JP5457675B2 - Therapeutic or preventive for atopic dermatitis - Google Patents

Description

  The present invention relates to a pharmaceutical composition for preventing and / or treating atopic dermatitis comprising low molecular weight hyaluronic acid as an active ingredient.

Atopic dermatitis is a chronic skin disease with itching caused by various stimuli on allergic constitution called atopic predisposition, such as allergic asthma, allergic rhinitis, dermatitis urticaria . The diagnostic criteria of the Japanese Dermatological Association define skin diseases mainly with eczema with itching and atopic dermatitis that repeat exacerbations and remissions, and many patients have an atopic predisposition, but have an atopic predisposition. I don't need it. Therefore, in a broad sense, it may be referred to as “atopic dermatitis” for diagnosis including contact dermatitis. In general, it exhibits chronic eczema symptoms over a wide area, becomes dry and blows white powder on the surface, and is accompanied by strong itching. Some patients may have red eczema, nodules, etc.
Tissue fluid leaches out from the wet phase, causing severe pain. When it becomes chronic, it becomes rough like goosebumps, and the skin may gradually become thicker or a lump-like wart-like rash may occur.

  Atopic dermatitis develops in early childhood and often remits spontaneously before reaching adulthood. However, there are an increasing number of cases that carry over to adults, and cases that develop or recur after adulthood. There is no way to fundamentally treat allergic symptoms of atopic dermatitis, and the goal is to alleviate the symptoms by symptomatic treatment with anti-itch drugs such as steroids and antihistamines. Treatment that suppresses excessive immunity by administration of external or internal use steroids or protects the dried part with a moisturizing agent such as petrolatum is the mainstream.

  However, the steroid agent, which is said to have the highest therapeutic effect, may cause a phenomenon called rebound that rebounds more strongly than the original symptom, or may reduce the effect (tachyphylaxis) due to long-term continuous use. In addition, it has been known that continuous use of a steroid topical agent for a long period of time may cause disorders such as skin atrophy, induction of skin infection, and dilation of capillaries. Control is difficult. There has been a demand for the development of therapeutic agents with fewer side effects than steroids.

In order to prevent drying due to atopic dermatitis, therapeutic agents containing hyaluronic acid as a moisturizer have been developed. For example, Patent Document 1 discloses a therapeutic drug for atopic dermatitis containing ε-polylysine as an active ingredient and hyaluronic acid as a humectant. Patent Document 2 describes that a sheet in which hyaluronic acid and hydrolyzed silk are contained in a silk nonwoven fabric is used for preventing and treating atopic dermatitis. Patent Document 3 discloses an external preparation for skin containing borage seed oil and vegetable ceramide extracted from wheat germ as main components and hyaluronic acid as a functional component. Patent Document 4 describes a skin external preparation containing eicosapentaenoic acid or docosahexaenoic acid and hyaluronic acid as a humectant. However, hyaluronic acid is used only as a moisturizing agent and is not contained as an active ingredient in the treatment of atopic dermatitis. Hyaluronic acid is so-called high molecular weight hyaluronic acid (for example, weight average molecular weight of about 900,000) and does not contain low molecular weight hyaluronic acid, particularly hyaluronic acid tetrasaccharide.
JP 2005-035914 A JP 2003-212715 A JP 2002-053428 A JP 2000-095683 A

  An object of the present invention is to provide a therapeutic agent having a high therapeutic effect that can be substituted for a steroid agent in the prevention and / or treatment of atopic dermatitis.

  The present inventor has found that low molecular weight hyaluronic acid, particularly hyaluronic acid tetrasaccharide, has an effect of suppressing inflammation due to atopic dermatitis, and can reduce symptoms of atopic dermatitis by administration.

  That is, the present invention provides a therapeutic or prophylactic agent for atopic dermatitis comprising low molecular weight hyaluronic acid as an active ingredient.

  Further, in the present invention, low molecular weight hyaluronic acid is disaccharide (molecular weight about 400), 3 sugar (molecular weight about 600), 4 sugar (molecular weight about 800), 5 sugar (molecular weight about 1000) or 6 sugar (molecular weight 1200). There may be. The low molecular weight hyaluronic acid is preferably a hyaluronic acid tetrasaccharide (molecular weight of about 800).

  ADVANTAGE OF THE INVENTION According to this invention, in the prevention and / or treatment of atopic dermatitis, the therapeutic agent with a high therapeutic effect with few or no side effects can be provided.

It is a graph which shows the result of having measured the left auricle thickness of the atopic dermatitis model mouse in Example 1. FIG. 2 is a color photograph taken of an affected area of an atopic dermatitis model mouse in Example 1. FIG. It is a graph which shows the result of having observed the pruritus action of the atopic dermatitis model mouse in Example 1 (times / 15 minutes). 6 is a color photograph taken of an affected area of a patient in Example 2.

Hyaluronic acid is a high-molecular long-chain polysaccharide composed of disaccharide repeating units of D-glucuronic acid and N-acetyl-D-glucosamine, while oligosaccharides are also known.
The low molecular weight hyaluronic acid of the present invention is hyaluronic acid having 2 saccharides, 3 saccharides, 4 saccharides, 5 saccharides or 6 saccharides (molecular weight of about 400 to about 1200). Hyaluronic acid tetrasaccharide is particularly desirable.

  The low molecular weight hyaluronic acid contained in the drug according to the present invention basically includes a disaccharide unit in which the 1-position of β-D-glucuronic acid and the 3-position of β-DN-acetyl-glucosamine- are bonded. As long as it contains at least one disaccharide or more and is basically composed of β-D-glucuronic acid and β-DN-acetyl-glucosamine—one or more disaccharide units Saccharides in which those elements are bound to the bound ones may be used, and derivatives thereof such as those having a hydrolyzable protecting group such as an acyl group may also be used. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, usually those in which the 4th and 5th carbons of glucuronic acid are unsaturated.

  Specific examples of the low molecular weight hyaluronic acid used in the present invention include those extracted from natural products such as animals, those obtained by culturing microorganisms, and those synthesized chemically or enzymatically. Can be used. For example, it can be obtained by known extraction and purification methods from biological tissues such as chicken crown, corpse, skin, and joint fluid. It can also be produced by a fermentation method using Streptococcus bacteria or the like.

  In the present invention, low molecular weight hyaluronic acid such as a disaccharide consisting of one disaccharide unit and a derivative thereof can be used. Preferably, low molecular weight hyaluronic acid of about 2 to 20 sugars can be mentioned, more preferably low molecular weight hyaluronic acid of 2 to 6 sugars (molecular weight of about 400 to about 1200), most preferably hyaluronic acid tetrasaccharide. is there.

  Specifically, the low molecular weight hyaluronic acid is obtained by converting hyaluronic acid into a low molecular weight by a known method such as an enzymatic decomposition method, an alkaline decomposition method, a heat treatment method, and an ultrasonic treatment method (Biochem., 33 (1994) p6503-6507). It is preferable to produce it by the method to synthesize | combine, the method of synthesize | combining chemically or enzymatically (Glycoconjugate J., (1993) p435-439, WO93 / 20827). For example, as an enzymatic degradation method, hyaluronic acid degrading enzymes (hyaluronidase (derived from testicles), hyaluronidase (derived from Streptomyces), hyaluronidase SD, etc.), chondroitinase AC, chondroitinase ACII, chondroitinase ACIII, chondroitinase ABC, etc. A method of producing hyaluronan oligosaccharides by causing an enzyme that degrades hyaluronan to act on hyaluronan (Shinsei Chemistry Laboratory "Carbohydrate II-Proteoglycans and Glycosaminoglycans" p244-248, published in 1991, see Tokyo Kagaku Dojin) Is mentioned.

  As an alkali decomposition method, for example, a base such as about 1N sodium hydroxide is added to a hyaluronic acid solution, heated for several hours to lower the molecular weight, and then neutralized by adding an acid such as hydrochloric acid. And a method for obtaining low molecular weight hyaluronic acid. The hyaluronic acid used in the present invention includes a pharmacologically acceptable salt form, and a pharmaceutically acceptable salt thereof can be used as necessary in the preparation. For example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, amine salts such as tri (n-butyl) amine salt, triethylamine salt, pyridine salt and amino acid salt Can do.

  The agent of the present invention can be used without particular limitation, such as hyaluronic acid having a specific molecular weight alone or a mixture of hyaluronic acids having different molecular weights. The therapeutic agent for atopic dermatitis of the present invention comprises hyaluronic acid as an active ingredient, and by administering an effective amount thereof to mammals including humans, symptoms of atopic dermatitis without adversely affecting the living body Can be improved or prevented.

  The agent of the present invention can be applied to the affected area and the surrounding skin with low molecular weight hyaluronic acid or a salt thereof as it is or together with a carrier, excipient, or other additives as necessary. Alternatively, it can be formulated into an arbitrary dosage form and administered into tissue (injection), orally or enterally.

  Examples of the dosage form of the drug applied to the skin include ointments, lotions and creams. The base of these external preparations for skin is not particularly limited as long as it is a base that can generally be melted, blended or dispersed uniformly in an external preparation. For example, petroleum jelly, castor oil, silicon, squalane, sodium acrylate, behenyl alcohol, glycerol monostearate, stearyl alcohol, ethanol, batyl alcohol, phenoxyethanol, 1,3-butylene glycol, isopropyl myristate, stearic acid, lecithin, purified water, etc. However, it is not limited to these.

  Moreover, the chemical | medical agent of this invention adds additives, such as an antioxidant, antiseptic | preservative, a wetting agent, a thickener, a buffering agent, an adsorbent, a solvent, an emulsifier, a stabilizer, surfactant, as needed. be able to.

  As for the dosage, an appropriate amount of an ointment containing about 0.05 to 5% of low-molecular hyaluronic acid can be applied to the affected area and its surroundings several times a day. For example, hyaluronic acid tetrasaccharide can be applied to affected areas at a concentration of 1%. When used for the prevention of atopic dermatitis, it is applied in the same way to a place where atopic dermatitis is likely to develop.

  The drug of the present invention can be used as a therapeutic agent for suppressing the progression of symptoms of atopic dermatitis or alleviating the symptoms by being applied to the affected area, and can be applied around the affected area. , Applied to those who have a predisposition to atopic dermatitis, those who have atopic dermatitis, those who are likely to develop atopic dermatitis, such as those who have atopic dermatitis in their family, and prevention of recurrence of atopic dermatitis Can be used as a prophylactic.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[Example 1: Confirmation of therapeutic effect of HA4 using atopic dermatitis model mouse]
Inflammation of the left auricle was produced in NC mice by sensitization and induction with PiCl, and the effect of the HA4-containing coating agent on this was examined. As a result, it was confirmed that inflammation scores such as skin dryness, crust formation / bleeding, hair loss, pruritus behavior, and auricular thickness associated with inflammation were reduced.

<Creation of dermatitis model mouse>
Twelve male 9-week-old NC / Nga Slc mice were obtained from Japan SLC Co., Ltd., and the appearance was inspected at the time of acceptance to confirm that there were no abnormalities in all cases. Breeding is performed in a plastic cage (14.5 × 26.0 × 12.5 cm) in a breeding room set at a temperature of 22 ± 2 ° C., a relative humidity of 55 ± 15%, a ventilation rate of 12 times / hour, and a light / dark time of 12 hours. ) Were reared one by one. The feed was a solid feed Roden Lab Diet EQ (Japan SLC, Inc., Lot No. AUG24062), and the drinking water was ingested freely every day with public tap water. After 9 days of acclimatization, 10 animals with good weight gain, general condition and handling site during the acclimatization period were selected and randomly divided into 2 groups of 5 animals.

  In all cases, 0.05% per mouse was applied to the abdomen of a 5% PiCl / ethanol 4: acetone 1 solution using a disposable tuberculin syringe on the abdomen of a mouse previously removed with a hair clipper, and 0.02 mL per limb. Applied. Furthermore, using a disposable tuberculin syringe, after 4 days of sensitization, 1% PiCl olive oil solution was added to the left auricle of the mouse at 0.02 mL, and the inflammation of the left auricle was confirmed three times at weekly intervals. A skin inflammation model was created by inducing application.

<Preparation of test substance>
Specifically, HA4, which is a test substance, is prepared by the method of Tawada et al. (Tawada A, Masa T, Oonuki Y, Watanabe A, Matsuzaki Y, Asari A. Large-scale preparation, purification, and characterization of hyaluronan oligosaccharides from 4-mers. to 52-mers. Glycobiology. 2002; 12 (7): 421-6.). Moreover, what was mix | blended as shown in Table 1 was used for HA4 compounding coating agent which is a test substance. As a control substance, a base-only coating agent not containing HA4 was used. Of the components contained in this drug, those other than HA4 are general base materials and are not known to be effective for skin diseases such as atopic dermatitis.

<Administration of test substance>
In group 1, 1 hour before the third induction, the test substance listed in Table 1 HA4 combination coating agent (manufactured by Glucose Science Laboratory Co., Ltd .: Lot No. 060607, HA combination amount 1 mg / ml) is 1 Once a day, 0.02 mL was applied and administered to the left auricle using a disposable tuberculin syringe. The administration of the HA4 combination coating agent was carried out for 5 weeks from the third induction. The remaining 1 group was administered with the control substances listed in Table 1 (manufactured by Glucose Science Laboratory: Lot No. 060607) as a negative control (Table 2).

<Evaluation>
The general state of each mouse was observed at least once a day, and the body weight was measured at least once a week on the sensitization date, the test end date, and other days. Furthermore, Student-t test was performed about the body weight and the score of the following evaluation items.

(1) Measurement of auricle thickness Before the sensitization, before each induction application and before each administration of the test substance, the thickness of the left auricle of the mouse was measured using a micrometer (manufactured by Mitutoyo). Was measured. For reference, the right auricle was also measured.
(2) Observation of skin symptoms The following items were observed and evaluated for the left auricle of mice just before each induction application and once a week from the test substance administration start date, just before administration.
Observation items: Redness, edema, hair loss, dryness, crust formation / bleeding, ulcer / tissue loss, etc. Evaluation score: Asymptomatic (score 0), mild (score 1), moderate (score 2), severe (score 3)
(3) Observation of pruritic behavior The pruritic behavior of the applied part was observed for about 15 minutes from 1 hour after each induction application and 1 hour after each administration once a week from the test substance administration start date, and the number of scratches was counted. Photographs were taken of the pinna of all mice on the test end date.

<Test results and discussion>
There was no significant difference in body weight in the test substance group relative to the control group, and no abnormality was observed except that both the control group and the test substance group showed atopic skin inflammation.

1. Auricular thickness measurement (Figure 1)
The left auricle increases after 1 week of sensitization in both the control group and the test substance group, and after 2 weeks, both groups reach about 2.1 times the thickness at the time of sensitization. She had symptoms of atopic dermatitis.
On the day after the administration of HA4 over 5 weeks (36th day of administration), the control group was 1.48 times the same as the test substance group and the test substance group was 1.39 times the same. Significantly decreased. In addition, although the left auricle thickness of the test substance group showed a low value compared with the control group at each measurement time point of 9, 16, 23, and 30 days after the start of administration, no significant difference was observed. The thickness of the right auricle measured as a reference was 5% lower than that of the control group on the day following the 35th day of administration, and the test substance group showed a low value.

2. Observation of skin symptoms (redness)
In the control group, 2/5 cases were observed in the left auricle skin at the second induction, all cases at the third induction, 2/5 cases from the control group on the ninth day of administration, and mild redness was observed.
In the test substance group, mild redness was observed in 3/5 cases during the second induction and in all cases during the third induction. Mild redness was observed in 1/5 cases of the test substance group on the 9th day of administration, and mild redness was observed in 1/5 cases of the test substance group on the 23rd day of administration.

(edema)
In all groups, mild edema was observed in the 3rd induction / administration day 1, day 9 and day 16 of administration.
(Dry)
In the control group, mild dryness was observed in all cases at the 2nd and 3rd induction, 2/5 cases on the 9th day of administration, 1/5 cases on the 16th and 23rd day of administration, and mild dryness. Admitted.
In the test substance group, 4/5 cases at the second induction, all cases at the third induction, and 1/5 cases on the ninth day of administration, mild drying was observed.

(Crust formation / bleeding)
Mild in all cases in the control group at the second induction, moderate in 1/5 cases at the third induction, mild in 3/5 cases, 4/5 cases on the 9th day of administration, 1 / day on the 16th day of administration 5 cases, 2/5 cases of mild crust formation / bleeding were observed on the 23rd day of administration.
All cases of the test substance group at the second induction, 3/5 cases at the third induction, 2/5 cases on the 9th day of administration, and 1/5 cases on the 16th day of administration. It was.
There were no skin ulcers or tissue defects in all cases.

(Hair loss)
Hair loss was observed in all cases in both groups at the third induction and on the ninth day of administration. On the 16th day of administration, 4/5 cases were observed in the control group and 2/5 cases in the test substance group, and the test substance group showed a significantly low value at a level of 5% relative to the control group. A photograph of the left pinna of typical individuals in the control group and the test substance group on the 30th day of administration is shown in FIG.

  As described above, there are examples in which the test substance group shows a low value with respect to the control group in each item of redness, edema, hair loss, dryness, crust formation / bleeding, and the hair loss on the 16th day of administration shows a significantly low value. Indicated. In all cases, ulcers and tissue defects were not observed in the inflamed sites.

3. Observation of Pruritus Behavior FIG. 3 shows a graph summarizing the observation results of pruritus behavior for the control group and the test substance group. In both groups, the number of pruritus at the first induction was high at the second induction, and only the test substance group was higher at the third induction.
From day 9 to day 35 of administration, the number of pruritus gradually decreased in both groups except for the high value of the control group on day 30 of administration. The test substance group on the 23rd, 30th, and 35th days after administration (36th day) showed a lower value than the control group, but no significant difference was observed.

  As described above, administration of a test substance to inflammation produced by PiCl sensitization / induction in NC mice reduced inflammation scores such as hair loss, pruritus behavior and auricle thickness. These results mean that the administration of HA4 alleviates symptoms in atopic dermatitis model mice, and strongly suggests that HA4 is effective in the treatment of atopic dermatitis.

  In addition, in the right auricle thickness on the opposite side to which the test substance was applied, the auricular thickness was significantly increased compared to the control group. This is because the applied HA4 was transferred from the skin to the blood, Suggests acting on the other side. This strongly suggests that the treatment for atopic dermatitis with systemic symptoms is successful.

[Example 2: Confirmation of therapeutic effect of HA4 in patients with atopic dermatitis]
As the agent of the present invention, the HA4 combination coating agent described in Table 1 was used.

  A 16-year-old female (height 169 cm, weight 50 kg) suffering from atopic dermatitis was applied as a volunteer to the cream. The patient was diagnosed with atopic dermatitis at an early age and was not completely cured. The patient presents with mild, chronic lesions such as infiltrative erythema, prurigo, crust, scratches, papules and pruritus. The patient was applied the above cream on the inside of the elbow for 1 week after bathing in the morning and evening. One week after administration, chronic lesions and pruritus disappeared (FIG. 4). Other than hyaluronic acid tetrasaccharide, it is a general base material and is not known to be effective in the treatment of atopic dermatitis. Therefore, this effect is considered to be an action of hyaluronic acid tetrasaccharide, and it was suggested that hyaluronic acid tetrasaccharide is effective in the treatment of atopic dermatitis.

Claims (1)

A therapeutic or preventive agent for atopic dermatitis comprising purified and isolated hyaluronic acid tetrasaccharide as an active ingredient.