JPH06135843A - Agent for skin - Google Patents

Abstract

PURPOSE:To obtain an agent for skin capable of protecting dermatopathy caused by pathogenic microorganisms, etc. CONSTITUTION:An agent for skin comprises a cell of a microorganism having adhesivity to the surface of skin or an extract of the cell. A substance adherent to epidermal cell is bonded to the surface of skin and prevents infection and fixation of pathogenic organisms. Consequently, the agent is effective for preventing and treating infectious diseases of skin and in the field of medicine and cosmetic.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the protection of various skin disorders, in particular skin, containing microorganisms or bacterial cell extracts of microorganisms having adhesion to the skin surface, for example, microorganisms belonging to the genus Staphylococcus. The present invention relates to a skin agent for the purpose of protection against infection.

[0002]

BACKGROUND OF THE INVENTION Chemotherapy with antibacterial agents, antifungal agents, antiviral agents, etc. for the protection of skin infection of pathogenic microorganisms such as bacteria, fungi and viruses, and especially for the treatment of diseases caused by these pathogenic microorganisms. External preparations containing agents are widely used. However, such conventional external preparations and the like still have serious problems such as side effects during use and appearance of drug-resistant bacteria. Furthermore, the desired effect is not always satisfactory.

The skin is the largest organ accounting for 16% of human body weight, which is three times as large as that of the liver. The epidermis, which is the outermost layer of the three-layered skin, is covered with chemically relatively stable keratinized cells, and the healthy epidermis protects against pathogenic microorganisms such as bacteria and mold, chemicals and physical damage. In addition, it also has a function as a barrier against ultraviolet rays.

A large number of microorganisms, which are symbiotic bacteria, are colonized on normal skin such as human cheeks to form a skin flora. The presence of microorganisms on the skin surface can be understood as an ecosystem. That is, the types and amounts of the microorganisms constituting the skin flora are maintained while maintaining a complicated relationship such as an interaction between the microorganisms and between the microorganism and the skin, and an antagonistic effect.

However, when the balance of such an ecosystem is lost, there are a wide variety of skin pathogenic microorganisms that cause skin lesions beyond physicochemical and biological barriers. In addition, intractable resistant bacterial infections such as methicillin-resistant Staphylococcus aureus (MRSA), etc. are also serious, because skin abnormalities due to internal causes such as diabetes mellitus, internal infections, or infections at injured sites Has become a problem.

[0006]

Staphylococcus is divided into coagulase-positive Staphylococcus aureus and coagulase-negative bacterial species (S. epidermidis, etc.). Staphylococcus aureus is one of the passage flora in normal skin or nasopharynx and often causes infection. It is widely distributed on the skin surface in doctors and nurses in hospitals, hemodialysis patients, diabetics, intravenous drug administration, and patients with psoriasis and atopic dermatitis. In particular, in the lesioned area, infection by this bacterium is remarkable.

The virulence of pathogenic microorganisms is represented by invasiveness to host tissues and toxin production. Invasion of pathogenic microorganisms begins with the attachment of tissue to the cell surface. Regarding this attachment or adhesion, there is a specific involvement of the surface substance on both the microbial side and the host tissue side.

A few examples of microorganisms causing skin lesions will be given. Staphylococcus aureus is a typical bacterial species that causes suppurative infection. The skin diseases caused by this fungus are: halebroidery, cellulitis, flunkel (▲ set ▼), folliculitis, carbunkel (▲ you ▼), wound infection, purulent sweating gland inflammation, impetigo, staphylococcal burn-like skin syndrome. , Etc. It is also a causative bacterium of pyelonephritis, otitis media, mastitis, etc. Staphylococcus aureus is a bacterial species that is seen as a member of the indigenous flora or as one of the passage flora, and does not normally express pathogenicity. It is the causative bacterium of a disease that presents a serious medical condition.

[0009] Tenfu is a chronic superficial dermatomycosis, in which pale brown spots or pale depigmented spots are formed and acrid rice bran-like desquamation. The causative organism of this disease is Malassezia furfur
( Malassezia furfur ), it has been reported that this bacterium is also involved in the development of folliculitis, interstitial rash, seborrheic dermatitis, psoriasis, atopic dermatitis and the like. The bacterium is highly isolated from healthy skin such as the back, head, chest, upper arms, lower legs, and hands, especially from seborrheic parts. As a clinical symptom due to infection with this bacterium, light brown spots or depigmented spots frequently occur, which may be accompanied by edema, redness and itching, which is a major cosmetic problem.

Opportunistic infectious bacteria such as Candida and Pseudomonas aeruginosa develop infections due to impaired balance between the virulence of pathogenic bacteria and the infection resistance of the host. Cancer or diabetes, renal failure, patients with basic diseases such as immunodeficiency or anticancer agents, immunosuppressants or steroids administration and administration of radiation, and decrease in the immune resistance of the body due to irradiation, etc., significantly increase fungal infections,
This is a serious clinical problem.

[0011] Such infectious diseases have more complicated problems in treatment and prevention such as the appearance of drug resistant bacteria including opportunistic infectious diseases, and effective and safe methods are required.

An object of the present invention is to provide a dermatological agent capable of exerting a useful effect in the prevention or treatment of microbial infections by preventing the above-mentioned skin disorders, particularly preventing the adhesion of pathogenic microorganisms. is there.

[0013]

Means for Solving the Problems As a result of research for the above-mentioned purpose, the present inventors have found that a microorganism having an ability to adhere to the skin surface or an extract thereof is a pathogenic microorganism such as Staphylococcus aureus on the skin surface. The present invention has been accomplished by finding that the adhesion and fixing of the toner can be prevented.

The microorganism used in the present invention is not limited to the genus, species, strains, etc., and has the ability to adhere to the skin surface, and the microorganism itself or the extract is another pathogenic bacterium. Any one can be used in the present invention as long as it prevents adhesion of microorganisms such as fungi to the skin surface. For example, a coagulase-negative staphylococcus, which is a normal resident bacteria on human cheek, is a good example thereof, and its novel strain Staphylococcus sp. SCRC-763.
1 ( Staphylococcus sp. SCRC-7631) has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as a microbe research institute number.

The method for separating the strain and the mycological properties are shown below.

Separation Method A normal cotton is swabbed with a commercially available sterile cotton swab moistened with sterilized physiological saline to collect indigenous bacteria on the cotton swab. Disperse this by stirring in an appropriate dispersion such as sterile physiological saline. If necessary, the solution is serially diluted 10-fold, and a part of the diluted solution is evenly spread on, for example, a tryptic soy agar plate medium (Diff Corporation, USA). Alternatively, staphylococci can be selectively separated reliably by using a mannitol salt agar plate medium (Eiken Kagaku Co., Ltd.), which is a selective medium for Staphylococcus . The plate medium is aerobically cultured in a 37 C incubator for 24-48 hours to form bacterial colonies. A part of the expressed single colony is thinly applied to a plate medium of the same species and cultured in the same manner to obtain a single colony again. The same operation is repeated to obtain a pure strain.

The mycological properties of staphylococci (coagulase-negative Staphylococcus sp.) Isolated from facial skin using the mannitol salt agar plate medium according to the above-mentioned separation method are shown (Table 1).

[0018]

[Table 1] Table 1 Gram stain + Bacterial morphology Spherical obligate aerobic-facultative anaerobic + Colony on Tryptic Soy agar white, opaque Growth on mannitol salt agar + Colony on mannitol salt agar white, opaque adhesion to Man'nitotto degradable over catalase + coagulase over human buccal epithelial cells +

In carrying out the present invention, microorganisms having an adhesive property to the skin surface, for example, resident coagulase-negative Staphylococcus aureus on human cheeks are cultured by a conventional method, for example, in a liquid medium or an agar plate medium, Are collected by centrifugation, washed, and suspended or dried to obtain a bacterial cell powder.

The medium used in this case is not particularly limited, and examples of the agar plate medium are shown in Table 2.
The GAM liquid medium shown in Table 3 can be prepared as an example of the medium and liquid medium shown in.

[0021]

[Table 2]

[0022]

[Table 3] Table 3 (Composition of GAM broth "Nissui" 1 liter 59.0 g) Peptone 10.0 g Soybean peptone 3.0 g Proteose peptone 10.0 g Digested serum powder 13.5 g Yeast extract 5.0 g Meat extract 2.0 g Liver extract powder 1.2 g Glucose 3.0 g Phosphorus Potassium dihydrogen acid 2.5 g Sodium chloride 3.0 g Soluble starch 5.0 g L-Cysteine hydrochloride 0.3 g Sodium thioglycolate 0.3 g pH 7.3 ± 0.1 (15 minutes at 115C. High-pressure steam sterilization)

(Culturing method) Microorganisms that adhere to the skin surface (for example, coagulase-negative Staphylococcus sp.)
Inoculate 3 ml of sterilized GAM liquid medium and incubate at 37C for 10
Aerobic static culture (preculture) for 16 hours, approximately 10 ⁹ cells /
Obtain ml of culture.

1 ml of this bacterial solution is inoculated into 1 liter of a sterilized GAM liquid medium, or this bacterial solution is applied on an agar plate and cultured at 37C for 10 to 48 hours. Cultivation in a liquid medium can achieve better growth by stirring or shaking during the culture.

(Collecting, Washing and Drying Method) The culture solution obtained as described above is centrifuged at 8000 to 12000 rpm to obtain bacterial cells (precipitate), and the bacterial cells are subjected to the same procedure. Wash twice with saline (0.85% NaCl). Alternatively, the cells on the agar plate are suspended in physiological saline and washed twice in the same manner. The washed bacterial cells are appropriately dried by a freeze-drying method or the like to obtain bacterial cell powder. The obtained bacterial cells may be live bacterial cells or dead bacterial cells appropriately treated.

(Isolation Method of Attachment Inhibitor) Epidermal-adherent cells showing good growth on an agar plate medium were suspended in physiological saline and collected by centrifugation at 3000 to 10000 rpm for 15 minutes. And wash with saline. The cells are suspended in phosphate-buffered physiological saline (PBS), the cell concentration is adjusted appropriately, and then lysozyme is added to 1 mg / ml. If necessary, lysostaphin may be added appropriately. Further, the microbial cell may be a live microbial cell or a killed microbial cell by appropriate treatment.

This suspension is stirred at 37C for 10 to 10 minutes.
After reacting for 16 hours, a supernatant is obtained by centrifugation at 10,000 rpm for 15 minutes. The obtained supernatant is dialyzed against distilled water and the dialyzed solution is freeze-dried to obtain a dry powder.

(Experimental Method for Attaching Bacterial Cells to Epidermal Cells) One side of a double-sided adhesive tape is strongly pressed against a human cheek to exfoliate and collect keratinized cells on the epidermis. This tape is cut into an appropriate size (5 mm square), and the side free of cells is attached to a slide glass and fixed.

The concentration of the bacterial solution obtained as described above is appropriately adjusted using physiological saline or PBS (OD ₆₆₀ =
1 to 10), the bacterial solution is overlaid on epidermal cells fixed on a slide glass.

After leaving at room temperature (25C) for 10 to 20 minutes,
Epidermal cells are washed with physiological saline or PBS to remove non-adherent bacteria. Then counted adhering bacteria were stained overlaid with crystal violet staining solution thereto, epidermal cells one per (7.4 × 10 ^{over 6} cm ²⁾ or to calculate the number of attached bacteria per given area.

(Adhesion Inhibition Experimental Method) (1) The substance isolated from the adherent bacterial cells by the above-mentioned method is added to the adherent bacterial suspension, and the bacterial solution is counted according to the adherent experimental method. . (2) The substance isolated from the adherent bacterial cells by the above-mentioned method is dissolved in physiological saline or PBS, and this is overlaid on the epidermal cells and allowed to stand at room temperature (25C) for 10 to 20 minutes. The epidermal cells with selective saline or PBS. Then, the number of adherent bacteria is counted according to the adhesion experiment method. The adhesion inhibition rate is expressed by the following equation. Adhesion inhibition rate (%) = (1-number of adherent bacteria when treated / number of adherent bacteria without treatment) x 100

[0032]

【Example】

Example 1 (Isolation of Adhesion Inhibiting Substance) Coagulase-negative Staphylococcus sp. SCR was added to the agar plate medium (Petridish having an inner diameter of 9 cm) described above (Table 2).
0.1 ml of GAM liquid culture solution of C-7631 was uniformly applied, and cultured aerobically at 37C for 40 hours.

10 ml of PBS was used for each agar plate.
Suspended in. The cells of this suspension were collected by centrifugation at 3000 rpm for 20 minutes. After the cells were washed twice by centrifugation under the same conditions, 5 ml of PB was added per agar plate.
Suspended in S, to which was added 5 mg lysozyme.

This suspension was stirred at 37C for 10 hours.
Then, this was centrifuged at 10,000 rpm for 15 minutes to obtain a supernatant, which was filtered through a 0.45 μm membrane filter, and the filtrate was dialyzed against distilled water. The dialyzed solution was freeze-dried to obtain 45 mg of dry powder per agar plate.

Example 2 (Experimental example of cell attachment to epidermal cells) Coagulase-negative Staphyl obtained according to the method described above.
OD of the concentration of the physiological saline suspension of ococcus sp.
Adjusted to ₆₀₀ 10.

This bacterial solution was layered on human buccal epidermal keratinized cells, allowed to stand at room temperature (25C) for 20 minutes, and then the epidermal keratinized cells were washed with physiological saline. Then, the cells were stained with a crystal violet stain and the number of adherent cells of 10 to 20 epidermal cells was counted under a microscope to calculate the number of adherent cells per cell (Table 4). As a control, epidermal cells that did not overlay the bacterial suspension were similarly stained and observed under a microscope.

[0037]

[Table 4] Table 4 Number of adherent bacteria per epidermal cell (on average)

Example 3 (Adhesion Inhibition Experimental Example) Staphylococcus aureus (S. aureus IFO12732)
Was inoculated into the GAM liquid medium described above (Table 3), 16 at 37C
After culturing for a time, the cells were collected by centrifugation at 3000 rpm for 15 minutes. This was washed once with physiological saline and then suspended in PBS to adjust the bacterial concentration to OD ₆₀₀ 10.

The dry powder preparation prepared from the adherent bacterial cells in Example 1 was used as the S. Added to Aureus cell suspension (1% w
/ V), and according to the adhesion experimental method shown in Example 2,
The adherence to human cheek epidermal keratinocytes was examined.

S. Aureus cells adhered to the buccal epidermal cells and the surface of the adhesive tape in the same manner. When the preparation isolated from the adherent cells was added, both the adherence to the epidermal cells and the surface of the adhesive tape were significantly inhibited. The results are shown in Table 5.

[0041]

[Table 5] Table 5 Number of adherent bacteria (average) Adhesion inhibition rate (per 6.25 x 10 ^-6 cm ² ) (%) Control epidermal cells 86.1 tape 82.3 Treatment (added) epidermal cells 4.7 94.5 tape 2.2 97.3

Example 4 (Experimental Example of Inhibition of Adhesion) Fresh Coagulase-Positive S Isolate from Skin Surface of Human Buccal Inflammation
After culturing taphylococcus sp. SA-1 and SA-3 in a GAM liquid medium, collecting and washing, a PBS suspension was prepared.

According to Example 2, the sample obtained in Example 1 was added to this bacterial solution (1% w / v), and the adhesion of the test strain to the buccal epidermal cells was examined. As shown in Table 6,
A significant inhibition of adhesion was observed by adding the standard preparation obtained in Example 1.

[0044]

[Table 6] Table 6 Number of adherent bacteria (average) Adhesion inhibition rate (per 1x10 ^-6 cm ² ) (%) Control (non-added) SA-1 20.9 SA-3 15.6 Treatment (added) SA-1 1.4 93.3 SA-2 1.1 93.0

Example 5 (Adhesion Inhibition Experimental Example) Freshly isolated strain coagulase-positive S from the skin surface of human cheek inflammation
Culture taphylococcus sp. SA-1 in GAM liquid medium,
After collecting and washing the cells, a PBS suspension was prepared.

The preparation obtained in Example 1 was dissolved in PBS (1%
w / v), and this was layered on human buccal epidermal keratinocytes, left at room temperature (25C) for 15 minutes, and then washed with PBS. Then, an adhesion experiment was conducted according to Example 2. The results are shown in Table 7. By treating the epidermal cells with the bacterial cell preparation, the adhesiveness of the SA-1 strain was significantly inhibited.

[0047]

[Table 7] Table 7 Number of adherent bacteria (average) Adhesion inhibition rate (per 1x10 ^-6 cm ² ) (%) Control 18.4 Treatment 4.5 75.4

Example 6 (Formulation Example) Table 8 shows an ointment formulation example containing the preparation obtained in Example 1.

[0049]

[Table 8] Table 8 Specimen obtained in Example 1 1-10% Base 90-99

Example 7 (prescription example) Table 9 shows a prescription example of a skin preparation containing the preparation obtained in Example 1.

[0051]

[Table 9] Table 9 Specimen obtained in Example 1 1.0% stearyl alcohol 7.0 squalane 5.0 reduced lanolin 2.0 polyoxyethylene cetyl ether 5.0 propylene glycol 5.0 purified water 75.0

Examples of formulation of the dermatological agent of the present invention are given in Examples 6 and 7, but these are examples and can be appropriately changed as necessary, and are not limited to the examples. If necessary, moisturizers, surfactants, antioxidants, lubricants, preservatives, pigments, fragrances, and other components can be added and blended. The dermatological formulation includes powders and liquids. , Emulsion, cream, ointment and the like can be appropriately selected.

[0053]

EFFECTS OF THE INVENTION It is possible to produce a dermatological agent having a high effect for the purpose of preventing skin infection. By using this dermatological agent, doctors and nurses in hospitals, hemodialysis patients, patients with basic diseases such as diabetes and renal failure, intravenous administration of drugs, patients with psoriasis and atopic dermatitis, further aging, overwork, Particularly, the preventive effect and therapeutic effect of severe skin infections caused by a decrease in the immune system of the living body such as administration of anticancer agents, immunosuppressants, steroids, etc., immunodeficiency, irradiation, etc. can be expected.

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[Procedure amendment]

[Submission date] November 27, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

The microorganism used in the present invention is not limited to the genus, species, strains, etc., and has the ability to adhere to the skin surface, and the microorganism itself or the extract is another pathogenic bacterium. Any one can be used in the present invention as long as it prevents adhesion of microorganisms such as fungi to the skin surface. For example, a coagulase-negative staphylococcus, which is a normal resident bacteria on human cheek, is a good example thereof, and its novel strain Staphylococcus sp. SCRC-763.
No. 1 ( Staphylococcus sp. SCRC-7631) has been deposited at the Institute of Microbial Science and Technology, the Agency of Industrial Science and Technology as Microorganism Research Institute No. 13217 .

Claims (4)

[Claims] 1. A skin preparation containing a microbial cell or a microbial cell extract having adhesion to a skin surface. 2. The agent for skin according to claim 1, which is intended to prevent skin infection. 3. The skin preparation according to claim 1, wherein the microorganism is a microorganism belonging to the genus Staphylococcus. 4. A microorganism belonging to the genus Staphylococcus is Staphylococcus sp. The skin agent according to claim 3, which is SCRC-7631.