JP2001019704A - Inhibitor of neutrophile activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an inhibitor of neutrophile activity, capable of effectively inhibiting the productivity of an active oxygen by the neutrophile without perishing normal cells, and capable of curing various kinds of diseases such as inflammation caused by the excess release of the active oxygen by including chitosan oxide compound derived from a specific chitosan. SOLUTION: This inhibitor contains a chitosan oxide compound comprising (A) a structural unit of formula I and (B) a structural unit of formula II, regulated so that the molar proportion of the unit A based on the units A+B may be >70 mol%. The conversion of CH2OH groups in the formulas of the units A and B to COOX groups (X is H, Na or K) is 1-100 mol%, and the rate of acetylation of NH2 group in the formula of the unit A is 40-95%. The weight average molecular weight of the chitosan oxide is >=5,000, and the solubility of the chitosan oxide in water at pH 7 at 25 deg.C is >=0.1 g/100 g. The chitosan oxide can be applied to the whole preparation because of the water-solubility.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a neutrophil activity inhibitor, and more particularly to a neutrophil activity inhibitor using a water-soluble oxidized chitosan compound.

[0002]

2. Description of the Related Art Living organisms have an infection defense mechanism to protect themselves from pathogenic microorganisms. Its function is roughly divided into the innate immune system and the acquired immune system. When pathogenic microorganisms invade, the innate immune system works first, but phagocytes, that is, neutrophils, are members of the innate immune system, and pathogens, mainly It plays a role in eliminating bacteria from living organisms. Adhesion and chemotaxis are the steps in which neutrophils flowing in the blood vessels exit the blood vessels and reach the pathogen, and phagocytosis and sterilization are the steps in eliminating the pathogen.

[0003] Neutrophils eliminate pathogens through these steps, and essentially function as host defenses and exert useful effects.

When neutrophils sterilize, they release several types of active oxygen toward pathogenic microorganisms, which exert an extremely powerful bactericidal action. However, if active oxygen is released to surroundings other than pathogenic microorganisms, tissue damage will occur. Active oxygen can be removed by an antioxidant enzyme such as superoxide dismutase (hereinafter abbreviated as SOD), and it can be removed by SOD in excess of germicidal activity, if it is in a small amount. Causes cancer and aging. From this, promotion of neutrophil activation may lead to side effects in some cases.

[0005] Therefore, excess active oxygen of neutrophils is removed.
Numerous suppressive substances have been studied and used. For example, in vivo substances include vitamin C (ascorbic acid), vitamin E, ubiquinol, and carotenoids. However, vitamin C has a low molecular weight and is water-soluble, so it is excreted from the body immediately and its effect cannot be sustained. Vitamin C may cause nausea and diarrhea if overdose. Vitamin E, ubiquinol, and carotenoids are fat-soluble and can only exist in cell parts and are hardly absorbed. It is said that excessive intake may cause side effects such as rash, anorexia and diarrhea.

[0006] Examples of in vitro substances used as anti-inflammatory agents include sulfa drugs, tetracycline hydrochloride, colchicine and the like. However, the sulfa drug produces resistant bacteria and may cause liver damage and kidney damage. There is concern that tetracycline hydrochloride may cause nausea, anorexia, diarrhea, stomatitis, liver and blood disorders. Colchicine may also cause abdominal pain, rash, fever, blood disorders, and peripheral neuritis.

[0007]

Accordingly, the present inventors have found that chitin, which is present abundantly in crustaceans such as crabs and shrimps, and skeletal substances of insects such as beetles and crickets, and chitin that is present in living organisms such as fungi and cell walls. The present inventors have discovered that an oxidized chitosan compound produced as a raw material effectively acts as a neutrophil activity inhibitor, and have completed the present invention based on such findings.

[0008]

That is, the present invention provides a compound of the general formula

[0009]

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And the general formula

[0011]

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An oxidized chitosan compound derived from chitosan represented by the formula: wherein the oxidation rate of a CH ₂ OH group to a COOX group (X represents H, Na or K) is 1
-100 mol%, wherein the acetylation ratio of NH ₂ groups to NHCOCH ₃ groups is 40 to 95 mol%, the weight average molecular weight is 5,000 or more, pH 7, temperature 25 ° C.
The present invention relates to a neutrophil activity inhibitor comprising an oxidized chitosan compound having a solubility in water of 0.1 g / 100 g or more.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The oxidized chitosan compound used in the present invention can be produced by various methods. A detailed production method is described in Japanese Patent Application No. 11-60162. It is as follows.

First, chitosan is converted to acetate. As a method for converting to acetate, acetic acid is added in an amount of 1.5 to 4 moles, more preferably 2 to 3 moles, based on the number of moles of amino groups of chitosan used, and is added at about 15 to 40 ° C, more preferably 20 to 2
Stir at 5 ° C for 1 to 3 hours (provided that the deacetylation rate is 1
In the case of 00 g of chitosan Xg, the number of moles of amino groups of chitosan is calculated by Xg / 161 g (molecular weight of constituent sugar of chitosan). The concentration of acetic acid used at this time is 0.1.
The concentration is 5 to 10 molar (the molar concentration is mol / l), more preferably 1 to 5 molar.

Next, this is dried at 50 to 100 ° C. to obtain chitosan acetate. Hydrochloric acid can be used in place of acetic acid. The conditions for use are the same as those for acetic acid, but acetic acid is preferred from the viewpoint of ease of oxidation, which will be described in detail later. The reason for drying the acetate is to minimize the water content. Next, concentrated acetic acid, that is, 8
0.0-99.8% by weight, more preferably 95.0-9
Add 9.8% by weight of acetic acid and stir well. The reason for using concentrated acetic acid here is that when chitosan acetate is suspended in concentrated acetic acid, oxidation of the carbon at the 6-position described later is easily performed.

[0016] That is, although the oxidation of CH ₂ OH groups carbons Tsugiki 6-position is oxidized through the oxidation reaction step of the CH ₂ OH → CHO → COOH, CH ₂ OH → reaction CHO the reaction the smaller water This is because the speed is high. Therefore, the chitosan acetate may be concentrated using an evaporator or the like without drying as described above. In short, it is desirable to reduce the water content as much as possible before performing the oxidation reaction step in consideration of economy.

Next, perchloric acid is added to the suspension to convert the chitosan acetate into chitosan perchlorate. At this time, the solution generally becomes jelly-like. Hydrochloric acid instead of perchloric acid,
Sulfuric acid may be used, but if the concentration, the amount of addition, and the degree of reaction are not sufficiently controlled, the carbon at the second and third positions may be oxidized in the oxidation reaction described below, which is not desirable. The amount of perchloric acid used is 1 to 3 moles, more preferably 1.5 to 2 moles, per mole of amino groups of chitosan acetate.
Double mole is better. The concentration of perchloric acid used is preferably 4 to 8 molar, more preferably 5 to 7 molar. More preferably, the concentration is around 6 molar. If the molar ratio deviates from the above range, the chitosan structure may be broken and ring opening may occur. A conversion time to chitosan perchlorate, in other words, a reaction time of 1 hour is sufficient. The reason for conversion to chitosan perchlorate is that acetate ions (CH ₃ CO 2)
O ⁻ ) is replaced by perchlorate ion (ClO ₄ ⁻ ) with stronger acidity.
And to form a stable jelly-like or gel-like perchlorate to prevent oxidation of carbons at the 2- and 3-positions in the subsequent oxidation reaction.

Next, an oxidizing agent is added to this chitosan perchloric acid suspension (the gel state becomes a suspension state by stirring) to oxidize carbon at the 6-position, that is, CH ₂ OH groups. As the oxidizing agent, sodium permanganate, potassium permanganate, hydrogen peroxide, sodium hypochlorite, potassium hypochlorite, nitric acid, etc. can be used, but chromic anhydride is most preferred from the viewpoint of reaction efficiency. desirable. The amount of chromic anhydride used depends on the desired oxidation rate, but when a 50 mol% oxidation rate is desired, it is 1.5 to 4 moles, more preferably 2 to 3 moles, of the CH ₂ OH group. The oxidation reaction temperature is normal temperature, and the oxidation reaction time is 5 to 6 hours. When a 100 mol% oxidation rate is desired, the reaction is desirably carried out slowly at a temperature of preferably 0 ° C. or less, but it is recommended to adopt a production method described later.

Next, the obtained oxidized chitosan is filtered off. Filtering methods include centrifuge, filter press,
Any filtration method such as a vacuum filter can be adopted.

The chitosan oxide obtained by the separation by filtration is dissolved in an alkaline solution. Preferred examples of the alkali agent to be used include sodium hydroxide, potassium hydroxide, alkanolamine, and an aqueous ammonia solution. Of these, sodium hydroxide and potassium hydroxide are particularly desirable.
The amount of the alkali agent used is such that the pH of the chitosan oxide solution becomes 12 or more, and the concentration of chitosan oxide is approximately 0.001 to 0.01 molar, preferably 0.02, with water.
Adjust so as to have a concentration of 04 to 0.006 molar.

Next, methanol is added to the aqueous alkali solution of chitosan oxide. The use ratio of methanol to oxidized chitosan varies depending on the chitosan oxidation rate, the acetylation rate described later, and the like.
When 0 mol% and an acetylation rate of 95 mol% are desired, the amount is 150 to 500 parts of methanol based on 100 parts of chitosan oxide. The concentration of methanol used is 90 to 98
% By weight is preferred. Here, the reason for using methanol is to improve the acetylation rate. When methanol is not used, it is presumed that acetic anhydride causes hydrolysis and the acetylation rate does not improve. In addition, ethanol, isopropanol, butanol and the like can be used in addition to methanol, but methanol has a large polarity and the acetylation reaction efficiency can be maximized.

Next, acetic anhydride is added to the methanol solution to acetylate the amino group of the chitosan oxide. The amount of acetic anhydride to be added may be determined according to the desired acetylation rate. For example, when an acetylation rate of 80 mol% is desired, acetic anhydride is 0.9 times as much as the number of moles of amino groups of chitosan oxide. What is necessary is just to add mol. When an acetylation ratio of 80 mol% or more is desired, it is desirable to use acetic anhydride in an amount of 1 mol or more with respect to the number of amino groups of the oxidized chitosan. The reaction time of the acetylation reaction varies depending on the acetylation rate, oxidation rate, etc., and cannot be unconditionally limited,
Acetylation is completed in about 6 hours to 2 days.

The acetylated chitosan compound thus acetylated is separated by filtration to obtain an alkaline solution having a pH of 12 or more, and then desalted by dialysis, ultrafiltration ion exchange resin or the like. When it is not necessary to be a high-purity product,
It may be dried as it is by any drying means such as spray drying and standing drying.

As the method for producing the oxidized chitosan compound of the present invention, the methods described above are the best from the viewpoints of yield, workability, availability of raw materials, simplicity of production equipment and other economical factors. The chitosan compound can be produced by other methods. That is, any method may be used as long as it has the above-mentioned properties.

According to the present invention, the neutrophil activity inhibitor of the present invention functions to suppress the ability of cells to produce active oxygen. Assuming that neutrophils usually produce a value of 10 reactive oxygen species, neutrophils always have the first protection in the blood against bacteria with a capacity of 10.

However, when further stimulated by bacteria, IL (i
nterleukin) 1,8, TNF (tumer necrosis facter) and the like increase the ability of neutrophils to produce active oxygen. For example, neutrophils temporarily have a capacity to produce 1000 active oxygen. In this case, activated neutrophils will function as 100 cells of normal neutrophils.

However, in this case, damage to normal cells also increases rapidly, leading to diseases such as inflammation. That is, an object of the present invention is to suppress the neutrophil-activating enzyme-producing ability in such a case.

With regard to the action and effect of the neutrophil activity inhibitor of the present invention, the higher the oxidation rate, the higher the effect of suppressing the production of active oxygen. The higher the acetylation rate, the higher the production inhibitory effect. Although the mechanism of action of the inhibitor of the present invention is not clear, it is thought that the carboxy group of the inhibitor of the present invention acts on neutrophil receptors to suppress the production of active oxygen. Therefore, it is presumed that the higher the oxidation rate, the higher the effect of suppressing active oxygen. The higher the acetylation rate, the higher the inhibitory effect is due to the improvement in water solubility, or because neutrophils are negatively charged around pH 7 in living organisms, increasing the acetyl group is more likely to increase chitosan. It is considered that the amount of NH ₃ ⁺ (cation of amino group) becomes smaller and the recognition of the carboxy group by the receptor in neutrophils is not hindered by NH ₃ ^+, so that the higher the acetylation rate, the higher the inhibitory effect.

Next, regarding the method of applying the neutrophil activity inhibitor of the present invention, the oxidized chitosan compound used in the present invention is water-soluble and can be applied to all agents. In particular, it is suitable for ointments and injections, and when it is made into tablets, it shows excellent medicinal effects. When used as an ointment, 2 to 5 g of the inhibitor of the present invention is mixed with 100 g of an ointment base such as propylene glycol, white petrolatum, and stearyl alcohol, which are components of a hydrophilic ointment, and thinly applied to the affected part. When administering by injection,
About 0.5 ml of the inhibitor of the present invention is added to 100 ml of a commonly used injection solution such as distilled water, physiological saline, and 5% glucose solution.
~ 2 g can be mixed and dissolved and administered by subcutaneous injection, intramuscular injection or intravenous injection. Injection preparations take less time to be absorbed than in internal use, and can be administered in a small amount. Generally, if oral administration is 1, subcutaneous injection is about 1/2 and intramuscular injection is about 1
/ 3, intravenous injection may be about 1/4.

The inhibitor of the present invention is administered in the following cases. For example, when a wound is treated with a wound dressing, when inflammation appears between the wound and normal cells as the healing progresses, or when neutrophils have excessive activity, SIRS (Sys
temic inflammatory response syndrom (systemic inflammatory response syndrome). Note that SI
RS can occur in cases of infection, surgery, trauma, burns, acute pancreatitis, and the like. Hereinafter, examples of the present invention will be described further.

[0031]

EXAMPLES [Example 1] Neutrophil production of active oxygen was measured by a chemiluminescence method. As a measuring device, Berthold Biolumat LB9505 was used.
The chemiluminescence value was measured at 7 ° C. HBSS (Hanks balanc
ed salt solution) 350 μl and 50 μl of the inhibitor of the present invention (weight average molecular weight 64,000, oxidation rate 20%, acetylation rate 70 mol%, solubility 13.3 g / 100 g-water) (concentration of the inhibitor of the present invention 10 mg / ml) ) And 100 μl of dog whole blood
Was agitated and incubated for 15 minutes. After changing the activity of neutrophils by incubation for 15 minutes, 20 μl of a luminol solution (2 mg / ml), which is a chemiluminescent substance, was added, and 50 μl of zymosan (10 mg / ml) was added. The amount of active oxygen produced when the sphere engulfed the zymosan was measured. Comparative substance (weight average molecular weight: 58,000, oxidation rate: 0%, deacetylation rate: 50 mol% chitosan, solubility: 6.5 g / 100 g-water)
RI of the neutrophil chemiluminescence value when 1 (DAC50 concentration 10 mg / ml) was added and the neutrophil chemiluminescence value when the inhibitor of the present invention was added were calculated and compared. Note that RI was obtained by the following equation. RI (Relative Intensity) (%) = (Peak count when the inhibitor of the present invention or a comparative substance and zymosan are added /
The peak count when zymosan was added) × 100 The neutrophil active oxygen-producing ability RI of the inhibitor of the present invention was 62%, and the RI of DAC50 used for comparison was 470%.
Met. The inhibitor of the present invention successfully suppressed active oxygen production. In addition, the number of dead neutrophil cells incubated with the inhibitor of the present invention was counted using tryphan blue staining that can selectively stain dead cells, but no dead cells were found. In addition, when the presence or absence of the hemagglutination effect of the inhibitor of the present invention was examined by a blood agglutination test and microscopic observation, no erythrocyte agglutination was observed. In the hemagglutination test, 100 μl of dog whole blood and 50 μl of the inhibitor of the present invention were added to 400 μl of HBSS and incubated for 15 minutes, and then the presence or absence of red blood cell aggregation was observed by visual observation. Further, observation with an optical microscope (400 times) was performed.

[Examples 2 to 5] In the same manner as in Example 1,
Inhibitor of the present invention (Example of the present invention) 50 μl (concentration 10 mg / m
l) Addition and similar compounds (Comparative Example) 50 μl (concentration 1)
(0 mg / ml) addition was measured for RI. The results are shown in Table 1.

[0033]

[Table 1]

[0034] D-Culcose and D-Quorconic acid are special grade products manufactured by Wako Pure Chemical Industries, Ltd., cellulose is a powdered reagent manufactured by Hanoi Tesque Co., Ltd., and Dextrin and polyquinochitosan are Wako Pure Chemical Industries, Ltd. Reagents were used. Others were synthesized by the inventor.

[0035]

Industrial Applicability The neutrophil activity inhibitor of the present invention is an oxidized chitosan compound produced from chitin which is abundant in nature, and is capable of effectively suppressing neutrophil active oxygen production without killing normal cells. And can cure various diseases such as inflammation caused by excessive release of the active enzyme.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4C086 AA01 AA02 EA23 MA01 MA04 NA14 ZA59 ZA89 ZB08 ZB11 ZB32 4C090 AA09 BA47 BD05 BD31 BD35 BD36 BD37 CA34 CA39 DA09 DA23

Claims (1)

[Claims] 1. A compound of the general formula And the general formula And (a) / ((a) + (b)) (molar ratio)> 70%, wherein the oxidized chitosan compound is derived from chitosan, wherein CH _{2 in} the formulas 1 and ₂ The oxidation rate of an OH group to a COOX group (where X represents H, Na or K) is 1 to 100 mol%, and NH _{2 in} the formula
An acetylation ratio of the group to an NHCOCH ₃ group of 40 to 95 mol%, and a weight average molecular weight of 5,000 or more;
And a solubility in water at pH 7 and a temperature of 25 ° C. of 0.1
g / 100 g or more, a neutrophil activity inhibitor comprising an oxidized chitosan compound.