JP3342672B2 - Epidermal thickening inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for controlling epidermal hyperplasia.

[0002]

2. Description of the Related Art The skin has an appearance and appearance.
It has an important function as a boundary wall between a living body and the outside world. The skin intrinsically prevents the loss of water and various biological components from tissues, controls the evaporation of water on the body surface, and maintains the body temperature constantly, while the physical and chemical stimuli (temperature , Humidity, ultraviolet rays, and various chemical substances). Thus, the skin plays a very important role in the vital activity of living organisms due to its physiological functions.

However, when changes in the external environment or changes in physiological functions (due to diseases or aging) occur, dysfunction of the skin tissue occurs, and epidermal thickening, parakeratosis, and epidermal lipid metabolism due to enhanced turnover of the skin. Abnormalities are often induced. Such interactions of internal and external factors result in various vulnerable skin problems such as dry or greasy skin and dandruff.

Heretofore, the main methods for preventing and improving these skin problems have been to apply a synthetic or natural moisturizing component to prevent the skin from drying and increase the moisturizing ability of the skin, or to apply a blood circulation promoting agent. Attempts have been made to improve the skin blood circulation promoting effect.

[0005]

However, the above-mentioned conventional methods not only evaluate the effectiveness of prevention and improvement of various skin troubles, but also the continuity of the effects, and the stability of drugs.
It has many problems in terms of safety and the like. In particular, since the conventional method generally replenishes moisture on the surface of the stratum corneum or partially replenishes moisturizing components, its effect or effect is only temporary and has a permanent improvement effect. Can not be expected. Therefore, a prompt solution to this problem has been desired.

[0006]

Means for Solving the Problems The present inventors have found that sphingosine, which is a physiologically active ingredient present in the epidermis, suppresses epidermal hyperplasia caused by ultraviolet rays and various components.

[0007] That is, the present invention provides an epidermal hyperplasia inhibitor comprising sphingosine.

The sphingosine used in the present invention preferably has 12 to 24 carbon atoms in the carbon chain. In addition, whether a natural (D (+) form) optically active substance or a non-natural (L (+) form) optically active substance is used, or a mixture of a natural form and a non-natural form is used. Is also good. The relative configuration of the above compound may be a natural configuration, another non-natural configuration, or a mixture thereof. A specific example of sphingosine is represented by the following equation (1).

[0009]

Embedded image

[0010] Sphingosine is obtained by extracting sphingolipid or the like from an appropriate tissue (for example, bovine brain), hydrolyzing and extracting with an organic solvent.

Also, the Journal of the American Chemical Society (J. Am. Chem. So
c. 95, 4098 (1973), Journal of Lipid Research (J. Lipid Re).
s. 19, 250 (1978), Tetrahedron Letters.
29, 239 (1988), tetrahedron (T
etrahedron) 42, 5961 (1986)
Year)).

The amount of sphingosine added to the epidermal hyperplasia inhibitor of the present invention is not particularly limited, but when it is used as an emulsified skin external preparation, it is usually 0.001% of the total composition.
To 5% by weight (hereinafter expressed as%), particularly 0.01 to 1% by weight.
% Is preferred. When used as an oily external preparation for skin based on a liquid hydrocarbon such as squalane, it is usually 0.01 to 10%, particularly preferably 0.05 to 2%.

The epidermal thickening inhibitor of the present invention can take various forms such as an external preparation for medicated skin, an external preparation for cosmetics, and a cosmetic. Examples of the medicated skin external preparation and the cosmetic skin external preparation include various ointments containing a pharmaceutically active ingredient. The ointment may be any of those based on an oily base and those based on an oil / water or water / oil type emulsified base. The oily base is not particularly limited and includes, for example, vegetable oil, animal oil, synthetic oil, fatty acid, and natural or synthetic glyceride. The medicinal component is not particularly limited, and for example, an analgesic / inflammation agent, an antipruritic, a bactericidal disinfectant, an astringent, an emollient, a hormonal agent, and the like can be used as needed.

When used as cosmetics, oils, humectants, ultraviolet absorbers, alcohols, chelating agents, pH adjusters, preservatives, thickeners, pigments, and the like commonly used as cosmetic ingredients are used. Flavors and the like can be arbitrarily combined and blended. As cosmetics, various uses and forms, for example, water / oil, oil / water emulsified cosmetics, creams,
It can be used as a cosmetic emulsion, lotion, oily cosmetics, lipstick, foundation, skin cleanser, hair tonic, hair styling agent, hair restorer, hair restorer and the like.

The epidermal thickening inhibitor of the present invention can be prepared into the above various forms by a conventional method.

[0016]

EFFECTS OF THE INVENTION The agent for controlling epidermal hyperplasia of the present invention has a remarkable inhibitory effect on epidermal hyperplasia caused by ultraviolet rays and various other factors.

[0017]

【Example】

Reference Example 1 Inhibitory effect of sphingosine derivative on DNA synthesis of epidermal keratinocytes (1) Method a) Culture of human keratinocytes Epidermal keratinocytes are human normal keratin cells sold by Kurabo Industries, Ltd. Purified cells (trade name: Epipack) were purchased and used. The cells were maintained and passaged using a medium for human normal keratinocytes (trade name: K-GM) sold by the company. b) Measurement of DNA synthesis (thymidine incorporation measurement) Keratinocytes cultured in a proliferating state in a 24-well plate were used. First, the medium in each well was removed by suction, and 450 μl of K-GM to which no pituitary extract was added was added to replace the medium. Thereafter, a sphingosine derivative was added. Further, 0.2 μCi / ml [ ³ H] thymidine was added over time, followed by incubation for 4 hours. After that, the supernatant was removed by suction, washed three times with PBS (-), and then washed 5 times.
00 μl of 2N NaOH was added. After incubation at 37 ° C. for 10 minutes, the same amount of 2N HCl was added for neutralization, and 4 ml of ice-cooled 10% trichloroacetic acid was added.
Let stand for minutes. After collecting the precipitate with a glass filter, the precipitate was washed three times with 3 ml of ice-cooled 10% trichloroacetic acid. After washing the filter once with 3 ml of ice-cold ethanol, the glass filter was air-dried and its radioactivity was measured with a liquid scintillation counter to calculate the amount of thymidine incorporated into the cells.

(2) Results The results are shown in FIG.

FIG. 1 shows C ₁₂ dihydrosphingosine (2
- amino-1,3-dodecanediol), C ₁₂ dimethyl dihydrosphingosine [2- (dimethylamino) -
1,3-dodecanediol], C ₂₀ dihydrosphingosine (2-amino-1,3-eicosanediol Sanji ol), and C ₂₀ dimethyl dihydrosphingosine [2- (dimethylamino) -1,3-eicosanediol Sanji ol] (C ₁₂ etc. indicate the carbon number of the carbon chain) at 10 μM (upper),
And [ ³ H] thymidine incorporation rate (control%) when 100 μM (lower) was added. In particular, C
When 100 μM each of ₁₂ dihydrosphingosine and C ₁₂ dimethyldihydrosphingosine was added, [ ³
H] A remarkable decrease in the thymidine incorporation rate was observed.

From the above, it has been clarified that the rate of incorporation of thymidine is significantly reduced by the addition of the sphingosine derivative, that is, the DNA synthesis of human epidermal keratinocytes is significantly inhibited.

Example 1 Effect of sphingosine and sphingosine derivatives on transglutaminase activity of epidermal keratinocytes (1) Measurement of transglutaminase activity Keratinocytes cultured in a 6-well plate in a proliferating state were used. The medium in each well was removed by suction, and 2 ml of K-GM to which no pituitary extract was added was added to replace the medium.
Thereafter, sphingosine or dimethyl sphingosine was added. After 24 hours, each well was diluted with PBS (-) for 3 hours.
After washing twice, the cells were peeled and collected with a rubber policeman. The obtained cell suspension was centrifuged at 2,500 rpm for 10 minutes to collect a sediment. Add buffer solution (a) [10
mM Tris-HCl buffer, 10 mM DTT,
0.5 mM EDTA; pH 7.4]
Sonication was performed by ultrasonic twice for 1 minute. The resulting suspension was ultracentrifuged at 25,000 rpm for 30 minutes,
The supernatant was obtained. After dispensing a certain amount of the supernatant, each reaction solution [300 mM Tris-HCl buffer,
pH 8.1, 60 mM CaCl ₂ 100 μl, 30 mM
100 μl of DTT, 100 μl of distilled water containing 540 μg of dimethyl casein, 50 μl of 12 mM putrescine,
2.5 μCi [ ¹⁴ C] putrescine 50 μl, distilled water 10
0 μl mixed solution] and incubated at 37 ° C. for 1 hour. Then 10% trichloroacetic acid 600
After adding μl and leaving the mixture to stand for 30 minutes, the precipitate was collected using a 0.45 μm nitrocellulose membrane. The membrane was washed with 15 ml of 5% ice-cold trichloroacetic acid (1 ml).
% Putrescine), the radioactivity on the membrane was calculated with a liquid scintillation counter.

(2) Results FIG. 2 shows the transglutaminase activity (control%) when sphingosine and dimethyl sphingosine were added at 10 μM, respectively. The addition of sphingosine or dimethyl sphingosine increased the transglutaminase activity of the epidermal keratinocytes by 1.8 to 2.2 times compared to the control. Therefore, it was revealed that sphingosine and dimethyl sphingosine have a keratinocyte differentiation-inducing activity and suppress epidermal parakeratosis.

Example 2 Inhibitory effect of sphingosine on epidermal hyperplasia (1) Method After shaving the auricle of 25 4 to 6-week-old white guinea pigs, ultraviolet rays (UVB: 2 to 3 MED) were irradiated. Immediately after irradiation, a squalane solution containing 0.05% sphingosine was applied once a day in 50 μl portions for one week. One week later, the pinna of the guinea pig was excised, and a section of skin tissue was prepared. Each tissue sample was photographed under a microscope, and the thickness of the epidermis was measured to examine the inhibitory effect of each evaluation sample on thickening due to ultraviolet rays. (2) Results FIG. 3 shows the skin thickness obtained by ultraviolet irradiation (control) and ultraviolet irradiation + sphingosine addition.
Sphingosine was found to suppress epidermal thickening.

Example 3 Production of W / O cream

[0026]

(1) Sphingosine 0.1 (2) Cholesterol 0.5 (3) Cholesteryl isostearate 1.0 (4) Polyether-modified silicone 1.5 (5) Cyclic silicone 20.0 (6) Methylphenylpolysiloxane 2.0 (7) Methylpolysiloxane 2.0 (8) Magnesium sulfate 0.5 (9) 55% ethanol 5.0 (10) Carboxymethylchitin (Chitin Liquid, manufactured by Ichimaru Falcos) HV) 0.5 (11) Purified water balance

(1) to (7) were heated to 80 ° C. to dissolve, and (8) to (11) were added thereto and mixed uniformly.
A / O cream was prepared.

Example 4 Production of an ointment

[0029]

(% By weight) (1) sphingosine 0.5 (2) white petrolatum balance (3) cholesteryl isostearate 3.0 (4) liquid paraffin 10.0 (5) glyceryl ether 1.0 (6) Glycerin 10.0

(1) to (6) were heated to 80 ° C. to dissolve and then cooled to prepare an ointment.

Each of the W / O creams and ointments obtained in Examples 3 and 4 has an inhibitory effect on epidermal turnover enhancement such as parakeratosis, epidermal hyperplasia, and lipid metabolism abnormality, and normal skin function. Recovery and homeostasis.

[Brief description of the drawings]

FIG. 1 is a graph showing a change in the thymidine incorporation rate due to addition of a sphingosine derivative in Reference Example 1.

FIG. 2 is a diagram showing a change in transglutaminase activity caused by addition of sphingosine or a sphingosine derivative in Example 1.

FIG. 3 is a graph showing changes in skin thickness due to ultraviolet irradiation and addition of sphingosine in Example 2.

──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Tsutomu Fujimura 4594 Hanawa, Kaimachi-cho, Haga-gun, Tochigi Prefecture (72) Inventor Yukihiro Ohashi 30-19, Koshidocho, Utsunomiya-shi, Tochigi (72) Inventor Akira Kawamata Hanabo, Utsunomiya-shi, Tochigi JP-A-1-22810 (JP, A) JP-A-2-169511 (JP, A) JP-A-3-83908 (JP, A) JP-A-62-178 120308 (JP, A) JP-A-61-260008 (JP, A) JP-A-4-193815 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 7 / 00-7 / 50 CA (STN)

Claims (1)

(57) [Claims] An epidermal hyperplasia inhibitor comprising sphingosine.