JP2009234945A - Sphingomyelin liposome containing h-egf and cosmetic and drug incorporating the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a technique of delivering an active ingredient to below the epidermal horny layer and to the dermis by only its direct application to the skin and to develop a technique of enabling caring for live tissues below the horny layer. <P>SOLUTION: Into the sphingomyelin liposome, h-EGF is incorporated (to be entangled). To live tissues below the horny layer, the h-EGF can be supplied to promote the division and growth of cells and by thoroughly supplying sphingomyelin which comes to a raw material of an intercellular lipid to divided cells, the cells can sufficiently produce a lipid. By these synergistic effects a beautiful and healthy horny layer is formed, and moveover, such effects can be obtained by only applying the sphingomyelin liposome containing h-EGF on to the skin. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a technique that contributes to improving skin health, and in particular, relates to EGF, sphingomyelin, and cosmetics and pharmaceuticals containing them.

  EGF (Epidermal Growth Factor) translated as epidermal growth factor or epidermal growth factor, epidermal growth factor, etc. is a protein discovered in 1962 by Dr. Stanley Cohen, an American biologist. It is a peptide. EGF is a growth factor of target cells (target cells) that promotes DNA and RNA synthesis, promotes cellular gene replication and transcription, accelerates cell division, and promotes cell growth and differentiation. Because of these functions, EGF is known to have a significant effect on wound repair and ulcer healing in clinical medicine, and in the field of beauty and skin care, It is known to improve skin health and prevent skin aging, such as increasing skin firmness and elasticity, and eliminating problems such as spots, freckles, and rough skin.

Humans have the ability to produce EGF, but their secretion decreases with age. Thus, it has been proposed to obtain EGF's skin conditioning effect by blending EGF into cosmetics and replenishing the skin from the outside. (See claims 1 and 2 of Japanese Patent Publication No. 4-69123)
Japanese Examined Patent Publication No. 4-69123

  However, no matter how much EGF is applied from above the skin, it is difficult to penetrate the EGF into the skin. The structure of the skin is such that the dermis covers the subcutaneous tissue and the epidermis covers it, and the epidermis is divided into the horny layer, granule layer, spiny layer, and basal layer from the surface side. . In other words, the outermost surface of the skin is the stratum corneum, but EGF is very difficult to pass through the stratum corneum.

  The stratum corneum has a thickness of about 20 microns and serves as a barrier to prevent bacteria and dirt from entering the skin, and also serves as a moisturizer to prevent moisture inside from escaping outside. . Because of this barrier function, no matter how much an active ingredient such as EGF is applied from above the skin, it is blocked by the stratum corneum, so it is very difficult to reach the layers below the stratum corneum.

  Moreover, the cells of the stratum corneum are actually cells that have already died. Epidermis cells are born in the basal layer, which are gradually pushed up to the surface into spiny layer, granule layer, and stratum corneum, and finally become plaque and peel off. It takes about 28 days for a human to be around 25 years old to be separated from the stratum corneum after being born in the basal layer. Epidermal cells die at the top of the granule layer and die. It takes about 14 days from the time of birth to apoptosis, and about 14 days from the time of apoptosis until peeling off as plaque. These days are known to gradually increase as aging progresses. While epidermal cells are born in the basal layer and migrate to the granular layer, the cells synthesize lipids such as ceramide and accumulate inside. When cells in the granule layer undergo cell death (apoptosis), ceramide and the like accumulated inside are released, so the stratum corneum has a structure in which dead cells float in lipid. This intercellular lipid is a source of moisture for the skin. When there are few intercellular lipids, the moisture of the skin is lost and the skin becomes dry. In particular, women use lotion, milky lotion, cream, etc. to keep the moisture of the skin. However, no matter how much active ingredient is applied over the skin, it only cares for tissue that has already died.

  Since care for the stratum corneum is important for maintaining skin health, it is of course meaningful to apply cosmetics to the skin as before. However, as described above, the inventor thought that the stratum corneum was composed of cells that had already died, and no matter how much care was taken for dead tissue, it would not be a real solution. In other words, if you really want to improve your skin health, you must care for the living cell structure. In order to improve the quality of the stratum corneum consisting of dead cells, it will first need to be healthy when the cells are alive. As described above, lipids such as ceramide, which is the source of the stratum corneum barrier function and moisture retention function, are synthesized when cells are alive. You will then have to take care to allow enough ceramide synthesis while your cells are alive. Skin elasticity is created by the dermis layer under the epidermis retaining collagen, elastin and moisture. If you do not care for the dermis layer, it will not be a true solution to keep your skin young. Therefore, it is considered that care of the epidermis structure and dermis layer under the stratum corneum is more important for improving skin health than caring for the stratum corneum. Conventionally, however, the active ingredient cannot be delivered through the stratum corneum barrier and below the stratum corneum. For this reason, the conventional skin cosmetics are exclusively intended for keratin care, and the purpose has to be limited to keratin care.

  The present invention has been made in view of such circumstances and considerations, and has developed a technique for delivering an active ingredient to the subepidermal horny layer and dermis of the skin by simply applying it directly to the skin. It is to develop a technology that makes it possible to care for living tissues.

  The present applicant has found that the above-mentioned problems can be solved by incorporating an active ingredient into liposomes obtained by making sphingomyelin into liposomes.

Liposomes in which sphingomyelin is made into liposomes have a very small diameter of 100 nm to 1 μm and can pass through the stratum corneum and reach the underlying granular layer, spiny layer, basal layer, and even the underlying dermis. (See prior patent application WO 2007/145276 by the applicant). However, it was unclear whether or not the sphingomyelin liposome can pass through the stratum corneum even when another component is contained therein.
WO2007 / 145276

  The present applicant, through experiments, shows that even when another component is contained in the sphingomyelin liposome, it can penetrate through the stratum corneum and penetrate into the epidermis structure below the stratum corneum and further reach the dermis. I confirmed. Then, the inventor found that the sphingomyelin liposomes that reached the epidermis structure or dermis structure under the stratum corneum were gradually broken in these structures, and then the components incorporated in the liposomes were released into the structure. It was confirmed experimentally.

  Accordingly, when the active ingredient is incorporated into the sphingomyelin liposome and applied from above the skin, the active ingredient can be supplied to living skin cells through the stratum corneum.

The active ingredient can be EGF, for example. EGF is preferably EGF (human EGF, h-EGF) having the same structure as EGF synthesized in the human body. h-EGF can be obtained from skin, hair, etc., but can also be artificially synthesized using genetic engineering techniques. There is WO85 / 00369 (Japanese translation is Japanese translation No. 60-501737) as a literature on a method for synthesizing h-EGF by genetic engineering.
WO85 / 00369

  It is preferable that the sphingomyelin to be made into a liposome has a very high purity. High-purity sphingomyelin with a purity of 98% or more is commercially available from NOF Corporation under the trade names COATSOME NM-10 and COATSOME NM-70. A similar high-purity sphingomyelin is commercially available from the applicant, Shalom Co., Ltd. under the trade name “Sphingomyelin Nature”.

  According to the present invention, an active ingredient can be delivered deep into the skin by transdermal absorption, and the active ingredient can be delivered to living tissue. For example, h-EGF can be delivered to the epidermis and dermis structures below the stratum corneum. When h-EGF is delivered to living cells, h-EGF functions are exerted, cell division is activated, cell growth and differentiation are promoted, and new skin cells are produced one after another, while old cells are Discarded promptly. Thus, the skin can remain youthful indefinitely, has increased gloss and elasticity, and can be quickly repaired if there is a wound. As the metabolism of the epidermis is activated, young healthy epidermal cells can undergo apoptosis into keratinocytes, so that a beautiful and strong stratum corneum can be formed. In addition, the formation of healthy dermis improves the water retention function and elasticity of the skin.

  It must be emphasized many times that the cell growth promoting effect of h-EGF cannot be exerted on dead cell partners such as corneocytes. No matter how much h-EGF is applied to a cell that has already died, it is impossible to revive the cell, so cell division and growth cannot occur. And since there was no conventional means for h-EGF to pass through the stratum corneum, it is impossible to deliver h-EGF below the stratum corneum by application on the skin, and the effect of h-EGF on percutaneous absorption It was impossible to get. In the past, the excellent wound healing effect of h-EGF has been confirmed when the stratum corneum is broken and the underlying tissue is exposed.

  The present invention makes it possible for the first time in the world to deliver h-EGF to living cell tissues below the stratum corneum by transdermal absorption. To the best of the applicant's knowledge, there has never existed a technique that enables the effect of h-EGF to be manifested simply by applying it from the skin.

  Furthermore, the present invention utilizes liposomes made with sphingomyelin to deliver active ingredients such as h-EGF. Sphingomyelin is a precursor of ceramide and is used to make ceramide in granular and spinous layers. When the liposome according to the present invention is applied to the skin, the permeated liposome is broken under the stratum corneum, and sphingomyelin is supplied to the epidermis cells under the stratum corneum. For this reason, the cells in the granule layer can make a large amount of ceramide before it undergoes apoptosis and accumulate it inside, and can release a large amount of ceramide when it undergoes apoptosis and changes into corneocytes. Therefore, a fresh and smooth stratum corneum containing a large amount of intercellular lipid is formed. When the amount of intercellular lipid is large, not only the skin looks clean, but also the moisture retention and barrier ability are excellent.

  That is, according to one embodiment of the present invention characterized in that the sphingomyelin liposome contains (entangles) h-EGF, by supplying h-EGF to living tissue below the stratum corneum, In addition to promoting division and growth, by supplying enough sphingomyelin as a raw material for intercellular lipids to the divided cells, it is possible for the cells to generate enough lipids, and these synergistic effects make them beautiful. The effect that a healthy stratum corneum is formed can be obtained. And the convenience which was not imagined before that such an effect can be acquired only by application on skin is provided.

  Because of such effects, the liposome according to the present invention can be used in general for cosmetics and pharmaceuticals used for skin and hair care, and the present invention includes such cosmetics and pharmaceuticals in its scope.

Embodiments of the present invention include the following.
(1) A liposome in which sphingomyelin is made into a liposome, wherein an active ingredient is incorporated therein.
(2) The liposome according to claim 1, wherein the active ingredient is h-EGF.
(3) A liposome preparation comprising the liposome of claim 1 or 2.
(4) A cosmetic comprising the liposome according to claim 1 or 2.
(5) A pharmaceutical comprising the liposome according to claim 1 or 2.
(6) A method for producing a liposome, which is a liposome in which sphingomyelin is formed into a liposome, wherein the active ingredient is incorporated therein, wherein the active ingredient is dissolved in ethanol. And a step of adding water in which sucrose is dissolved to emulsify and stirring.

  Some of the preferred embodiments of the present invention are specified in the appended claims. However, the embodiments of the present invention are not limited to those explicitly described in the claims, specification and drawings, and can take various forms without departing from the spirit of the present invention. . The present invention includes in its scope all novel and useful configurations that can be taught from these documents, whether explicitly disclosed in the claims, the description, and the figures of the present application.

  Hereinafter, examples of embodiments of the present invention will be described in detail.

[Production method of sphingomyelin liposome containing h-EGF]
(1) Obtaining Sphingomyelin As Sphingomyelin used in the examples of the present invention described here, “Sphingomyelin Nature” (trademark) manufactured by Shalom Co., Ltd. is used. Sphingomyelin nature is a sphingomyelin produced using milk as a raw material, and is an extremely high-purity sphingomyelin with a purity of 98% or more. Hereinafter, sphingomyelin is sometimes referred to as SPM.

(2) Obtaining h-EGF
h-EGF can be purchased from several companies, including Appeloa, China. As shown in FIG. 1, as a result of measuring the activity of these h-EGFs through measurement of cell proliferation activity, it can be determined that the EGF activities of each manufacturer are equivalent in consideration of the specific activity (FIG. 2). .

(3) Method for producing h-EGF-containing SPM liposomes
Sphingomyelin liposomes incorporating h-EGF can be produced, for example, by the following production method.
Dissolve 2.0 g of sphingomyelin with 4.0 g of ethanol ↓ at 65 ℃
Add EGF 1.0mg and sucrose 9.0g water and emulsify ↓ at 65 ℃, 10000rpm, 20min
Measure up to 100 g with water for injection ↓
Sizing with two stacked membrane filters (0.2 μm) ↓ at 65 ° C, 0.8 to 1.0 MPa, repeated 3 times
Sphingomyelin liposome solution containing EGF

  This completes the EGF-containing sphingomyelin liposome solution. It is preferable to measure the average particle diameter and pH.

(4) EIA quantification
EIA quantification was performed using R & D systems “Quantikine Human EGF Immunoassay” Cat No. DEG00.

  FIG. 3 shows the results of quantification of the simple EGF aqueous solution and the EGF-containing SPM liposome solution diluted so as to have the same EGF concentration by EIA. When diluted with water and diluent, only about half the signal of free EGF aqueous solution was shown, but when diluted after breaking liposomes with EtOH, a signal almost equivalent to free EGF was shown. This test result shows that about half of the charged EGF was taken up by the SPM liposome in the above production method.

(5) Examination of SPM concentration of SPM liposomes containing h-EGF
Prepare EGF-containing sphingomyelin liposome solutions with SPM concentrations of 1, 2, 5% and constant EGF concentration, and quantitate each EGF-containing SPM liposome solution diluted so that these EGF concentrations are equal by EIA did.

  FIG. 4 shows the result of quantifying the EGF-containing SPM liposome solution diluted so that the EGF concentration of each liposome solution becomes equal by EIA. It is estimated that the amount of EGF taken into the SPM liposome is the amount of the signal when diluted with EtOH and the signal when diluted after breaking the liposome with EtOH, depending on the SPM concentration, The estimated amount of EGF incorporated into SPM liposomes is increasing. In addition, there is almost no difference in the average particle diameter of each liposome liquid.

(6) Activity measurement The activity of EGF-containing SPM liposomes was measured through measurement of cell proliferation activity.

(6-1) Cells: Balb / 3T3 clone A31 (ATCC, No. CCL163)

(6-2) Method:
Collect Balb / 3T3 cells, make a cell suspension with a concentration of 3 × 10 ⁴ cells / mL in DMEM medium containing 10% FBS, add 100 μL / well to a 96-well plate, and maintain at 37 ° C and 5% CO ₂ Incubate for 18-24 hours. On the next day, the supernatant of each well is removed, and DMEM medium containing 0.4% FBS is added at 100 μL / well and cultured for 18 to 24 hours.
Add 100 μL / well of each sample solution diluted with 0.4% FBS-containing DMEM medium to the wells in culture. After about 72 hours of culturing at 37 ° C and 5% CO ₂ , cell proliferation was measured by the WST-1 method (Cell Counting Kit; DOJINDO, Cat. No. 345-06463). The concentration (EC50) at the time of giving is calculated, and the EGF activity of each sample is compared.

(6-3) Results The results are shown in FIG. No activity was observed in the placebo (SPM) SPM liposomes, and the EGF-containing SPM liposomes (EGF-SPM) showed about 1/2 the activity of free EGF (EGF). The trend was similar to the EIA result.

(7) Storage stability test of EGF-containing SPM liposomes
For EGF-containing SPM liposomes and EGF aqueous solution, the EGF content before and after storage at 5, 25, and 40 ° C for 1 month, 3 months, and 4 months was determined using the EGF-EIA kit (R & D systems “Quantikine Human EGF Immunoassay” Cat No. DEG00). And quantified. The test results are shown in FIG.

  The activity after 3 months storage at 5 ° C. and 40 ° C. was measured by the WST-1 method. FIG. 7 shows the test results for Free EGF, and FIG. 8 shows the test results for EGF-containing SPM liposomes.

  As seen in FIGS. 6 and 8, when stored at 5 ° C., both activity and EIA signals are generally stable for at least 3 months.

[Drop test on stratum corneum of calcein-containing SPM liposomes]
In order to show experimentally that h-EGF can be delivered to the dermis by percutaneous absorption by incorporating SPM liposomes, calcein, which is a fluorescent material, is incorporated into SPM liposomes and calcein-containing SPM Liposomes were dropped from above the stratum corneum and an experiment was conducted to examine the state of penetration.

(1) Production of calcein-containing liposomes
Calcein-containing SPM liposomes were produced by the following production method in the same manner as the production method for h-EGF-containing SPM liposomes.
Dissolve 1.0 g of sphingomyelin with 2.0 g of ethanol ↓ at 65 ℃
Emulsified by adding water in which 0.0623 g of calcein and 9.0 g of sucrose were added ↓ at 65 ° C, 10000 rpm, 20 min
Measure up to 100 g with water for injection ↓
Sizing with two stacked membrane filters (0.2 μm) ↓ at 65 ° C, 0.8 to 1.0 MPa, repeated 3 times

The calcein-containing SPM liposome solution produced as described above was concentrated as follows.
Ultrafiltration concentration ↓ 50000cut off, at 4 ℃
Sepharose CL-4B gel filtration ↓ at 4 ℃, eluent: PBS
Pool SPM liposome fractions, replace solvent with 9% sucrose aqueous solution ↓ PD-10 column ultrafiltration concentration ↓ 50000 cut off, at 4 ℃
Thus, a concentrated calcein-containing SPM liposome solution from which free calcein has been removed is completed. The calcein-containing SPM liposome solution can hardly fluoresce because of fluorescence quenching, but TritonX-100 (final concentration 1%) is added to the solution to completely destroy the liposomes and all calcein The fluorescence intensity when is released to the outside is about 4 to 5 times the fluorescence intensity when the liquid is directly measured.

(2) Test and Results Concentrated calcein-containing SPM liposomes from which free calcein produced as described above was removed were dropped onto the human three-dimensional skin model (using TESTSKIN ^™ LSE-high) from above the stratum corneum for 6 hours. After the lapse, photographs were taken with an electron microscope. A photograph is shown in FIG.

  Referring to FIG. 9, there is something that appears white on the screen, but this is calcein. It can be seen that calcein is present not only in the epidermis under the stratum corneum but also in the dermis layer even though it is dripped from the stratum corneum. In particular, see the part indicated by the white arrow.

  It should be noted that the calcein shown is that the SPM liposome was broken and released to the outside. If SPM liposomes are not broken, calcein is not visible. As mentioned above, this photo was taken 6 hours after dropping, but if more time was taken, more SPM liposomes were broken and more calcein was deposited in the substratum tissue. It may have been reflected.

  The photograph in FIG. 9 shows that the components incorporated into the SPM liposome are delivered through the stratum corneum to the epidermal tissue and dermis layer below the stratum corneum.

  More specifically, the cosmetics included in the scope of the present invention can be, for example, the following cosmetics: white, white powder, white paper, white powder, white powder, white powder, white paste, water Interesting, cosmetics for skin, lotion, skin lotion, soft lotion, astringent lotion, mucous lotion, hand lotion, shaving lotion, medicated lotion, lotion, moisturizer, essence, cream, oily cream , Medium oil cream, Weak oil cream, Cleansing cream, Cold cream, Hizenic cream, Vanishing cream, Hand cream, Shaving cream, Bleaching cream, Foundation cream, Medicated cream, Lip cream, Milky lotion, Skin lotion, Skin milk Burn cream, sunscreen cream , Sunburn (for), sunscreen (for), finishing cosmetics, foundation, face color, concealer, makeup base, makeup base, pre-makeup, red, lipstick, lipstick, ripple rouge, lip color, lip Pencil, Lip Gloss, Lip Liner, Eye Makeup, Eye Shadow, Eye Color, Eye Liner, Eyebrow, Eyebrow Pencil, Eyebrow Brush, Mascara, Eyelash Cosmetic, Nerima, Cheek Cosmetic, Blusher, Cheek Color, Cheek Loose, Body Makeup, Hairdressing, Hair Nourishing, Scalp, Hair Coloring, Hair Washing, Hair Cosmetics, Hair Oil, Color Rinse, Cold Perm Solution, Suki Oil, Setting Lotion, Hair Dye, Hair Decoloring Agent, Chick, Permanent liquid, bottle oil, hair cream, hair spray Hair tonic, hair fixer, hair lacquer, hair rinse, balum, pomade, body powder, talcum powder, bath powder, perfume powder, baby powder, natural nail powder, bath cosmetic, bath oil, bath salt, bubble bath, foam bath, Cosmetics for packs.

Test result of Balb / 3T3 cell activation effect by EGF (WST-1 method) Results of EGF cell proliferation activity test by each company EIA quantitative test results of EGF-containing SPM liposome solution Results by SPM concentration of EGF-containing SPM liposomes Test result of Balb / 3T3 cell activation effect of EGF-containing SPM liposome (WST-1 method) Storage stability test results of EGF-containing SPM liposomes (EIA) EGF storage stability test results (activity) Storage stability test results (activity) of EGF-containing SPM liposomes Electron micrograph showing results of penetration test of calcein-containing SPM liposomes

Claims (6)

  A liposome in which sphingomyelin is made into a liposome, wherein an active ingredient is incorporated therein.   The liposome according to claim 1, wherein the active ingredient is h-EGF.   A liposome preparation comprising the liposome according to claim 1 or 2.   A cosmetic comprising the liposome according to claim 1 or 2.   A pharmaceutical comprising the liposome according to claim 1 or 2. A method for producing a liposome, characterized in that it is a liposome in which sphingomyelin is made into a liposome, and an active ingredient is incorporated therein.
A method comprising the steps of dissolving sphingomyelin with ethanol, adding the active ingredient and water in which sucrose is dissolved, emulsifying and stirring.