JPH08165243A - Anti-inflammatory agent - Google Patents

Abstract

PURPOSE: To easily obtain an anti-inflammatory agent virtually free from side effects, having sufficient anti-inflammatory action, containing, as active ingredi ent, a sulfated chitinoligosaccharide. CONSTITUTION: This anti-inflammatory agent contains, as active ingredient, a sulfated chitinoligosaccharide of the formula [(n) is 0-8] and/or a salt thereof. This compound, by acting as a ligand of selektin, hampers the adhesion between leukocyte and hemangioendothelial cells via selektin, a factor contributing to inflammation, thereby curing an inflammatory disease. This agent can be made into a preparation in the form of an agent for external use such as oral agent, ointment or spray, or injection, etc., its dose, in terms of the sulfated chitinoligosaccharide, being 0.1-1g, 10-100mg, 50-300mg, or 30-150mg, a day per adult, for oral, intravenous, intramuscular, or subcutaneous/intracutaneous administration, respectively, in one to several portions. For an agent for external use, the concentration of the sulfated chitinoligosaccharide is pref. set at 0.1-1wt.%.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-inflammatory agent for treating inflammatory diseases based on a new mechanism of action.

[0002]

2. Description of the Related Art Conventionally, glucocorticoids, which are adrenocortical hormones, or various non-steroidal anti-inflammatory agents have been used as therapeutic agents for inflammatory diseases such as pneumonia, hepatitis and nephritis. These have been developed based on antiprostaglandin actions such as phospholipase A2 inhibition, lipoxygenase inhibition, and cyclooxygenase inhibition, for the purpose of suppressing or blocking the inflammatory reaction and preventing complications due to inflammation. Had been used.

On the other hand, in recent years, the induction of inflammation is caused by a molecule that promotes adhesion between leukocytes and vascular endothelial cells, and it has become clear that a specific adhesion molecule is deeply involved in this reaction. That is, specific adhesion molecules are present on leukocytes and on activated vascular endothelial cells activated by interleukin 1 and TNF (tumor necrosis factor) released from inflammatory sites, and these adhesion molecules interact with each other. By the action
It has become clear that leukocytes, particularly neutrophils, adhere to vascular endothelial cells, and infiltration of leukocytes is initiated in the inflamed area.

These adhesion molecules, expressed on both leukocytes and vascular endothelial cells,
The integrin family is typical. In addition,
The selectin family causes adhesion during the early stages of leukocyte binding to vascular endothelial cells, and the integrin family is primarily responsible for the tighter binding that occurs subsequently.

Of these, the selectin family is
It has become clear that a sugar chain is used as its ligand.
The selectin family includes E-selectin (Bevilacqua, MP et al. (1987) Proc. N, which is present in vascular endothelial cells).
atl. Acad. Sci., USA, 84, 9238-9243), P-selectin expressed on activated platelets (Geng, JG et al. (199).
0) Nature, 343, 757-760), L-selectin present in leukocytes (Ley, K. et al. (1991) Blood, 77, 2553-255).
5) exist, but these sugar chain ligands are sialyl Le ^x or sialyl Le ^a (Lowe, JB et al. (1
991) cELL, 53, 475-484; Philipis, et. Al. (1990) S
cience, 250, 1130-1132; Magnani, JL81991) Glycob
iology, 1, 318-320).

[0006] Therefore, it is considered that the inflammation is suppressed by inhibiting the infiltration of leukocytes from vascular endothelial cells based on the knowledge about the mechanism of inflammation induction that has become clear in recent years. That is, in order to inhibit the selectin-mediated adhesion between leukocytes and vascular endothelial cells, attempts have been made to suppress inflammation by directly administering the selectin ligands sialyl Le ^x and sialyl Le ^a into the body. .

[0007]

However, although glucocorticoids have a strong anti-inflammatory effect, when they are given to a younger group, full moon-like face, dizziness, headache, vomiting, and more serious infections, digestion, etc. There is a problem that continuous administration is difficult because of side effects such as ductal bleeding, metabolic disorders, osteoporosis, and induction of thrombosis.

Further, there has been a problem that non-steroidal anti-inflammatory agents generally have weak efficacy.

Further, the method of directly administering sialyl Le ^x or sialyl Le ^a , which is a ligand for selectin, into the body has not been clarified, but its problem has not been approved yet. Sialyl Le ^x is enzymatically synthesized using sialyltransferase, but is difficult to synthesize. Also, sialyl Le
^x mainly binds to E-selectin and does not bind to L-selectin or P-selectin very strongly.

The present invention has been made in view of the above problems, and an object thereof is to provide an anti-inflammatory agent which has few side effects, has a sufficient anti-inflammatory action, and is easily manufactured.

[0011]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and investigated the rat
venom factor (hereinafter abbreviated as CVF), which is induced by intravenous administration of sulphate in a neutrophil-dependent and oxygen radical-mediated and P-selectin and / or L-selectin-dependent inflammation model. It has been found that a modified chitin oligosaccharide and / or a salt thereof is useful as an anti-inflammatory agent, since it significantly increases vascular permeability in the lung, hemorrhage associated with inflammation and myetoperoxidase (MPO) activity. The present invention has been completed.

That is, the anti-inflammatory agent of the present invention is characterized by containing a sulfated chitin oligosaccharide and / or a salt thereof as an active ingredient.

The present invention will be described in detail below with reference to preferred embodiments.

In the present invention, the sulfated chitin oligosaccharide is
It is preferable to use those obtained by chemically sulfiting chitin oligosaccharides. Chitin oligosaccharides include chitin, which is a natural polysaccharide obtained from the shells of crustaceans such as crab and shrimp.
It is an oligomer of N-acetylglucosamine (GlcNAc) which is chemically partially hydrolyzed with an acid, an alkali or the like or biochemically hydrolyzed by a microorganism or an enzyme. The degree of polymerization of chitin oligosaccharide is about 2 to 10,
It has higher solubility in water than chitin, which is a polysaccharide. Since chitin oligosaccharides have high-quality sweetness, they have been used as food materials for a long time, and therefore, they can be said to be highly safe for the human body.

For the sulfation of chitin oligosaccharides, a generally used method can be adopted, but it is preferable to use a sulfuric acid / trimethylamine complex as a sulfating agent. The ratio of chitin oligosaccharide and sulfating agent used is
It can be selected according to the desired sulfation ratio (or sulfur content) of the sulfated chitin oligosaccharide and the reaction conditions. For example, when the reaction is carried out at a temperature of 50 to 60 ° C. for several tens of hours to several days, it is preferable to use the sulfating agent in an amount twice the weight of the chitin oligosaccharide. When produced under such conditions, the sulfation rate is about 50-60% of the total hydroxyl groups of the chitin oligosaccharide.

Further, the purification of the sulfated chitin oligosaccharide can be carried out by the method which is usually employed for the purification of various modified polysaccharides. Specifically, the reaction mixture is concentrated under reduced pressure, desalted by dialysis against distilled water, treated with trifluoroacetic acid to remove trimethylamine, and then freeze-dried to obtain a white powder.

The general formula of the sulfated chitin oligosaccharide is shown in Chemical formula 1.

[0018]

Embedded image

The sulfated chitin oligosaccharide used in the present invention can also be produced by sulfating chitosan oligosaccharide by the same treatment.

In the present invention, salts of sulfated chitin oligosaccharides include sodium salt, potassium salt, calcium salt,
A divalent iron salt or the like can be used.

The anti-inflammatory agent of the present invention may contain a sulfated chitin oligosaccharide and / or a salt thereof as an anti-inflammatory substance, and the form thereof is an oral administration agent such as powder, tablets, capsules, ointment and the like. , Topical drugs such as sprays, intravenous, intraarterial,
It may be an intramuscular, subcutaneous or intradermal injection, etc., and it is preferable to add diluents, excipients and the like used for usual preparation of pharmaceuticals depending on the form. Incidentally, the diluent, the excipient, depending on the form of the drug to be manufactured, powders, solvents, suspensions and the like are used, these are,
Specifically, for example, lactose, cellulose, etc. are used as the powder, and water, ethyl alcohol, as the solvent.
Propylene glycol or the like is used, and as a suspending agent,
Polyoxyethylene sorbitol, sorbitan esters and the like are used.

The dose of the anti-inflammatory agent of the present invention per adult is 0.1 to 10 when orally administered as a sulfated chitin oligosaccharide.
1.0 g, 10-100 mg intravenously, 50-30 intramuscularly
In the case of 0 mg, subcutaneous or intradermal, 30 to 150 mg is preferably administered once or divided into several times a day. As an external medicine, for example, it can be applied locally on the outer skin as an ointment, or can be applied to the outer skin, throat, nasal cavity, etc. in the form of a spray, or can be used as an eye drop. In this case, the concentration of the sulfated chitin oligosaccharide in the external preparation varies depending on the dosage form and application site, but is preferably 0.1 to 1% by weight.

[0023]

The anti-inflammatory agent of the present invention contains a sulfated chitin oligosaccharide and / or a salt thereof as an active ingredient, and is neutrophil-dependent and neutrophil-dependent, which is induced by intravenous administration of CVF to rats. Oxygen radical mediated and P-selectin and / or L-selectin dependent inflammation model, when the sulfated chitin oligosaccharide and / or its salt is administered, increases vascular permeability of the lung and bleeding associated with inflammation. Also, since it significantly suppresses myeloperoxidase (MPO) activity, it can be said to have an excellent anti-inflammatory effect.

This is considered to be because the sulfated chitin oligosaccharide and / or its salt act as a ligand of selectin and inhibit the adhesion between leukocytes and vascular endothelial cells, which induces inflammation, via selectin.

That is, the substance screened by using the above-mentioned experimental animal model suppresses tissue infiltration due to the adhesion of leukocytes to vascular endothelial cells mediated by selectin, so that it is widely used in general inflammation such as adult respiratory distress syndrome (adult).
It is expected to be effective for the prevention or treatment of respiratory distress syndrome (ARDS) and the like, and therefore, the sulfated chitin oligosaccharide and / or its salt is expected to be effective for various inflammations.

Moreover, since chitin oligosaccharides have a high-quality sweetness, they have been conventionally used as food materials.
Therefore, it can be said that the safety of the human body is high.

Further, the sulfated chitin oligosaccharide and / or its salt can be mass-produced at low cost, which is economically advantageous.

[0028]

[Examples] In order to clarify the effect of the sulfated chitin oligosaccharide, which is an active ingredient of the anti-inflammatory agent of the present invention, examples will be described below, but the present invention is not limited thereto. Absent.

Example 1 Chitotetraose ([GlcNAc] ₄ ) which is a chitin oligosaccharide
200 mg and 400 mg of sulfuric acid / trimethylamine complex (manufactured by Aldrich) were dissolved in 6 ml of dimethylformamide, and the mixture was stirred in an oil bath at 50 to 60 ° C for 1 week.

The obtained reaction solution was concentrated under reduced pressure with a vacuum pump, the residue was dissolved in water, dialyzed against deionized water for 15 hours, and then freeze-dried. 2 ml of water was added to the obtained dried product.
Was dissolved in, and trifluoroacetic acid in an amount equivalent to 1.5 times the total number of hydroxyl groups of chitotetraose was added, and the mixture was stirred at room temperature for 1 hour. Subsequently, the reaction solution was dialyzed and then freeze-dried to obtain 190 mg of a sulfated chitin oligosaccharide ([GlcNAc] ₄ -SO ₃ H) having a sulfation ratio (sulfur content) of 7.2%.

Example 2 In Example 1, the chitin oligosaccharide chitotetraose was replaced with chitopentaose ([GlcNAc] ₅ ), and the same procedure as in Example 1 was followed except for the sulfation ratio (sulfur content ) Is 1
3.5% sulfated chitin oligosaccharide ([GlcNAc] ₅ -SO ₃ H) 200 m
got g.

Test Example (Evaluation Test for Anti-Inflammatory Action) (1) Evaluation Method of Pulmonary Inflammation and Preparation of Pulmonary Inflammation Model Animal [GlcNAc] ₄ -SO ₃ H and [GlcNAc] ₅ obtained in Examples 1 and 2 -SO
_In order to evaluate the anti-inflammatory effect of two types of sulfated chitin oligosaccharides of ₃ H, [GlcNAc] ₄ and [GlcNAc] ₅ before sulfation were used as Comparative Example 1 and Comparative Example 2, respectively. . These are the test substances.

Each test substance was added at a concentration of 1 mg / kg,
Dissolve in 1 mg bovine serum albumin / ml saline solution,
Ultrasonic treatment was carried out for 1 minute to prepare a test substance solution.

In addition, from Cobra (Naja naja) crude toxin, Ti
ll et al. (J. Clin. Invest., 69, 1126-1135 (1982))
The CVF was purified and isolated by.

Rat (male, Long Evans, 250-350g)
Then, the test substance solution was intravenously administered in an amount of 0.3 ml / animal, and then 20 U of CVF per 1 kg of body weight was intravenously administered together with ¹²⁵ I-bovine serum albumin (0.5 μCi) and ⁵¹ Cr-labeled rat erythrocytes. Rats develop lung inflammation by the administration of CVF.

Thirty minutes after the administration of CVF, the rats were anesthetized with 100 mg / kg body weight of ketamine hydrochloride and killed, and blood was collected from the dorsal aorta.

Then, at 30-minute intervals, the pulmonary vasculature was perfused through the right ventricle with 10 ml of pH 7.4 phosphate buffer. Then, the lung was removed, and the vascular system was perfused with 10 ml of sterile physiological saline, and then the amount of radioactivity remaining in the tissue was measured by a gamma scintillation counter.

The positive control rats were not treated with the test substance and were treated in the same manner as described above.
The test substance was not administered, and the same treatment as described above was carried out except that a phosphate buffer of pH 7.4 was used instead of CVF.

Pulmonary inflammation was measured by increased pulmonary vascular permeability and hemorrhage. The increase in vascular permeability was measured by determining the ratio of the amount of ¹²⁵ I-bovine serum albumin radioactivity present in lung tissue to the radioactivity present in 1 ml of venous blood obtained at the time of sacrifice. The bleeding was also measured by the radioactivity of ⁵¹ Cr-labeled rat erythrocytes present in the lung tissue with respect to the radioactivity present in 1 ml of vein blood obtained at the time of sacrifice.

The measured values of the test substance and the CVF-administered rat were used as the test substance value, the measured value of the positive control rat as the positive control value, and the measured value of the negative control rat as the negative control value, respectively.
The inhibitory activity of lung inflammation was calculated by the following formula 1.

[0041]

[Equation 1] Inhibitory activity against lung inflammation (%) = 100 × [(test substance value−negative control value) / (positive control value−negative control value)]

(2) Method for measuring tissue myeloperoxidase (hereinafter abbreviated as MPO) activity as an index of neutrophil infiltration The infiltration of neutrophils from blood vessels into tissues was examined as another index of inflammatory reaction. MP as a marker enzyme for neutrophils
O activity is present. Therefore, MPO activity as an index of neutrophil infiltration was measured.

A standard curve was prepared after adding a certain number of intraperitoneal neutrophils to glycogen-stimulated rats to the lungs of normal rats, homogenizing the tissues and then extracting (Warren et al.,
J. Clin. Invest., 84, 1873-1882, 1989).

The lung preparation obtained in (1) above was treated with 6 ml of a buffer solution (50 mM phosphate, pH 6.0) and "Polytron" (manufactured by Tokmar Co., set to dial 4) as a homogenizer. Use and homogenize for 40 seconds. Then
After centrifuging at 3000 xg at 4 ° C for 30 minutes, MPO activity in the supernatant is measured by measuring the change in absorbance at 460 nm caused by the consumption of H ₂ O _{2 in} the presence of O-dianidin. It was evaluated by

(3) Results Table 1 shows the results of measurement of vascular permeability in the lung, bleeding, and MPO activity.

[0046]

[Table 1] (In Table 1, <in parentheses indicates p-value, and NS means no significant difference.)

From the results shown in Table 1, the sulfated chitin oligosaccharides of Examples 1 and 2 were compared with the non-sulfated chitin oligosaccharides of Comparative Examples 1 and 2 in the lung inflammation caused by the administration of cobra venom (CVF). , Increased vascular permeability of the lung, hemorrhage associated with inflammation and myetoperoxidase (MPO) are significantly inhibited. Therefore, it can be seen that the sulfated chitin oligosaccharides of Examples 1 and 2 can be used as an anti-inflammatory agent.

(4) Acute toxicity test results The sulfated chitin oligosaccharides obtained in Examples 1 and 2 were tested for acute toxicity in rats.

As a result, the LD _{50 was} > 5 g / kg. Therefore, it can be said that the sulfated chitin oligosaccharide is highly safe.

[0050]

As described above, according to the anti-inflammatory agent of the present invention, the sulfated chitin oligosaccharide and / or its salt contained as an active ingredient acts as a selectin ligand to induce inflammation. The inflammatory diseases can be treated by inhibiting the selectin-mediated adhesion between leukocytes and vascular endothelial cells. Therefore, it can be said that the anti-inflammatory agent has a mechanism of action completely different from that of conventional anti-inflammatory agents. It should be noted that chitin oligosaccharide has been conventionally used as a food material, and is therefore highly safe for humans and has no side effects. In addition, sulfated chitin oligosaccharides and / or
Alternatively, the salt thereof can be easily mass-produced inexpensively and is economically advantageous.

Claims (1)

[Claims] 1. An anti-inflammatory agent comprising a sulfated chitin oligosaccharide and / or a salt thereof as an active ingredient.