WO2019054106A1 - Suppressor of extracellular matrix production and utilization thereof - Google Patents

Abstract

A suppressor of extracellular matrix production by epidermal growth factor receptor (EGFR)-expressing cells, said suppressor comprising an EGFR inhibitor as an active ingredient.

Description

Inhibitor of extracellular matrix production and use thereof

The present invention relates to an extracellular matrix production inhibitor and use thereof. More specifically, the present invention relates to an extracellular matrix production inhibitor and a screening method for a preventive or therapeutic agent for fibrotic diseases. Priority is claimed on Japanese Patent Application No. 2017-176017, filed September 13, 2017, the content of which is incorporated herein by reference.

Fibrotic diseases are diseases that occur in the respiratory system, digestive tract, liver, kidney, heart, mediastinum, pancreas, eye, skin, bone marrow, retroperitoneum, muscles, blood vessels and the like. Fibrosis is a state in which normal tissue is replaced by extracellular matrix in the repair process of tissue damage, and fibrosis progresses, and tissue in which extracellular matrix is excessively deposited loses its function.

For example, in the airways of chronic bronchial asthma patients, it is known that tissue fibrosis is observed, and establishment of a therapeutic method thereof is required (see, for example, Non-Patent Document 1).

Also, for example, eosinophilic sinusitis is a type of fibrotic disease that can cause multiple nasal polyps (nasal polyps) with marked eosinophil infiltration in the bilateral sinuses and nasal cavity, and its pathogenesis mechanism Has not been revealed. It is easy to relapse after surgical removal of the nasal swabs of eosinophilic sinusitis patients, and it has become a major clinical problem as refractory chronic sinusitis.

Under such circumstances, the present invention aims to provide a technique for effectively preventing or treating fibrotic diseases.

The present invention includes the following aspects.
[1] An inhibitor of extracellular matrix production by EGFR-expressing cells, which comprises as an active ingredient an Epidermal Growth Factor Receptor (EGFR) inhibitor.
[2] The inhibitor according to [1], wherein the EGFR-expressing cell is an eosinophil, an epithelial cell or a fibroblast.
[3] The inhibitor according to [1] or [2], wherein the extracellular matrix is osteopontin, tenascin, collagen, glycosaminoglycan, proteoglycan, fibronectin, heparan sulfate or hyaluronic acid.
[4] The inhibitor according to any one of [1] to [3], which is for the prevention or treatment of fibrotic diseases.
[5] The inhibitor according to [4], wherein the fibrotic disease is a disease involving infiltration of eosinophils into tissues.
[6] The fibrotic disease is a disease in the respiratory tract, digestive tract, liver, kidney, heart, mediastinum, pancreas, eye, skin, bone marrow, retroperitoneum, muscle or blood vessel, [4] or [5] Inhibitor as described in.
[7] The fibrotic diseases are eosinophilic sinusitis, allergic rhinitis, bronchial asthma, pulmonary fibrosis, eosinophilic esophagitis, allergic conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, skin fibrosis , Eosinophilic fasciitis, liver fibrosis, liver cirrhosis, renal fibrosis, myocardial fibrosis, fibrotic mediastinitis, pancreatic fibrosis, ocular fibrosis, retroperitoneal fibrosis, muscle fibrosis, vascular fibrosis or bone marrow The inhibitor according to any one of [4] to [6], which is fibrotic.
[8] A method of screening for a preventive or therapeutic agent for fibrotic diseases, which comprises contacting an EGFR-expressing cell with an EGFR ligand in the presence of a test substance, and an extracellular matrix in the EGFR-expressing cell contacted with an EGFR ligand. Measuring the amount of expression of said extracellular substance, wherein the amount of expression of said extracellular matrix is reduced as compared to the amount of expression in the absence of the test substance, said test substance preventing or treating fibrotic diseases Screening method to show that it is an agent.
[9] The screening method according to [8], wherein the EGFR-expressing cells are eosinophils, epithelial cells or fibroblasts.
[10] The screening method according to [8] or [9], wherein the extracellular matrix is osteopontin, tenascin, collagen, glycosaminoglycan, proteoglycan, fibronectin, heparan sulfate or hyaluronic acid.
[11] The screening method according to any one of [8] to [10], wherein the fibrotic disease is a disease accompanied by infiltration of eosinophils into a tissue.
[12] The fibrotic disease is a disease in the respiratory tract, digestive tract, liver, kidney, heart, mediastinum, pancreas, eye, skin, bone marrow, retroperitoneum, muscle or blood vessel, [8]-[11] The screening method as described in any of the above.
[13] The fibrotic diseases are eosinophilic sinusitis, allergic rhinitis, bronchial asthma, pulmonary fibrosis, eosinophilic esophagitis, allergic conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, skin fibrosis , Eosinophilic fasciitis, liver fibrosis, liver cirrhosis, renal fibrosis, myocardial fibrosis, fibrotic mediastinitis, pancreatic fibrosis, ocular fibrosis, retroperitoneal fibrosis, muscle fibrosis, vascular fibrosis or bone marrow The screening method according to any one of [8] to [12], which is fibrosis.

According to the present invention, it is possible to provide a technique for effectively preventing or treating fibrotic diseases.

(A) and (b) are representative photomicrographs showing the results of immunostaining of a tissue section of the nasal mucosa of a healthy subject and a tissue section of a nasal polyp from a patient with eosinophilic sinusitis in Experimental Example 1. It is. (C) is the graph which quantified the result of (a) and (b). (A) to (e) are graphs showing the measurement results of the expression level of the fibrosis related gene in Experimental Example 2. (F) is a graph which shows the measurement result of the expression level of AREG (amphiregulin) in Experimental example 2. It is a representative fluorescence-microscope photograph which shows the result of the immunostaining in Experimental example 3. FIG. 15 is a graph showing the results of quantitative RT-PCR of SPP1 (osteopontin) in Experimental Example 4. 15 is a graph showing the results of quantitative RT-PCR of SPP1 in Experimental Example 5. (A) and (b) are representative micrographs showing the results of Sirius red staining of mouse lung tissue sections in Experimental Example 6. (C) is the graph which quantified the result of (a) and (b). In experimental example 7, it is a fluorescence microscope picture which shows the result of having stained a mouse lung tissue section with an anti- osteopontin antibody and 4 ', 6- diamino 2-phenyl indole (DAPI). (A) to (d) are graphs showing the measurement results of the expression level of the fibrosis related gene in Experimental Example 2. It is a heat map which shows the expression level of the fibrosis related gene in eosinophils measured in Experimental example 9. In Experimental Example 10, it is a graph which shows the result of quantitative RT-PCR of Spp1 (osteopontin). (A) and (b) are representative micrographs showing the results of Sirius red staining of lung tissue sections of mice in Experimental Example 11. (C) is the graph which quantified the result of (a) and (b). (A) to (d) are graphs showing the measurement results of the expression level of the fibrosis related gene in Experimental Example 12. It is a representative fluorescence-microscope photograph which shows the result of the immunostaining in Experimental example 13. FIG.

[Inhibitor of extracellular matrix production]
In one embodiment, the present invention provides an inhibitor of extracellular matrix production by EGFR-expressing cells, which comprises an EGFR inhibitor as an active ingredient.

EGFR (epithelial growth factor receptor) is a type of tyrosine kinase type receptor. As ligands of EGFR, epidermal growth factor (EGF), amphiregulin, transforming growth factor-α (TGF-α), heparin binding EGF-like growth factor (Heparin-binding EGF- Known Growth Factor (HB-EGF), epiregulin and the like are known.

As described later in the Examples, the inventors clearly show that the production of extracellular matrix by EGFR-expressing cells can be significantly reduced in vitro and in vivo by administering the inhibitor of this embodiment. I made it.

The EGFR inhibitor is not particularly limited as long as it is a substance capable of blocking EGFR signaling, and includes tyrosine kinase inhibitors, EGFR antagonists, EGFR ligand specific binding substances and the like.

Here, the EGFR ligand-specific binding substance is a substance that specifically binds to the EGFR ligand and inhibits the binding of the EGFR ligand to the EGFR, and examples include antibodies, antibody fragments, aptamers, low molecular weight compounds and the like. . EGFR ligands include EGF, amphiregulin, TGF-α, HB-EGF, epiregulin and the like.

More specific EGFR inhibitors include, for example, erlonitib, gefinitib, ocimertinib, afatinib, vandetanib, lapatinib, neratinib, neratinib, nasultinib, namutinib, orumutinib, canltinib, cetuximab, panitumumab, dacomitinib, peritinib, losiletinib, valuritinib, otiritinib, Pogiotinib, defnetin, tyrphostin 9, BIBW-2948, ARRY-380, EAI045, AG-490, CP-724714, WZ4002, CUDC-101, AG-1478, PD153035 HCl, AC480, AEE788, AP26113-analog, OSI-420, WZ3146, WZ8040, AST-1306, TAK-285, WHI-P154, PD1 8393, CNX-2006, AG-18, AZ5104, AZD9291, CL-387785, AZD3759, and the like.

In addition, examples of EFGR-expressing cells include eosinophils, epithelial cells, fibroblasts and the like. In particular, eosinophils express EGFR, which is the first time the present inventors have revealed.

Moreover, as an extracellular matrix which the inhibitor of this embodiment suppresses production, osteopontin, tenascin, collagen, glycosaminoglycan, proteoglycan, fibronectin, heparan sulfate, hyaluronic acid and the like can be mentioned.

As described later in the Examples, the inventors revealed that the action of an EGFR ligand on an EGFR-expressing cell leads to an increase in the expression of extracellular matrix in the EGFR-expressing cell, leading to fibrosis of the tissue.

The inventors also demonstrate that administering the inhibitor of this embodiment can significantly reduce the expression of extracellular matrix by EGFR-expressing cells in vitro and in vivo, and suppress tissue fibrosis. I made it.

Therefore, it can be said that the inhibitor of this embodiment is for prevention or treatment of fibrotic diseases. That is, the inhibitor of this embodiment can be suitably used for the use of prevention or treatment of fibrotic diseases.

Here, the fibrotic disease is preferably a disease involving infiltration of eosinophils into tissues. As described later in the Examples, the inventors of the present invention have studied that eosinophils are associated with fibrosis, such as osteopontin, tenascin, collagen, etc., by the EGFR ligand amphiregulin acting on ephrocytes that are EGFR-expressing cells. It was revealed to increase the expression of gene (extracellular matrix). The fact that amphiregulin acts on eosinophils to promote the expression of extracellular matrix is the first to be revealed by the present inventors.

The inventors have also shown for the first time that memory Th2 cells produce amphiregulin by stimulation of interleukin (IL) -33, a cytokine associated with allergy.

That is, the inventors of the present invention found that memory Th2 cells produce amphiregulin by stimulation with IL-33, and amphiregulin stimulates eosinophils to produce extracellular matrix such as osteopontin, thereby causing eosinophils. We clarified for the first time the pathogenesis mechanism that fibrosis of the infiltrated tissue progressed.

Since the inhibitor of this embodiment suppresses the production of extracellular matrix, tissue that has already been fibrosed can not be restored. However, it is useful for treatment or prevention such as preventing exacerbation of symptoms of fibrotic diseases and preventing recurrence after fibrotic tissue is removed by surgery.

That is, the inhibitor of this embodiment is suitable for treatment or prevention of fibrotic diseases in the respiratory tract, digestive tract, liver, kidney, heart, mediastinum, pancreas, eye, skin, bone marrow, retroperitoneum, muscle, blood vessels, etc. ing. Here, the respiratory tract includes upper and lower airways. Upper airways include the nasal cavity, nasopharynx, pharynx, larynx. The lower airways include trachea, bronchi, bronchioles and lungs.

It is preferable that the fibrotic disease which the inhibitor of this embodiment can treat or prevent is a disease accompanied by infiltration of eosinophils into tissues, and in particular, eosinophil infiltration associated with allergic mechanism and the like It is preferable that the disease is accompanied by fibrosis as a result.

More specific fibrotic diseases that the inhibitor of the present embodiment can treat or prevent include eosinophilic sinusitis, allergic rhinitis, bronchial asthma, pulmonary fibrosis, eosinophilic esophagitis, Allergic conjunctivitis, spring catarrh, atopic keratoconjunctivitis, skin fibrosis, eosinophilic fasciitis, liver fibrosis, liver cirrhosis, liver cirrhosis, renal fibrosis, myocardial fibrosis, fibrous mediastinitis, pancreatic fibrosis, ocular fibrosis , Retroperitoneal fibrosis, myofibrosis, vascular fibrosis, myelofibrosis and the like.

Among these diseases, eosinophilic sinusitis, allergic rhinitis, bronchial asthma, pulmonary fibrosis, eosinophilic esophagitis, allergic conjunctivitis, spring catarrh, atopic keratoconjunctivitis, skin fibrosis, eosinophil Sclerosing fasciitis is a disease with eosinophil infiltration associated with allergic mechanisms and the resulting fibrosis.

In addition, liver fibrosis, liver cirrhosis, renal fibrosis, myocardial fibrosis, fibrotic mediastinitis, pancreatic fibrosis, ocular fibrosis, retroperitoneal fibrosis, myofibrosis, vascular fibrosis are accompanied by eosinophil infiltration, It is a disease with fibrosis as a result of chronic inflammation that is not limited to allergic mechanisms. For example, liver fibrosis / cirrhosis is a disease that progresses from viral chronic hepatitis and is known to be accompanied by eosinophil infiltration.

As described above, the inhibitor of the present embodiment can be said to be a preventive or therapeutic agent for fibrotic diseases. The agent for preventing or treating fibrotic diseases is preferably formulated as a pharmaceutical composition comprising the EGFR inhibitor described above and a pharmaceutically acceptable carrier.

The pharmaceutical composition may be in the form of oral use or in the form of parenteral use. As a dosage form used orally, for example, tablets, capsules, elixirs, microcapsules and the like can be mentioned. Examples of the dosage form used parenterally include injections, inhalants, suppositories, patches and the like.

Examples of pharmaceutically acceptable carriers include solvents such as sterile water and saline; gelatin, corn starch, tragacanth gum, binders such as gum arabic, excipients such as crystalline cellulose, and swelling agents such as alginic acid Can be mentioned.

The pharmaceutical composition may further comprise an additive. Additives include lubricants such as magnesium stearate; sweeteners such as sucrose, lactose and saccharin; flavoring agents such as peppermint and red mono oil; stabilizers of benzyl alcohol and phenol; buffers such as phosphate and sodium acetate Agents; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.

The pharmaceutical composition can be formulated by combining the above-mentioned carriers and additives as appropriate and mixing in the unit dose form required for generally accepted pharmaceutical practice.

Administration to patients is carried out, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, percutaneous, or orally according to methods known to those skilled in the art. sell. The dose varies depending on the weight and age of the patient, the condition of the patient, the administration method and the like, but one skilled in the art can appropriately select a suitable dose.

The dosage of the EGFR inhibitor varies depending on the type of EGFR inhibitor, the condition of the patient, etc., but in the case of oral administration, it is generally about 0.1 to 500 mg per day in adults (as 60 kg body weight) Preferably, about 1.0 to 250 mg, more preferably about 1.0 to 100 mg, may be suitably administered once a day or in several divided doses.

When administered parenterally, the dosage varies depending on the type of EGFR inhibitor, patient's condition, target organ, administration method, etc., but in the case of systemic administration, it is generally for adults (60 kg body weight) It is appropriate to administer about 0.1 to 500 mg, preferably about 1.0 to 250 mg, more preferably about 1.0 to 100 mg once a day or in several divided doses per day. It is considered to be. When topical administration is performed, in general, for adults (as body weight 60 kg), about 0.001 to 10 mg, preferably about 0.01 to 5 mg, more preferably about 0.02 to 2 mg per day, It is considered appropriate to administer once or several times a day.

The agent for preventing or treating fibrotic disease may be administered to a patient for the purpose of suppressing exacerbation of fibrotic disease. Alternatively, for example, after the affected area of fibrotic disease is removed by surgery and treated, it may be directly administered locally to the affected area to prevent recurrence.

[Method of screening for preventive or therapeutic agent for fibrotic diseases]
In one embodiment, the present invention provides a method of screening for a preventive or therapeutic agent for fibrotic diseases, which comprises contacting an EGFR-expressing cell with an EGFR ligand in the presence of a test substance, and contacting the EGFR ligand with the EGFR ligand. Measuring the expression level of extracellular matrix in the expressing cells, wherein the expression level of said extracellular matrix is reduced as compared to the expression level in the absence of the test substance, said test substance is a fiber The present invention provides a screening method which shows that the agent is a preventive or therapeutic agent for

In the screening method of the present embodiment, for example, a natural compound library, a synthetic compound library, an existing drug library and the like can be used as the test substance.

Moreover, cells derived from humans and cells derived from non-human animals can be used as EGFR-expressing cells, and more specifically, eosinophils, epithelial cells, fibroblasts and the like can be used. Eosinophils may be cells derived from peripheral blood mononuclear cells. Alternatively, eosinophils may be obtained by inducing differentiation of, for example, bone marrow blasts derived from non-human animals such as mice. The epithelial cells and fibroblasts may be primary cells or may be established cell lines.

EGFR ligands include EGF, amphiregulin, TGF-α, HB-EGF, epiregulin and the like. As the EGFR ligand, it is preferable to use one derived from the species of the EGFR-expressing cell to be used.

Moreover, as an extracellular matrix whose expression level is to be measured, osteopontin, tenascin, collagen, glycosaminoglycan, proteoglycan, fibronectin, heparan sulfate, hyaluronic acid and the like can be mentioned.

The method for measuring the amount of extracellular matrix expression is not particularly limited, and may be measured at the gene level or at the protein level. When the expression level of extracellular matrix is measured at the gene level, it can be measured, for example, by real-time RT-PCR, DNA microarray or the like. In addition, when the expression level of extracellular matrix is measured at the protein level, it can be measured, for example, by ELISA, western blotting, protein array, immunostaining, flow cytometry and the like.

The test substance is a preventive or therapeutic agent for a fibrotic disease when the expression level of extracellular matrix measured in the presence of the test substance is decreased compared to the expression level in the absence of the test substance Can. Here, the degree of reduction of the expression level of extracellular matrix may be, for example, a degree at which a significant difference is indicated.

The fibrotic disease targeted by the preventive or therapeutic agent screened by the screening method of the present embodiment is preferably a disease involving infiltration of eosinophils into tissues.

The fibrotic disease is preferably a disease in the respiratory tract, gastrointestinal tract, liver, kidney, heart, mediastinum, pancreas, eye, skin, bone marrow, retroperitoneum, muscle, blood vessels and the like.

More specific fibrotic diseases targeted by the preventive or therapeutic agent screened by the screening method of the present embodiment include eosinophilic sinusitis, allergic rhinitis, bronchial asthma, pulmonary fibrosis, eosinophils Sclerotic esophagitis, allergic conjunctivitis, spring catarrh, atopic keratoconjunctivitis, skin fibrosis, eosinophilic fasciitis, liver fibrosis, liver cirrhosis, liver fibrosis, renal fibrosis, myocardial fibrosis, fibrous mediastinitis, pancreatic fibers , Ocular fibrosis, retroperitoneal fibrosis, myofibrosis, vascular fibrosis, myelofibrosis and the like.

Other Embodiments
In one embodiment, the present invention provides a method for preventing or treating fibrotic diseases, comprising the step of administering an effective amount of an EGFR inhibitor to a patient in need of treatment.

In one embodiment, the present invention provides an EGFR inhibitor for the prevention or treatment of fibrotic diseases.

In one embodiment, the present invention provides the use of an EGFR inhibitor for producing a preventive or therapeutic agent for fibrotic diseases.

In these embodiments, EGFR inhibitors include those similar to those described above. Moreover, as the fibrotic diseases, the same ones as described above can be mentioned.

Amphiregulin elevates osteopontin expression by eosinophils
[Experimental Example 1]
Eosinophilic sinusitis (hereinafter sometimes referred to as "ECRS") is a chronic inflammatory disease of the upper respiratory tract, often forming nasal polyps in which eosinophils are markedly infiltrated. In this experiment, tissue sections of nasal polyps of ECRS patients were first stained with anti-fibronectin antibody. Also, as a control, tissue sections of the nasal mucosa of healthy subjects were similarly stained.

FIG. 1 (a) is a representative photomicrograph of a control, and FIG. 1 (b) is a representative photomicrograph of a histological section of a nasal polyp. FIG. 1 (c) is a graph showing the result of measuring the ratio of the area of the area stained with the anti-fibronectin antibody to the total area of the tissue (n = 3). In FIG. 1 (c), "****" indicates that there is a significant difference at P <0.0001.

As a result, it became clear that fibronectin was deposited in a large amount on nasal polyps of ECRS patients. On the other hand, no deposition of fibronectin was found in the nasal mucosa of healthy individuals.

[Experimental Example 2]
Subsequently, the expression level of fibrosis-related gene in nasal polyps of ECRS patients was measured by quantitative RT-PCR (n = 6). In addition, as a control, the expression level of fibrosis related gene in the nasal mucosa of healthy subjects was similarly measured (n = 5).

As a fibrosis related gene, SPP1 (osteopontin), TNC (tenascin C), COL1A1 (collagen type I α1 chain), ACTA2 (α-smooth muscle actin), and FN1 (fibronectin 1) were examined.

FIGS. 2 (a) to 2 (e) are graphs showing the results of measurement of the expression level of fibrosis related genes. In FIG. 2 (a) to (e), "***" indicates that there is a significant difference at P <0.001, and "****" indicates that there is a significant difference at P <0.0001. Show.

As a result, it was revealed that the expression level of the fibrosis related gene was significantly increased in nasal polyps of ECRS patients as compared with the control.

In addition, as a result of quantitative RT-PCR, in nasal polyps of ECRS patients, AREG (amphiregulin), IL-33 (interleukin 33), IL1RL1 (interleukin 1 receptor-like 1), Th2 in comparison with controls. It was also revealed that the expression of cytokines was significantly higher. The measurement result of the expression level of AREG is shown in FIG. 2 (f). In FIG. 2 (f), "****" indicates that there is a significant difference at P <0.0001.

[Experimental Example 3]
Subsequently, histological analysis of nasal polyps of ECRS patients was performed. Specifically, tissue sections of nasal polyps of ECRS patients were stained with anti-osteopontin antibody and anti-Siglec-8 antibody. Siglec-8 is a marker for eosinophils in humans. Also, as a control, tissue sections of the nasal mucosa of healthy subjects were similarly stained.

FIG. 3 is a representative fluorescence micrograph showing the results of immunostaining. In FIG. 3, the scale bar indicates 50 μm. “Merge” is the result of integrating the staining result with anti-osteopontin antibody and the staining result with anti-Siglec-8 antibody in the same visual field.

As a result, it was revealed that in nasal polyps of ECRS patients, a large amount of eosinophil infiltration and accompanying high density deposition of osteopontin are observed (n> 3).

[Experimental Example 4]
Subsequently, the expression levels of SPP1 (osteopontin) in eosinophils from nasal polyps of ECRS patients and in peripheral blood mononuclear cells (PBMCs) of the same patients were measured by quantitative RT-PCR (n = 5).

FIG. 4 is a graph showing the results of quantitative RT-PCR. In FIG. 4, “***” indicates that there is a significant difference at P <0.001.

As a result, it was revealed that eosinophils infiltrated into nasal polyps of ECRS patients significantly express osteopontin in comparison with eosinophils derived from peripheral blood of the same patients.

[Experimental Example 5]
Subsequently, eosinophils from peripheral blood of healthy individuals were cultured in the medium for 72 hours, and the expression level of SPP1 (osteopontin) was measured by quantitative RT-PCR. Here, a group to which nothing was added to the medium, a group to which amphiregulin was added to the medium, a group to which erlotinib was added to the medium, and a group to which amphiregulin and erlotinib were added to the medium were prepared (n = 4) . Erlotinib is a compound that selectively inhibits the tyrosine kinase of EGFR.

FIG. 5 is a graph showing the results of quantitative RT-PCR. In FIG. 5, “**” indicates that there is a significant difference at P <0.01, and “ns” indicates that there is no significant difference.

As a result, it was revealed that the expression of osteopontin by peripheral blood-derived eosinophils of healthy subjects was increased. Moreover, it became clear that addition of the EGFR inhibitor erlotinib suppresses the increase in the expression of osteopontin.

The above results indicate that eosinophils express osteopontin via EGFR signaling upon amphiregulin stimulation.

In addition, the inventors revealed that memory-like CD4 positive T cells produce amphiregulin. Thus, in nasal polyps of ECRS patients, subpopulations of memory-like CD4 positive T cells producing amphiregulin induce a tissue fibrotic response through the production of osteopontin by eosinophils It became clear.

<Amniregulin induces lung fibrosis by allergic inflammation>
[Experimental Example 6]
Subsequently, the inventors examined the role of amphiregulin in the fibrotic response of the lung due to allergic inflammation.

Specifically, first, OVA peptide and alum are intraperitoneally administered twice to Areg (amphiregulin) ^{+ / +} mice and Areg ^-/- mice (1st and 8th days from the start of the experiment) ), Followed by inhalation of OVA peptide (30, 33, 35, 37, 40 and 42 days from the start of the experiment). On the 43rd day from the start of the experiment, the lungs were excised from each mouse to make tissue sections, and the deposition of collagen was examined by Sirius red staining.

FIG. 6 (a) is a representative photomicrograph showing the staining results of tissue sections of Areg ^{+ / +} mice, and FIG. 6 (b) is a representative microscope showing staining results of tissue sections of Areg ^{− / −} mice It is a photograph. Scale bar indicates 100 μm. Further, FIG. 6 (c) is a graph in which the ratio of the area of the region stained with Sirius red is quantified (n> 3). In FIG. 6C, "***" indicates that there is a significant difference at P <0.001.

As a result, it was revealed that collagen deposition in the lung was observed in Areg ^{+ / +} mice. On the other hand, it became clear that deposition of collagen was significantly reduced in Areg ^{− / −} mice.

[Experimental Example 7]
Subsequently, lung tissue sections removed from each mouse of Experimental Example 6 were stained with anti-osteopontin antibody and observed with a fluorescence microscope. FIG. 7 is a fluorescence micrograph. The nuclei were also stained with 4 ', 6-diamino-2-phenylindole (DAPI) for comparison. In FIG. 7, “merge” is the result of integrating the staining result by the anti-osteopontin antibody and the staining result by DAPI in the same visual field.

As a result, it was revealed that osteopontin expression was significantly reduced in the lungs of Areg ^{− / −} mice, as compared to the lungs of Areg ^{+ / +} mice.

[Experimental Example 8]
Subsequently, the expression level of the fibrosis related gene in the lung isolated from each mouse of Experimental Example 6 was measured by quantitative RT-PCR (n = 3). As fibrosis related genes, Spp1 (osteopontin), Tnc (tenascin C), Col1a1 (collagen type I α1 chain), and Acta2 (α-smooth muscle actin) were examined.

FIGS. 8 (a) to 8 (d) are graphs showing the results of measurement of the expression level of fibrosis related genes. In FIG. 8 (a) to (d), “*” indicates that there is a significant difference at P <0.05, “**” indicates that there is a significant difference at P <0.01, “*” ** "indicates that there is a significant difference at P <0.001.

As a result, it was revealed that expression of fibrosis related genes was significantly reduced in Areg ^{− / −} mice as compared to Areg ^{+ / +} mice.

The above results indicate that amphiregulin is essential for the induction of pulmonary fibrotic response in allergic airway inflammation.

<Eosinophils express osteopontin via EGFR signaling by amphiregulin stimulation>
[Experimental Example 9]
Cells harvested from mouse bone marrow were stimulated with 100 ng / mL SCF and 100 ng / mL FLT3 ligand for 4 days. Subsequently, the cells were stimulated with 10 ng / mL of IL-5 for 10 days to obtain eosinophils.

Subsequently, amphiregulin was added to the culture medium of the obtained eosinophils, and the expression level of the fibrosis related gene was measured. In addition, as a control, eosinophils to which amphiregulin had not been added were used.

FIG. 9 is a heat map showing the measured expression levels of fibrosis related genes. Arrows indicate Spp1 (osteopontin). As a result, it was revealed that the addition of amphiregulin significantly increases the expression of osteopontin in eosinophils.

[Experimental Example 10]
Subsequently, amphiregulin was added to the culture medium of eosinophils obtained in Experimental Example 9, and the expression amount of Spp1 (osteopontin) was measured by quantitative RT-PCR. Here, the EGFR inhibitor erlotinib was added along with amphiregulin. As a control, a group to which phosphate buffer (PBS) was added instead of erlotinib was prepared, and the expression level of Spp1 was similarly measured.

FIG. 10 is a graph showing the results of quantitative RT-PCR. As a result, amphiregulin was found to increase Spp1 expression in a concentration-dependent manner. In addition, it became clear that erlotinib suppresses the increase in expression of Spp1 in eosinophils induced by amphiregulin.

From the above results, it became clear that eosinophils upregulate osteopontin expression via EGFR signaling upon amphiregulin stimulation.

Erlotinib Suppresses Lung Fibrosis In Vivo
[Experimental Example 11]
We investigated the effect of erlotinib on the fibrotic response of the lung due to allergic inflammation. Specifically, first, wild type mice were immunized intraperitoneally with OVA peptide and alum twice (on the first and eighth days from the start of the experiment), and then OVA peptide was inhaled (experiment (experiment) 30, 33, 35, 37, 40 and 42 days from the start)). In addition, 12.5 mg / kg / day of erlotinib was intraperitoneally administered at 30, 33, 35, 37, 40 and 42 days from the start of the experiment. As a control, a group to which PBS was administered instead of erlotinib was prepared. On the 43rd day from the start of the experiment, the lungs were excised from each mouse to make tissue sections, and the deposition of collagen was examined by Sirius red staining.

FIG. 11 (a) is a representative photomicrograph showing staining results of tissue sections of control mice, and FIG. 11 (b) is a representative photomicrograph showing staining results of tissue sections of erlotinib-administered mice . Scale bar indicates 100 μm. Further, FIG. 11C is a graph in which the ratio of the area of the region stained with Sirius red is quantified (n> 3). In FIG. 11 (c), "****" indicates that there is a significant difference at P <0.0001.

As a result, it was revealed that collagen deposition in the lungs was observed in control mice. On the other hand, in mice treated with erlotinib, collagen deposition was found to be significantly reduced.
[Experimental Example 12]
Subsequently, the expression level of the fibrosis related gene in the lung isolated from each mouse of Experimental Example 11 was measured by quantitative RT-PCR (n = 3). As fibrosis related genes, Spp1 (osteopontin), Tnc (tenascin C), Col1a1 (collagen type I α1 chain), and Acta2 (α-smooth muscle actin) were examined.

FIGS. 12 (a) to 12 (d) are graphs showing the results of measurement of the expression level of fibrosis related genes. 12 (a) to 12 (d), "*" indicates that there is a significant difference at P <0.05, "**" indicates that there is a significant difference at P <0.01, "*" ** "indicates that there is a significant difference at P <0.001.

As a result, it was revealed that in mice treated with erlotinib, the expression of fibrosis-related gene was significantly reduced as compared to control mice.
Experimental Example 13
Subsequently, lung tissue sections removed from each mouse of Experimental Example 11 were stained with an anti-osteopontin antibody and an anti-Siglec-F antibody. Siglec-F is a marker for eosinophils in mice.

FIGS. 13 (a) and 13 (b) are representative fluorescence micrographs showing the results of immunostaining, which are the result of integrating the staining results with anti-osteopontin antibody and the staining results with anti-Siglec-F antibody in the same field of view is there. The scale bar shows 50 micrometers in FIG. 13 (a) and (b).

As a result, it was revealed that osteopontin-positive cells co-localized with Siglec-F-positive cells in the lungs of control mice. This result further supports that eosinophils are the main source of osteopontin in the lung that has undergone a fibrotic response due to allergic inflammation. In addition, it was revealed that osteopontin-positive eosinophils disappeared in the lungs of erlotinib-administered mice.

The above results indicate that amphiregulin-EGFR mediated signaling induces the expression of osteopontin in eosinophils and promotes a fibrotic response in vivo.

According to the present invention, it is possible to provide a technique for effectively preventing or treating fibrotic diseases.

Claims (8)

1. An inhibitor of extracellular matrix production by an EGFR-expressing cell, which comprises an Epidermal Growth Factor Receptor (EGFR) inhibitor as an active ingredient.
2. The inhibitor according to claim 1, wherein the EGFR-expressing cells are eosinophils, epithelial cells or fibroblasts.
3. The inhibitor according to claim 1 or 2, wherein the extracellular matrix is osteopontin, tenascin, collagen, glycosaminoglycan, proteoglycan, fibronectin, heparan sulfate or hyaluronic acid.
4. The inhibitor according to any one of claims 1 to 3, which is for the prevention or treatment of a fibrotic disease.
5. The inhibitor according to claim 4, wherein the fibrotic disease is a disease accompanied by infiltration of eosinophils into tissues.
6. The inhibitor according to claim 4 or 5, wherein the fibrotic disease is a disease in the respiratory tract, digestive tract, liver, kidney, heart, mediastinum, pancreas, eye, skin, bone marrow, retroperitoneum, muscle or blood vessel. .
7. The fibrotic diseases include eosinophilic sinusitis, allergic rhinitis, bronchial asthma, pulmonary fibrosis, eosinophilic esophagitis, allergic conjunctivitis, vernal keratoconus, atopic keratoconjunctivitis, skin fibrosis, eosinophil With bulbar fasciitis, liver fibrosis, liver cirrhosis, renal fibrosis, myocardial fibrosis, fibrotic mediastinitis, pancreatic fibrosis, ocular fibrosis, retroperitoneal fibrosis, myofibrosis, vascular fibrosis or myelofibrosis The inhibitor according to any one of claims 4 to 6, which is present.
8. A method of screening a preventive or therapeutic agent for a fibrotic disease, comprising
   Contacting an EGFR-expressing cell with an EGFR ligand in the presence of a test substance;
   Measuring the amount of extracellular matrix expressed in said EGFR-expressing cells contacted with an EGFR ligand.
   A screening method, wherein the decrease in the expression level of the extracellular matrix compared to the expression level in the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for a fibrotic disease.

Images