JPH08268906A - Pulmonary fibrosis-preventing agent - Google Patents

Abstract

PURPOSE: To obtain a preventing agent containing a hepatocyte growth factor as an active ingredient, exhibiting excellent suppressing action on onset of pulmonary fibrosis and useful from preventing evolution from interstitial pneumonia such as cataplectic interstitial pneumonia, iatrogenic interstitial pneumonia or interstitial pneumonia due to collagen diseases to pulmonary fibrosis. CONSTITUTION: This preventing agent contains a hepatocyte growth factor(HGF) as an active ingredient. HGF is obtained by intraperitoneally administering carbon tetrachloride to rat, delivering rat lever kept in a state of hepatitis, grinding the lever and purifying the ground lever by a protein purification method such as gel column chromatography using S-Sepharose, heparin Sepharose, high-performance liquid chromatography. The formulation of the preventing agent is an injection, an inhalant, a suppository or an oral medicine. Furthermore, the preventing agent is preferably administered in a daily dose of 0.05-500mg, especially 1-100mg as HGF amount.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preventive agent for pulmonary fibrosis. More specifically, HGF (Hepatocyte Growth Factor)
The present invention relates to a pulmonary fibrosis preventive agent containing as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION Pulmonary fibrosis refers to a pathological condition that causes diffuse fibrosis in the lungs and causes cough, shortness of breath, diffuse pulmonary shadow, restrictive ventilation disorder, diffusion disorder, hypoxemia and the like.
This condition is a transition from interstitial pneumonia, and interstitial pneumonia can often be the pre-stage. The prognosis of interstitial pneumonia is poor, and many of them progress to pulmonary fibrosis. If they change to pulmonary fibrosis, about half of them die within 4 years after the onset. Here, interstitial pneumonia generally refers to a disease in which the main stroma of the lesion is the interstitium of the alveolar region (interstitium around the alveolar walls and respiratory bronchioles). The following are known as the etiological factors of interstitial pneumonia.・ The cause is unknown 1. Idiopathic interstitial pneumonia 2. Bronchiolitis obliterans interstitial pneumonia (BIP) 3. Detachable interstitial pneumonia (DIP) 4. 4. Lymphocytic interstitial pneumonia (LIP) 5. Giant cell interstitial pneumonia ・ The cause is clear 1. Inorganic dust: Asbestos, silica, talc, beryllium, etc. 2. Drugs (iatrogenic interstitial pneumonia): anti-cancer / immunosuppressive agents (peplomycin, bleomycin, cyclophosphane,
2. Nitrosoureas, interferons, etc.), antibiotics / chemotherapeutic agents (cephems, tetracyclines, etc.), gold preparations, etc. Other physical / chemical substances: X-rays, cobalt, enzymes, paraquat, mercury vapor, perchlorethylene, etc. 4. Infectivity: virus, mycoplasma, attenuated bacteria, etc. 5. Granulomatous interstitial pneumonia: Extrinsic allergic alveolitis (farmer pneumonia, sugar cane pneumonia, bird-rearing pneumonia, etc.), etc. Collagen disease (collagen interstitial pneumonia): progressive systemic sclerosis, rheumatoid arthritis, polymyositis / dermatomyositis, mixed connective tissue disease, systemic lupus erythematosus, etc. 7. Metabolic disorders: uremia, lungs, etc.

On the other hand, HGF is known to increase the labeling index of bronchial epithelial cells and to be involved in cell proliferation, and to promote the proliferation of alveolar epithelial cells to repair lung injury (Japanese Patent Laid-Open No. Hei 10 (1999) -242242). 6-172207).

[0004]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned conventional problems. That is, an object of the present invention is to provide a novel agent for preventing pulmonary fibrosis.

[0005]

The present invention has been made to solve the above problems, and the gist thereof is (1) H
A pulmonary fibrosis preventive agent comprising GF as an active ingredient, (2) a pulmonary fibrosis preventive agent according to (1) above, which prevents progression of interstitial pneumonia to pulmonary fibrosis, and ( 3)
The pulmonary fibrosis preventive agent according to (2) above, wherein the interstitial pneumonia is idiopathic interstitial pneumonia, iatrogenic interstitial pneumonia or collagenous interstitial pneumonia.

The HGF used in the present invention may be purified by various methods as long as it is purified to the extent that it can be used as a medicine. HGF
Various methods are known as the purification method of. For example, obtained by extracting and purifying from liver, spleen, lung, bone marrow, organs such as brain, kidney, placenta, etc. of blood cells such as rat, bovine, horse, sheep, blood cells such as platelets and leukocytes, plasma, serum, etc. (FEBS Letters, 224 , 312, 198
7, Proc. Natl. Acad. Sci. USA, 86 , 5844, 1989, etc.). In addition, HGF can also be obtained by culturing primary culture cells or cell lines that produce HGF and separating and purifying them from the culture (culture supernatant, cultured cells, etc.). Alternatively, a gene encoding HGF is incorporated into an appropriate vector by a genetic engineering technique, and this is inserted into an appropriate host for transformation, and the desired recombinant HGF is obtained from the culture supernatant of this transformant. (For example, Nature, 342 , 440,
1989, JP-A-5-111383, Biochem. Biop
hys. Res. Commun., 163 , 967, 1989, etc.). The above-mentioned host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells can be used.

More specifically, as a method for extracting and purifying HGF from living tissue, for example, carbon tetrachloride is intraperitoneally administered to a rat, and the liver of a hepatitis rat is excised and crushed, and S- It can be purified by an ordinary protein purification method such as gel column chromatography using sepharose or heparin sepharose, and HPLC. In addition, an animal cell, such as a Chinese hamster ovary (CHO) cell, a mouse C127 cell, etc., which is obtained by incorporating a gene encoding the amino acid sequence of human HGF into a vector such as bovine papillomavirus DNA using a gene recombination method, It can be obtained from the culture supernatant by transforming monkey COS cells and the like. H thus obtained
As long as GF has substantially the same effect as HGF, a part of its amino acid sequence is deleted or substituted by another amino acid, another amino acid sequence is partially inserted, N
One or more amino acids may be bound to the terminal and / or C-terminal, or the sugar chain may be similarly deleted or substituted.

The preventive agent of the present invention prevents pulmonary fibrosis.
Preferable diseases to be the subject of the present invention include pulmonary fibrosis that develops from interstitial pneumonia, and particularly preferably idiopathic interstitial pneumonia, iatrogenic interstitial pneumonia or collagenous interstitial Pulmonary fibrosis that develops from pneumonia is mentioned.

The preventive agent of the present invention includes humans as well as mammals (eg, cow, horse, pig, sheep, dog, cat, etc.).
It is applied to the treatment and prevention of interstitial pneumonia.

The prophylactic agent of the present invention may take various dosage forms (eg, liquid preparation, solid preparation, capsule preparation, etc.), but generally, only HGF as an active ingredient or an injection and inhalation together with a common carrier thereof. It is a drug, suppository, or oral preparation. The injection can be prepared by a conventional method, for example, HG
It can be prepared by dissolving F in an appropriate solvent (eg, sterilized water, buffer solution, physiological saline, etc.), sterilizing by filtering with a filter, and then filling an aseptic container. . The HGF content in the injection is usually adjusted to about 0.0002 to 0.2 (w / v%), preferably about 0.001 to 0.1 (w / v%). Further, as the oral drug, for example, tablets, granules, fine granules,
It is formulated into a dosage form such as powder, soft or hard capsule, liquid, emulsion, suspension, syrup, etc., and these formulations can be prepared according to a conventional method of formulation. Suppositories can also be prepared by a conventional pharmaceutical method using a conventional base (eg, cocoa butter, laurin butter, glycerogelatin, macrogol, Witepsol, etc.). Further, an inhalant can also be prepared according to a conventional method for formulation. The HGF content in the preparation can be appropriately adjusted depending on the dosage form, the disease to be applied and the like.

Upon formulation, a stabilizer is preferably added. Examples of the stabilizer include albumin, globulin, gelatin, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. Furthermore, the formulation of the present invention may contain additives necessary for formulation, for example, excipients, solubilizing agents, antioxidants, soothing agents, isotonic agents and the like. In the case of a liquid preparation, it is desirable to remove water by freezing, or freeze-drying, and then save. The lyophilized preparation is used by re-dissolving it with distilled water for injection or the like at the time of use.

The prophylactic agent of the present invention can be administered by an appropriate administration route depending on its formulation form. For example, it can be administered in the form of an injection, such as vein, artery, subcutaneous, intramuscular, etc. The dose is appropriately adjusted depending on the patient's symptoms, age, weight, etc.
The dosage is from mg to 500 mg, preferably from 1 mg to 100 mg, and it is suitable to administer this once or several times a day in divided doses.

[0013]

The preventive agent of the present invention can suppress the onset of pulmonary fibrosis and prevent pulmonary fibrosis.

[0014]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Method C57BL / 6 female mouse 8-12 weeks old body weight 18-22
g was used. Bleomycin (hereinafter referred to as BL
M) and HGF were co-administered (HGF administration group) and BLM alone was administrated (control group), and the inhibitory effects of HGF on drug-induced lung injury caused by BLM were compared and examined. Details of the administration method and test method and the results are shown below.

BLM Administration Method BLM is an osmotic minipump model 2001 manufactured by alza.
, BLM 100 mg / kg per mouse
Was administered. Alza Osmotic Mini Pump Model 20
01 has the ability to continuously discharge the drug solution in the pump for one week. BLM was dissolved using physiological saline as a solvent so that the total dose of the drug solution by the osmotic minipump 200 μl was adjusted to the above dose of BLM in the mouse. The back of a mouse anesthetized by intraperitoneal administration of thiopental sodium 50 mg / kg was shaved and incised, the subcutaneous tissue of the back was peeled off, this BLM solution was injected into an osmotic minipump, and the minipump was placed subcutaneously in the mouse for 1 week. Continuous BLM subcutaneous administration was performed.

HGF administration method HGF is an osmotic minipump model 1007D manufactured by alza.
Was used to administer 50 μg of human recombinant HGF per mouse. The mini pump model 1007D also has the capability of continuously discharging the chemical solution for one week. HGF was dissolved using physiological saline as a solvent so that HGF was 50 μg per 100 μl of minipump, and the same solution was injected into the minipump. Mice were opened under anesthesia with thiopental sodium, a minipump was inserted into the abdominal cavity, and HGF was intraperitoneally administered for 1 week continuously.

Test Method In both the HGF-administered group and the control group, two and four weeks after the start of administration, the chest was opened under anesthesia by intraperitoneal administration of thiopental sodium, blood was removed from the inferior vena cava and sacrificed, and the lung was excised.
Next, the left lung was fixed with 10% formalin under negative pressure for 48 hours, and the sagittal specimen was embedded in paraffin. A tissue section having a thickness of 3 μm was prepared from this paraffin block with a microtome, elastica-Masson staining was performed, and histopathological examination was performed.

In order to examine the organizational findings more objectively, As
hcroft score (Ashcroft T et al .: Simple method o
f estimating severity of pulmonary fibrosis on an
umerical scale. J Clin Pathol 41 (4): 467-70, 1988)
And point counting method (Snider GJ et al .: Chron
ic interstitial pulmonary fibrosis produced in ham
sters by endotracheal bleomycin Am Rev Respir Dis
117: 1099-1108, 1978) for the quantitative evaluation of lung tissue injury. Aschroft's score is obtained by scoring the lung fibrosis in a lung tissue sample at a magnification of 100 times, dividing the degree of fibrosis of the lung into 0-8 stages from mild to severe, and scoring the entire visual field. It is a method of evaluating the degree of fibrosis of the entire lung by averaging. In the point counting method, in a lung tissue specimen, a 100 × 100 grid is attached to an eyepiece lens at a magnification of 200 × under a microscope, and 5 points are selected, and it is evaluated whether or not there is fibrosis at each 5 points in each visual field. Each selected point is assigned to the subpleural region, perivascular region, or bronchiolar region.
The onchiolar region) and the alveolar region (alveolar region) are classified into four regions, each of which is tabulated, and the ratio of the count showing fibrosis to the total count of each region is shown.

The amount of collagen in the lung was examined by the amount of hydroxyproline in the right lung. After slaughtering the mouse, the right lung was excised, dehydrated and dried in diethyl ether for 48 hours or more, homogenized, 1 ml of 6N hydrochloric acid was added, and the mixture was kept at 110 ° C. for 2 hours.
Hydrolysis was performed for 4 hours. The amount of hydroxyproline in the sample was quantified by the HPLC method. 3 mice per group / week
The experiment was conducted by using each animal. Statistical processing is 2 level nest
ed ANOVA and ANOVA were used.

Results The histopathological examination was carried out on changes in the lungs of mice due to lung injury due to BLM and the suppressive effect on lung injury due to HGF coadministration. Fig. 1 shows a normal mouse (Fig. 1a) and BL as a control group.
The lung tissue specimens at 2 weeks and 4 weeks of the M single administration group and the HGF and BLM simultaneous administration group (HGF administration group) are shown. BL
At 2 weeks after administration of M alone (Fig. 1b), the alveolar tissue retained the overall structure, but there were some fibrosis under the pleura and accompanying disappearance of air spaces, and in some cases, the fibrous region was close to the fibrosis. It was connected to the pulmonary vein branch. In the fiber area and its surrounding area, II
Type cells, foam macrophages and a few lymphocytes were scattered. Thus, fibrosis just below the pleura was prominent, and destruction of the alveolar structure was observed, but no change in bees or lungs was observed. In the 2nd week of the HGF-administered group (Fig. 1c), there were some areas where subpleural fibrosis occurred, but most of them had a normal structure. In addition, the degree of fibrosis was lighter than that of the BLM-only administration group, and the appearance of foam macrophages was small. Thus, there was some subpleural fibrosis, but it was mild, and most had normal alveolar structure.

At the 4th week after the administration, the BLM administration group (see FIG.
In d), strong fibrosis occurred mainly in the subpleural area, and the fibrotic region formed a large wedge-shaped lesion and partially had a bee or lung structure. Although there was a normal part in the area away from the pleura, there were some areas around the blood vessels and airways where fibrosis occurred as in the subpleural area. Foam macrophage infiltration was prominent around the fibrosis. In this way, extensive fibrosis occurs continuously from the subpleura to the surrounding airways and blood vessels running in the central axis of the lung lobe, and almost no normal alveolar structure is observed. On the other hand, in the HGF-administered group (Fig. 1e), wedge-like fibrosis occurred in places under the pleura, but the degree of the fibrosis was lighter than that of the administration of BLM alone, and there were few bees or lung-like structures. It was However, a large number of foam macrophages were found around it. Fibrosis was mild even around the airways and blood vessels. In this way, wedge-shaped fibrosis is observed under the pleura, but its extent does not extend to the central axis of the lung lobe, but remains in the superficial layer. Thickening of the alveolar wall and infiltration of inflammatory cells were not observed in the area remote from the pleura. Histopathologically, suppression of lung injury and fibrosis was observed in the HGF-administered group compared to the BLM-alone administration group, which was used as the control group at both the second and fourth weeks after administration.

In order to more objectively judge the degree of histopathological lung injury, Ashcroft's score and numerical comparison by the point counting method were performed for comparison. FIG. 2 shows the Ashcroft scores at 2 weeks and 4 weeks after the start of administration. Two
The values in the HGF-administered group were lower than those in the control group at both the 4th and 4th weeks, indicating that fibrosis was suppressed. At the fourth week, ANOVA revealed a significant difference between the two groups at a significance level of 5%. FIG. 3 is a comparison by the point counting method at the 4th week. In each of the subpleural, perivascular, peribronchiolar, and alveolar regions, the level of fibrosis was lower in the HGF-administered group than in the control group.
A difference was observed between the two groups at a significance level of 5%.

FIG. 4 shows the amount of collagen in the lung shown by the amount of hydroxyproline in the right lung. The amount of collagen increased with time in each group, and the HGF-administered group showed a lower value than the control group at both 2 weeks and 4 weeks, suggesting that the HGF-administered group had a lower degree of fibrosis. .

[Brief description of drawings]

FIG. 1 BLM (bleomycin) or HGF + BLM
Histopathological specimens of lungs of mice administered with
Is a picture of. In the figure, a is a histopathological specimen of normal lung of a mouse; b is a histopathological specimen of the second week after administration of BLM alone; c is H.
Histopathological specimen 2 weeks after simultaneous administration of GF and BLM; d is BL
Histopathological specimen 4 weeks after administration of M alone; e is HGF / BLM
It is a histopathological specimen 4 weeks after the simultaneous administration.

FIG. 2 is a diagram showing Ashcroft scores at 2 weeks and 4 weeks after the start of administration.

FIG. 3 is a diagram showing comparison by a point counting method at 4 weeks after the start of administration.

FIG. 4 is a graph showing the amount of collagen in the lung as indicated by the amount of hydroxyproline.

Claims (3)

[Claims] 1. A prophylactic agent for pulmonary fibrosis, which comprises HGF as an active ingredient. 2. The preventive agent for pulmonary fibrosis according to claim 1, which prevents the progression of interstitial pneumonia into pulmonary fibrosis. 3. The preventive agent for pulmonary fibrosis according to claim 2, wherein the interstitial pneumonia is idiopathic interstitial pneumonia, iatrogenic interstitial pneumonia or collagenous interstitial pneumonia.