JP2000256196A - Anti-inflammatory and anti-allergic medicine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-inflammatory and anti-allergic medicine which is highly safe and inhibits the excessive active oxygen concentration of human neutrophils and a high calcium concentration in the cytoplasm of the neutrophils, which cause the aggravation of the inflammation and the allergy. SOLUTION: This anti-inflammatory and anti-allergic medicine contains a water-soluble chitosan salt or water-soluble chitosan derivative salt which has a charged amino group content of 25-100%. The water-soluble chitosan salt having a charged amino group content of 25-100% includes a chitosan- glycolic acid salt formed from a glycolic acid and chitosan having a deacetylation degree of 25-100%. The salt is especially not limited but includes a pyridone carboxylic acid salt, a glutamic acid salt, a lactic acid salt, an ascorbic acid salt and a hydrochloride.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an anti-inflammatory and anti-allergic agent.

[0002]

2. Description of the Related Art Conventionally, various anti-inflammatory and anti-allergic agents have been developed as internal medicines and external preparations.
It is used.

[0003]

However, at present, very few conventional anti-inflammatory agents and anti-allergic agents are highly safe from the viewpoint of side effects and the like.

For example, steroids and the like have been used as external preparations for a long time, but they only suppress inflammation locally, and when inflammation recurs after healing, the symptoms are worse than before administration. There are cases.

On the other hand, anti-inflammatory agents that suppress the production of prostaglandins have been used as oral drugs by oral administration, but they have side effects such as anorexia and gastrointestinal symptoms such as heartburn.

[0006] In recent years, inflammatory cells derived from phagocytes such as neutrophils have been activated by excessive active oxygen and an increase in Ca ²⁺ concentration as an allergic deterioration factor (Tadaomi Takenawa,
Others: Leukocytes and host defense, Kodansha 145, 1990) have been suggested.

[0007] For example, it is known that neutrophil-derived excessive active oxygen and neutrophil activation are one of the deteriorating factors in various human inflammatory skin diseases (Yoshiki Miyachi: Japanese clinical practice 46). : 2252, 1988).

[0008] Regarding inflammatory acne and pimples,
It has been suggested that excessive reactive oxygen produced from neutrophils that reached the hair follicle wall by acnes-derived migration factors and the like may be a factor in the onset and exacerbation (Hirohiko Akamatsu, et al .: Reactive Oxygen and Free Radicals 3: 306, 1992).

[0009] From the above-mentioned viewpoints, the present invention provides a safety method for suppressing excessive active oxygen in human neutrophils and an increase in the concentration of calcium in the cytoplasm of neutrophils, which are one of the causes of inflammation and allergy. It is an object of the present invention to provide highly anti-inflammatory and anti-allergic agents.

[0010]

Means for Solving the Problems The present invention has been made to solve such problems, and the means for solving the problems is that the content (ratio) of charged amino groups is 25 to 25%.
That is, the composition contained 100% of a water-soluble chitosan salt or a water-soluble chitosan derivative salt.

[0011] Chitosan has a moisturizing property, an anticholesterol effect and the like and is excellent in safety, and thus has been put to practical use as a raw material for cosmetics and a functional food material.

Further, it is suggested to have various physiologically active functions such as a wound healing effect, an antibacterial property, an immunostimulatory action, and the like.

Recently, water-insoluble powders and sponges of chitosan have been put to practical use as veterinary wound dressings because of their excellent wound healing effect on animals.

Since chitosan and its derivatives are expected to have new physiological functions and have high biocompatibility, application research is being actively conducted.

By deacetylating chitin obtained from crustaceans and filamentous fungi, it is possible to obtain a biocompatible compound having an amino group at C-2 (carbon atom at position 2) (substitution degree of about 0.4 to 1.0). Chitosan, which is an excellent functional material, can be obtained.

The present inventors have found that the content of charged amino groups is 25%.
~ 100% water-soluble chitosan salt and water-soluble chitosan derivative salt significantly suppresses activation due to excessive active oxygen and Ca ²⁺ concentration derived from human neutrophils involved in inflammation and allergic diseases It has also been found that they exert anti-inflammatory and anti-allergic effects.

It is important to note that the presence of a certain amount of charged amino groups derived from a water-soluble chitosan salt and a chitosan derivative salt having chitosan as a basic skeleton acts on human neutrophils to suppress excessive active enzyme production, In order to contribute to the suppression of activation due to an increase in Ca ²⁺ concentration, the content of charged amino groups due to salt formation is 25 to 100%.

As a water-soluble chitosan salt having a charged amino group content of 25 to 100%, chitosan glycolate salt formed by coexistence of chitosan having a degree of deacetylation of 25 to 100% and glycolic acid is used. No.

The kind of the salt is not particularly limited, and examples thereof include pyrrolidone carboxylate, glutamic acid, lactate, ascorbate, and hydrochloride.

As a water-soluble chitosan derivative salt having chitosan as a basic skeleton having a charged amino group content of 25 to 100%, O-acylated chitosan glycolate, partial N-
Acylated chitosan glycolate, sulfated chitosan
Examples include glutamate, carboxymethylated chitosan glycolate, phosphorylated chitosan glycolate, and glycolated chitosan glycolate.

The type of the salt is not particularly limited, and examples thereof include glycolic acid, pyrrolidone carboxylate, glutamic acid, lactate, ascorbate, and hydrochloride.

The molecular weight is not particularly limited,
In the case of high molecular weight, the average molecular weight is preferably about 5,000 to 100,000, and in the case of low molecular weight, the average molecular weight is preferably 3,000 or less.

Although there is no particular limitation on the blending concentration,
There is an advantage that a sufficient effect is exhibited even at a low concentration of 0.001% to 0.1%.

The water-soluble chitosan salt having a charged amino group and the chitosan derivative salt of the present invention act on human neutrophils and cause excess neutrophils derived from neutrophils, which is one of the causes of inflammation and allergy. It exerts anti-inflammatory and anti-allergic effects by suppressing an excessive increase in calcium concentration in cells of active oxygen and neutrophils.

The anti-inflammatory and anti-allergic agents of the present invention are highly safe, have high biocompatibility and are water-soluble, so that external skin preparations such as liquid, cream, gel, emulsion, and ointment, and basic cosmetics , Quasi-drugs, pharmaceuticals, drops,
It can be widely used for health drinks and the like.

When coexisting with an active oxygen scavenger (active oxygen eliminating component) such as vitamin C, β-carotene, SOD, etc., there is also an advantage that the effect is synergistically enhanced.

[0027]

Embodiments of the present invention will be described below.

Example 1 Inhibitory Effect of Production of Reactive Oxygen from Human Neutrophils Chitosan salts having a charged amino group content of 25 to 100% (average molecular weight: 800,000, 1000), chitosan derivatives (average molecular weight: 100,000) ~ 800000) to the following experimental system from 1 to 100 μg /
mL and evaluated.

Each chitosan derivative was prepared by a conventional method (Hiran
o, S et al: Carbohydr. Res., 137, 205, 1985, etc.) and adjusting the degree of substitution such as sulfation or acylation so that the content of charged amino groups is 25 to 100%. Set.

The charged amino group content was determined by using a colloid titration method (Toei, K., T. Kohata: Anal. Chem. Acta, 83, 59, 1976) and the like.

From the venous blood of eight healthy persons, Ficoll-Hypaque
The neutrophil layer mixed with erythrocytes in the upper layer was separated by the gradient density method, neutrophil viability was maintained according to the literature, red blood cells were removed, and neutrophils were harvested (Niwa Y, et. al: Arch Dermatol 121: 73,1985).

For O ² -measurement, a neutrophil free solution in which neutrophils were suspended in a Krebs Ringer phosphate (hereinafter abbreviated as KRP) buffer containing 5% glucose, and intracellular C
For a ²⁺ concentration measurement, 0.1 mM neutrophils at 10 ⁷ ratio of
Neutrophil free solution suspended in 1 mL of KRP buffer containing CaCl ₂ (Ozaki Y, et al: Biochem Biophys Acta 972: 11
3, 1988).

After neutrophils were stimulated with zymosan opsonized with test sera, Johnston et al. (Johnston RB Jr.
et al: J Clin Invest 57: 836, 1976) was measured O ^2- in the method of.

More specifically, 1 × 10 ⁶ neutrophils were cultured with 1 mg / mL of opsonized zymosan for 10 minutes, and then 0.1 mM ferricytochrome C (type I)
II, Sigma), and the cells were further cultured at 37 ° C. for 30 minutes.

Thereafter, the absorbance (wavelength: 550 nm) was measured using a Beckman spectrophotometer, and O ²⁻ produced from the reduced amount of ferricytochrome C was calculated.

In this embodiment, chitosan glycolate having a charged amino group content of 85% is used, and the concentration is 1 μm.
Active oxygen production was determined for three concentrations, g / mL, 10 μg / mL, and 100 μg / mL.

The results are shown in Table 1.

[0038]

[Table 1]

The active oxygen production was determined for 100 μg / mL samples of various chitosan derivatives having different charged amino group contents.

Table 2 shows the results.

[0041]

[Table 2]

In Tables 1 and 2, the value of the degree of active oxygen production is represented by the relative value of the average value of the results of the tests performed three times, and the value without the chitosan derivative as 100%. .

As shown in Table 1, the chitosan glycolate having a charged amino group content of 85%, which is the chitosan derivative of this example, was 1 μg / mL, 10 μg / mL, and 100 μg / mL in a concentration-dependent manner. It was confirmed that the production of active oxygen derived from human neutrophils, which is excessively produced by zymosan opsonized in S. cerevisiae, was significantly suppressed.

As shown in Table 2, chitosan glycolate, phosphorylated chitosan glycolate, partially N-propionylated chitosan glycolate having a charged amino group content of 25 to 100%, O- Various chitosan derivatives of this example such as propionylated chitosan glycolate, glycolated chitosan glycolate, carboxymethylated chitosan glutamate, etc. are derived from human neutrophils which are excessively produced by opsonized zymosan Was confirmed to significantly suppress the production of active oxygen, suggesting that it has anti-inflammatory and anti-allergic effects.

For the carboxymethylated chitosan / glutamate, the charged amino group content of 4
2% and a charged amino group content of 27% which is a comparative example
A clear difference in active oxygen production was observed between the two.

On the other hand, the effect of removing active oxygen generated in the cell-free xanthine-xanthine oxidase system (scavenging effect) was not recognized, but conventional active oxygen scavenger components (vitamin C, SOD, astaxanthin, β-carotene) were used. And the anti-inflammatory agent of the present invention,
It has also been found that the coexistence of an antiallergic agent synergistically enhances the effect of suppressing the production of active oxygen derived from human neutrophils.

Example 2 Inhibitory Effect of Ca ²⁺ Concentration Derived from Human Neutrophils The same sample as in Example 1 was used in the following experimental system at 1 to 100 μm.
g / mL was added and evaluated.

Fura2-AM (Sigma, MO,
USA) according to the literature (Ozaki Y, et al: Bioche
m Biophys Acta 972: 113, 1988).

That is, 1 mM Fura was added to 10 mL of neutrophil free solution.
After adding 10 μL of 2-AM and incubating at 37 ° C. for 30 minutes, the cells were washed with KRPbuffer, the cells were suspended again in the solution, and 1.5 mL of the suspension was taken and 10 ⁻⁶ M fMLP (Sigma)
340 nm and 380 nm of the cell suspension using a fluorescence spectrophotometer (Hitachi F-4000).
The fluorescence intensity during resting was measured at 510 nm.

The maximum fluorescence (Fmax) when intracellular Fura2 binds to Ca ²⁺ and the minimum fluorescence (Fmin) when not bound to 10% Triura
30 μL of ton-X-100 and 30 μL of 100 mM EGTA (pH 9.3)
Was added to each sample.

From the above, the concentration of Ca ²⁺ is given by the following equation:
It was calculated from the ratio of the fluorescence intensity of Fura2 at the excitation wavelengths of 340 nm and 380 nm.

[0052]

In the above formula, R is F 340 nm and F 380 nm
And R_maxCan convert Fura2 to Ca by Triton method etc.²⁺
And R when sufficiently bonded to_minMeans EGTA
Ca below²⁺R when not bound, and Kd is Ca²⁺And Fura2
Where b is Ca ²⁺F 380nm in the absence of
Ca²⁺The ratio to F 380 nm in the presence is shown.

The unit of the numerical value calculated by the above equation is:
nM.

In this embodiment, first, a chitosan glycolate having a charged amino group content of 80% is used, and the concentration is 1 μm.
The intracellular Ca ²⁺ concentration was determined for three concentrations, g / mL, 10 μg / mL, and 100 μg / mL.

Table 3 shows the results.

[0057]

[Table 3]

Intracellular Ca ²⁺ concentrations were determined for 100 μg / mL samples of various chitosan derivatives having different charged amino group contents.

Table 4 shows the results.

[0060]

[Table 4]

In Tables 3 and 4, the values of the intracellular Ca ²⁺ concentration are represented by the relative values of the average of the results of the tests performed three times, and the value without the chitosan derivative as 100%. It is.

As shown in Table 3, chitosan glycolate having a charged amino group content of 85% in this example was 1 μm.
g / mL, 10 μg / mL, 100 μg / mL and 10 ^-6 M fM
It was confirmed that the intracellular Ca ²⁺ concentration of LP-stimulated human neutrophils was significantly suppressed.

As shown in Table 4, chitosan glycolate, phosphorylated chitosan glycolate, partially N-propionylated chitosan glycolate having a charged amino group content of 25 to 100%, and O- Various chitosan derivatives of the present example, such as propionylated chitosan glycolate, glycol chitosan glycolate, carboxymethylated chitosan glutamate, and the like, are used to induce intracellular Ca ²⁺ of human neutrophils stimulated by 10 ⁻⁶ M fMLP. It was confirmed that the concentration was significantly suppressed, suggesting that it has anti-inflammatory and anti-allergic effects.

Example 3 Efficacy for Dermatitis Patients with Inflammatory / Allergic Skin Symptoms

Forty dermatitis patients with dermatitis of any two or more of desquamation, papules, erythema papules, erythema, flushing, rosacea, and pustules (average age: 2
8.5 years old: against allergic contact dermatitis, acne vulgaris, psoriasis, rosacea dermatitis, xerosis, seborrheic dermatitis)
A lotion containing an effective amount of the component of the present invention was applied to the diseased site twice a day for 4 weeks to evaluate the effectiveness.

The state of each symptom of the inflammatory site before and after use was 4: high, 3: moderate, 2: mild, 1: slight, 0:
Evaluation was made on a five-point scale with no symptoms.

The reduction effect was determined from the change in the symptom average score.

The composition of the lotion used is as follows.

Ingredients Weight (%) Ethanol 7.0 Polyethylene glycol 2.0 Glycerin 6.0 Paraben 0.2 85% Chitosan glycolate containing charged amino groups 0.1 60% Succinylated chitosan glycol containing charged amino groups Acid salt 0.05 Purified water Remaining

Table 5 shows the test results.

[0071]

[Table 5]

As is clear from Table 5, at the end of the test, all the symptoms at the inflammatory site were significantly reduced as compared with the start of the test.

It was confirmed that the reduction effect further increased in proportion to the observation period.

No side effects were observed.

Two months after discontinuation of use, a placebo lotion (without the component of the present invention) was again given to the same skin inflammation patient
Was similarly applied, and the effectiveness was evaluated.

As a result, no reduction effect as seen in the above example was observed in all the symptoms after 1 to 4 weeks of use.

Next, formulation examples of various formulations containing anti-inflammatory and anti-allergic agents containing the chitosan derivative salt of the present invention will be shown.

(Formulation Example 1) Lotion Component Weight (%) Ethanol 3.0 Polyethylene Glycol 3.0 Paraben 0.2 50% Charged Amino Group-Containing Phosphorylated Chitosan Glycolate 0.15 Purified Water Remaining

(Formulation Example 2) Lotion Component Weight (%) Ethanol 10.0 Polyethylene glycol 2.0 Glycerin 5.0 Paraben 0.3 80% Chitosan glycolate containing 80% charged amino group 0.2 Rosemary extract 1 Remaining purified water

(Formulation Example 3) Cream or hydrophilic ointment Ingredient Weight (%) Cetanol 5.0 Vaseline 2.5 Liquid paraffin 17.0 POE (15) monostearylsorbitan 2.0 Glycerin 7.5 Paraben 0.1 65% Charged amino group-containing phosphorylated chitosan / glutamate 0.4 Purified water

(Formulation Example 4) Emulsion Component Weight (%) Cetanol 2.0 Ethanol 7.5 Liquid Paraffin 3.5 POE (15) Monostearylsorbitan 2.5 Natural Vitamin E 0.05 Glycerin 7.5 Paraben 0 .05 75% charged amino group-containing chitosan / lactate 0.1 95% charged amino group-containing partially N-stearoylated chitosan / glycolate 0.02 purified water

(Formulation Example 5) Essence Component Weight (%) Ethanol 5.0 Hydroxymethyl Cellulose 0.5 Glycerin 10.0 Polyethylene Glycol 3.0 Paraben 0.3 80% Charged Amino Group-Containing Glycol Chitosan Hydrochloride 0.3 Purified water balance

Formulation Example 6 Instillation or Injection Component Weight (%) Physiological saline 99.7 Glucose 0.2 70% charged amino group-containing carboxymethylated chitosan / hydrochloride 0.1

(Prescription Example 7) Health Drink or Pharmaceutical Drink Ingredient Weight (%) Vitamin C 0.1 Ginseng Extract 3.0 Fructose 2.0 Sucrose 1.0 Fragrance Trace 70% O-propionylation containing charged amino group Chitosan / glutamate 0.05 80% charged amino group-containing chitosan / lactate 0.02 Paraben 0.05 Purified water

[0085]

As described above, the present invention provides an anti-inflammatory or anti-allergic agent containing a water-soluble chitosan salt or a water-soluble chitosan derivative salt having a charged amino group content of 25 to 100%. Is a highly safe anti-inflammatory that suppresses excessive activation of human neutrophils and activation of neutrophils due to an increase in calcium concentration in the cytoplasm, which is one of the causes of inflammation and exacerbation of allergies Agent and neutrophil allergic agent.

As a result, the charged amino group content was 25 to 10
A water-soluble chitosan salt or a water-soluble chitosan derivative salt of 0% is used as an anti-inflammatory and anti-allergic agent, and can be widely used as an external preparation for skin and the like.

Continuation of the front page (72) Inventor Jira Jino 1-3-1 Muroya, Nishi-ku, Kobe Pierce Co., Ltd. Central Research Laboratory (72) Inventor Toshinaga Niwa 4-6 Asahicho, Tosashimizu-shi, Kochi F-term (reference) 4C086 AA01 AA02 EA23 MA01 MA04 NA14 ZB11 ZB13 4C090 AA09 BA47 BB17 DA23

Claims (1)

[Claims] 1. An anti-inflammatory or anti-allergic agent comprising a water-soluble chitosan salt or a water-soluble chitosan derivative salt having a charged amino group content of 25 to 100%.