JPH06172207A - Pulmonary injury-treating agent - Google Patents

Abstract

PURPOSE:To provide a pulmonary injury-treating agent useful for preventing/ treating the pulmonary injuries. CONSTITUTION:The pulmonary injury-treating agent contains HGF (hepatic cell-growing factor). In the treating agent, the active ingredient HGF can stimulate the growth of the epithelial cells of alveoli and regenerate injured lungs. The treating agent is useful for preventing and treating chronic and acute pulmonary diseases (e.g. pneumonia, pulmonary emphysema, pulmonary tuberculosis, chronic obstructive pulmonary diseases, coniosis, or deglutition).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a therapeutic agent for lung injury, and more particularly to a therapeutic agent for lung injury containing HGF (Hepatocyto Growth Factor) as an active ingredient.

[0002]

2. Description of the Related Art HGF is a protein found by the present inventors as a factor for proliferating mature hepatocytes from regenerated liver rat serum in vitro (Biochem Biophys Re.
S Commun, 122, 1450, 1984). We further
Succeeded in isolating HGF from rat platelets (Pro
c. Natl. Acad. Sci, 83 , 6489,1986, FFBS Letter, 2
2 , 311, 1987), and its amino acid sequence was partially determined. Furthermore, the present inventors succeeded in cloning human and rat-derived HGF cDNA based on the elucidated HGF amino acid sequence, and recombining this cDNA into animal tissues to obtain hepatocyte growth factor as a protein ( Human HGF: Nature, 342 , 440, 1989; Rat HGF: Pr
oc. Natl. Acad. Sci, 87 , 3200, 1990).

The molecular weight of HGF is 82-85 kD by SDS-polyacrylamide gel electrophoresis. Rat HG
The F molecule has a heterodimer structure in which an α chain consisting of 463 amino acid residues and a β chain consisting of 233 amino acid residues are crosslinked by one disulfide bond, and both α and β chains have 2 chains.
There are individual glucosamine-type sugar chain binding sites. Human HG
F also has almost the same physiological activity and consists of an α chain consisting of 463 amino acid residues and a β chain consisting of 234 amino acid residues. There are four kringle structures in the α chain, which are similar to the fibrinolytic enzyme plasmin, and the amino acid sequence of the β chain is about 3 times that of the plasmin B chain having serine protease activity
It has a homology of 7%. Rat HGF and human HGF
The amino acid sequence homology is 91.6% in α chain, β
It has a very high homology of 88.9% in the chains and its activities are completely compatible.

HGF, which was discovered as a factor that specifically proliferates liver parenchymal cells, has been shown to exhibit various activities in vivo according to recent research results by the present inventors and other researchers. However, expectations are growing for its application not only as a research target but also as a therapeutic drug for humans and animals. The present inventors have clarified that HGF acts widely as a growth factor not only on hepatocytes but also on epithelial cells, and achieved several inventions. In Japanese Patent Application No. 2-158841, HGF promotes the proliferation of renal proximal tubule cells, so that the application development as a therapeutic agent for renal diseases is also described.
-419158, HGF is melanocyte,
By promoting the growth of normal epithelial cells such as keratinocytes, it can be used as an epithelial cell promoter to treat wounds and skin ulcers.
The application and development of hair root cells as a proliferative agent were accomplished and the details thereof were disclosed. In particular, HGF is more suitable for practical use because it does not have the canceration action and cancer cell growth activity found in many other growth factors such as EGF. Furthermore, the present inventors have disclosed that in Japanese Patent Application No. 3-140812, HGF human liver cancer-derived HepG2 cell line and lymphoblastic cancer-derived IM9.
It has been disclosed that it can be used as an antitumor agent by utilizing the cancer cell growth inhibitory activity of cell lines and the like.

More important point in considering the practicality of HGF as a medicine is that HGF promotes the growth of only cells that have entered the G1 phase, that is, the growth phase, and acts on the cells in the G0 phase, that is, the quiescent phase. Do not do it. This is
It means that proliferation and regeneration of injured tissue is promoted, but it has no effect on undamaged tissue. Therefore, it is considered that even if HGF is excessively administered or HGF reaches the non-affected part via blood or the like, it does not induce canceration in normal tissues or cause excessive proliferation.

[0006]

As described above, HGF promotes the proliferation of not only hepatocytes but also epithelial cells in a wide range and has an activity of suppressing the proliferation of cancer cells.
It is expected that GF acts in healing tissue damage. H
GF-producing cells are not epithelial cells themselves, but Kup in the liver
It is known to be produced mainly by mesenchymal cells such as ffer cells and sinusoidal endothelial cells, capillary endothelial cells in the kidney, and alveolar macrophages and vascular endothelial cells in the lung. It has been clarified that a so-called paracrine mechanism in which HGF is supplied according to the above is established. However, when the liver or kidney is injured, HGF production is increased even in an uninjured organ, such as the lung, and therefore it is considered that HGF is also supplied by the so-called endocrine mechanism.

As described above, the HGF produced in the lung contributes to the tissue repair of at least remote organs such as the liver and the kidney, but whether HGF contributes to the regeneration of the lung when the lung is damaged. It has not been revealed. Examples of lung injury diseases include pneumonia, emphysema, pulmonary tuberculosis, chronic obstructive pulmonary disease, etc., but in particular, injury due to fibrosis of the lung (eg, pneumoconiosis), illness of patients with weakened immunity and the elderly Since swallowing pneumonia and the like may have a poor prognosis, it is strongly desired to establish preventive and therapeutic methods. The present inventor has found that the HGF receptor is present in the lungs, and most of the target cells for HGF are epithelial cell lines.
Thinking that HGF will contribute to the regeneration of the lungs when the lungs themselves are injured, and that it will be effective in the prevention and treatment of the above diseases,
The effect of HGF on the lungs when acute lung injury occurred was examined. As a result, the injury causes HG in the lungs.
It was found that F is newly produced, that HGF promotes DNA synthesis in lung epithelial cells via the lung HGF receptor, and that human blood HGF concentration is significantly elevated in patients with lung disease. It was found that administration of HGF to injured animals increased cell proliferation in lung tissue and promoted repair of lung injury. The present invention has been made based on such findings, and an object of the present invention is to provide a therapeutic agent for lung injury, which is useful for the prevention / treatment of lung injury.

[0008]

Means for Solving the Problems The therapeutic agent for lung injury of the present invention made to solve the above problems comprises HGF as an active ingredient. The HGF having the above-mentioned constitution, which is the active ingredient of the present invention, can be prepared by various methods as long as it is purified to the extent that it can be used as a medicine. Various methods are known as methods for preparing HGF, and examples thereof include liver, spleen, and lung of mammals such as rats, cows, horses, and sheep.
It can be obtained by extraction and purification from organs such as bone marrow, brain, kidney and placenta, blood cells such as platelets and white blood cells, plasma and serum. In addition, HGF can also be obtained by culturing primary culture cells or cell lines that produce HGF and separating and purifying from culture (culture supernatant, cultured cells, etc.). Alternatively, a gene encoding HGF is inserted into an appropriate vector by a genetic engineering method, and this is inserted into an appropriate host for transformation, and the desired recombinant HG is obtained from the culture of this transformant.
F can be obtained (eg Nature, 342 , 440, 198
See 9). The above host cells are not particularly limited, and various host cells conventionally used in genetic engineering techniques,
For example, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant or animal cells and the like 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 rat in a hepatitis state 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 papilloma virus DNA using a gene recombination method. Transforming monkey COS cells,
It can be obtained from the culture supernatant.

The HGF thus obtained has a part of its amino acid sequence deleted or substituted with another amino acid, a part of another amino acid sequence inserted, or has 1 amino acid at the N-terminal and / or C-terminal. Alternatively, two or more amino acids may be linked, or the sugar chain may be similarly deleted or substituted. Examples of the HGF-similar drug include, for example, JP-A-3-130091 and International Publication WO90 / 106.
The substances described in Japanese Patent Publication No. 51, etc. are mentioned, and these are applicable to the present invention and are included in the scope of the present invention.

The formulations of the present invention may be formulated into various forms (eg,
Liquid, solid, capsule, etc.), but generally, it is made into an injection with HGF which is the active ingredient alone or with a conventional carrier, or an external preparation with a conventional carrier,
Suppositories, inhalants, and nasal agents. The injection can be prepared by a conventional method. For example, HGF is dissolved in an appropriate solvent (eg, sterilized water, buffer solution, physiological saline, etc.) and then sterilized by filtering with a filter or the like, and then sterilized. It can be prepared by filling a conventional 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%). In addition, the topical drug is, for example, formulated into a dosage form such as an ointment, a gel or a liquid, and the HGF content in the formulation can be appropriately adjusted according to the disease to which the topical drug is applied, the site to which it is applied, and the like. . Suppositories, inhalants and nasal preparations can be prepared according to conventional methods for formulation. Upon formulation, a stabilizer is preferably added, and examples of the stabilizer include, for example,
Examples thereof include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol and the like. Furthermore, the formulation of the present invention may contain additives necessary for formulation, for example, excipients, solubilizers, 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 by adding distilled water for injection or the like at the time of use. The preparation of the present invention can be administered by an appropriate administration route depending on the form of the preparation. For example, it can be administered in the form of an injection such as vein, artery, subcutaneous or intramuscular. The dose is
It is adjusted according to the patient's symptoms, age, weight, etc.
Usually, it is 0.01mg to 100mg as HGF.
It is appropriate to administer in divided doses.

[0012]

INDUSTRIAL APPLICABILITY In the therapeutic agent of the present invention, HGF, which is an active ingredient, promotes the growth of alveolar epithelial cells and can regenerate damaged lungs. Therefore, the therapeutic agent of the present invention is useful for the prevention and treatment of chronic and acute lung diseases (for example, pneumonia, emphysema, pulmonary tuberculosis, chronic obstructive pulmonary disease, pneumoconiosis pneumonia, etc.).

[0013]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. The materials and methods used in the following experiments are as follows.

Treatment of animals: Adult Wistar male rats (weight 150-250 g) were anesthetized with ether. The neck was incised and the trachea was exposed. 18 gauge elaster to cause acute lung injury
A needle was pierced into the trachea, and then 0.1 M HCl was infused at a rate of 2 ml / kg body weight mainly into the left lung.

Northern blot analysis: Total RNA was isolated using the AGPC method from rats in which acute lung injury was caused by HCl. 10 μg of total RNA is 1.
Used for gel electrophoresis on 0% agarose / formaldehyde gel and Hybond-N membrane filter
Moved to (Amercham). Rat HGF cDNA Eco
The RBC-1 clone encoding the RI portion and the 5′-portion, and the fourth Kringle region of the α-chain, the entire β-chain of HGF, and part of the 3′-noncoding region were labeled Mega.
Labeling was performed using the prime DNA labeling system (Amercham) according to the manufacturer's instructions. Hybridization was carried out at 42 ° C. with 50% (volume / volume) formaldehyde, 5 × SSPE (1 × SSPE was 0.15 of NaCl).
M, 10 mM sodium phosphate buffer (pH 7.4)
And 1 mM of EDTA), 2 × Denhardt's solution, 0.5% SDS and 50 μg / ml salmon sperm DNA
In a solution consisting of over 20 hours. The filter used is 0.2 × SSPE-0.1% SDS,
It was washed for 15 minutes at 65 ° C., dried and then autoradiographed on Fuji X-ray film.

Partial purification treatment of HGF taken from the lungs: To purify the HGF action, the lungs were purified by 10 mM Tris-HCl, 0.3 M NaCl, and protease inhibitors (1 mM PMSF, 1 mM monoiodoacetate and 1 mM). 0.01% Tw including EDTA)
Homogenized in 4 parts buffer consisting of een80. After centrifuging at 105,000 g for 60 minutes, the supernatant is dialyzed against 100 times the above buffer,
And applied to a heparin-sepharose column equilibrated in the same buffer. The column was washed with 6 parts of the same buffer, then 2 M NaCl 2 in buffer.
Eluted by part. The eluate was 2 mM HEPES-
Dialyzed against 100 times a buffer consisting of NaOH (pH 7.2) and 20 mM NaCl, freeze-dried,
Then it was dissolved in 0.3 part of distilled water. These samples were passed through a 0.22 μm pore size filter (Millipore) and used for assay of HGF activity.

Assay of HGF activity: HGF activity was determined by measuring the stimulatory effect on DNA synthesis of rat hepatocytes in primary culture. Adult rat hepatocytes were isolated using in situ collagenase perfusion. The separated cells were subjected to 6.2 on a 24-well plastic dish (Corning) coated with type I collagen.
Seeded at a density of 5 × 10 ⁴ cells / 0.5 ml / ² cm ² , 5% bovine serum, 10 ⁻⁵ M insulin and 10 ⁻⁹ M
Cultivated in William's medium containing dexamethasone for 3 hours, then the medium contains 10 ^-9 M insulin, 10 ^-9 M dexamethasone and 0.1 μg / ml aprotinin, and serum-free William's medium. Was changed to. Two
Samples were added to the culture after 0 hours and incubated for an additional 20 hours. Next, the cultured cells are [ ¹²⁵ I]
Pulsed labeled with 1 μCi of deoxyuridine for 6 hours, phosphate buffer (PBS) and 10%
Washed with trichloroacetate and 1M N
Solubilized with aOH. The transfer of [ ¹²⁵ I] -deoxyuridine to the nucleus was counted using a γ-counter. One unit is a value corresponding to half the maximum value of the stimulatory effect of 10 ng / ml EGF on DNA synthesis in cultured rat hepatocytes.

Efficacy Assay for Tissue Change and Labeling Index: 5-Bromo-2'-deoxyuridine (BrdU, Sigma) was injected intraperitoneally into rats at 50 μg / kg body weight, killed after 1 hour and then lungs were removed. 1
4 μm thick sections were removed from paraffin and stained with hematoxylin and eosin to examine lung histological changes due to HCl embedded in 0% ethanol for 24-48 hours and then embedded in paraffin. To assay the labeling index, sections were denatured in DNA by immersion in 2M HCl for 1 hour at room temperature (RT) and in 0.1M borate buffer (pH 8.5). Neutralized, blocked with 5% horse serum in PBS, then diluted 1/50 with PBS. Anti-BrdU monoclonal antibody (Beckton
Dickinson) and incubated for 1 hour at room temperature. Sections are washed with PBS then 1/20
Biotin-conjugated horse anti-mouse IgG diluted to 0
Incubated with the vector at room temperature for 1 hour. After washing, the tissues were diluted 1/100 and treated with alkaline phosphatase streptavidin (Dak).
o) and incubated for 1 hour at room temperature. Enzyme reaction is 0.2M Tris-HCl (pH 8.2), 250
μg / ml 1-levamisole (Sigma), 0.01% fresh fuchsin (Merck), 0.01% sodium nitrite and 250 μg / ml naphthol AS-BI phosphate (Sigm
It was started in a buffer consisting of 0.25% N, N-dimethylformamide containing a). Sections were washed with PBS, fixed with 10% formalin and counterstained with hematoxylin. The labeling index was counted within the 3-6 independent regions of 3 individuals in the cells of the bronchial epithelium and the walls of the alveoli.

[ ¹²⁵ I] -bound to the cell membrane from the lung
Efficacy assay for HGF: Cell membranes isolated from rat lungs were prepared by centrifugation on a percoll gradient as described below. The lungs are 0.25M sucrose, 10
mM Tris-HCl (pH 7.5) and 2 mM ED
Homogenize in STE buffer consisting of TA, then 3
Centrifuge at 1,000 g for 0 seconds. The supernatant was centrifuged for 10 minutes at 5,000 g and then resuspended in STE buffer. The crude membrane fraction obtained is perc
Layered on oll solution (17: 1) and centrifuged for 20 minutes at 10,000 g. 50 μg of cell membrane was produced by chlorination-T method by iodination of recombinant human HGF with [ ¹²⁵ I] -HGF for 1 hour 1
Incubated in 100 ml of 100 ml binding buffer at 0 ° C. This buffer is 10 mM HEPE
S, with 6.4 nM unlabeled HGF, or without [ ¹²⁵ I] -HGF in Hanks' 0.2% bovine serum albumin. Membrane is 1
Centrifuge for 0 min at 12,000 g at 4 ° C., resuspend with 10 μl of binding buffer and then transfer to a new tube. [ ¹²⁵ I] binding to the membrane-
HGF was counted on a γ-counter. All binding experiments were performed in conjunction with supporting experiments.

Assay of HGF concentration in human blood: The effect of the sandwich RIA method for measuring the HGF concentration in human blood was confirmed. Briefly, beads coated with anti-human-HGF monoclonal antibody were incubated with 1 / 2-diluted human serum overnight at room temperature with shaking. After washing, the beads were incubated with [ ¹²⁵ I] -conjugated human HGF monoclonal antibody for 4 hours at room temperature with shaking. The beads were washed, transferred to another tube, and then [ ¹²⁵ I] bound to the beads was counted in a gamma counter. H
The GF concentration was determined from a standard curve with recombinant human HGF.

Example 1 HGF mRNA levels in rats with hydrochloric acid lung injury
Zan-blot analysis RNA was extracted from the lungs of rats with hydrochloric acid lung injury over time by the AGPC method, and the expression level of HGF mRNA was examined by Northern blot in accordance with the above method. The result is shown in FIG. As shown in FIG. 1, in the rat lung, a relatively strong expression of HGF mRNA was observed even in the normal state, and the expression level was more than double that in the normal state only 3 hours after administration of hydrochloric acid. After a slight decrease with time, it increased again, and after 12 hours, it was more than three times the normal value. HGF mRNA decreased to about twice the normal level after 24 hours, and thereafter remained at the same level for at least 1 week.

Example 2 Changes in HGF activity in lungs of rats with hydrochloric acid lung injury After HGF was isolated from the lungs of rats with hydrochloric acid lung injury over time and partially purified by heparin-sepharose chromatography, HGF activity was measured by the method described above. The result is shown in FIG. HGF in normal lung
Activity was 67 U / g tissue, but lung H after administration of hydrochloric acid
The GF activity rapidly increased, and the HGF activity 6 hours after the injury became about 130 U / g tissue. HGF activity decreased slightly after 12 hours but increased again after 24 hours.
After that, the HGF activity gradually decreased, and a biphasic course was shown in which it returned to almost normal level after 1 week.

Example 3 Hydrochloric Acid Lung Labeling Index of Rat Lungs
At each elapsed time after aging hydrochloric lung injury of the scan, Brd in vivo,
The proportion of cells incorporating U, that is, cells in S phase, was examined by a labeling index and shown in FIG. The labeling index in bronchial epithelial cells (indicated by ● in the figure) was 0.95% in the normal state, but 1 after administration of hydrochloric acid
It started to increase from 2 hours and reached a peak at 12.95% after 24 hours. After that, the labeling index gradually decreased, and after one week, it returned to almost the same level as in normal conditions. On the other hand, the labeling index of the cells that make up the alveoli (marked with a circle in the figure) was 1.42% in the normal state,
It increased gradually from 24 hours after the administration of hydrochloric acid, and peaked at 11.74% after 48 hours. After that, it gradually decreased, and returned to almost normal level one week later.

Example 4 Hydrochloric acid [ ¹²⁵ ] on the cell membrane of lungs of rats
I] -HGF binding amount specific HGF binding amount per unit protein of cell membrane (PM) fractions obtained from the left lung at various elapsed times after injury by transtracheally administering hydrochloric acid to the left lung I checked. [ ¹²⁵ I] -HG
The specific binding amount of F to the cell membrane fraction is about 1 of the total binding amount.
It was 0%, and analysis was possible. The result is shown in FIG. Each value shown in FIG. 4 is the average of two independent experiments. As shown in FIG. 4, the specific binding amount of HGF was about 68% at 3 hours after the administration of hydrochloric acid, and about 26% at 6 hours.
At 2 hours it fell below the limit of detection and the level persisted until 48 hours. After that, the specific binding is 7
Approximately 24% of the normal time after 2 hours and approximately 8 of the normal time after 1 week
Recovered to 8%. In Scatchard analysis shown in Inset, Kd value in normal lung was 58 pM, Bmax was 1530 site / μg
protein, Kd value in lung 3 hours after administration of hydrochloric acid was 51p
M and Bmax were 1050 site / μg protein. That is, it was shown that the decrease in the specific binding amount of [ ¹²⁵ I] -HGF to the cell membrane fraction was not due to the change in the Affinity of the receptor for HGF but to the decrease in the number of receptors. It was FIG. 5 shows liver (marked with Δ in the figure), kidney (marked with ▲ in the figure), spleen (marked with ○ in the figure).
The mark () indicates the specific binding amount of [ ¹²⁵ I] -HGF in the cell membrane fraction, but the specific binding amount of HGF decreases after administration of hydrochloric acid only in the lung (marked with ● in the figure). No change in organs. That is, it was revealed that HGF produced in the lung binds only to the receptor of the lung itself.

Example 5 Measurement of HGF Concentration in Blood of Healthy Subjects and Pulmonary Disease Patients The HGF concentration in blood of healthy subjects and lung disease patients was measured by the sandwich RIA method. The result is shown in FIG. As shown in FIG. 6, with respect to 197 healthy subjects, the blood HGF concentration determined by the sandwich RIA method was 0.33 ± 0.10 ng / ml. In contrast,
The blood HGF concentration of 22 patients with various lung diseases was 1.22.
It was as high as ± 1.01 ng / ml. Among them, one case with silicosis was 4.10 ng / ml, and one case with pulmonary suppuration was 2.
The value was extremely high at 84 ng / ml. For 7 patients with pneumonia, the distribution was 1.06-2.80 ng / ml.

Example 6 Effect of administration of HGF on lung-injured rats The effect of HGF administration on lung-injured rats was tested by the following method. Materials and Methods Wistar Male Rat (SPF) Weight 150-250
Under anesthesia with g of pentobarbital, the neck was incised to expose the trachea. Stick a 20 gauge elastane needle into the trachea to cause acute lung injury, then
0.01 M hydrochloric acid / physiological saline was infused mainly into the left lung at a rate of 2 ml / kg body weight. HGF is 0.25% BS
A and 0.01% Tween 80, Ca ²⁺ and M
Phosphate buffered saline without g ²⁺ (PBS (-))
Was dissolved as a solvent to a concentration of 1 mg / ml. 2.5 mg / g of this HGF solution immediately after administration of hydrochloric acid and 12 hours later
It was administered from the tail vein at a rate of kg body weight / dose, and the solvent alone was similarly administered to the control group. The experiment was performed with 10 animals per group. 22 hours after administration of hydrochloric acid, 5-bromo-2 '
-Deoxyuridine (BrdU) and 5-fluoro-2 '
A solution containing deoxyuridine in a ratio of 10: 1 (Cell
Proliferation kit labeling reagent (Amersham) is injected into the abdominal cavity of the rat at a rate of 10 ml / kg body weight,
Two hours later, under ether anesthesia, blood was removed from the abdominal artery and sacrificed. Next, the left lung is treated with 4% paraformaldehyde / PBS.
The cells were fixed with (-) for 3 to 5 hours, and then the region near the hilum was cut out and embedded in paraffin. A 2 μm-thick lung section was cut out from this paraffin block with a microtome, attached to a silane-coated slide glass, and deparaffinized with xylene. With respect to the lung tissue section thus prepared, BrdU incorporated into cell nuclei was immunostained using Cell proliferation kit (Amersham), and the lung tissue section was double-stained with hematoxylin. Next, using a microscope (20
Under observation (0 times), the one in which the nucleus was dyed in black was counted as BrdU positive cells, and the ratio (%) of the number of positive cells in all cells was determined and used as a labeling index. The spleen was treated in the same manner as the lung, and a section was made so as to cross the central part of the spleen, and this section was stained in the same manner as the lung, and individuals without BrdU-positive cells were not reliably administered the labeling reagent intraperitoneally. It was removed from consideration as a thing. For the statistical processing, Student's t-test was used.

Results Changes in the labeling index due to HGF administration after lung injury due to hydrochloric acid were examined in bronchial epithelial cells in the lung. The result is shown in FIG. 7. Here, the number of rats for which the labeling index was analyzed was 8 in both the HGF-administered group and the control group. The labeling index of bronchial epithelial cells in the control group was 4.15 ±
It was 1.33%. In contrast, the HGF-administered group had a significant increase of 8.36 ± 0.92% (p <0.
001). The labeling index of bronchial epithelial cells in the vicinity of the hilum of untreated normal rats was examined in 4 rats and found to be 0.71 ± 0.08%.
The increase in the labeling index of bronchial epithelial cells in the rat with lung injury caused by hydrochloric acid due to HGF administration showed an increase in cell proliferation in the injured lung tissue, and was considered to indicate the action of promoting lung injury repair by HGF. . This suggested the possibility of HGF as a therapeutic agent for lung injury.

Formulation Example 1 A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg of polysorbate 80 in 100 ml of physiological saline was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed to give a lyophilized formulation. Got

Formulation Example 2 0.02M phosphate buffer (containing 0.15M NaCl and 0.01% polysorbate 80, pH 7.4) 10
HGF 1mg and human serum albumin 100m in 0ml
An aqueous solution containing g was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed to obtain a lyophilized preparation.

Formulation Example 3 HGF 1 mg, sorbitol 2 g, glycine 2 g and polysorbate 80 10 m in 100 ml of distilled water for injection.
A solution containing g was aseptically prepared, dispensed in 1 ml aliquots, lyophilized and sealed to obtain a lyophilized preparation.

[Brief description of drawings]

FIG. 1 HGFmR in rats with hydrochloric acid lung injury
It is a figure which shows the northern blot of NA.

FIG. 2 is a diagram showing changes in HGF activity in the lungs of rats suffering from hydrochloric acid lung injury.

FIG. 3 is a view showing a time-dependent change in labeling index of lungs of rats suffering from hydrochloric acid lung injury.

FIG. 4 Cell membrane (P of the lung of a rat suffering from hydrochloric acid lung injury)
It is a figure which shows the change in the amount of [ ¹²⁵ I] -HGF binding to M).

FIG. 5: Liver of a rat suffering from hydrochloric acid lung injury (in the figure, Δ
FIG. 4 is a diagram showing the specific binding amount of [ ¹²⁵ I] -HGF in the cell membrane (PM) fractions of (), kidney (▴ in the figure) and spleen (∘ in the figure). In the figure, ● indicates the amount of [ ¹²⁵ I] -HGF bound to the lung cell membrane.

FIG. 6 is a diagram showing measurement results of HGF concentration in blood of healthy subjects and patients with lung diseases.

FIG. 7 is a graph showing the effect of administration of HGF on rats suffering from hydrochloric acid lung injury.

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[Procedure amendment]

[Submission date] July 14, 1993

[Procedure Amendment 1]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

Claims (1)

[Claims] 1. A therapeutic agent for lung injury, which comprises HGF as an active ingredient.