JPH11269076A - Anti-fibrillating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an anti-fibrillating agent, namely, a medicament for inhibiting the progression of fibrillation. SOLUTION: This anti-fibrillating agent contains a keratan sulfate oligosaccharide or a pharmaceutically acceptable salt thereof as active ingredient. This agent is usable for the prevention or progression inhibition of the following diseases: pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary artery restenosis after percutanecus transluminal coronary vasodilative operation, interstitial myocarditis, interstitial bladder cystitis, glomerular nephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus naphropathy, interstitial nephritis, obstructed kidney due to ureteral obstruction, skin cicatrization after burn, and fibrosis associated with poisoning and the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical use of keratan sulfate oligosaccharide, and more particularly, to an antifibrotic agent containing keratan sulfate oligosaccharide as an active ingredient.

[0002]

2. Description of the Related Art In chronic inflammation such as interstitial pneumonia and cirrhosis, the production and secretion of active oxygen and proliferative cytokines from inflammatory cells are enhanced by some kind of stimulus, thereby increasing the proliferation of fibroblasts and extracellular matrix. Overproduction of protein occurs, leading to irreversible fibrosis. Chronic inflammation is treated with anti-inflammatory agents such as steroids, but there is no drug that suppresses the progression of fibrosis, and an effective treatment method is required.

[0003] In addition, pulmonary fibrosis may be caused as a side effect by administration of a drug such as an antitumor agent, and a drug that effectively prevents or suppresses such side effect (fibrosis) is desired.

[0004]

As described above, a drug which prevents or suppresses fibrosis is expected. Therefore, an object of the present invention is to provide a pharmaceutical product that prevents fibrosis and suppresses the progress of fibrosis, that is, an anti-fibrotic agent.

[0005]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that they have been used as active ingredients of anti-inflammatory agents, anti-allergic agents, immunomodulators, differentiation inducers, and apoptosis inducers. The inventors have found that keratan sulfate oligosaccharides, which are known to be useful (WO 96/16973), have an antifibrotic effect, and have completed the present invention.

[0006] That is, the present invention provides an antifibrotic agent (hereinafter, also referred to as the antifibrotic agent of the present invention) containing a keratan sulfate oligosaccharide or a pharmaceutically acceptable salt thereof as an active ingredient. The anti-fibrotic agent of the present invention is preferably pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, percutaneous transluminal coronary angioplasty;
PTCA) after coronary restenosis, interstitial myocarditis, interstitial cystitis, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive renal sclerosis, HIV nephropathy, IgA nephropathy, lupus nephropathy, It is used as an agent for preventing or inhibiting the progression of fibrosis associated with one or more diseases selected from the group consisting of interstitial nephritis, obstructed kidney due to ureteral obstruction, skin burn after burn and intoxication.

[0007] The antifibrotic agent of the present invention is more preferably used for fibrosis caused by drug administration. The drug is preferably an antitumor agent, an antibiotic, an antibacterial, an antiarrhythmic, an anti-inflammatory, an antirheumatic, interferon or Shosaikoto.

[0008] The antifibrotic agent of the present invention is even more preferably a prophylactic or progress inhibitor of pulmonary fibrosis associated with administration of an antitumor agent. The antitumor agent is preferably an antitumor antibiotic.

[0009]

Embodiments of the present invention will be described below. The antifibrotic agent of the present invention contains keratan sulfate oligosaccharide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0010] The keratan sulfate oligosaccharide used in the antifibrotic agent of the present invention is not particularly limited as long as it is an oligosaccharide derived from keratan sulfate.
It is preferably a decomposition product of keratan sulfate that can be obtained by decomposition with an N-acetylglucosaminidase-type keratan sulfate degrading enzyme.

The keratan sulfate oligosaccharide may contain a sialic acid residue and / or a fucose residue. Usually, sialic acid residues bind to galactose residues at α2,3 or α2,6 glycosidic bonds, and fucose residues bind to galactose residues at α1,3 glycosidic bonds.

[0012] Preferably, the keratan sulfate oligosaccharide is a di- to pentasaccharide oligosaccharide having a sulfated N-acetylglucosamine residue at a reducing end, wherein at least two hydroxyl groups in one molecule are sulfated. Keratan oligosaccharides,
In particular, at least Gal (6S) -GlcNAc (6S) (Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, and 6S represents a 6-O-sulfate group). The above keratan sulfate oligosaccharide containing a disaccharide as a constitutional unit.

More preferably, the keratan sulfate oligosaccharide comprises a tetrasulfated N-acetyllactosamine tetrasaccharide represented by the formula (I) and a disulfated N-acetyllactosaminedisaccharide represented by the formula (II): Chosen from sugar. Gal (6S) β1-4GlcNAc (6S) β1-3Gal (6S) β1-4GlcNAc (6S) ・ ・ ・ ・ ・ ・ (I) Gal (6S) β1-4GlcNAc (6S) ・ ・ ・ ・ ・ ・ ・ ・ ・・ ・ ・ ・ ・ ・ ・ ・ ・ ・ (II) (In the formula, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, and 6S represents a 6-O-sulfate group.)

The keratan sulfate oligosaccharide may consist of a single species or may be a mixture. The keratan sulfate oligosaccharide used in the present invention may be prepared by adding an endo-β-N-acetylglucosaminidase-type keratan sulfate-degrading enzyme, for example, keratanase (II) derived from a bacterium belonging to the buffer solution of keratan sulfate, preferably highly sulfated keratan sulfate. (Japanese Unexamined Patent Application Publication No.
No. 57182) or a keratan sulfate-degrading enzyme derived from Bacillus circulans KsT202 (WO 96/16166), followed by degradation, followed by fractionation of the resulting degradation product. . The oligosaccharide obtained can be separated and purified by a usual separation and purification method, for example, concentration by ethanol precipitation, gel filtration and separation and purification by anion exchange chromatography.

The keratan sulfate used as the raw material of the keratan sulfate oligosaccharide used in the present invention is mainly galactose or galactose-6-sulfate and N-acetylglucosamine-
It is composed of 6-sulfuric acid and disaccharide repeating structures, and the sulfuric acid content varies depending on animal species and organs. Usually, cartilage fish such as sharks, whales, cartilage of mammals such as cattle, bone and cornea, etc. Manufactured from raw materials.

The keratan sulfate used as a raw material is not particularly limited as long as it is generally available, but is not particularly limited. Highly sulfated keratan sulfate in which a galactose residue as a constituent sugar is sulfated (per constituent disaccharide) It is preferable to use a highly sulfated keratan sulfate containing 1.5 to 2 molecules of a sulfate group as keratan polysulfate). The position of the sulfate group of the galactose residue is preferably at position 6.
Such highly sulfated keratan sulfate can be obtained, for example, from proteoglycans of cartilaginous fish such as sharks. Also, commercially available products can be used.

The keratan sulfate oligosaccharide used in the present invention includes those having an ionized state and those having a structure to which a proton is added. Also, pharmaceutically acceptable salts include, for example, sodium salts, potassium salts, alkali metal salts such as lithium salts, alkaline earth metal salts such as calcium salts, salts with inorganic bases such as ammonium salts, or diethanolamine Among salts with organic bases such as salts, cyclohexylamine salts and amino acid salts, pharmaceutically acceptable salts can be used, but are not limited thereto.

The keratan sulfate oligosaccharide obtained as described above can be blended as an active ingredient in a dosage form described later to obtain a desired antifibrotic agent.

The diseases to which the present invention is applied are diseases accompanied by fibrosis in mammals including humans, and the antifibrotic agents of the present invention are used for preventing or inhibiting the progression of fibrosis associated with these diseases. Can be administered to a subject to be treated. This prophylactic or progress inhibitor can be administered for the purpose of preventing fibrosis associated with the disease, preventing (maintaining) progress, and the like.

The above-mentioned diseases include, specifically, pulmonary fibrosis,
Cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation (PTCA), interstitial myocarditis, interstitial cystitis, glomerulonephritis, vasculitis, diabetic nephropathy , Hypertensive renal sclerosis, HIV nephropathy, IgA nephropathy, lupus nephropathy,
One or more diseases selected from the group consisting of interstitial nephritis, obstructed kidney due to ureteral obstruction, fibrosis associated with burned skin and poisoning (eg, paraquat poisoning) can be mentioned. Among these diseases, pulmonary fibrosis, fibrosis associated with glomerulonephritis and vasculitis are particularly suitable for application of the antifibrotic agent of the present invention.

Furthermore, the antifibrotic agent of the present invention is also effective against fibrosis caused by drug administration. Examples of drugs that may cause fibrosis include antitumor agents, antibiotics, antibacterial agents, antiarrhythmic agents, anti-inflammatory agents, antirheumatic agents, interferons, Sho-saiko-to, and the like. It can be more preferably applied to fibrosis caused by administration of these drugs.

In particular, the antifibrotic agent of the present invention is preferably a prophylactic or progress inhibitor of fibrosis associated with administration of an antitumor agent, and a prophylactic or inhibitor of progression of pulmonary fibrosis associated with administration of an antitumor agent. More preferably, there is. Examples of the antitumor agent include bleomycin, methotrexate, procarbazine, melphalan, busulfan, mitomycin C, fluorouracil, vinca alkaloid, azathioprine, cytosine arabinoside, cyclophosphamide, chlorambucil, nitrosourea compound, etoposide, dinostatin and the like. . Among antitumor agents, antitumor antibiotics, especially pulmonary fibrosis caused by administration of bleomycin, especially interstitial lung disease (among others, interstitial pneumonia)
This is a particularly preferable disease to which the antifibrotic agent of the present invention is applied.

However, the disease names listed here are examples, and the application range of the keratan sulfate oligosaccharide preparation of the present invention should not be limited to these exemplified diseases.

The antifibrotic agent of the present invention may contain, as an active ingredient, a substance having an antifibrotic action other than the keratan sulfate oligosaccharide, together with the keratan sulfate oligosaccharide.

The “substance having an anti-fibrotic action other than keratan sulfate oligosaccharide” as used herein means that serious side effects are caused by combination or administration of the substance with keratan sulfate oligosaccharide, Is not particularly limited as long as it does not inhibit the intrinsic antifibrotic action of the other substance.

It is also possible to combine the anti-fibrotic agent of the present invention with an anti-inflammatory agent, or to administer the anti-fibrotic agent of the present invention in combination with an anti-inflammatory agent. Applicable anti-inflammatory agents include, for example, steroids such as prednisolone, and anti-inflammatory enzymes such as superoxide dismutase, but are not limited thereto.

The antifibrotic agent of the present invention is effective against fibrosis caused by drug administration,
The anti-fibrotic agent of the present invention may be combined with a drug that may cause fibrosis (for example, the aforementioned drugs),
It is also possible to administer the anti-fibrotic agent of the present invention in combination with an agent that may cause fibrosis (for example, the aforementioned agents). Of course, before administering drugs that can cause fibrosis (for example, the drugs mentioned above),
The antifibrotic agent of the present invention can be administered in advance.

In the present invention, any dosage form can be appropriately selected as the dosage form of the antifibrotic agent of the present invention, depending on the nature, progress, administration method, etc. of the target disease.

That is, the antifibrotic agent of the present invention can be administered by injection (intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, etc.), oral, transdermal, inhalation, etc., depending on the administration method. Can be formulated as appropriate.

The dosage form that can be selected is not particularly limited. For example, injection preparations (solutions, suspensions, emulsions, solid preparations for dissolution, etc.), tablets, capsules, granules, powders, liquids, liposomes It can be widely selected from agents, ointments, gels, external powders, sprays, inhalation powders and the like. In addition, in preparing these formulations, conventional excipients, stabilizers, binders, lubricants, emulsifiers, osmotic pressure regulators, pH regulators, other colorants,
Components usually used in medicines such as disintegrants can be used.

The amount of the keratan sulfate oligosaccharide which is an active ingredient in the antifibrotic agent of the present invention and the dosage of the antifibrotic agent of the present invention are determined by the method of administration of the preparation, the dosage form, the purpose of use and the specific patient It is a matter to be determined individually according to symptoms, patient weight, and the like, and is not particularly limited. However, the clinical dose of keratan sulfate oligosaccharide is generally about 300 to 300 per day.
1500 mg / human / day. In addition, the administration interval of the above preparation can be about once a day,
It can also be administered in divided doses.

[0032]

The present invention will be described below in more detail with reference to Test Examples and Formulation Examples. However, these should not limit the technical scope of the present invention.

[0033]

[Test Example 1] Examination of effect on bleomycin-induced mouse pulmonary fibrosis (interstitial pneumonia) model In pharmacological test example 1 of the present drug, keratan sulfate tetrasaccharide (hereinafter, referred to as formula (I)) as a keratan sulfate oligosaccharide is used. L4
A solution of a sodium salt of keratan sulfate disaccharide (hereinafter, referred to as L4) represented by the formula (II) was used. The properties of this L4L4 solution were a colorless and transparent aqueous solution, the pH was about 6 to 7, and the osmotic pressure ratio to distilled water was about 1. The L4 solution was a colorless and transparent aqueous solution, the pH was about 6 to 7, and the osmotic pressure ratio to physiological saline was about 1.

<Experimental Method> A model was prepared by injecting bleomycin at a dose of 150 mg / kg intravenously once into 6-week-old male ICR mice. L4 L4 or a solution of L4 was intramuscularly administered at doses of 1, 5, and 25 mg / kg for 13 consecutive days from the day after bleomycin administration (12 to 15 mice per group). Two weeks after the administration of bleomycin, the right lung was subjected to bronchoalveolar lavage three times with physiological saline to obtain a bronchoalveolar lavage fluid (BALF). The evaluation items were the total number of cells and total protein (protein amount) in the bronchoalveolar lavage fluid, the amount of hydroxyproline in the right lung (corresponding to the amount of collagen), the wet weight of the left lung and the lung fibrosis score (pathological findings). did. The amount of protein is BCA Protein
Using an Assay kit (manufactured by PIERCE), the amount of hydroxyproline was determined by the method of Woessner et al. (Woessner, LF, 1961, Arch.
Biochem. Biophys., 93: 440-447).
The lung fibrosis score was determined by the method of Yumoto et al. (Hisao Yumoto, Katsutoshi Takahashi, Akira Matsuda, Hamao Umezawa, 1983, Cancer and Chemotherapy, 10: 255
0-2557).

<Results> (1) Effect of L4L4 A significant suppression effect on lung wet weight was observed at 25 mg / kg administration (FIG. 1). The total number of cells in the bronchoalveolar lavage fluid was significantly suppressed by administration of 5 and 25 mg / kg (FIG. 2). Protein content in bronchoalveolar lavage fluid is 25 mg / k
g showed a low value (Fig. 3). The hydroxyproline amount and the pulmonary fibrosis score were significantly inhibited by the administration of 25 mg / kg (FIGS. 4 and 5). From this, L4L
No. 4 was shown to have an antifibrotic effect.

(2) Effect of L4 Lung wet weight showed a low value at 25 mg / kg administration (FIG. 6). The total number of cells in bronchoalveolar lavage fluid was 1, 5 and 2
A significant inhibitory effect was observed at 5 mg / kg administration (FIG. 7). The amount of protein in the bronchoalveolar lavage fluid showed a low value at 25 mg / kg administration (FIG. 8). The hydroxyproline amount and pulmonary fibrosis score showed low values at 25 mg / kg administration (FIGS. 9 and 10). This indicated that L4 has an antifibrotic effect.

In FIGS. 1 to 9, * indicates that the value was significant at the level of p <0.05 relative to the control in Dunnett's multiple comparison test, and ** indicates that the level was significant at the level of p <0.01. Show.

[0038]

[Test Example 2] Examination of effects on glomerulonephritis and vasculitis in MRL mice In pharmacological test example 2 of the present drug, keratan sulfate disaccharide (hereinafter referred to as L4) represented by formula (II) as keratan sulfate oligosaccharide
) Was used. This L4 solution is a colorless and transparent aqueous solution and has a pH of about 6 to
7. The osmotic pressure ratio to physiological saline was about 1.

<Experimental method> A 10-week-old male MRL (lpr / l
pr) mouse (Izui S., et al., Mechanism of genetic con
trol of murine systemic lupus erythematosus., In S
ystemic Lupus Erythematosus, pp.3-12, Ed.Peter A.
Miescher, Springer-Verlag Berlin, 1995), and male C57BL / 6 mice were used as a control. The group composition of the mice is shown below. The grouping was performed on the day before the administration of the test substance in principle. Blood was collected from the orbital venous plexus during the pre-breeding period, and blood creatinine (CR
Using E) and blood urea nitrogen (BUN) as indexes, the average value and standard deviation of each group were assigned so that there was no difference between the groups. The standard deviation was set to be as small as possible.

[0040]

[Table 1] 群 Group No. Test substance Mouse L4 dose L4 dose Concentration L4 dose Number of mice (mg / kg) (mg / ml) (ml / kg) Male ─────────────────────────── １ 1 PBS MRL--2.5 7 2 L4 MRL 2.5 1 2.5 73 3 L4 MRL 5 2 2.5 74 4 L4 MRL 10 4 2.5 75 5 No treatment C57BL / 6----5- ───────────────────────────────── PBS indicates phosphate buffered saline.

A PBS solution of the test substance was administered intramuscularly for 35 consecutive days between 11 and 16 weeks of age. After the end of the administration period, blood was collected from the posterior aorta. The collected blood was applied to an automatic clinical chemistry analyzer (COBAS MIRA S; sold by Roche Japan), and CR
E was measured.

In the same group composition as above (12 mice in each group), administration was carried out in the same manner as described above. After the administration period, the kidney was excised, and histopathological examination was performed on glomeruli and renal arteries. Was.

The glomeruli were: (1) Only a slight increase in snare thickening and cell proliferation was observed, and the microscopic change group (Mini
mal change (MC) and the glomerular image determined to be classified as type (MC), and (2) the Bowman's capsule and vascular cavity almost completely disappear, and the glomerulus is buried in cellular components and deposits,
Glomerular image whose function is presumed to be abolished (WHO
Classification of diffuse glomerulonephritis (Diffuse Glomerulonephri
tis) Proliferative Glomeruloneph
ritis); DP]. M
The number of glomeruli showing C and DP was counted, and the appearance rate (%) was calculated by dividing each count by the total number of glomeruli.

FIG. 11 shows a normal glomerular image (upper left), a glomerular image of MC (upper right), and a glomerular image of DP (lower left) (a photograph showing the form of an organism. Photo magnification: × 500. The bar at the bottom right of the photograph indicates 10 μm).

As shown in FIG. 11, glomerular lesions gradually shift from MC to DP in which the Bowman's capsule and vascular lumen disappeared due to the proliferation of monocytes and mesangial cells, the deposition of fibrin and immune complexes, and the like. It is thought that it goes. That is, MC is closer to normal than DP, and M
It turns out that fibrosis accompanying glomerulonephritis is more advanced in DP than in C.

The renal arteries are classified into the following grades 1 to 4
And the number of occurrences of each was counted. The histological image of each of Grades 1 to 4 is shown in FIG. 12 (photograph showing the morphology of the organism. Photo magnification: × 204. The bar at the lower right of each photograph indicates 50 μm).

Grade 1: almost normal renal artery image (upper left) Grade 2: inflammatory cell infiltration around blood vessels (upper right) Grade 3: inflammatory cell infiltration around blood vessels and disruption of adventitia-media ( Vacuolar degeneration and fibrin deposition) (lower left) Grade 4: Inflammatory cell infiltration around blood vessels, adventitia
Medial rupture (vacuolar degeneration, fibrin deposition and smooth muscle cell proliferation) and inflammatory cell infiltration in the intima-media (lower right)

Grade 1 is most normal and grade 4
Indicates that fibrosis associated with vasculitis is most advanced.

<Results> The blood creatinine (CRE) concentration was
A significant decrease was observed in all L4 administration groups (group numbers 2 to 4) (FIG. 13). This indicates that L4 administration tends to restore renal function, suggesting that fibrosis is suppressed.

The appearance rate of MC in the glomerulus
In the four administration groups (Group Nos. 2 to 4), the number was increased as compared with the PBS administration group (Group No. 1) (FIG. 14). In addition, the appearance rate of DP in glomeruli was determined for all L4 administration groups (group numbers 2 to
4) also decreased as compared with the PBS administration group (Group No. 1), and a significant decrease was observed particularly in the L4 10 mg / kg administration group (Group No. 4) (FIG. 15). From these, L
No. 4 was shown to have an antifibrotic effect.

The histological images in the renal artery were observed, particularly in the L4 5 mg / ml administration group (Group No. 3) and the 10 mg / ml administration group.
(Group No. 4) markedly reduced grade 4;
A more normal tissue image was observed (FIG. 16). This also indicates that L4 has an antifibrotic effect.

In FIG. 13, * indicates that the significance was at the level of p <0.05 in the Williams multiple comparison test, and ** indicates
Significance is shown at the level of p <0.01. FIG.
In the figure, * indicates that the significance was at the level of p <0.05 in Bonferroni's multiple comparison test.

[0053]

[Test Example 3] Investigation of effect on diabetic nephropathy in spontaneously diabetic mice (db / db) In pharmacological test example 3 of the present invention, the sodium salt of L4 was used as a keratan sulfate oligosaccharide. The sodium salt of L4 was dissolved in distilled water for injection to a concentration of 6%, and then dissolved in physiological saline to a predetermined concentration before use. Captopril (ACE inhibitor; Sankyo Co., Ltd.) was used as a positive control. Captopril was dissolved in distilled water for injection to a predetermined concentration and used.

<Experimental Method> C57BL (db / db) mice (34-week-old female mice) which develop diabetes and whose urinary albumin excreted in 24 hours is 2700-6000 μg or more are used for the experiments. Was. In addition, C57 which is healthy as a normal group
BL mice (10 week old female mice) were used. The group composition of the mice is shown below.

[0055]

[Table 2] Group name Test substance Dosage Administration method Number of mice (Substance / fluid / body weight) Female ──────────────────────────────────── Control group Physiological saline Liquid 2ml / kg back subcutaneous 10 L4 administration group L4 5mg / 2ml / kg back subcutaneous 10 L4 administration group L4 10mg / 2ml / kg back subcutaneous 10 L4 administration group L4 20mg / 2ml / kg back subcutaneous 10 positive control group captopril 3mg / 10ml / kg Oral 10 Normal group---10 ────────────────────────────────────

Each test substance was administered subcutaneously in the back six times a week for 8 weeks in a blind. To the positive control group, captopril was similarly orally administered 6 times a week for 8 weeks.

At the time of grouping and every two weeks after the start of drug administration, urine is stored in a metabolic cage for 24 hours, and after measuring the urine volume, the albumin concentration in urine is measured by microalbumin HA Test Wako (Wako Pure Chemical Industries, Ltd.). The amount of urinary albumin excreted during 24 hours was calculated from the urinary albumin concentration and urine volume. The results are shown in FIG.

Eight weeks after the administration of the drug, the urinary creatinine level was measured by an automatic clinical chemistry analyzer (COBAS MIRAS; sold by Nippon Roche), and the ratio between the urinary albumin level and the urine creatinine level was calculated. The results are shown in FIG.

In FIGS. 17 and 18, * indicates that the value was significant at the level of p <0.05 with respect to the control in Dunnett's multiple comparison test, and ** indicates that the value was significant at the level of p <0.01. Show.

<Results> A clear increase in urinary albumin was observed in the control group, and the amount of albumin excreted with time was increased in the control group (FIG. 17).

In the L4 administration group, the value began to decrease from 2 weeks after administration, and thereafter, in all groups, the value was lower than that in the control group. L4
A significant inhibitory effect was observed after 4 weeks and 8 weeks in the 20 mg / kg group.

In the positive control group, the value began to decrease from 4 weeks after administration, and thereafter showed a low value, although not significant (FIG. 1).
7).

The ratio of urinary albumin / urine creatinine at 8 weeks after administration was lower in all groups in the L4 administration group than in the control group, and significant inhibition was observed in the L4 20 mg / kg group. Was. A significant inhibitory effect was also observed in the positive control group as compared to the control group (FIG. 18).

In the case of diabetic nephropathy, it has been found that urinary albumin generally gradually increases in accordance with the disease state. Similarly, in the present model, a time-dependent increase in urinary albumin was observed, indicating that the progress of the disease state was gradually progressing. L4 rapidly suppressed and improved urinary excretion of albumin. The suppression effect of L4
The expression earlier than in the positive control administration group suggested that L4 might be more useful than the ACE inhibitor.

[0065]

[Formulation Examples] (1) Injection L4 was dissolved at a final concentration of 10 mg / ml in a physiological saline solution adjusted to pH 6.8 to 7.6 with phosphate according to a conventional method, and the solution was subjected to aseptic filtration. Each 1 ml was dispensed into a vial and sealed to produce an injection.

(2) Ointment L4L4 was dissolved at a final concentration of 10 mg / ml in a hydrophilic ointment of the Japanese Pharmacopoeia to prepare an ointment.

(3) Spraying agent L4 was dissolved at a final concentration of 10 mg / ml in a physiological saline solution adjusted to pH 6.8 to 7.6 with a phosphate according to a conventional method. 1 ml was dispensed into vials and sealed to produce a spray.

[0068]

Industrial Applicability According to the present invention, there is provided a pharmaceutical agent for preventing or suppressing the progress of fibrosis, that is, an anti-fibrotic agent. Since the antifibrotic agent of the present invention is particularly effective for fibrosis caused by drug administration, it can also be used as an agent for preventing / suppressing side effects due to drug administration.

[Brief description of the drawings]

FIG. 1 shows the wet lung weight of mice that received keratan sulfate tetrasaccharide (L4L4) at various doses.

FIG. 2 shows the total number of cells in bronchoalveolar lavage fluid (BALF) of mice that received keratan sulfate tetrasaccharide at various doses.

FIG. 3 shows the amount of protein in bronchoalveolar lavage fluid of mice to which keratan sulfate tetrasaccharide was administered at various doses.

FIG. 4 shows the amount of hydroxyproline in the lungs of mice to which keratan sulfate tetrasaccharide was administered at various doses.

FIG. 5 shows pulmonary fibrosis scores of the lungs of mice administered with keratan sulfate tetrasaccharide at various doses.

FIG. 6 shows the lung wet weight of the lungs of mice administered keratan sulfate disaccharide (L4) at various doses.

FIG. 7 shows the total number of cells in bronchoalveolar lavage fluid of mice that received keratan sulfate disaccharide at various doses.

FIG. 8 shows the amount of protein in bronchoalveolar lavage fluid of mice to which keratan sulfate disaccharide was administered at various doses.

FIG. 9 shows the amount of protein in bronchoalveolar lavage fluid of mice to which keratan sulfate disaccharide was administered at various doses.

FIG. 10 shows pulmonary fibrosis scores of the lungs of mice administered with keratan sulfate disaccharide at various doses.

FIG. 11 shows normal tissue images (N) and minute changes (M
inimal change) Histology (MC) and Diffuse proliferation (Diffuse pro
liferative) shows a histological image (DP) (photograph showing the form of an organism).

FIG. 12 shows histological images of grades 1 to 4 in the renal artery (photographs showing morphology of an organism).

FIG. 13 shows serum creatinine concentrations in mice administered keratan sulfate disaccharide at various doses.

FIG. 14 shows the incidence of MC in glomeruli of mice to which keratan sulfate disaccharide was administered at various doses.

FIG. 15 shows the appearance rate of DP in glomeruli of mice to which keratan sulfate disaccharide was administered at various doses.

FIG. 16 shows changes in histological grade in renal arteries of mice to which keratan sulfate disaccharide was administered at various doses.

FIG. 17 shows urinary albumin levels of mice to which keratan sulfate disaccharide was administered at various doses.

FIG. 18 shows the ratio of urinary albumin to urinary creatinine in mice administered with keratan sulfate disaccharide at various doses.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/00 613 A61K 31/00 613 617 617 617G // C07H 11/00 C07H 11/00

Claims (7)

[Claims] 1. An antifibrotic agent comprising keratan sulfate oligosaccharide or a pharmaceutically acceptable salt thereof as an active ingredient. 2. The anti-fibrotic agent according to claim 1, which is a prophylactic or progress inhibitor of fibrosis. 3. Pulmonary fibrosis, cirrhosis, arteriosclerosis, scleroderma, coronary restenosis after percutaneous transluminal coronary vasodilation, interstitial myocarditis, interstitial cystitis, glomerulonephritis, vascular Inflammation, diabetic nephropathy, hypertensive renal sclerosis, HIV nephropathy, IgA nephropathy,
Lupus nephropathy, interstitial nephritis, obstructed kidney due to ureteral obstruction, a preventive or progress inhibitor of fibrosis associated with one or more diseases selected from the group consisting of skin erosion and poisoning after burn, The anti-fibrotic agent according to claim 2. 4. The antifibrotic agent according to claim 1, wherein the fibrosis is fibrosis caused by administration of a drug. 5. The antifibrotic agent according to claim 4, wherein the drug is an antitumor agent, an antibiotic, an antibacterial agent, an antiarrhythmic agent, an anti-inflammatory agent, an antirheumatic agent, interferon or Shosaikoto. 6. The anti-fibrotic agent according to claim 4, which is a prophylactic agent or a progress-inhibiting agent for pulmonary fibrosis accompanying administration of the anti-tumor agent. 7. The antifibrotic agent according to claim 6, wherein the antitumor agent is an antitumor antibiotic.