segment has an amino acid sequence selected from the group consisting of: KNNLKECGLY (SEQ ID NO: 1); KNNLKDCGLF (SEQ ID NO: 2); KENLKDCGLF (SEQ ID NO: 3);

KNNLKECGLY

I I

I ε-NH (SEQ ID NO: 4);

KNNLKECGL-para-amino-F (SEQ ID N0:5); KQNLKECGLY (SEQ ID N0:6); KSNLKECGLY (SEQ ID N0:7);

KNNLKEVGLY (SEQ ID N0:8); and KENLKECGLY (SEQ ID NO:9).

According to one embodiment, the second segment has the amino acid sequence of

SEQ ID NO: 1. According to another embodiment, the second segment has the amino acid sequence of SEQ ID NO: 2. According to another embodiment, the second segment has the amino acid sequence of SEQ ID NO: 3. Each possibility represents a separate embodiment of the present invention.

According to further embodiments, the present invention provides complex molecule (e.g., a peptide or peptidomimetic molecule) having an amino acid sequence selected from the group consisting of:

AAVALLPAVLLALLAPKNNLKECGLY (SEQ ID NO: 10); AAVALLPAVLLALLAPKNNLKDCGLF (SEQ ID NO: 11); AAVALLPAVLLALLAPKENLKDCGLF (SEQ ID NO: 12); AAVALLPAVLLALLAPKQNLKECGLY (SEQ ID NO: 13); AAVALLPAVLLALLAPKNNLKEVGLY (SEQ ID NO: 14);

Succinyl- AAV ALLP AVLLALLA-Sar-KNNLKECGLY (SEQ ID NO: 15); Succinyl- VTVLALGALAGVGVGPKNNLKECGLY (SEQ ID NO: 16); Succinyl- AAV ALLP AVLLALLAPKSNLKECGLY (SEQ ID NO: 17); Succinyl- AAV ALLP AVLLALLAPKENLKECGLY (SEQ ID NO: 18); Succinyl- AAV ALLP AVLLALLAPKANLKECGLY (SEQ ID NO: 19);

Succinyl-AAVALLPAVLLALLAPKNNLKECGL-para-amino-F (SEQ ID NO: 20) ; Succinyl-AA V ALLP AVLLALLAPKQNLKECGLY (SEQ ID NO: 21); and Succinyl-AAV ALLP AVLLALLAPKNNLKEVGLY (SEQ ID NO: 22).

According to one embodiment, the complex molecule has the amino acid sequence of SEQ ID NO: 10. According to another embodiment, the second segment has the amino acid sequence of SEQ ID NO: 11. According to another embodiment, the second segment has the amino acid sequence of SEQ ID NO: 12. Each possibility represents a separate embodiment of the present invention.

The term "peptide" as used herein encompasses native peptides (degradation products, synthetic peptides or recombinant peptides), peptidomimetics (typically including non peptide bonds or other synthetic modifications), and the peptide analogues peptoids and semipeptoids, and may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.

The term "polypeptide" as used herein refers to a linear series of natural, non- natural and/or chemically modified amino acid residues connected one to the other by peptide bonds. The amino acid residues are represented throughout the specification and claims by either one or three-letter codes, as is commonly known in the art. A peptide mimetic or peptidomimetic as used herein is a molecule that mimics the biological activity of a peptide but is not completely peptidic in nature. Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of chemical moieties that closely resembles the three-dimensional arrangement of groups in the peptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems which are similar to the biological activity of the peptide.

Without wishing to be bound by theory, the present invention encompasses peptide, peptide analog and peptidomimetic compositions, which are capable of inhibiting AREG upregulation and/or secretion. Said peptide/peptidomimetic compositions are effective in situations where down regulation of AREG is desirable and where up-regulation of AREG is beneficial, including but not limited to non-allergic lung diseases, cancer and fibrosis.

There are clear advantages for using a mimetic of a given peptide rather than the peptide itself, because peptides commonly exhibit two undesirable properties: poor bioavailability and short duration of action. Peptide mimetics offer a route around these two major obstacles, since the molecules concerned are have a long duration of action. Small peptidomimetics of 3-6 amino acids exhibit improved patient compliance since they can be administered orally compared with parenteral administration for peptides or larger peptidomimetics. Furthermore there are problems associated with stability, storage and immunoreactivity for peptides that are not experienced with peptide mimetics.

Peptidomimetics are small molecules that can bind to proteins by mimicking certain structural aspects of peptides and proteins. They are used extensively in science and medicine as agonists and antagonists of protein and peptide ligands of cellular and other receptors, and as substrates and substrate analogs for enzymes. Some examples are morphine alkaloids (naturally-occurring endorphin analogs), penicillins (semi-synthetic), and HIV protease inhibitors (synthetic). Such compounds have structural features that mimic a peptide or a protein and as such are recognized and bound by other proteins. Binding the peptidomimetic either induces the binding protein to carry out the normal function caused by such binding (agonist) or disrupts such function (antagonist, inhibitor).

A primary goal in the design of peptide mimetics has been to reduce the susceptibility of mimics to cleavage and inactivation by peptidases. In one approach, one or more amide bonds have been replaced in an essentially isosteric manner by a variety of chemical functional groups. In another approach, a variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilized by a covalent modification, such as cyclization or by incorporation of γ-lactam or other types of bridges as disclosed for example in US patent 5,811,392. In US Patent 5,552,534, non-peptide compounds are disclosed which mimic or inhibit the chemical and/or biological activity of a variety of peptides. Such compounds can be produced by appending to certain core species, such as the tetrahydropyranyl ring, chemical functional groups which cause the compounds to be at least partially cross-reactive with the peptide. As will be recognized, compounds which mimic or inhibit peptides are to varying degrees cross- reactive therewith. Other techniques for preparing peptidomimetics are disclosed in US Patent 5,550,251 and US Patent 5,288,707, for example. Non-limiting examples of the use of peptidomimetics in the art include inhibitors of protein isoprenyl transferases (particularly protein farnesyltransferase and geranylgeranyltransferase) and anti-cancer drugs (US patent 5,965,539) inhibitors of p21 ras (US patent 5,910,478 ) and inhibitors of neurotropin activity (US patent 6,291,247).

As previously mentioned, the complex molecule of the present invention comprises a first segment competent for importation of said molecule into mast cells, and a second segment sufficient for ameliorating the symptoms of said amphiregulin-mediated disorder.

According to the present invention, the first segment is joined to the second segment through a linker. The linker is a crucial element of the present invention, and preferably it must impose certain conformational constraints at or near the junction of the two segments of the molecule. In one embodiment, the first segment is connected to the second segment through a linker. In another embodiment, the first segment is connected to the second segment through a direct bond. In one embodiment, the linker creates a conformational constraint by forming a bend. . In one embodiment, the linker creates a conformational constraint by forming a turn. According to certain currently most preferred embodiments the conformational constraint is selected from the group consisting of, a proline or proline mimetic, an N alkylated amino acid, a double bond or triple bond or any other moiety which introduces a rigid bend into the peptide backbone. Each possibility represents a separate embodiment of the present invention.

In addition to proline, specific examples of moieties which induce suitable conformations include but are not limited to N-methyl amino acids such as sarcosine, hydroxy proline, anthranilic acid (2-amino benzoic acid) and 7-azabicyloheptane carboxylic acid. For example, as depicted in SEQ ID NO: 15, the first segment is connected to the second segment through sarcosine (Sar), an N-methyl amino acid.

The linker which connects the first segment to the second segment is preferably a covalent bond. Conveniently, the covalent bond may be a peptide bond if at least one of the first and second segments is a peptide.

A range of methods of creating suitably constrained conformations at or near the junction of the complex molecules of the invention are well known in the art. Classical methods of introducing conformational constraints include structural alteration of amino acids or introduction of bonds other than a flexible peptide bond. In addition to other modes of conformational restriction, such as configurational and structural alteration of amino acids, local backbone modifications, short-range cyclization, medium and long range cyclizations (Hruby, V. J., Life Sci. 31, 189 (1982); Kessler, H., Angew. Chem. Int. Ed. Eng.,21, 512 (1982); Schiller, P. W., in The Peptides, Udenfriend, S., and Meienhofer, J. Eds., Volume 6 p. 254 (1984); Veber, D. F. and Freidinger, R. M., Trends in Neurosci. 8, 392 (1985); Milner- White, E. J., Trends in Pharm. Sci. 10, 70 (1989)) are useful to optimize the active conformations of the peptides according to the invention.

Therapeutically active peptides are cyclized to achieve metabolic stability, to increase potency, to confer or improve selectivity and to control bioavailability. The possibility of controlling these important pharmacological characteristics through cyclization of linear peptides prompted the use of medium and long range cyclization to convert natural bioactive peptides into peptidomimetic drugs, as is known in the art. Cyclization also brings about structural constraints that enhance conformational homogeneity and facilitates conformational analysis (Kessler, H., Angew. Chem. Int. Ed. Eng., 21, 512 (1982)).

Moreover, the combination of structural rigidification-activity relationship studies and conformational analysis gives insight into the biologically active conformation of linear peptides.

The therapeutic complex of the present invention can be manufactured in various ways. For example, if the therapeutic complex includes a peptide for at least one of the first segment and the second segment, or if the entire therapeutic complex is a peptide, then such a peptide could be manufactured by peptide synthetic methods which are well known in the art.

Alternatively, such a peptide could be produced by linking the signal sequence and the biologically active moiety through laboratory techniques for molecular biology which are well known in the art. According to another preferred embodiment of the present invention, a peptide could optionally be modified. For example, the N-terminus of the peptide could be modified by succinylation, addition of a sugar residue, or addition of stearic or palmitic acid. For example, a peptide having a succinyl group linked to its N-terminus has increased solubility. In addition, certain amino acids of the peptide could also be modified. For example, if the peptide includes a cysteine at amino acid 23, this cysteine could be replaced by another amino acid, including but not limited to, amino butyric acid, serine or other such amino acids. As another example, if the peptide includes a lysine at amino acid 17, this residue could be replaced by another amino acid, such as a neutral amino acid, or two amino acids such as a pair of glutamic acid residues. As yet another example, if the peptide includes a proline at amino acid 16, this residue could be replaced by another amino acid, such as a neutral amino acid, or two amino acids such as a pair of glutamic acid residues. Thus, the peptide could optionally be modified in order to enhance penetration into the cell or to enhance the pharmaceutical activity, for example.

Hereinafter, the term "biologically active" refers to molecules, or complexes thereof, which are capable of exerting an effect in a biological system. Hereinafter, the terms "fragment" or "segment" refer to a portion of a molecule or a complex thereof, in which the portion includes substantially less than the entirety of the molecule or the complex thereof. The term "decapeptide" as used herein refers to a peptide containing ten amino acids.

The therapeutic complex of the present invention can be manufactured in various ways. For example, if the therapeutic complex includes a peptide for at least one the first segment and the second segment, or if the entire therapeutic complex is a peptide, then such a peptide could be manufactured by peptide synthetic methods which are well known in the art.

Alternatively, such a peptide could be produced by linking the signal sequence and the biologically active moiety through laboratory techniques for molecular biology which are well known in the art.

By way of illustration, as a non-limitative example, a recombinant fusion protein could be prepared which would feature the peptide permeabilization sequence in the N- terminus and the C-terminal moiety of Gαt, G0U₃ or GaI₂, preferably including the last 10 amino acids, for production in bacteria. For this purpose, DNA sequences coding for the desired peptides are amplified by PCR and purified. After sequence verification, these DNA sequences are ligated and cloned in an appropriate vector. The resulting recombinant plasmid is expressed in E. coli and the recombinant proteins purified from bacterial extracts.

According to one embodiment, the molecules of the present invention are capable of inhibiting expression of amphiregulin gene. "Expression" is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s). As used herein, the terms "inhibit expression" and "inhibit upregulation" are meant reducing, diminishing or suppressing expression of the amphiregulin gene by any method known to the art. According to some embodiments, the molecule of the present invention is capable of inhibiting at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of amphiregulin upregulation. Each possibility represents a separate embodiment of the present invention.

According to one embodiment, the molecules of the present invention are capable of inhibiting amphiregulin secretion. According to some embodiments, the molecule of the present invention is capable of inhibiting at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% of amphiregulin secretion. Each possibility represents a separate embodiment of the present invention.

The term "amino acid" or "amino acids" is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2-aminoadipic acid, hydroxylysine, isodesmosine, nor- valine, nor-leucine, and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids. Natural aromatic amino acids, Trp, Tyr, and Phe, may be substituted for synthetic non-natural acids such as, for instance, tetrahydroisoquinoline- 3-carboxylic acid (TIC), naphthylalanine (NoI), ring-methylated derivatives of Phe, halogenated derivatives of Phe, and o-methyl-Tyr. One of skill in the art will recognize that individual substitutions, deletions or additions to a peptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a conservatively modified variant where the alteration results in the substitution of an amino acid with a similar charge, size, and/or hydrophobicity characteristics, such as, for example, substitution of a glutamic acid (E) to aspartic acid (D). Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, the term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. In general, analogs typically will share at least 50% amino acid identity to the native sequences disclosed in the present invention, in some instances the analogs will share at least 60% amino acid identity, at least 70%, 80%, 90%, and in still other instances the analogs will share at least 95% amino acid identity to the native polypeptides. Each possibility represents a separate embodiment of the present invention.

The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function of inhibiting ARGE expression and/or secretion as specified herein. Typically, the present invention encompasses derivatives of the peptides. The term

"derivative" includes any chemical derivative of the peptide having one or more residues chemically derivatized by reaction of side chains or functional groups. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxy 1 groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine.

In addition, a peptide derivative can differ from the natural sequence of the peptides of the invention by chemical modifications including, but are not limited to, terminal-NH₂ acylation, acetylation, or thioglycolic acid amidation, and by terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like. Peptides can be either linear, cyclic or branched and the like, which conformations can be achieved using methods well known in the art.

The peptide derivatives and analogs according to the principles of the present invention can also include side chain bond modifications, including but not limited to -CH₂- NH-, -CH₂-S-, -CH₂-S=O, O=C-NH-, -CH₂-O-, -CH₂-CH₂-, S=C-NH-, and -CH=CH-, and backbone modifications such as modified peptide bonds. Peptide bonds (-C0-NH-) within the peptide can be substituted, for example, by N-methylated bonds (-N(CH3)-C0-); ester bonds (-C(R)H-C-O-O-C(R)H-N); ketomethylene bonds (-CO-CH2-); α-aza bonds (-NH- N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)-CH2-); thioamide bonds (-CS-NH); olefinic double bonds (-CH=CH-); and peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at one or more of the bonds along the peptide chain and even at several (e.g., 2-3) at the same time.

The present invention also encompasses peptide derivatives and analogs in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. The peptide analogs can also contain non-natural amino acids. Examples of non- natural amino acids include, but are not limited to, sarcosine (Sar), norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala).

Furthermore, the peptide analogs can contain other derivatized amino acid residues including, but not limited to, methylated amino acids, N-benzylated amino acids, O- benzylated amino acids, N-acetylated amino acids, O-acetylated amino acids, carbobenzoxy-substituted amino acids and the like. Specific examples include, but are not limited to, methyl-Ala (MeAIa), MeTyr, MeArg, MeGIu, MeVaI, MeHis, N-acetyl-Lys, O-acetyl-Lys, carbobenzoxy-Lys, Tyr-O-Benzyl, Glu-O-Benzyl, Benzyl-His, Arg-Tosyl, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, and the like.

The peptides can be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). Alternatively, the peptides of the present invention can be synthesized using Standard solution methods well known in the art

(see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984) or by any other method known in the art for peptide synthesis.

As indicated above, the peptides of the invention may be synthesized or prepared by techniques well known in the art. In general, these methods comprise sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain bound to a suitable resin.

Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions conductive for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by the solid phase method, is cleaved from the solid support to afford the final peptide.

In the solid phase peptide synthesis method, the alpha-amino group of the amino acid is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain. Suitable protecting groups are t-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (alpha,alpha)- dimethyl-3 ,5dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t- butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC) and the like. The BOC protecting group is preferred.

In the solid phase peptide synthesis method, the C-terminal amino acid is attached to a suitable solid support. Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the solvent media used. Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like. The coupling reaction is accomplished in a solvent such as ethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art.

The peptides of the invention may alternatively be synthesized such that one or more of the bonds, which link the amino acid residues of the peptides are non-peptide bonds. These alternative non-peptide bonds include, but are not limited to, imino, ester, hydrazide, semicarbazide, and azo bonds, which can be formed by reactions well known to skilled in the art.

The peptides of the present invention, analogs, derivatives or fragments thereof produced by recombinant techniques can be purified so that the peptides will be substantially pure when administered to a subject. The term "substantially pure" refers to a compound, e.g., a peptide, which has been separated from components, which naturally accompany it. Typically, a peptide is substantially pure when at least 50%, preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the peptide of interest. Purity can be measured by any appropriate method, e.g., in the case of peptides by HPLC analysis.

Included within the scope of the invention are peptide conjugates comprising the peptides of the present invention derivatives, analogs, or fragments thereof joined at their amino or carboxy-terminus or at one of the side chains via a peptide bond to an amino acid sequence of a different protein. Additionally or alternatively, the peptides of the present invention, derivatives, analogs, or fragments thereof can be joined to another moiety such as, for example, a fatty acid, a sugar moiety, arginine residues, and any known moiety that facilitate membrane or cell penetration. Conjugates comprising peptides of the invention and a protein can be made by protein synthesis, e. g., by use of a peptide synthesizer, or by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the conjugate by methods commonly known in the art.

Pharmaceutical Compositions of the Invention

According to certain embodiments, the present invention provides pharmaceutical composition for the treatment or prophylaxis of an amphiregulin-mediated disorder in a subject, wherein the pharmaceutical composition comprises as an active ingredient a complex molecule of the present invention and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, without limitation, calcium carbonate, calcium phosphate, starch, cellulose derivatives, gelatin, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, vegetable oils, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the complex molecules described herein, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the formulation for proper administration to the subject.

Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal, or parenteral delivery, including intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

In a particularly preferred aspect, the compositions of the invention may be administered in the form of an inhalant as a powdered or liquid aerosol. Such a formulation may comprise the active agent solubilized in a micronized hydrophobic/hydrophilic emulsion. Aerosols and methods for the synthesis thereof are described in the art. Aerosols which are to be administered with inhalation appliances and which contain a molecule of the present invention are preferable in case that direct treatment of pulmonary symptoms is necessary.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofiuoromethane, dichloro- tetrafiuoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water- soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use. The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a "therapeutically effective amount" means an amount of active ingredients (e.g., the complex molecule of the invention) effective to prevent, alleviate, or ameliorate symptoms of an amphiregulin-mediated disorder or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any preparation used in the methods of the invention, the dosage or the therapeutically effective amount can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition (See, e.g., Fingl, E. et al. (1975), "The Pharmacological Basis of Therapeutics," Ch. 1, p.l.). Dosage amount and administration intervals may be adjusted individually to provide sufficient plasma or brain levels of the active ingredient to induce or suppress the biological effect (i.e., minimally effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks, or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Efficacy of the dosing regime may be determined by assessing for improved function in the patient. For example, in cases where the treatment is intended for treatment of a lung disease, efficacy of the dosing regime may be determined by assessing for improved lung function in the patient. This assessment may include viscoelasticity measurements of sputum, improvements in pulmonary function, including improvements in forced exploratory volume of sputum and maximal midexpiratory flow rate. The aforementioned therapeutic regime can be given in conjunction with adjunct therapies such as antibiotics, DNAse I or other current therapies for the treatment of hypersecretory pulmonary disease. If antibiotics are co-administered as part of the patient s therapy, bacterial quantitation following therapy can be included to assess the efficacy of the treatment by decreased bacterial growth, indicating decreased viscosity of mucus or sputum and increase of the mucus or sputum lung clearance.

Pulmonary function tests, as well as diagnostic tests for the clinical progression of pulmonary hypersecretory disease, are known to those individuals with skill in this art. Standard pulmonary function tests include airway resistance (AR); forced vital capacity (FVC); forced expiratory volume in 1 second (FEV(I)); forced midexpiratory flow; and peak expiratory flow rate (PEFR). Other pulmonary function tests include blood gas analysis; responses to medication; challenge and exercise testing; measurements of respiratory muscle strength; fibro-optic airway examination; and the like. Some basic procedures for studying the properties of mucus include rheology, e.g. with the use of a magnetic microrheometer; adhesivity to characterize the forces of attraction between an adherent surface and an adhesive system by measuring the contact angle between a mucus drop and a surface. Mucus transport by cilia can be studied using conventional techniques, as well as direct measurement, i.e. in situ mucus clearance. Transepithelial potential difference, the net result of the activity of the ion-transport system of the pulmonary epithelium, can be measured using appropriate microelectrodes. Quantitative morphology methods may be used to characterize the epithelial surface condition.

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may include labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as further detailed above.

Therapeutic Use

The present invention provides methods for treating or ameliorating an amphiregulin-mediated disorder in a subject, preferably a human. Lung disease or disorder

In one embodiment, the amphiregulin-mediated disorder is lung disorder. In a particular embodiment, the lung disorder is a non-allergic lung disease.

According to some embodiments, the non-allergic lung disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), goblet cell hyperplasia, chronic bronchitis and cystic fibrosis. In a particular embodiment, the lung disease is non-allergic asthma. Each possibility represents a separate embodiment of the present invention.

According to another embodiment, the lung disease is lung cancer. The term "allergy" is defined by a hypersensitivity reaction initiated by specific immunologic mechanisms. "Non allergic" hypersensitivity should be used when other mechanisms are involved, such as in hypersensitivity to aspirin (Johansson J. et al., A revised nomenclature for allergy. An EAACI position statement from the EAACI nomenclature task force; Allergy 2001;56: 813-824). Thus, as used herein, the terms "non-allergic asthma" and "intrinsic asthma" encompass asthma which is caused by viral infections, certain medications or irritants found in the air, which aggravate the nose and airways. Triggers of non-allergic asthma include, but are not limited to, airborne particles (e.g., coal, chalk dust), air pollutants (e.g., tobacco smoke, wood smoke), strong odors or sprays (e.g., perfumes, household cleaners, cooking fumes, paints or varnishes), viral infections (e.g., colds, viral pneumonia, sinusitis, nasal polyps), aspirin-sensitivity, and gastroesophageal reflux disease (GERD). As opposed to non-allergic asthma, allergic asthma is characterized by airway obstruction associated with allergies and triggered by substances called allergens. Triggers of allergic asthma include, but are not limited to, airborne pollens, molds, animal dander, house dust mites and cockroach droppings.

It is to be understood that the patient populations that are amenable to treatment according to the present invention are not those subjects having psychogenic or allergic asthma disclosed to be treatable with the known peptides in WO 00/78346 and WO 02/050097.

According to one embodiment, the present invention provides a method of treating pulmonary mucus hypersecretion. In general, the method comprises administering a therapeutically effective amount of a complex molecule, capable of inhibiting amphiregulin upregulation and/or secretion, to a subject suffering from airway hypersecretion of mucus. In some embodiments, the invention provides methods of treating hypersecretion of mucus in an airway of an individual by mucus-producing goblet cells. In other embodiments, the invention provides methods for reducing goblet cell hyperplasia in an airway of an individual. Any disease and particularly any pulmonary disease characterized by hypersecretion of mucus or accumulation of pathological levels of mucus may be treated by the methods described herein. Examples of pulmonary hypersecretory diseases that may be treated by this method include, but are not limited to, chronic obstructive lung diseases, such as chronic bronchitis, inflammatory diseases such as asthma (excluding psychogenic or allergic asthma), bronchiectasis, pulmonary fibrosis, COPD, diseases of nasal hypersecretion, e.g. nasal polyps; and other mucus-hypersecretory diseases. Genetic diseases such as cystic fibrosis, Kartagener syndrome, alpha- 1 -antitrypsin deficiency, familial non-cystic fibrosis mucus inspissation of respiratory tract, are intended to be included as well. Each possibility represents a separate embodiment of the present invention.

According to one embodiment, the method of the present invention further comprises administration of at least one anti- inflammatory agent for the treatment of a lung disease or disorder. According to certain embodiments the anti- inflammatory agent is selected from the group consisting of: corticosteroids, sodium cromolyn, IgE inhibitors, phosphodiesterase inhibitors, methylxanthines, beta-adrenergic agents, and leukotriene modifiers. According to one particular embodiment, the method of the present invention further provides administration of corticosteroids. Each possibility represents a separate embodiment of the present invention. Cancer

In another embodiment the amphiregulin-mediated disorder is cancer. According to additional embodiments, the cancer is an epithelial cell cancer. According to particular embodiments the epithelial cell cancer is selected from the group consisting of prostate cancer, lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, colon cancer, brain cancer, head and neck cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, rectal cancer, colorectal cancer, genital-urinary cancer, bladder cancer and glioblastoma. Each possibility represents a separate embodiment of the present invention.

According to one embodiment, the cancer is a lung cancer. In certain embodiments, the lung cancer is selected from the group consisting of small cell lung carcinoma, lung adenocarcinoma, squamous cell lung carcinoma or non-small cell lung carcinoma. According to one embodiment, the lung cancer is non-small cell lung carcinoma.

According to one embodiment, the cancer is an EGFR-associated cancer. As used herein, the terms "EGFR-associated cancer" refers to tumor cells and neoplasms which over-express EGFR and/or tumor cells characterized by EGFR excessive activation.

According to particular embodiments, the cancer is characterized by epidermal growth factor receptor (EGFR) over-expression. A cell which over-expresses EGFR is one which produces significantly higher levels of EGFR compared to a normal cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. Overexpression of EGFR may be determined diagnostically by evaluating its protein levels (or nucleic acid encoding it) in the patient, e.g. in a biopsy or by various diagnostic assays such as the IHC, immunoenzyme, Western blot, ligand binding, FISH, southern blotting, PCR or in vivo assays known in the art.

According to another embodiment, the cancer is characterized by EGFR excessive activation. A cancer "characterized by excessive activation" of an EGFR is one in which the extent of EGFR activation in cancer cells significantly exceeds the level of activation of that receptor in non-cancerous cells of the same tissue type. Such excessive activation may cause and/or be caused by the malignant state of a cancer cell. In some embodiments, the cancer will be subjected to a diagnostic or prognostic assay to determine whether amplification and/or overexpression of an EGFR is occurring which results in such excessive activation of the EGFR. Fibrosis disorders

According to another embodiment, the AREG-mediated disease is a fibrotic disorder or fibrotic condition. According to another embodiment, the fibrotic disorder or condition is primary fibrosis or secondary fibrosis.

Chronic inflammation may lead to clinical conditions and disorders associated with primary or secondary fibrosis, such as systemic sclerosis, graft-versus-host disease (GVHD), pulmonary and hepatic fibrosis. Thus, suppression of AREG (e.g., inhibition of AREG upregulation and/or secretion) may be effective in reducing fibrosis. Fibrotic processes are the primary cause or the end result of the healing process of multiple inflammatory and physical injuries, as well as aging. These conditions and disorders can best be interpreted in terms of perturbations in cellular functions, a major manifestation of which is excessive collagen deposition. According to certain embodiments, the present invention provides peptide or peptidomimetic molecules for treating or preventing a fibrotic disorder or fibrotic related conditions selected from the group consisting of:

1) diseases caused by, or associated with, excessive collagen type I deposits or fibrosis, including primary or secondary diffuse, local, focal, or organ-specific fibrosis following inflammation in a variety inflammatory disorders (e.g., degenerative arthritis, liver cirrhosis due to alcohol or chronic active hepatitis, systemic lupus erythematosis, SLE, Sjogren syndrome, and diffuse systemic sclerosis with scleroderma, primary and secondary pulmonary and broncho-alveolar fibrosis, fibrous dysplasia, cystic fibrosis, myelosclerosis, etc.; 2) fibrosis around foreign bodies (i.e., mechanical implants: artificial pancreas devices with insulin or insulin-secreting islets; artificial valves; artificial blood vessels, silicon implants, etc.; 3) systemic, organ or site-specific fibrosis post-irradiation, fibrosis secondary to chronic inflammatory disorders and cytotoxic agents;

4) scar formation and/or intimal thickening due to surgical vascular interventions (i.e., coronary artery bypass, vascular surgery and artificial vascular implants); 5) cutaneous, ocular and mucosal superficial or deep and destructive fibrosis and/or scar formation due to systemic diseases, infections (i.e., acne vulgaris, trachoma, etc.) or burns, ionizing irradiation, or other physico-chemical trauma;

6) chronic graft vs. host disease following allogeneic bone marrow transplantation with secondary local, organ-specific or systemic fibrosis and/or scleroderma; and 7) scar formation following surgical incisions and cosmetic surgical interventions.

Furthermore, topical compositions to prevent cutaneous, ocular and mucosal scar formation and for clinical application for cosmetic purposes are also provided by the present invention.

Collagen molecules are an integral part of fibrosis elements or supramolecular structures in extracellular spaces, which function as major components of various connective tissues. Of 13 distinct collagen types, representing at least 21 individual gene products, skin contains especially type I (85%) and type III (15%) collagen. Three representative major clinical conditions associated with fibrosis as a major pathogenesis of the disease are described below: a) Primary or secondary systemic sclerosis (scleroderma)

A chronic disorder, characterized by fibrotic and inflammatory changes in skin and internal organs, is approximately 10 times more prevalent than the incidence, at approximately 100 per million in the population and in some cases up to 290 per million.

Skin biopsies of scleroderma patients exhibited elevated levels of collagen type I gene expression and fibroblasts recovered from these patients revealed increased synthesis of collagen and fibronectin. Skin and subcutaneous tissues mainly are involved; this involvement can extend to tendons and muscles. Various internal organs can be affected

(blood vessels, gastro-intestinal tract, lungs, heart, kidneys). Cutaneous and/or systemic sclerosis, indistinguishable from primary scleroderma, may complicate chronic graft vs. host disease, following allogeneic bone marrow transplantation. b) Pulmonary fibrosis

Interstitial lung diseases are heterogenous disorders, characterised by diffuse involvement of lung parenchyma mostly progressive and often fatal. The most common etiology includes chronic inhalation of dusts containing silica, as in miners and asbestos workers. Pulmonary fibrosis or broncho-alveolar fibrosis may be primary or secondary to bone marrow transplantation due to radiation, high dose chemotherapy, infectious complications, or associated with immune or autoimmune etiology. Pulmonary fibrosis may also result from chemotherapy or paraquat poisoning. In most cases, abnormally high levels of collagen type I synthesis were observed. c) Hepatic fibrosis

Increased hepatic connective tissue is relatively common. Depending on its extent and distribution, hepatic fibrosis may be either of little clinical consequence or devastating, fatal cirrhosis. Major etiologies of hepatic fibrosis in the West include alcoholic-nutritional diseases, viral hepatitis, and autoimmune etiology (i.e., secondary to chronic active hepatitis or primary biliary cirrhosis). Other underlying diseases are iron overload, copper metabolism disorders, extra hepatic biliary obstruction, primary biliary cirrhosis and chronic congestive heat failure. Another important cause in millions worldwide is hepatic chistosomiasis. Excessive production of collagen is usual in liver fibrosis, although the pattern of collagen deposition may be distinctive for each disease.

In addition to the above, there are many other possible clinical indications, in which the prevention of collagen type I associated fibrosis is desired, and which can now be treated with the compositions of the present invention.

Thus, the present invention provides compositions and methods for:

1) treatment of post-inflammatory fibrosis, such as myocardial fibrosis, post- Coxsackievirus B viral disease, or bacterial endocarditis; post-inflammatory fibrosis of the pleura; 2) inhibition of AREG in cirrhosis, including primary billiary cirrhosis as well as alcoholic and post-hepatitic cirrhosis, and cirrhosis due to iron overload copper metabolism disorders, extra hepatic billiary obstructions and chistosomiasis;

3) inhibition of AREG in dermatology disorders, such as buleous pemphigoid, erysipelas, erythema multiforme, hydroa vicciniforme, acne keloidalis;

4) inhibition of AREG for treatment of lung fibrosis, including autoimmune lung disease, idiopathic fibrosis, post-inflammatory fibrosis, vasculitis, radiation and chemotherapy- induced fibrosis, fibrosis as part of interstitial pneumonitis;

5) inhibition of fibrosis in diseases of the musculo-skeletal system, including arthritis and tendenitis, bursitis and frozen shoulder;

6) inhibition of AREG for decreasing post-surgery scar formation, including heart surgery, plastic surgery, vascular surgery, orthopedic surgery and opthalmologic surgery, including filtration operation for glaucoma;

7) 8) inhibition of fibrosis due to foreign body reactions, including artificial implants such as artificial pancreas, artificial hip or other joints, anastomosis in heart aorta and other microvascular surgery or artificial valves; and 9) treatment of CNS lesions due to primary or secondary fibrosis, such as glial scar formation or amyotropic lateral sclerosis.

According to particular embodiments, the fibrotic disease is selected from the group consisting of fibrosis and remodeling of lung tissue in chronic obstructive pulmonary disease, fibrosis and remodeling of lung tissue in chronic bronchitis, fibrosis and remodeling of lung tissue in emphysema, lung fibrosis and pulmonary diseases with a fibrotic component, fibrosis and remodeling in asthma, fibrosis in rheumatoid arthritis, virally induced hepatic cirrhosis, radiation-induced fibrosis, post angioplasty restenosis, chronic glomerulonephritis, renal fibrosis in patients receiving cyclosporine and renal fibrosis due to high blood pressure, diseases of the skin with a fibrotic component, and excessive scarring. Each possibility represents a separate embodiment of the present invention.

According to particular embodiments, lung fibrosis and pulmonary diseases with a fibrotic component are selected form the group consisting of idiopathic pulmonary fibrosis (IPF), giant cell interstitial pneumonia (GIP), sarcodosis, cystic fibrosis, respiratory distress syndrome (ARDS), granulomatosis, silicosis, drug-induced lung fibrosis (for example, induced by drugs such as bleomycin, bis-chloronitrosourea, cyclophosphamide, amiodarone, procainamide, penicillamine, gold or nitrofurantoin), silicosis, asbestosis, systemic scleroderma. Each possibility represents a separate embodiment of the present invention.

According to another embodiment, the fibrotic disease is an organ fibrosis selected from the group consisting of liver fibrosis (i.e., hepatic fibrosis), lung fibrosis, kidney fibrosis and skin fibrosis. Each possibility represents a separate embodiment of the present invention. According to other embodiments the AREG-mediated disorder is psoriasis.

Evaluation of the efficacy of the peptides and peptidomimitics of the present invention in the treatment or prophylaxis of a fibrotic disorder may be achieved, for instance, by evaluation of Collagen and Non-Collagen Protein Synthesis. A non-limiting example, is detecting the amount of radiolabeled collagen in fibroblast cells incubated with various concentrations of the molecules of the invention and [³H] proline (B. Peterkofsky, et al., Biochemistry, Vol. 10, pp. 988-994 (1971)).

The pharmaceutical compositions of the present invention are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the particular active agent employed; the metabolic stability of the active agent; the age, body weight, general health, sex, and diet of the subject; the mode of administration; and the severity of the particular disease.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

Materials and Methods

Cells

HMC-I cells, a human mast cell leukemia cell line, and the Jurkat T cell lymphoma line were each maintained in suspension culture, in RPMI supplemented with 10% FCS (Invitrogen-Gibco), 2 mM L-glutamine, sodium pyruvate, and 1% of a penicillin- streptomycin-nystatin mixture (Biological Industries, Bet-Haemek, Israel).

Human non-small cell lung carcinoma cell line H 1299 were grown in RPMI supplemented with 10% FCS (Biological Industries, Bet-Haemek, Israel) and penicillin- streptomycin-nystatin mixture in a humidified atmosphere of 5% CO2 at 37°C.

Preparation of T cell membranes

Jurkat T cells were activated with 75 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Calbiochem Merck) for 60 min at 37⁰C. Activated and resting Jurkat membranes were isolated as described in (Baram et al., 2001). Aliquots of the isolated membranes were stored at -70⁰C.

Activation of mast cells and AREG determination After serum starvation for 18-20 h, HMC-I cells were washed twice with Tyrode's buffer (137 mM NaCl, 2.7 mM KCl, ImM MgCl₂, 1.0 mM CaCl₂, 5.6 mM glucose, 1 mg/ml BSA, 20 mM HEPES, pH 7.4) and resuspended to 2x10⁶ cells/ml with the same buffer. The cells were preincubated with ALLl peptide for 1 h at 37⁰C, prior to stimulation with adenosine receptor agonists or activated Jurkat membranes. Following 20 h incubation at 37⁰C, the supernatants were separated from the cells and AREG levels in the supernatants were monitored by ELISA (R&D Systems, Minneapolis, MN).

A3 receptor antagonist (e.g., MRS 1220 (Sigma- Aldrich)) was administered 15 min before stimulation of the cells. In any experiment, which included direct activation of adenosine receptors, 1.5 U/ml of adenosine deaminase (ADA) were added to the cells. As the adenosine receptor agonist and antagonist used were dissolved in DMSO, the same DMSO concentrations were used in vehicle controls (final DMSO concentration not exceeding 0.1%). Reaction were terminated by placing tubes on ice, followed by a brief spin (14,000xg, 20 seconds) at 4⁰C. Cell pellets were lysed by the addition of a lysis buffer (buffer A comprising 50 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 50 mM NaF, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM EDTA, 2 mM EGTA, ImM PMSF, protease inhibitor mixture, phosphatase inhibitor mixture) and incubation for 20 min on ice. The lysates were then centrifuged for 15 min at 14,000 x g. Cell lysates were mixed with X5 concentrated Laemmli sample buffer, boiled, and subject to SDS-PAGE and Western blotting.

Expression microarray analysis

Following preincubation with ALLl and 3 h activation, total RNA was extracted from duplicate samples of 6x10⁶ HMC-I cells using the RNeasy Mini Kit (RNeasy Mini Kit; Qiagen Inc., Valencia, CA), according to the manufacturers protocol. Gene expression was screened for by using the Affymetrix Human Genome U133A 2.0 array (Affymetrix, Santa Clara, CA), which contains probe set for about 23,000 genes and expressed sequence tags. This was performed by the Microarray Unit at the Weizmann Institute of Science (Rehovot, Israel). Data analysis was performed by RACE gene expression platform (Remote Analysis Computation for gene Expression data, http://race.unil.ch/). Lists of statistically significant (p<0.05) up- or down-regulated genes by at least 2-fold expression (compared to the non-treated cells) were generated, and these were then further analyzed and subdivided into functional categories with the bioinformatic analysis resource DAVID (Database for Annotation, Visualization and Integrated Discovery, http://david.abcc.ncifcrf.gov/).

MTT cell proliferation assay Cells were seeded at 10⁵/well in 96-well plates in a final volume of 100 μl, and treated with the different HMC-I supernatants or with membranes of activated T cells (30 μg/ml) for 2 and 5 days. Ten μl of MTT (5 mg/ml) was then added to each well and incubated for 1 h at 37 "C. The reaction was blocked by the addition of 110 μl of HCl 0.07 M diluted in isopropanol and O.D. was measured at 560 nm using a SpectraMax 190 microplate reader (Molecular probes).

EXAMPLE 1

Human mast cells are activated by membranes of PMA-stimulated T cell

Exposure of mast cells to membranes derived from activated T cells generates a series of signaling events culminating in exocytosis of the secretory granules.

HMC-I cells were incubated for 20 hours with increasing concentrations of membranes isolated from PMA-activated Jurkat T cells. Supernatants were collected for measurement of β-hexoseaminidase (β-Hex) release. Following incubation, HMC-I released, dose-dependently, up to 25% of their granule content, measured by the activity of β-hexoseaminidase, a marker enzyme of mast cell granules (Fig. IA). Exocytosis was evident only when the cells were incubated with membranes of activated (T*m), but not resting (r-Tm) T cells (Fig. IB).

HMC-I cells (2xlO⁶/ml) were incubated for the indicated time periods (5, 10, 30 minutes or overnight (o.n.)) with activated T cells. Cell activation was measured by Western blotting using anti phospho-tyrosine, anti phospho-ERKl/2 and anti tubulin antibodies, as indicated. Contact with membranes derived of activated T cells also provoked the propagation of signaling networks. These included protein tyrosine phosphorylation (Fig. 1C) and ERK1/2 activation (Fig. ID), which were detected as early as 5 min after exposure to the activated membranes.

EXAMPLE 2

Gi3 and endogenous adenosine contribute to cell signaling initiated upon contact with activated T cell membranes

T cell membranes-induced signaling is partially inhibited by ALLl, a cell permeable peptide that comprises the C-terminal end of Gαi₃ fused to an importation sequence, therefore indicating the contribution of Gi3 to T cell membranes-induced signaling.

HMC-I cells were preincubated with 200 μM ALLl (FIG. 2A), increasing concentrations of ALLl (FIG. 2C) or 1.5 U/ml adenosine deaminase (ADA; FIG. 2E). Cells were then stimulated for 5 min with activated T membranes (T*m). HMC-I activation was measured by Western blotting using anti phospho-tyrosine and anti tubulin (FIG. 2A), anti pohspho-ERKl/2 and anti ERK2 (FIG. 2C) or anti phospho-MEKl/2 or anti MEK 1/2(FIG. 2E). The intensities of the bands were quantified and the average relative (phosphorylated/total) pixel densities were calculated and plotted (FIGs. 2B, D and F, respectively).

Consequently, ALLl inhibited the phosphorylation (by >50%) of a subset of tyrosine phosphorylated proteins, including pp34, pp45, pp55 and pp83 (Fig. 2A-B), phosphorylation of ERK1/2 (Fig. 2C-D) and phosphorylation of MEK1/2 (Fig. 2E-F). Inhibition was dose-dependent displaying an IC50 value of 37 μM (Fig. 2D). Similarly, inclusion of adenosine deaminase (ADA; 1.5 U/ml), an enzyme that breaks down endogenous adenosine during the contact period with the T cell membranes, inhibited MEK 1/2 activation by 50% (Fig. 2E-F), therefore suggesting that endogenous adenosine partially mediates the signaling induced by activated T cell membranes. EXAMPLE 3

Activation of mast cells by Cl-IBMECA, an adenosine receptor (A3R) agonist, is inhibited by ALLl

HMC-I cells express three members of the adenosine receptor family: The A2a,

A2b and A3 (Feoktistov et al., 2003). The A2a and A2b receptors are coupled to Gs, while the A3 R interacts with Ptx sensitive G proteins (Fredholm et al., 2001; Linden, 2001). This places the A3 adenosine receptor in the position of serving the Gi3 -coupled GPCR that is activated in activated mast cells during inflammatory conditions associated with the formation of endogenous adenosine.

This notion was substantially strengthened by the observations that exposure of HMC-I cells (2xlO⁶/ml) to Cl-IBMECA (100 nM), a highly selective agonist for the A3R, resulted in phosphorylation of MEK1/2 (Fig. 3A-B) and ERK1/2 (Fig. 3C-D). Phosphorylation was rapid, reaching maximal phosphorylation as soon as 30 seconds (MEK 1/2) or 1 min (ERK 1/2) of A3 receptors activation. Importantly, pre-incubation of HMC-I cells with 200 μM ALLl (Ih at 37⁰C) totally inhibited Cl-IBMECA mast cell activation (Fig. 3A -D).

EXAMPLE 4 Induction of AREG expression and inhibition by ALLl

Microarray analysis performed on 23,000 different genes, comparing non-treated cells to cells treated with Cl-IBMECA (100 nM, 3 h), indicated genes whose expression was modified by this agonist. Among those genes, including for example IL-8 (Fig. 4A) and the growth factors CSFl/2 and VEGFA (Fig. 4B), the gene for AREG was up- regulated by 11 fold (Fig. 4A-B). Surprisingly, AREG up-regulation was totally prevented by ALLl (200 μM, 1 h at 37⁰C). Expression of IL-8, CSFl and CSF2 and VEGFA were inhibited as well (Fig. 4A-B).

EXAMPLE 5

AREG is induced by Cl-IBMECA and activated T cell membranes Up-regulation of AREG was validated by RT-PCR. Exposure of HMC-I cells to Cl-IBMECA (100 nM, 3 h) or activated T cell membranes (T*m, 20 μg/ml) increased AREG mRNA levels (Fig. 5), consistent with the results obtained by the Microarray analysis (Example 4).

EXAMPLE 6 AREG secretion is partially inhibited by Gi3 or A3 R inhibition

Contact with activated T cell membranes induced AREG secretion (Fig. 6). ALLl partially inhibited AREG secretion and so did the specific A3 receptor antagonist MRS 1220 or adenosine deaminase (ADA).

EXAMPLE 7

Supernatants derived from activated mast cells stimulate proliferation of non small cell lung carcinoma (NSCLC)

Cancer is often linked with inflammation, raising the possibility that mediators released from inflammatory cells, such as mast cells, may affect tumor growth. Human NSCLC line H 1299 was exposed for 5 days to supernatants derived from resting or activated mast cells. While supernatants derived from resting cells had no effect on the proliferation or survival of the H 1299 cells, supernatants derived from T cell activated mast cells markedly stimulated the proliferation of the lung cancer cells as indicated by an MTT assay (Fig. 7).

EXAMPLE 8

H1299 NSCLC cell proliferation is inhibited upon incubation with either supernatant of ALLl -treated mast cells or direct exposure of ALLl to NSCLC cells

H1299 NSCLC cells were either untreated (Fig. 8; None), exposed to supernatant derived of HMC-I cells that were incubated with ALLl (200 μM, 2Oh; sup of resting

HMC + ALL-I) or exposed directly to ALLl (200μM). As indicated by an MTT assay,

NSCLC cell proliferation was markedly inhibited upon incubation with supernatant of ALLl -treated mast cells. Furthermore, limited proliferation of NSCLC cells was observed following ALLl treatment. These results indicate a dual effect of ALLl. ALLl counteracts the stimulatory effect on proliferation of the activated mast cell derived supernatants and in addition, it has a direct effect on NSCLC cells, inhibiting directly their proliferation.

EXAMPLE 9 Factors which facilitate Cl-IBMECA-induced AREG secretion

HMC-I cells were incubated with Cl-IBMECA (10OnM) to activate the A3 R, and a Ca²⁺ ionophore (0.1 μM; ION) to elevate cytosolic Ca²⁺ levels. In addition, HMC-I cells were incubated with both Cl-IBMECA (10OnM) and Ca²⁺ ionophore. As indicated in Figure 9, the combined presence of Cl-IBMECA and Ca²⁺ ionophore increased AREG secretion by 8 fold.

Steroids are often used to treat allergic and asthmatic patients. In order to examine whether steroids, and particularly dexamethasone, effect AREG secretion, activated HMC- 1 cells (T*m) were incubated with dexamethasone (DEX; lμm), Cl-IBMECA (10OnM), or both dexamethasone and Cl-IBMECA. While Cl-IBMECA upregulates AREG expression, it fails to trigger detectable AREG secretion. However, as indicate din Figure 10, in the presence of DEX, AREG secretion is significantly increased. In addition, AREG secretion is dependent on cytosolic levels of cAMP. As indicated in Figure 11, in the combined presence of Cl-IBMECA (10OnM) to activate the A3R and a cell permeable cAMP analog 8-Br-cAMP (1 mM), AREG secretion was increased by 6 fold in a dose-dependent fashion. Therefore, elevation of cytosolic levels of cAMP facilitates Cl-IBMECA-induced AREG secretion

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

References

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Claims

1. A pharmaceutical composition comprising a peptide or peptidomimetic molecule having at least a first segment competent for importation of said peptide or peptidomimetic molecule into cells, and a second segment derived from the C- terminal sequence of a Ga protein, wherein the second segment is capable of inhibiting amphiregulin upregulation and/or secretion, wherein the first segment is joined to the second segment through a linker, and a pharmaceutically acceptable carrier, for the treatment or prophylaxis of an amphiregulin-mediated disease or disorder in a subject.

2. The pharmaceutical composition according to claim 1, wherein the amphiregulin- mediated disease or disorder is other than psychogenic or allergic asthma.

3. The pharmaceutical composition of claim 1, wherein said second segment is selected from the group consisting of a peptide, a peptidomimetic, or a polypeptide.

4. The pharmaceutical composition of claim 3, wherein said second segment is a peptide, having a cyclic conformation stabilized by bonds selected from the group consisting of hydrogen bonds, ionic bonds and covalent bonds.

5. The pharmaceutical composition of claim 4, wherein said first segment is a peptide.

6. The pharmaceutical composition of claim 5, wherein said linker is a covalent bond.

7. The pharmaceutical composition of claim 6, wherein said covalent bond is a peptide bond.

8. The pharmaceutical composition of claim 6, wherein said linker provides a bend or a turn at or near the junction between the two segments.

9. The pharmaceutical composition of claim 3, wherein said second segment has an amino acid sequence selected from the group consisting of: a decapeptide derived from Gαi₃ having the sequence KNNLKECGLY (SEQ ID NO: i); a decapeptide derived from Gαi₂ having the sequence KNNLKDCGLF (SEQ ID NO:

2); a decapeptide derived from Gαt having the sequence KENLKDCGLF (SEQ ID NO: 3);

KNNLKECGLY ε-NH (SEQ ID NO: 4); KNNLKECGL-para-amino-F (SEQ ID NO:5); KQNLKECGLY (SEQ ID NO:6); KSNLKECGLY (SEQ ID NO:7); KNNLKEVGLY (SEQ ID NO:8); and KENLKECGLY (SEQ ID NO:9).

10. The pharmaceutical composition of claim 7, wherein said molecule is a peptide having an amino acid sequence selected from the group consisting of: AAVALLPAVLLALLAPKNNLKECGLY (SEQ ID NO: 10); AAVALLPAVLLALLAPKNNLKDCGLF (SEQ ID NO: 11); AAVALLPAVLLALLAPKENLKDCGLF (SEQ ID NO: 12);

AAVALLPAVLLALLAPKQNLKECGLY (SEQ ID NO: 13);

AAVALLPAVLLALLAPKNNLKEVGLY (SEQ ID NO: 14);

Succinyl-AAVALLPAVLLALLA-Sar-KNNLKECGLY (SEQ ID NO: 15);

Succinyl- VTVLALGALAGVGVGPKNNLKECGLY (SEQ ID NO: 16); Succinyl- AAV ALLP AVLLALLAPKSNLKECGLY (SEQ ID NO: 17);

Succinyl- AA V ALLP AVLLALLAPKENLKECGLY (SEQ ID NO: 18);

Succinyl- AAV ALLP AVLLALLAPKANLKECGLY (SEQ ID NO: 19);

Succinyl-AAVALLPAVLLALLAPKNNLKECGL-para-amino-F (SEQ ID NO: 20) ;

Succinyl-AAV ALLP AVLLALLAPKQNLKECGLY (SEQ ID NO: 21); and Succinyl-AAV ALLP AVLLALLAPKNNLKEVGLY (SEQ ID NO: 22).

11. The pharmaceutical composition of claim 1, wherein said amphiregulin-mediated disease or disorder is a lung disease.

12. The pharmaceutical composition of claim 11, wherein the lung disease is a non- allergic lung disease.

13. The pharmaceutical composition of claim 12, wherein said non-allergic lung disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), goblet cell hyperplasia, chronic bronchitis, non-allergic asthma and cystic fibrosis.

14. The pharmaceutical composition of claim 1, wherein said amphiregulin-mediated disease or disorder is cancer.

15. The pharmaceutical composition of claim 14, wherein the cancer is an epithelial cell cancer.

16. The pharmaceutical composition of claim 15, wherein the epithelial cell cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, colon cancer, brain cancer, head and neck cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, rectal cancer, colorectal cancer, genital-urinary cancer, bladder cancer and glioblastoma.

17. The pharmaceutical composition of claim 14, wherein the epithelial cell cancer is lung cancer.

18. The pharmaceutical composition of claim 17, wherein the lung cancer is selected from the group consisting of: small cell lung carcinoma, lung adenocarcinoma, squamous cell lung carcinoma or non-small cell lung carcinoma.

19. The pharmaceutical composition of claim 17, wherein the lung cancer is non-small cell lung carcinoma.

20. The pharmaceutical composition of claim 1, wherein said amphiregulin-mediated disorder is fibrosis.

21. A method for the treatment or prophylaxis of an amphiregulin-mediated disease or disorder in a subject, said method comprising administering to said subject a therapeutically effective amount of a therapeutic composition comprising a peptide or peptidomimetic molecule having at least a first segment competent for importation of the peptide or peptidomimetic molecule into cells, and a second segment derived from the C-terminal sequence of a Ga protein, wherein the second segment is capable of inhibiting amphiregulin upregulation and/or secretion, wherein the first segment is joined to the second segment through a linker.

22. The method of claim 21, wherein the amphiregulin-mediated disease or disorder is other than psychogenic or allergic asthma.

23. The method of claim 21, wherein said second segment is selected from the group consisting of a peptide, a peptidomimetic, or a polypeptide.

24. The method of claim 23, wherein said second segment is a peptide, having a cyclic conformation stabilized by bonds selected from the group consisting of hydrogen bonds, ionic bonds and covalent bonds.

25. The method of claim 24, wherein said first segment is a peptide.

26. The method of claim 25, wherein said linker is a covalent bond.

27. The method of claim 26, wherein said covalent bond is a peptide bond

28. The method of claim 26, wherein said linker provides a bend or a turn at or near the junction between the two segments.

29. The method of claim 23, wherein said second segment has an amino acid sequence selected from the group consisting of:

KNNLKECGLY (SEQ IDNO: 1); KNNLKDCGLF (SEQ IDNO: 2); KENLKDCGLF (SEQ IDNO: 3); KNNLKECGLY I ε-NH (SEQ IDNO: 4);

KNNLKECGL-para-amino-F (SEQ ID NO:5); KQNLKECGLY (SEQ IDNO:6); KSNLKECGLY (SEQ IDNO:7); KNNLKEVGLY (SEQ IDNO:8); and KENLKECGLY (SEQ IDNO:9).

30. The method of claim 27, wherein said molecule is a peptide having an amino acid sequence selected from the group consisting of: AAVALLPAVLLALLAPKNNLKECGLY (SEQ ID NO: 10); AAVALLPAVLLALLAPKNNLKDCGLF (SEQ ID NO: 11); AAVALLPAVLLALLAPKENLKDCGLF (SEQ ID NO: 12);

AAVALLPAVLLALLAPKQNLKECGLY (SEQ ID NO: 13);

AAVALLPAVLLALLAPKNNLKEVGLY (SEQ ID NO: 14);

Succinyl- AAVALLP AVLLALLA-Sar-KNNLKECGLY (SEQ ID NO: 15);

Succinyl-AA V ALLP A VLLALLAPKENLKECGLY (SEQ ID NO: 18);

Succinyl-AAV ALLP AVLLALLAPKANLKECGLY (SEQ ID NO: 19);

Succinyl-AA V ALLP AVLLALLAPKNNLKECGL-para-amino-F (SEQ ID NO: 20) ;

Succinyl- AAVALLP AVLLALLAPKQNLKECGLY (SEQ ID NO: 21); and Succinyl-AAV ALLP AVLLALLAPKNNLKEVGLY (SEQ ID NO: 22).

31. The method of claim 21 , wherein said amphiregulin-mediated disease or disorder is a lung disease.

32. The method of claim 31 , wherein the lung disease is a non-allergic lung disease.

33. The method of claim 32, wherein said non-allergic lung disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), goblet cell hyperplasia, chronic bronchitis, cystic fibrosis and non-allergic asthma.

34. The method of claim 21, wherein said amphiregulin-mediated disease or disorder is a cancer.

35. The method of claim 34, wherein the cancer is an epithelial cell cancer.

36. The method of claim 35, wherein the epithelial cell cancer is selected from the group consisting of prostate cancer, lung cancer, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, colon cancer, brain cancer, head and neck cancer, cancer of the nervous system, kidney cancer, retina cancer, skin cancer, liver cancer, pancreatic cancer, rectal cancer, colorectal cancer, genital-urinary cancer, bladder cancer and glioblastoma.

37. The method of claim 36, wherein the epithelial cell cancer is a lung cancer.

38. The method of claim 37, wherein the lung cancer is selected from the group consisting of: small cell lung carcinoma, lung adenocarcinoma, squamous cell lung carcinoma or non-small cell lung carcinoma.

39. The method of claim 38, wherein the lung cancer is non-small cell lung carcinoma.

40. The method of claim 21, wherein said amphiregulin-mediated disease or disorder is fibrosis.

41. The method of claim 20, wherein the fibrosis is selected form the group consisting of hepatic fibrosis, lung fibrosis, kidney fibrosis and skin fibrosis.

42. Use of a peptide or peptidomimetic molecule having at least a first segment competent for importation of said molecule into cells, and a second segment derived from the C-terminal sequence of a Ga protein, wherein the second segment is capable of inhibiting amphiregulin upregulation and/or secretion, wherein the first segment is joined to the second segment through a linker, for the preparation of a medicament for the treatment or prophylaxis of an amphiregulin-mediated disease or disorder in a subject.