JP2008222682A - Medicine for treatment of fibrotic lung disease, agent for suppressing airway mucous secretion cell hyperplasia and medicine for treatment of airway embolism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicine for the treatment of fibrotic lung disease, containing glycyrrhizin and having high therapeutic effect and low side action, which is different from a conventional treating medicine, an agent for suppressing the hyperplasia of airway mucous secretion cell, and a medicine for the treatment of airway embolism. <P>SOLUTION: The invention provides a medicine for the treatment of fibrotic lung disease, an agent for suppressing the hyperplasia of airway mucous secretion cell, and a medicine for the treatment of airway embolism containing glycyrrhizin and/or its pharmacologically allowable salt as active components. The pharmacologically allowable salt is preferably glycyrrhizin ammonium salt, alkali metal salts such as a glycyrrhizin sodium salt or a potassium salt or a glycyrrhizin choline salt, and a calcium salt, a magnesium salt, an aluminum salt, etc., are also usable. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a therapeutic agent for fibrotic lung disease, an agent for inhibiting hyperplasia of airway mucus-secreting cells, and a therapeutic agent for airway embolism, which contain glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.

Respiratory diseases are not only accompanied by systemic deterioration of symptoms when the symptoms become serious, but also cause serious distress to the affected person by not being able to breathe freely.
For example, the lung is an organ that performs gas exchange by taking oxygen from breathed air into the blood and releasing carbon dioxide outside the body, and this gas exchange is performed in hundreds of millions of alveoli. When fibrotic lung disease develops and the site of the disease spreads over a wide area, the gas exchange described above cannot be performed sufficiently, so the oxygen concentration in the blood decreases chronically, causing respiratory failure and often died. It reaches. Here, fibrotic lung disease is widely seen as one of the end conditions of inflammatory lung disease. For example, pulmonary fibrosis can be mentioned as a representative example, but in chronic bronchitis, etc. May also be accompanied by lung fibrosis.

  Pulmonary inflammation is mainly divided into alveolar pneumonia, where the air cavity is the main site of inflammation, and interstitial pneumonia, where the alveolar wall including the alveolar epithelium is the main site of inflammation. For example, pulmonary fibrosis is one in which fibrous connective tissue proliferates around the alveolar walls and bronchioles, resulting in hard atrophy. This is considered to be a result of the balance between collagen production and degradation being lost due to overproduction of collagen and suppression of degradation at the site of inflammation. In addition to alveolar pneumonia and interstitial pneumonia, it is known to shift from scarring granulomas seen in pulmonary tuberculosis, silicosis and other pneumoconiosis, and active oxygen and drug administration are also risk factors. It is known to get.

  For the treatment of fibrotic lung diseases such as pulmonary fibrosis, administration of steroids, immunosuppressive agents and the like is generally performed for the purpose of removing risk factors and suppressing inflammation. However, symptomatic treatment is the main and the effect is not sufficient, and the therapeutic agents used are not suitable for long-term continuous use due to strong side effects, and there is no effective therapeutic agent at present.

On the other hand, since the respiratory tract airways are directly exposed to the outside air, they are susceptible to invasion by foreign matter such as dust and microorganisms in the atmosphere. Thus, biological defense mechanisms such as mucus secretion, mucus cilia transport and cough are developed in the airway. Failure of these defense mechanisms is closely related to the onset, chronicity, and intractability of various respiratory diseases including chronic obstructive pulmonary disease (COPD).
For example, airway inflammation can cause excessive mucus secretion and cause progressive airway embolism. Mucus is produced by goblet cells and submucosal gland cells and is secreted into the airway lumen after degranulation. Among them, goblet cells are mucus-secreting cells present in the airway epithelium. It is known that goblet cell hyperplasia occurs in the central airways and peripheral airways when suffering from airway diseases, resulting in excessive secretion of mucus. In the peripheral airway, since many goblet cells exist in the narrow airway epithelium, airway embolism is particularly likely to occur. In addition, excessively secreted mucus causes the problem of reduced clearance of itself and may cause pulmonary infections due to, for example, the generation of bacterial colonies in the mucus.

  Such airway embolism associated with excessive secretion of mucus is found in various respiratory diseases such as chronic bronchitis, acute asthma, cystic fibrosis, bronchiectasis and chronic obstructive pulmonary disease. For the treatment, steroidal anti-inflammatory drugs, antibiotics, expectorants, bronchodilators and the like that have been developed for the purpose of treating acute respiratory diseases in the past are generally used.

However, steroidal anti-inflammatory drugs have problems such as side effects that cannot be ignored such as the occurrence of immunodeficiency due to long-term administration. Antibiotics also have problems with the emergence of resistant bacteria and opportunistic infections for chronic respiratory diseases. On the other hand, the effectiveness of current expectorants is far from satisfying both patients and doctors. In addition, bronchodilators are also administered for therapeutic purposes, but satisfactory therapeutic effects are not obtained as with expectorants.
That is, if the ideal therapeutic drug is to have a certain effect and few side effects, steroidal anti-inflammatory drugs and antibiotics are side effects, while current expectorants and bronchodilators are effective. It is hard to say that it is sufficient as a therapeutic drug.

In view of the above, there is a strong demand for the development of novel therapeutic agents for the above-mentioned diseases that have few side effects and high therapeutic effects, in place of these conventional therapeutic agents.
For example, glycyrrhizin has been used as a therapeutic agent for liver disease or an antiallergic disease agent for many years (see Non-Patent Documents 1 to 4).

In addition, glycyrrhizin has been confirmed to be a therapeutic agent with few side effects from its many years of use, and has an established reputation for safety. One of the few side effects observed at the time of administration is hypokalemia, but it is known that it is relatively easy to suppress by combined use with the antialdosterone drug spironolactone.
Therefore, it is very useful if a highly safe drug that has been used in this manner and has been confirmed to have few side effects can be used as a therapeutic drug for the above-mentioned diseases.
Hiroshi Suzuki et al: About the therapeutic effect of strong neominophagen C on chronic hepatitis, Ayumi Medicine, Vol. 102, 562 (1977) Yano, Y. et al .: Examination of the therapeutic effect of Glicyrone Tablets No. 2 for chronic hepatitis by double-blind method, Linyi and Research, Vol. 66, 2629 (1989) Strong Mino C Clinical Research Group: Clinical study of strong neominophagency for drug eruption, poisoning eruption and urticaria, West Japan Dermatology, Vol. 56, 603 (1994) Kunio Minami: Experience using glycyrone tablets in the field of dermatology, Linyi and Research, Vol. 78, 187 (2001)

However, glycyrrhizin has never been studied as a therapeutic agent for fibrotic lung disease.
In addition, the action of glycyrrhizin on mucus-secreting cells present in the respiratory tract has not been studied so far, and glycyrrhizin has not been studied as a therapeutic agent for airway embolism.
Therefore, the subject of the present invention is to use glycyrrhizin, a novel therapeutic agent for fibrotic pulmonary disease, an agent for inhibiting hyperplasia of airway mucus-secreting cells, which has a high therapeutic effect and few side effects, unlike conventional therapeutic agents And to provide a therapeutic agent for airway embolism.

  As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that glycyrrhizin, which is known to have low side effects and high safety, is fibrosis including pulmonary fibrosis. The present invention was found to be extremely effective in the treatment of pulmonary diseases, and further to suppress goblet cell hyperplasia and relieve airway embolism.

That is, the first invention of the present invention is a therapeutic agent for fibrotic lung disease containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.
A second aspect of the present invention is the fibrotic lung according to the first aspect, wherein the fibrotic lung disease is pulmonary fibrosis and chronic bronchitis with or without emphysema. It is a remedy for diseases.
A third invention of the present invention is the therapeutic agent for fibrotic lung disease according to the first or second invention, wherein the pharmaceutically acceptable salt is a monoammonium salt.
The fourth invention of the present invention is an airway mucus-secreting cell hyperplasia inhibitor containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.
A fifth invention of the present invention is the airway mucus secretory cell hyperplasia inhibitor according to the fourth invention, wherein the airway mucus secreting cells are goblet cells.
A sixth invention of the present invention is the airway mucus-secreting cell hyperplasia inhibitor according to the fourth or fifth invention, wherein the pharmaceutically acceptable salt is a monoammonium salt.
The seventh invention of the present invention is a therapeutic agent for airway embolism containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.
According to an eighth aspect of the present invention, the airway embolism is any of acute bronchitis, chronic bronchitis, bronchial asthma, cystic fibrosis, bronchiectasis, pulmonary tuberculosis, pneumoconiosis, sinusitis and chronic obstructive pulmonary disease. The therapeutic agent for airway embolism according to the seventh invention, which is derived from the above.
A ninth invention of the present invention is the therapeutic agent for airway embolism according to the seventh or eighth invention, wherein the pharmaceutically acceptable salt is a monoammonium salt.

  The present invention shows an effective therapeutic effect with few side effects on fibrotic lung disease. In addition, the present invention has few side effects, suppresses hyperplasia of airway mucus-secreting cells, and exhibits an effective therapeutic effect on airway embolism.

Hereinafter, the present invention will be described in detail.
In the present invention, the fibrotic lung disease refers to, for example, pulmonary fibrosis and inflammatory lung disease accompanied by lung fibrosis. For example, chronic bronchitis can be mentioned as long as it is other than pulmonary fibrosis, but the therapeutic agent of the present invention is also effective for chronic bronchitis accompanied by emphysema. Among these, the therapeutic agent of the present invention is particularly effective for pulmonary fibrosis.

  In the present invention, airway embolism refers to those mainly caused by excessive secretion of mucus, specifically, for example, acute bronchitis, chronic bronchitis, bronchial asthma, cystic fibrosis, bronchiectasis, pulmonary tuberculosis, Mention may be made from any of pneumoconiosis, sinusitis and chronic obstructive pulmonary disease.

The therapeutic agent for fibrotic lung disease of the present invention contains glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient. The therapeutic agent for fibrotic lung disease of the present invention suppresses the production of collagen in the lung parenchyma by suppressing the expression level of collagen mRNA, and suppresses lung fibrosis.
Moreover, the airway mucus secretory cell hyperplasia inhibitor and airway embolism therapeutic agent of the present invention contain glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention suppresses the excessive secretion of mucus by suppressing the hyperplasia of mucus-secreting cells such as goblet cells present in the airways, and has the effect of alleviating airway embolism.

The glycyrrhizin used in the present invention can be obtained, for example, by extracting from licorice, but commercially available products can also be used.
Further, the pharmaceutically acceptable salt thereof is obtained by allowing glycyrrhizin and an inorganic or organic base to act at a certain molar ratio, and preferable examples include glycyrrhizin monoammonium salt, glycyrrhizin diammonium salt; Examples thereof include alkali metal salts such as monosodium salt, glycyrrhizin disodium salt, glycyrrhizin monopotassium salt and glycyrrhizin dipotassium salt; or glycyrrhizin choline salt. In addition to these, calcium salts, magnesium salts, aluminum salts, and the like can also be used. Among these, glycyrrhizin monoammonium salt is particularly preferable.
These may be used alone or in combination of two or more.

The dosage form of the therapeutic agent of the present invention is not particularly limited. Generally, it is an oral agent such as a tablet, a powder, a granule, a capsule, a fine granule, and a liquid medicine, or a parenteral such as an inhalant, a suppository, or an injection. It can be used as an agent.
These can be produced by a conventionally known method.

  As described above, the oral preparations can be tablets, powders, granules, capsules, fine granules, liquids and the like. These preparations include excipients, lubricants, plasticizers, surfactants, binders, disintegrants, wetting agents, stabilizers, flavoring agents, coloring agents, flavoring agents, buffers, which are usually used in the production of these preparations. An agent etc. can be mix | blended and it can manufacture according to a conventional method.

Examples of excipients include lactose, glucose, D-mannitol, fructose, dextrin, starch, sodium chloride, sodium bicarbonate, calcium carbonate, sodium alginate, ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, silicic anhydride, and kaolin. Etc.
Examples of the lubricant include magnesium stearate, calcium stearate, stearic acid, talc, corn starch and macrogol.
Examples of the binder include gelatin, gum arabic, cellulose ester, and polyvinyl pyrrolidone.

Examples of the plasticizer include polyethylene glycol, propylene glycol, glycerol, triacetin, medium chain fatty acid triglyceride, acetyl glycerol fatty acid ester and triethyl citrate.
Examples of the binder include chickenpox, licorice extract, tragacanth, simple syrup, gelatin and the like.

Examples of the disintegrant include starch, agar, carmellose calcium, carmellose, and crystalline cellulose.
Examples of the wetting agent include gum arabic, polyvinyl pyrrolidone, methyl cellulose, carmellose sodium, hydroxypropyl cellulose and the like.

Examples of the corrigent include sucrose, honey, sodium saccharin, mint, eucalyptus oil, cinnamon oil and the like.
Examples of the colorant include iron oxide, β-carotene, chlorophyll, and a water-soluble edible dye.
Examples of the fragrance include lemon oil, orange oil, dl- or l-menthol.

When used as a parenteral preparation such as an inhalant or injection, the solvent includes distilled water for injection, a sterile non-aqueous solvent, or a suspension. As the base of the non-aqueous solvent or suspending agent, it is preferable to use propylene glycol, polyethylene glycol, glycerin, olive oil, corn oil, ethyl oleate, or the like.
In addition to these, a buffer, an antiseptic, an antioxidant, and the like can be appropriately added as necessary as optional pharmaceutically acceptable components within a range not impeding the effects of the present invention.

As the administration method, either oral or parenteral can be selected.
As a therapeutic agent for fibrotic lung disease, the dose varies depending on the age, symptoms, etc. of the patient. In the case of oral administration, the amount of glycyrrhizin or a salt thereof is preferably 25 to 1000 mg / person per day for each adult. More preferably, it is 150 to 225 mg / person. Such a range of glycyrrhizin can be administered once or divided into several times a day. In addition, the parenteral dosage is preferably 25 to 1000 mg / person as the amount of glycyrrhizin or a salt thereof per adult day. Such a range of glycyrrhizin can be administered once or divided into several times a day.

  As airway mucus secretory cell hyperplasia inhibitor or airway embolism therapeutic agent, the dosage varies depending on the patient's age, symptoms, etc. Preferably it is 25-1000 mg / person, More preferably, it is 150-225 mg / person. Such a range of glycyrrhizin can be administered once or divided into several times a day. In addition, the parenteral dosage is preferably 25 to 1000 mg / person as the amount of glycyrrhizin or a salt thereof per adult day. Such a range of glycyrrhizin can be administered once or divided into several times a day.

Hereinafter, the present invention will be described in more detail with specific examples. However, the present invention is not limited to the following examples.
[Example 1]
◎ Fiberous pulmonary disease treatment <Materials and procedures>
First, materials and experimental procedures used in this example are shown below.
(Experimental animals)
In this example, since the histopathological findings were very similar to human pulmonary fibrosis, a rat bleomycin-induced pulmonary fibrosis model was used.
As rats, SD male rats 8 weeks old (250-280 g) (manufactured by Charles River Japan Co., Ltd.) were used. During the habituation and testing period, these animals were housed with free access to chow and drinking water in an environment at room temperature of 25 ° C.

(Used drug)
As a therapeutic agent for fibrotic lung disease containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient of the present invention, Glicyrone Injection No. 1 (registered trademark, manufactured by Minofagen Pharmaceutical Co., Ltd. Abbreviated as GL). The components in 1 ampoule (2 ml) of Glicyrone Injection No. 1 are as shown below.

◎ Glycylon Injection No.1 glycyrrhizin monoammonium salt 40mg (in terms of glycyrrhizin)
Ammonia Appropriate amount Sodium chloride Appropriate amount

In this example, dexamethasone 21-phosphate (manufactured by Sigma, hereinafter abbreviated as DEX), which is a steroidal anti-inflammatory drug, was used as a drug for comparison.
In addition, bleomycin (manufactured by Nippon Kayaku Co., Ltd., hereinafter abbreviated as BLM) was used as bleomycin, a drug that induces pulmonary fibrosis in rats. All other chemicals used commercially available special grade reagents.

(Preparation of BLM-induced pulmonary fibrosis model)
Rats were immobilized by anesthesia with ketamine hydrochloride (100 mg / kg; intraperitoneal administration), the trachea was exposed after midline incision, and then BLM (4 mg / 0.5 mL / kg) was injected into the trachea with a microsyringe. Then, after the incision was sutured, the rats were reared with free food and drinking water until the treatment day to prepare a BLM-induced pulmonary fibrosis model.

(Drug administration)
GL or DEX was administered to the prepared BLM-induced pulmonary fibrosis model, and the suppression effect of pulmonary fibrosis was compared. At this time, as a control, a group to which physiological saline (0.9% sodium chloride) (6.75 mL / kg) was administered instead of these drugs (hereinafter abbreviated as BLM single administration group) was also provided. Administration was carried out by the following two methods (administration methods 1 and 2).

(Administration method 1)
A single dose of GL is 15, 45 and 135 mg / kg (subcutaneous administration), a single dose of DEX is 1 mg / kg (subcutaneous administration), and a single dose of physiological saline is 6.75 mL / kg. kg. These were administered on a schedule of daily administration by subcutaneous administration once a day for 4 weeks immediately after BLM administration, and samples were collected 4 weeks later and examined histopathologically and biochemically. In addition, the GL administration group at this time is classified into the GL (15) 4W group, the GL (45) 4W group, and the GL4W (135) group according to the dose, and these are collectively referred to as the GL4W group and the DEX administration group as the DEX4W group. In the following, each is abbreviated. In addition, the BLM-induced pulmonary fibrosis model administered was 5 animals in the GL (15) 4W group, 5 animals in the GL (45) 4W group, 5 animals in the GL4W (135) group, 6 animals in the DEX4W group, BLM There were 5 in the single administration group.

(Administration method 2)
A single dose of GL was 135 mg / kg (subcutaneous administration), a single dose of DEX was 1 mg / kg (subcutaneous administration), and a single dose of physiological saline was 6.75 mL / kg. These were administered in a schedule of daily administration by subcutaneous administration once a day for 2 weeks from 2 weeks after BLM administration, and samples were collected 2 weeks later and examined histopathologically and biochemically. The GL administration group at this time is abbreviated as GL2-4W group, the DEX administration group as DEX2-4W group, and the following is abbreviated. In addition, the number of BLM-induced pulmonary fibrosis models administered was 5 in the GL2-4W group, 5 in the DEX2-4W group, and 5 in the BLM single administration group.

(Sample collection)
After completion of the drug administration, the rat was laparotomized under anesthesia, exsanguinated from the abdominal aorta, bronchoalveolar lavage was performed through a tracheal cannula, and a lavage fluid (hereinafter referred to as BALF) was collected. The lungs after lavage were removed, and the left lobe was fixed with 10% neutral buffered formalin solution (pH 7.8), embedded in paraffin, and hematoxylin / eosin stained as a 5 μm thick section with a microtome. After performing, it observed under the optical microscope. On the other hand, total RNA was extracted and purified from the right lobe, upper lobe and middle lobe using TRIzol reagent (manufactured by Invitrogen).

(Biochemical analysis of BALF components)
As an index of inflammation, the number of inflammatory cells and protein concentration in BALF were measured. The protein concentration was measured using Protein Assay Dye Reagent (manufactured by Bio-Rad). In addition, the protein concentration of bovine serum albumin solution (0 to 250 μg / mL, hereinafter abbreviated as BSA) was measured by the same method, and a calibration curve was created from the obtained absorbance value to obtain a BSA equivalent amount. Quantified.

(Measurement of collagen type 1 mRNA in lung parenchyma)
Using 1 μg of total RNA extracted from the right lung as a template, reverse transcription was performed according to a conventionally known method to synthesize cDNA. The obtained cDNA was amplified by a real-time PCR method using iTaq SYBER Green Supermix with ROX (manufactured by Bio-Rad), and the amount of collagen type 1 mRNA was measured. At the same time, GAPDH mRNA was amplified and used as an internal standard.

(Measurement of collagen content in lung parenchyma)
The lower right lobe of the lung was finely cut, and a part of the obtained section was fractionated, added with 0.5 M acetic acid solution containing 1% protease inhibitor cocktail, and stirred at 4 ° C. for 24 hours. Next, centrifugation was performed at 15000 × g for 60 minutes, the supernatant was collected, and the amount of collagen was measured. For the measurement, Sircol Soluble Collagen Assay kit (manufactured by Biocolor) was used. The amount of collagen was corrected with the total amount of protein.

(Statistical processing)
Data are shown as mean ± standard error (mean ± SEM). For statistical analysis, ANOVA (Student-Newman-Keuls) was used and was considered significant with p <0.05.

<Result>
The results are shown below.
(BLM single administration group)
In the lungs of rats injected with BLM intratracheally, a large number of macrophages, neutrophils and lymphocytes were observed around the blood vessels one week after the injection, and it was considered that inflammation was clearly induced. Two weeks after the injection, thickening, i.e., fibrosis, is considered to be caused by fibroblasts in the alveolar walls around the blood vessels and bronchioles, and this fibrosis becomes remarkable with the passage of time, and after 4 weeks, it becomes extensively fibrotic. Was observed.

  The number of inflammatory cells in BALF was significantly increased to 2 to 4 times that of healthy rats at 1, 2, 3 and 4 weeks after BLM injection. Infiltration of neutrophils and an increase in the amount of protein in BALF were significant. In contrast, the amount of collagen in the lung parenchyma became prominent at 3 and 4 weeks after administration of BLM, and it was considered that acute airway inflammation had occurred prior to fibrosis.

(Administration Method 1 Action 1 of GL on BLM-induced pulmonary fibrosis 1)
In administration method 1, samples were collected and examined histopathologically and biochemically.
When the lung tissue was examined histopathologically, tissue thickening and macrophage infiltration, which were considered to be fibrosis, were observed extensively in the BLM single administration group. In the DEX4W group, inflammatory cell infiltration and interstitial thickening were observed mainly in the vicinity of blood vessels, but the extent was mild compared to the BLM alone administration group. In contrast, in the GL (15) 4W group, inflammatory cells infiltrated around the bronchioles and capillaries, but the thickness of the stroma was slight. Furthermore, in the GL (45) 4W group and the GL (135) 4W group, although slight infiltration of inflammatory cells was seen around the blood vessels, the interstitial thickening was hardly seen, which was similar to that in healthy rats. It was.

FIG. 1 shows the measurement results of the number of inflammatory cells and protein concentration in BALF measured as an index of inflammation. FIG. 1A is a graph showing the number of inflammatory cells in BALF, and FIG. 1B is a graph showing the protein concentration in BALF. In FIG. 1, “*” indicates that there is a significant difference with respect to healthy rats, and “*” indicates that there is a significant difference with respect to the BLM single administration group.
From FIG. 1 (a), the following was confirmed. That is, in the BLM alone administration group, the total number of inflammatory cells increased about 3 times that of healthy rats. In the DEX4W group, there was no significant decrease in the number of inflammatory cells. In contrast, in the GL4W group, the number of inflammatory cells decreased depending on the dose, and in the GL (135) 4W group, it decreased to about 45% of the BLM alone administration group. When the number of cells was examined for each leukocyte type, all cell types decreased in the GL4W group, whereas in the DEX4W group, only a decrease in the number of lymphocytes was significant, and the number of other cells was not affected. .

  Moreover, the following was confirmed from FIG.1 (b). That is, in the BLM alone administration group, the protein concentration in BALF increased about twice that in healthy rats, and in the DEX4W group, no obvious decrease in the protein concentration was observed. In contrast, in the GL4W group, a clear decrease in the protein concentration was observed at any dose.

The effect on collagen production was examined by the amount of collagen in the lung parenchyma. The result is shown in FIG. In FIG. 2, “*” indicates that there is a significant difference with respect to healthy rats.
The amount of collagen in the group administered with BLM alone was slightly higher than that in healthy rats but significantly increased. On the other hand, there was no significant difference between healthy rats in the DEX4W group or GL4W group. Moreover, the tendency for GL to suppress the production of coralasein tended to depend on the dose.
In the DEX4W group, a significant suppression of weight gain of rats was observed, but in the GL4W group, such a suppression of weight gain was not observed at any dose.

(Administration Method 2-Action 2 of GL on BLM-induced pulmonary fibrosis)
From the above results, a remarkable inhibitory effect on lung fibrosis by BLM was observed particularly in the GL (135) 4W group in the GL4W group. However, it was unclear whether the inhibitory effect was obtained as a result of suppressing the initial inflammation caused by BLM or the fibrosis that occurred following the initial inflammation. . Therefore, the administration method 2 in which the drug was administered after BLM was administered to rats to cause initial inflammation was performed in parallel with the administration method 1, and the results were compared. However, in the administration method 1, GL was only the GL (135) 4W group.

Samples were taken and examined histopathologically and biochemically.
Infiltration of inflammatory cells around blood vessels was remarkably suppressed in the GL (135) 4W group, and the above-mentioned results were reproduced. On the other hand, in the GL2-4W group, infiltration of inflammatory cells around the blood vessels was observed, but the degree was mild compared to the BLM single administration group. Stromal thickening was slightly observed in the GL2-4W group, but hardly occurred in the GL (135) 4W group. In the DEX2-4W group and the DEX4W group, infiltration of inflammatory cells around the blood vessels and interstitial thickening were reduced as compared with the BLM alone administration group.

On the other hand, when the effect on the number of inflammatory cells and protein concentration in BALF was confirmed, the results shown in FIG. 3 were obtained. 3A is a graph showing the number of inflammatory cells in BALF, and FIG. 3B is a graph showing the protein concentration in BALF. In FIG. 3, “*” indicates that there is a significant difference with respect to healthy rats, and “*” indicates that there is a significant difference with respect to the BLM single administration group. That is, in both the GL (135) 4W group and the GL2-4W group, significant suppression of increases in the total white blood cell count, macrophage count, neutrophil count, and protein concentration was observed.
On the other hand, in both the DEX2-4W group and the DEX4W group, although the remarkable suppression of the increase in the number of inflammatory cells was observed, the protein concentration was further increased as compared with the BLM alone administration group.

In addition, collagen mRNA in lung parenchyma was amplified by a real-time PCR method, and its expression level was measured. The results are shown in FIG. In addition, the vertical axis | shaft of the graph in FIG. 4 shows the mRNA expression level ratio of collagen with respect to the mRNA expression level of GAPDH which is an internal standard as stated above.
In the BLM single administration group, the expression level of collagen mRNA was remarkably increased to about 13 times that of healthy rats. In contrast, in both the GL2-4W group and the GL (135) 4W group, the mRNA expression level was similar to that in healthy rats. That is, it was confirmed that GL suppressed at the level of collagen production mRNA. The same applies to the DEX2-4W group and the DEX4W group.

As described above, the protein concentration and neutrophil count in BALF are markedly increased after 1 week from the intratracheal injection of BLM and decreased thereafter, while interstitial thickening is observed after 2 weeks from the BLM injection. It was observed locally and after 4 weeks, it was confirmed to be widely and clearly observed. That is, it is considered that a pathological condition is formed by two phases of initial inflammation that occurs 1 to 2 weeks after administration of BLM and subsequent lung fibrosis. In contrast, in both the GL4W group and the GL2-4W group, increase in protein concentration in BALF, which is an index of airway inflammation, and the number of inflammatory cells are remarkably suppressed, and collagen production in the lung parenchyma Was significantly suppressed at the mRNA level. That is, it was confirmed that GL suppresses fibrosis occurring at least following initial inflammation by BLM.
Thus, although GL has an excellent inhibitory effect on pulmonary fibrosis, unlike conventional therapeutic agents, GL does not have serious side effects at the time of administration, and therefore glycyrrhizin and / or its pharmaceutically acceptable. It was suggested that the salt is extremely effective as a therapeutic agent for fibrotic lung disease.

[Example 2]
◎ Airway mucus-secreting cell hyperplasia inhibitor, airway embolism drug <Materials and procedures>
First, materials and experimental procedures used in this example are shown below.
(Experimental animals)
In this example, a mouse LPS-induced acute bronchitis pathological model was used.
As a mouse, an ICR male rat 8 weeks old (manufactured by Charles River, Japan) was used. During the habituation and testing period, these animals were housed with free access to chow and drinking water in an environment at room temperature of 25 ° C.

(Used drug)
As an airway mucous secretory cell hyperplasia inhibitor and an agent for treating airway embolism containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient of the present invention, Glicyrone Injection No. 1 (registered trademark, stock), which is a subcutaneous injection Company Minophagen Pharmaceutical, hereinafter abbreviated as GL) was used. The components in 1 ampoule (2 ml) of Glicyrone Injection No. 1 are as shown below.

◎ Glycylon Injection No.1 glycyrrhizin monoammonium salt 40mg (in terms of glycyrrhizin)
Ammonia Appropriate amount Sodium chloride Appropriate amount

In this example, as a comparison drug, expectorant carbocysteine (manufactured by Sigma, hereinafter abbreviated as s-CMC) and steroidal anti-inflammatory drug dexamethasone 21-phosphate (manufactured by Sigma, hereinafter, DEX) (Abbreviated) was used.
Moreover, E. coli-derived lipopolysaccharide (manufactured by Sigma, hereinafter abbreviated as LPS) was used to induce acute bronchitis by hyperplasia of goblet cells in mice. All other chemicals used commercially available special grade reagents.

(Preparation of LPS-induced acute bronchitis pathological model)
Mice were fixed under anesthesia with pentobarbital (50 mg / kg, intraperitoneal administration), and 2 mg / ml LPS was administered from the oral cavity into the trachea with 100 μg / animal using a microsyringe. Then, the mice were reared with free food and drinking water until the treatment day, and an LPS-induced acute bronchitis pathological model was prepared.

(Drug administration method)
S-CMC, DEX or GL was administered to mice before and after LPS administration to compare and examine the inhibitory effect on goblet cell formation. Specifically, s-CMC, DEX, and GL were administered once a day immediately before LPS administration and every day after LPS administration. The single dose of s-CMC is 100 and 500 mg / kg (po), the single dose of DEX is 1 mg / kg (subcutaneous administration), and the single dose of GL is 135 mg / kg (subcutaneous administration) It was. At this time, as a control, instead of these drugs, physiological saline (0.9% sodium chloride) (6.75 mL / kg) was administered (hereinafter abbreviated as LPS single administration group), LPS and drugs. No administration group (hereinafter abbreviated as healthy mice) was also provided.
And 7 days after LPS administration, the trachea was removed and examined histopathologically and biochemically.

In addition, the s-CMC administration group at this time is abbreviated below as the s-CMC (100) administration group and the s-CMC (500) administration group according to the dose.
The LPS-induced acute bronchitis pathological model administered was 4 in any of the s-CMC (100) administration group, the s-CMC (500) administration group, the DEX administration group, the GL administration group, and the LPS single administration group. There were animals.

(Histopathological examination of mouse trachea)
The mouse was exsanguinated under pentobarbital (50 mg / kg, intraperitoneal administration) anesthesia, and the trachea was removed. The excised trachea was immersed in 10% neutral formalin overnight, fixed and dehydrated, and embedded in paraffin. And the structure | tissue was made into the thin sliced piece of thickness 6 micrometers using the microtome (made by Leica). The obtained section was deparaffinized and then subjected to AB-PAS staining and observed under a microscope. In the quantitative analysis of the number of goblet cells, the whole cell or a part of the cell observed in the epithelial fragment of cricoid cartilage side of about 3 mm in length approximately 2 to 5 mm from the thyroid cartilage of each trachea was observed. The number of AB-PAS staining positive cells that exhibited a bluish purple color was counted. The obtained data was expressed as the number of goblet cells / nm by subtracting the total number of cells counted by the length of the section.

(Statistical processing)
Data are shown as mean ± standard error (mean ± SEM). For statistical analysis, ANOVA (Student-Newman-Keuls) was used and was considered significant with p <0.05.

<Result>
The results are shown below.
(LPS single administration group)
The number of goblet cells in the trachea after LPS administration was measured daily. The results are shown in FIG. The number of goblet cells increased depending on the number of days of breeding of mice after LPS administration, and almost reached a plateau between 5 and 9 days. On the other hand, the number of goblet cells in the trachea did not increase in healthy mice bred similarly without administration of LPS. In FIG. 5, “*” indicates that there is a significant difference with respect to day 0 after administration.

(Drug administration group)
As shown in FIG. 6, in the s-CMC (100) administration group, no significant effect of suppressing the increase in the number of goblet cells was observed, and in the s-CMC (500) administration group, the LPS alone administration group A slight but significant effect of suppressing the increase in the number of goblet cells was observed.
On the other hand, in the DEX administration group and the GL administration group, a remarkable effect of suppressing the increase in the number of goblet cells was observed, and the number of goblet cells decreased to about ½ that of the LPS alone administration group. In FIG. 6, “*” indicates that there is a significant difference with respect to the LPS single administration group.

With s-CMC, a significant inhibitory effect on the number of goblet cells was confirmed, but the dose was extremely high and the effect was not sufficient. In contrast, GL showed a remarkable effect of suppressing the increase in the number of goblet cells comparable to DEX. In this case, although GL is a high dose, it is advantageous in that there are almost no side effects.
Thus, glycyrrhizin and / or a pharmaceutically acceptable salt thereof is extremely effective for suppressing goblet cell hyperplasia and effective as an agent for inhibiting airway mucus-secreting cell hyperplasia or as a therapeutic agent for airway embolism. It was suggested.

  INDUSTRIAL APPLICABILITY The present invention can be used in the medical field as a therapeutic agent for fibrotic lung disease, an agent for inhibiting hyperplasia of airway mucus-secreting cells, and a therapeutic agent for airway embolism.

It is a graph which shows the effect of the chemical | medical agent administration with respect to inflammation in the administration method 1 of Example 1, (a) is a graph which shows the inflammatory cell number in BALF, (b) shows the protein concentration in BALF, respectively. It is a graph which shows the effect of the medicine administration with respect to inflammation in the administration method 1 of Example 1, and is a graph which shows the collagen amount in lung parenchyma. It is a graph which shows the effect of the chemical | medical agent administration with respect to inflammation in the administration method 2 of Example 1, (a) is a graph which shows the inflammatory cell number in BALF, (b) shows the protein concentration in BALF, respectively. It is a graph which shows the effect of the chemical | medical agent administration with respect to inflammation in the administration method 2 of Example 1, and is a graph which shows the collagen mRNA expression level in lung parenchyma. It is a graph which shows the change of the number of goblet cells in the mouse trachea after LPS administration in Example 2. It is a graph which shows the effect of the medicine administration in Example 2, and is a graph which shows the number of goblet cells in the mouse | mouth trachea of the 7th day after LPS administration.

Claims (9)

  A therapeutic agent for fibrotic lung disease comprising glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.   The therapeutic agent for fibrotic lung disease according to claim 1, wherein the fibrotic lung disease is pulmonary fibrosis and chronic bronchitis with or without emphysema.   The therapeutic agent for fibrotic lung disease according to claim 1 or 2, wherein the pharmaceutically acceptable salt is a monoammonium salt.   An inhibitor of airway mucus-secreting cell hyperplasia containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.   The airway mucus-secreting cell hyperplasia inhibitor according to claim 4, wherein the airway mucus-secreting cells are goblet cells.   The airway mucus-secreting cell hyperplasia inhibitor according to claim 4 or 5, wherein the pharmaceutically acceptable salt is a monoammonium salt.   A therapeutic agent for airway embolism containing glycyrrhizin and / or a pharmaceutically acceptable salt thereof as an active ingredient.   The airway embolism is derived from any of acute bronchitis, chronic bronchitis, bronchial asthma, cystic fibrosis, bronchiectasis, pulmonary tuberculosis, pneumoconiosis, sinusitis and chronic obstructive pulmonary disease. 7. The therapeutic agent for airway embolism according to 7.   The therapeutic agent for airway embolism according to claim 7 or 8, wherein the pharmaceutically acceptable salt is a monoammonium salt.

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