WO2011086891A1 - Composition for non-invasively facilitating the removal of skin pigment - Google Patents

Abstract

Disclosed is a composition capable of actively removing skin pigment. The composition contains ibuprofen piconol as an active ingredient, may optionally contain other ingredients as desired, and can be an effective topical drug for the treatment of chromatosis caused by ultraviolet radiation, wounds, burns, acne, etc..

Description

Composition for non-invasively promoting skin pigment removal

The present invention relates to a composition for non-invasively promoting the removal of skin pigments. In particular, the present invention relates to a composition for non-invasively promoting the removal of melanin in the skin.

[Pigmentation such as spots, freckles, sunburn, etc., is caused by the deposition of melanin on the skin. Melanin is produced in melanocytes by the action of tyrosinase and the like using tyrosine as a raw material. Such pigmentation is particularly cosmetically unfavorable for women.

«Kojic acid, arbutin, ellagic acid, vitamin C and the like are well known as whitening agents for preventing pigmentation. These compounds suppress melanin production by inhibiting the activity of tyrosinase. In addition, non-steroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid and mefenamic acid have been reported to suppress melanin production by suppressing the expression of tyrosinase or by inhibiting the activity of tyrosinase (for example, Non-patent documents 1, 2).

Sato, K., et al., "Down-regulation of tyrosinaserosexpression by acetylsalicylic acid in murine B16 melanoma", Biol. Pharm. Bull., Vol.31 (2008), No.1, pp.33-37. Kazuomi Sato, 2 others, “Inhibitory effect of NSAIDs on melanogenesis of B16 melanoma”, Abstracts of the 129th Annual Meeting of the Japanese Pharmaceutical Association, p.223.

However, the conventional whitening agent suppresses melanin production and prevents pigmentation, and the pigment deposited on the skin cannot be positively removed.

An object of the present invention is to provide a composition capable of positively removing skin pigments.

The present inventor has found that ibuprofen piconol can positively and noninvasively remove skin pigments, and has further studied and completed the present invention.

That is, this invention relates to the following compositions.
[1] A composition for non-invasively promoting removal of skin pigment, comprising ibuprofen piconol.
[2] The composition according to [1], which is a composition for promoting the removal of melanin from the skin.
[3] The composition according to [1] or [2], which is an external preparation for skin.

According to the present invention, a composition capable of positively and non-invasively removing skin pigments can be provided. Therefore, according to the present invention, it is possible to provide an external preparation for skin that is effective for treatment of pigmentation caused by ultraviolet irradiation, trauma, burns, acne, etc., and removal of tattoos.

It is a photograph which shows the change of the application | coating area | region of a guinea pig back part. It is a graph which shows the change of the L * value of the application | coating area | region of a guinea pig back part. It is a graph which shows the change of the a * value of the application | coating area | region of a guinea pig back part. It is a graph which shows the result of a tyrosinase inhibition test. FIG. 5A is a photograph of the skin of an untreated mouse, and FIG. 5B is a photograph of the skin of a mouse to which an emulsion containing no IBP is applied. 6A is a photograph of the skin of a mouse to which an emulsion containing 10% IBP was applied, and FIG. 6B is a photograph of the skin of a mouse to which a retinoic acid preparation was applied. FIG. 7A is a graph showing the percentage of basal cells expressing Ki-67, and FIG. 7B is a graph showing the average thickness of the epidermis of each individual.

The composition of the present invention is a composition for actively and non-invasively removing skin pigments, and is characterized by containing ibuprofen piconol as an active ingredient. The composition of the present invention may contain other optional components.

In the present specification, the “skin pigment” is not particularly limited, and examples thereof include melanin and pigments artificially fixed in the skin. “Non-invasive removal of skin pigment” includes discharging the pigment from the skin, decomposing or changing the pigment in the skin, and promoting these.

Ibuprofen piconol (2-pyridylmethyl (RS) -2- (4-isobutylphenyl) propionate) is known as a non-steroidal anti-inflammatory drug (NSAIDs) effective for acute eczema, contact dermatitis, atopic dermatitis, etc. However, the action of removing skin pigment has not been known so far. The inventor has found that ibuprofen piconol can positively remove skin pigments. As shown in the Examples, ibuprofen piconol is considered to have little tyrosinase inhibitory action. Therefore, the pigment removal action of ibuprofen piconol is not a passive removal of melanin gradually due to skin turnover while inhibiting melanin production, but actively removes skin pigment (eg melanin) It is estimated that.

The content of ibuprofen piconol in the composition of the present invention is not particularly limited, but is preferably 0.01 to 20% by weight or less, particularly preferably in the range of 1 to 10% by weight. Although ibuprofen piconol contained in the composition of the present invention may be produced by using a known method, a commercially available product may be used.

The composition of the present invention can be used, for example, as a pharmaceutical, a quasi-drug, or a cosmetic. In addition, the composition of the present invention can be administered by any known administration route. For example, the composition of the present invention can be used as a parenteral or oral administration agent. Examples of parenteral dosage forms include extracts, plasters, spirits, suppositories, suspensions, tinctures, ointments, poultices, liniments, lotions, aerosols, eye drops, injections Agents and the like. Examples of dosage forms for oral administration include tablets, capsules, powders, fine granules, solutions, troches, jellies and the like. The composition of the present invention can also be in the form of a cosmetic composition such as lotion, cream, lotion, emulsion, foam, foundation, pack, skin cleanser, shampoo, rinse, conditioner. . A preferred embodiment of the composition of the present invention includes an external preparation for skin.

Formulation can be performed by known formulation techniques, and appropriate formulation additives can be added to the formulation. Examples of formulation additives include excipients, binders, disintegrants, lubricants, fluidizers, suspending agents, emulsifiers, stabilizers, moisturizing (wetting) agents, preservatives, solvents, dissolution aids. Agents, preservatives, corrigents, sweeteners, pigments, fragrances, propellants and the like. The formulation additive may be appropriately selected within a range not impairing the effects of the present invention, and an appropriate amount may be added.

The dose of the composition of the present invention may be appropriately set according to the sex, age, symptom, administration method, number of administrations, and administration timing of the recipient. Indications include, for example, patients with liver spots, senile pigment spots, Ota nevus, sparrow egg spots, inflammatory pigmentation, frictional dermatosis, petal pigment spots, or the prevention of these diseases Is a healthy person. Preferably, it is a patient with pigmentation.

The composition of the present invention is useful as a pigmentation treatment agent because it actively removes skin pigment. As shown in Examples, melanin was removed by applying the composition of the present invention to the skin, the amount of melanin in the skin was reduced, and the skin became white. Thus, the compositions of the present invention can be used to remove skin pigments.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

[Example 1]
1. Preparation of Formulation According to a method commonly used in the art, an emulsion (base) was prepared by mixing materials having the composition shown in Table 1 below.

Ibuprofen piconol (hereinafter referred to as “IBP”) was mixed with the prepared emulsion so that the final concentration was 10% by mass, and an emulsion containing 10% by mass IBP was prepared as a preparation of the example. In addition, IBP-free emulsion (negative control) and retinoic acid formulation (positive control; water-dispersible gel whose base is known to have no pharmacological effect on the skin) were also prepared as comparative preparations. .

2. Melanin removal promotion effect test To examine whether the composition of the present invention has a pigment removal promotion effect, a melanin removal promotion effect test was conducted.

Four colored guinea pigs were prepared by irradiating colored guinea pigs with melanin pigment-producing cells (Weiser-Maples, 11 weeks old, male; Tokyo Experimental Animal Co., Ltd.) with ultraviolet rays. Specifically, the back of each individual was shaved and UV-A (11 J / cm ² per day; 617 μW / cm ² × 5 hours) and UV-B (33 mJ / cm ² per day; 0.55 mW) / Cm ² × 1 minute) was repeated once a day for 5 days a week (from Monday to Friday) for a little less than 6 weeks (28 times in total). The UV-A lamp used was FL20S · BLB-A (wavelength range 315 to 380 nm, peak wavelength 352 nm; Toshiba Corporation), and the UV-B lamp used UVM-28 (peak wavelength 302 nm; UVP). . UVX Digital Radiometer (UVP) was used as the UV illuminance measuring instrument. After the irradiation period, the sample was left for one week in order to suppress the effects of inflammation caused by ultraviolet irradiation.

The application area with a size of 2 cm × 2 cm was set to 6 sections per animal on the back of each individual where pigmentation occurred. After 30 minutes have passed since the hair of each individual's application area is cut, washed with warm water, and excess water is wiped off, a preparation (10% IBP-containing emulsion, retinoic acid preparation, or IBP-free emulsion) is applied to each application area. 30 mg was applied once a day. This formulation was applied 11 times in total for 15 days (1, 2 weeks: Monday to Friday, 3 weeks: Monday). For comparison, an uncoated area (only ultraviolet irradiation) was also provided.

FIG. 1 is a photograph of the application start date, the guinea pig back formulation application area on the seventh, eleventh and fifteenth days after the start of application. As shown in these photographs, in the area where the retinoic acid preparation and the emulsion containing 10% IBP were applied, significant brightness recovery was observed on the 11th day after the start of application. In the region where the retinoic acid preparation was applied, inflammation with redness was observed, but in the region where the 10% IBP-containing emulsion was applied, no such inflammation was observed. On the other hand, in the area where the IBP-free emulsion was applied, neither lightness recovery nor inflammation was observed as in the non-application area (only UV irradiation).

As an index of melanin production in the skin of each individual, the lightness (L ^* value) of the skin was measured using a color difference meter (CR-400; Minolta). The measured increase in skin brightness was used as an index of the degree of recovery from pigmentation. The L ^* value indicates that the larger the value, the whiter the color. The color difference meter used in this example employs the L ^* a ^* b ^* color system. In the L ^* a ^* b ^* color system, lightness is represented by L ^* , and chromaticity indicating hue and saturation is represented by a ^* and b ^* . a ^* and b ^* indicate the color direction, a ^* indicates the red direction, -a ^* indicates the green direction, b ^* indicates the yellow direction, and -b ^* indicates the blue direction.

FIG. 2 is a graph showing changes in L ^* values in each application region from the formulation application start date (n = 6). The L ^* value of each application area was measured at 10 locations per application area, and the average value was adopted. In the area where the retinoic acid preparation was applied, significant brightness recovery was observed on the seventh day from the application start date. In the region where the 10% IBP-containing emulsion was applied, significant brightness recovery was observed on the ninth day from the application start date. On the other hand, in the area | region which apply | coated the IBP non-containing emulsion, the big difference with the non-application area | region was not recognized.

Comparing the results of the region where the 10% IBP-containing emulsion was applied and the non-application region on the 15th day from the start of the formulation application, it can be seen that the L ^* value was significantly increased by applying the 10% IBP-containing emulsion. . Moreover, when the result of the area | region which apply | coated the emulsion containing 10% IBP and the area | region which applied the emulsion which does not contain IBP is compared, it turns out that L ^* value becomes large remarkably by containing 10% IBP.

Further, as an index of inflammation in the skin of each individual, the redness (a ^* value) of the skin was measured using the above-described color difference meter. The measured change in skin redness was used as an index of the degree of skin inflammation. As described above, the a ^* value indicates that the larger the value, the stronger the redness.

FIG. 3 is a graph showing a change in a ^* value in each application region from the formulation application start date (n = 6). The a ^* value of each coating area was measured at 10 locations per coating area, and the average value was adopted. In the region where the retinoic acid preparation was applied, redness increased significantly from the 4th day to the 11th day after the application start date. On the other hand, in the area where the 10% IBP-containing emulsion was applied and the area where the IBP-free emulsion was applied, no significant difference was observed from the non-application area.

Comparing the results of the area where the retinoic acid preparation was applied on the 9th and 11th day from the start date of the preparation application and the area where the 10% IBP-containing emulsion was applied, the a ^* value was remarkably increased by applying the retinoic acid preparation You can see that

From the above results, it was confirmed that IBP remarkably accelerates the removal of skin pigments without causing a strong skin inflammatory reaction (side effects) known for retinoic acid. At the start of IBP application, the inflammatory reaction caused by ultraviolet irradiation has subsided (confirmed by the a ^* value), so the skin pigment removal promoting effect by IBP is due to actions other than the anti-inflammatory action of IBP.

3. Tyrosinase inhibition test To examine whether the whitening effect of the composition of the present invention is due to suppression of melanin production, a tyrosinase inhibition test was performed. The tyrosinase inhibition test is described in a paper (Roh, JS et al., “Inhibitory Effects of Active Compounds Isolated from Safflower (Carthamus tinctorius L.) Seeds for Melanogenesis”, Biol. Pharm. Bull., Vol. 27 (2004), No. 12, pp. 1976-1978.).

First, a predetermined amount of IBP was dissolved in a 0.1% ethanol aqueous solution to prepare an IBP aqueous solution (concentration: 0 to 1000 μM). As a positive control, kojic acid (114-00493; Wako Pure Chemical Industries, Ltd.), known as a tyrosinase inhibitor, was dissolved in water to prepare an aqueous kojic acid solution (concentration: 0 to 1000 μM).

To 10 μL of the sample solution (IBP aqueous solution or kojic acid aqueous solution), 65 mM of 2.5 mM L-DOPA (Sigma) and 105 μL of 0.1 M phosphoric acid buffer (pH 6.8) were added. After standing at room temperature for 10 minutes, the absorbance (405 nm) was measured. Next, 20 μL of 200 U / mL mushroom tyrosinase (T3824; Sigma) was added to the mixture. After standing at room temperature for 10 minutes, the absorbance (405 nm) was measured. The enzyme activity of tyrosinase was calculated from the change in absorbance.

FIG. 4 is a graph showing the results of a tyrosinase inhibition test. From this graph, it can be seen that the activity of tyrosinase is inhibited when the concentration of kojic acid increases, but the activity of tyrosinase does not change even when the concentration of IBP increases. That is, it can be seen that IBP has no inhibitory effect on tyrosinase. From this result, it is suggested that the melanin removing action by IBP is not due to suppression of melanin production but due to positive removal of melanin.

4). Cell Proliferation Activity Test To examine whether the whitening effect of the composition of the present invention is due to the promotion of skin turnover, the cell proliferation activity of IBP was examined.

The cell proliferation activity was evaluated by counting the number of cells expressing the cell proliferation marker Ki-67. Ki-67 is localized around the nucleus of cells in the G1 phase, in the nucleus of cells in the S and G2 phases, and in the chromosome of cells in the M phase. On the other hand, Ki-67 is not expressed in G0 phase cells. For this reason, Ki-67 is often used as a cell proliferation marker. By using this Ki-67, transition from a non-proliferating cell population to a proliferating cell population can be detected.

A coating area of 2 cm × 3 cm was set on the back of four hairless mice (HR-1, 5 weeks old, male; Nippon SLC Co., Ltd.). After washing the application area of each individual with lukewarm water, 30 mg of the above preparation (10% IBP-containing emulsion, retinoic acid preparation or IBP-free emulsion) was applied once a day. This formulation was applied for 5 days. For comparison, no treatment (cleaning only) was also performed.

On the 5th day, the last application was performed, and after 3 days had passed, the skin (0.5 cm × 1 cm) of the application area of each individual was collected. The collected skin was fixed overnight with a 10% neutral formalin solution. Each skin is wrapped in a paraffin block using a sealed automatic fixed embedding device (ETP-150CV; Sakura Finetech Japan Co., Ltd.) and a paraffin embedding block preparation device (Tissue-Tek TEC; Sakura Finetech Japan Co., Ltd.). Buried. Next, a 4 μm-thick section was prepared from the paraffin block using a microtome (ROM-380; Daiwa Kogyo Co., Ltd.). The obtained section was attached to a slide glass and dried at 40 ° C. overnight, and then the paraffin was removed.

The antigen-stimulation treatment was performed by immersing the slide glass with the section attached in 10 mM citrate buffer (pH 6.0) and heating the citrate buffer using an autoclave. After naturally cooling at room temperature, the slide glass was immersed in 3% H ₂ O ₂ (MeOH) for 30 minutes and washed with a phosphate buffer (PBS; pH 7.2), so that the intrinsic components contained in the section were included. Sex peroxidase was inactivated.

50 μL of blocking solution (5% BSA-PBS) was placed on the section and allowed to stand at room temperature for 30 minutes in a wet box (blocking). After removing the blocking solution, 50 μL of the primary antibody solution was placed on the section and allowed to stand overnight at 4 ° C. in a wet box (primary antibody reaction). The primary antibody solution used was an anti-mouse Ki-67 rat antibody (Dako) diluted 50-fold with 1% BSA-PBS. After washing the section with PBS, 50 μL of the secondary antibody solution was placed on the section and allowed to stand at room temperature for 1 hour in a wet box (secondary antibody reaction). In the secondary antibody solution, a labeled polymer (simple stain mouse MAX-PO (Rat); Nichirei Co., Ltd.) bound with Fab ′ of a secondary antibody (animal species: goat) and peroxidase was added with 1% BSA-PBS. Those diluted twice were used. After washing the sections with PBS, color was developed using DAB, nuclear staining (hematoxylin staining) was performed, and the cells were encapsulated.

The immunostained sections were observed using an optical microscope (Biozero; Keyence Corporation), and image data of each section was obtained. Using image analysis software (Photoshop; Adobe Systems Inc.), the total number of basal cells contained in each section and the number of basal cells expressing Ki-67 were measured, and Ki-67 was expressed in each section. The percentage of basal cells that were present was determined. The total number of basal cells contained in each section was 316 to 1316 (no treatment: 316 to 1316, IBP-free emulsion: 611 to 1171, 10% IBP-containing emulsion: 351 to 476, retinoic acid preparation: 350 ~ 1053).

In addition, using image analysis software (Photoshop; Adobe Systems Inc.), the thickness of the epidermis at 10 locations (each different section) was measured for each skin piece, and the average value and standard deviation (SD) of the thickness of the epidermis Was calculated.

FIG. 5 and FIG. 6 are photographs of the immunostained sections. FIG. 5A is a photograph of the skin of an untreated individual, and FIG. 5B is a photograph of the skin of an individual to which an IBP-free emulsion is applied. FIG. 6A is a photograph of the skin of an individual to which an emulsion containing 10% IBP is applied, and FIG. 6B is a photograph of the skin of an individual to which a retinoic acid preparation is applied. As shown in these photographs, Ki-67-positive basal cells (arrows) were observed in the individuals applied with the 10% IBP-containing emulsion or retinoic acid preparation compared to the untreated individuals and the non-IBP-containing emulsion. Number) increased. In addition, in the individuals to which the retinoic acid preparation was applied, the epidermis was thicker than in the other individuals.

FIG. 7A is a graph showing the percentage of basal cells expressing Ki-67 in each individual. As shown in this graph, about half of the basal cells were Ki-67 positive in the individuals to which the 10% IBP-containing emulsion or retinoic acid preparation was applied. On the other hand, about 20% of the basal cells were Ki-67 positive in the untreated individuals and the individuals to which the IBP-free emulsion was applied. Since Ki-67 is a marker for cell proliferation, it can be seen that cell proliferation is actively carried out in individuals to which a 10% IBP-containing emulsion or retinoic acid preparation was applied. In addition, since the number of Ki-67 positive cells did not increase in individuals coated with an IBP-free emulsion, it is considered that the promotion of cell proliferation is an effect of IBP.

FIG. 7B is a graph showing the average thickness of the epidermis of each individual. As shown in this graph, the epidermis was significantly thickened in the individuals to which the retinoic acid preparation was applied, but the epidermis was not thickened in the individuals to which the 10% IBP-containing emulsion was applied. Retinoic acid preparations are used as a whitening agent and an anti-wrinkle agent in hospital prescriptions. Retinoic acid is said to promote skin turnover and thicken the epidermis. In contrast, the 10% IBP-containing emulsion promoted cell growth but did not thicken the epidermis. This suggests that the whitening effect by IBP is due to a mechanism different from that of retinoic acid.

[Example 2]
IBP was mixed with the emulsion having the composition shown in Table 1 so that the final concentration was 1% or 3%, and a skin external preparation consisting of a 1% IBP-containing emulsion and a skin external preparation consisting of a 3% IBP-containing emulsion were prepared.

This application claims priority based on Japanese Patent Application No. 2010-004250 filed on Jan. 12, 2010. The contents described in the application specification and the drawings are all incorporated herein.

Since the composition of the present invention can remove skin pigments non-invasively, it is effective as an external skin preparation effective for treatment of pigmentation caused by, for example, ultraviolet irradiation, trauma, burns, acne, and removal of tattoos. Useful.

Claims (3)

A composition for non-invasively promoting the removal of skin pigments, including ibuprofen piconol. The composition according to claim 1, which is a composition for promoting removal of melanin from the skin. The composition according to claim 1, which is an external preparation for skin.

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