JP2009298752A - Skin care preparation composition for external use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a skin care preparation composition for external use that has extremely low skin irritation, excellent epidermal cell growth promotion action and is simply usable. <P>SOLUTION: The skin care preparation composition for external use comprises ADP and a xanthine derivative. The skin care preparation composition comprises 0.01-100 mM of ADP and 0.0005-5 mass% of the xanthine derivative. In the skin care preparation composition, preferably the xanthine derivative is caffeine and/or theophylline. In the skin care preparation composition for external use, preferably the composition is an epidermal cell growth promoter for external use. The leave-on type cosmetic comprises the skin care preparation composition for external use. The epidermal cell growth promotion method comprises applying an skin care preparation composition containing ADP and a xanthine derivative to the skin surface. In the method, the skin care preparation composition for external use contains 0.01-100 mM of ADP and 0.0005-5 mass% of a xanthine derivative. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to an external preparation for skin, and particularly to use as a low skin irritation epidermal cell proliferation promoter by blending ADP and a xanthine derivative.

Conventionally, ATP and ADP have been known to have epidermal cell proliferation action (Non-patent Document 1). The effect is present in epidermal cells, it is thought to be that ATP and ADP have through P2Y ₁ and P2Y ₂ is a subtype of ATP receptors that bind. It is also known that ADP is particularly agonists of P2Y _1.
In addition, it is known that glycolic acid generally used as a chemical peeling agent causes proliferation of epidermal cells, and the mechanism has been reported (Patent Document 1). In Patent Document 1, glycolic acid (a kind of α-hydroxy acid) binds to a TRPV1 (VR1) receptor present in epidermal cells, whereby Ca ions in the epidermal cells increase, and ATP is released from the epidermal cells to the outside of the cells. Has been shown to bind to the P2Y receptor and thus induce proliferation. The same document also reports that lactic acid induces extracellular release of ATP in mouse epidermal cells.
That is, they suggest that epithelial cell proliferation is enhanced by a certain concentration of extracellular ATP.

  However, since the TRPV1 receptor is activated by stimulation with an acid such as a chemical peeling agent, it is known to cause skin irritation, redness, tingling, and pain depending on the acid concentration that can be used. In addition, due to the nature of irritating the skin, the drug concentration adjustment is extremely delicate, and the application method such as washing away the drug immediately after a strictly set application time requires management. Is recommended by specialists. Therefore, it has been desired to develop a drug that has little irritation and is excellent in skin cell proliferation effect and can be easily used like a normal cosmetic.

By the way, in HaCaT cell line (established human keratinocyte), adenylate kinase involved in ATP conversion of ADP, F ₁ F ₀ ATP synthase, and nucleotide diphosphokinase (NDPK) exist, and skin cell (HaCaT) culture occurs. It has been reported that an increase in ATP concentration was observed when ADP was added to the system (Non-patent Document 2).
Further, it is disclosed that a cosmetic or pharmaceutical composition containing an effective amount of ADP can increase the ATP level of skin fibroblasts and prevent skin aging (Patent Document 2).
From these findings, it is considered that ATP can be released to the outside of the cell without irritating the skin by supplying ADP to keratinocytes instead of the above-described conventional chemical peeling agent (acid).

However, when extracellular ATP is applied to HaCaT cell line, the concentration of extracellular ATP decreases with time, so the presence of ATP-degrading enzyme has also been suggested (Non-patent Document 2). That is, in human keratinocytes, there are an enzyme that converts ADP into ATP and an enzyme that degrades ATP. Therefore, even when ADP is applied extracellularly and converted to ATP, the amount of ATP released is reduced by the action of the ATP-degrading enzyme, and the ATP concentration that contributes to cell growth can be maintained. There wasn't.
Greig AVH etal ,: Purinergic receptors are part of a functional signaling system for proliferation and differentiation of humab epidermal keratinocytes. J Invest Dermatol 120: 1007-1015, 2003 Burrell, HE, Wlodarski, B., et al.,: Human Keratinocytes Release ATP and UtilizeThree Mechanisms for Nucleotide Interconversion at the Cell Surface.J. Biol. Chem., Vol. 280, No. 33, pp. 29667-29676, 2005 JP 2006-262806 A JP-T-2001-503447

Due to the above problems, an external preparation for skin that can increase extracellular ATP concentration without stimulating inflammation to the skin and activate proliferation of epidermal cells has not been known yet.
The present invention has been made in view of such circumstances, and provides an external preparation for skin that has extremely low skin irritation, has an excellent epidermal cell proliferation promoting action, and can be easily used. The purpose is to provide.

As a result of intensive studies conducted by the present inventors to achieve the above object, a skin external preparation containing a specific amount of ADP and a xanthine derivative causes ATP degradation inhibition and an increase in extracellular ATP concentration. The inventors have found that the proliferation of epidermal cells is promoted, and have completed the present invention.
That is, the skin external preparation composition according to the present invention is characterized by containing ADP and a xanthine derivative.
In the skin external preparation composition, it is preferable to contain 0.01 to 100 mM of ADP and 0.0005 to 5% by mass of a xanthine derivative.
In the skin external preparation composition, the xanthine derivative is preferably caffeine and / or theophylline.
Moreover, in the said skin external preparation composition, it is suitable that this composition is an epidermal cell proliferation promoter.
In the skin external preparation composition, it is preferable that the mixing ratio of ADP: xanthine derivative is 1: 1 to 5: 1 by mass ratio.
Moreover, the leave-on-type cosmetic according to the present invention is characterized by including the above-mentioned skin external preparation composition.
Moreover, the epidermal cell proliferation promoting method according to the present invention is characterized in that a skin external preparation composition containing ADP and a xanthine derivative is applied to the skin surface.
In the epidermal cell growth promoting method, it is preferable that the skin external preparation composition contains 0.01 to 100 mM of ADP and 0.0005 to 5% by mass of the xanthine derivative.

  According to the present invention, it is possible to activate the proliferation of epidermal cells without inflammatory stimulation. Since the external preparation composition for skin of the present invention does not cause irritation to the skin, it is provided as a leave-on type cosmetic that has been difficult with conventional chemical peeling agents, and anyone can use it easily. Further, the composition is useful for anti-aging, whitening, anti-wrinkle, anti-dullness, wound healing, etc. by promoting the proliferation of epidermal cells.

Hereinafter, the present invention will be described in detail.
It is well known that intracellular ATP plays a very important role as energy in the body, for example, in causing muscle contraction, biosynthesis of biological materials, ion transport and concentration, and the like. On the other hand, for extracellular ATP, G.G. It is found by Burnstock et al. That it plays a role as a signal transmitter, and ATP released outside the cell acts on peripheral cells as a signal transmitter, and various physiological effects such as cell proliferation and pain are expressed. It has become clear. A conventional chemical peeling agent utilizes the role of extracellular ATP as a signal transmitter, and gives an inflammation stimulus to the VR1 receptor of epidermal cells by an acid as a main component. It promotes release and induces cell proliferation. That is, chemical peeling agents are based on activating the epidermal cell proliferative function by inflammatory stimulation.
On the other hand, the skin external preparation composition according to the present invention contains ADP and a xanthine derivative. In the skin external preparation composition, ADP is mainly used for ATP synthesis, and the xanthine derivative mainly suppresses the decomposition of ATP. Both these actions increase the epidermal extracellular ATP concentration and promote cell proliferation. That is, the present invention does not require inflammatory stimulation during cell proliferation. Therefore, since the present invention does not require an inflammatory stimulus for cell proliferation, it basically does not contain a substance (chemical peeling component) that acts on the VR-1 receptor by the inflammatory stimulus.

Hereinafter, the mechanism by which the proliferation of epidermal cells is promoted by the present invention will be described.
ATP analogs such as ATP and ADP are involved in various cellular processes such as proliferation through activation of P2 receptors, which have two subgroups: P2X (ion channel built-in type) and P2Y (G protein-coupled type). It is known to do. The expression of the P2 receptor subtype depends on the species and cell type. In normal human keratinocytes, the presence of P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ , P2Y ₁₁ , P2Y ₁₂ , P2Y ₁₃ is confirmed. .
For example, ATP released from neighboring cells under circumstances such as tissue damage increases intracellular Ca concentration by binding to the P2Y receptor. Thereafter, ATP is immediately degraded to ADP, AMP, or adenosine by a degrading enzyme, and the activity is calmed down.
In such an epidermal cell proliferation mechanism, the present invention intends to positively induce activation of P2Y receptor by ATP by applying an external preparation containing ADP to the skin.

When the ADP concentration in the epidermal cells becomes 0.01 mM or more due to the supply of ADP, the amount of ATP released to the outside of the cell is remarkably increased. Therefore, although ATP production becomes more active as ADP is supplied to epidermal cells, the concentration of ADP in the cells is preferably about 0.01 to 100 mM in consideration of the influence on the living body.
When the ADP concentration in the epidermal cells is set to the above-mentioned level by applying the skin external preparation composition according to the present invention, the ADP concentration in the composition is 0.01 to 100 mM, preferably 0.01 to 10 mM. It is preferable to mix. Although the compounding mass in a composition can be suitably adjusted according to the said concentration range, Preferably it is 0.0005-5 mass%.
In keratinocytes (epidermal cells), the presence of ectoadenylate kinase (AK), ectoF ₁ F ₀ ATP synthase, and ectonucleotide diphosphokinase (NDPK) are known as enzymes that catalyze the ATP synthesis reaction. ADP supplied to keratinocytes is converted to ATP by the following reaction (Pi represents phosphoric acid).

In addition, when ATP is directly blended into the skin external preparation composition without going through the above reaction, there is a concern about the influence on the biological system due to excessive supply of ATP for various adjustments. Therefore, in the present invention, it is preferable that ADP is supplied to the epidermal cells and the living body produces the necessary ATP through the adjustment of the synthetic enzyme. Usually, when ADP is supplied to epidermal cells, ADP is converted to ATP, and the release of ATP to the outside of the cell is also activated.

Further, keratinocytes include ATP-degrading enzymes that hydrolyze ATP into ADP and AMP together with the ATP synthase.
The present inventors have confirmed that three types (NTPDases 2, 3, and 4) are expressed in human keratinocytes among four types of NTPDases known as ATP-degrading enzymes. In addition, 5′-nucleotidase is known as an enzyme that further degrades AMP into adenosine. That is, in keratinocytes, the following ATP decomposition reaction is performed with enzyme adjustment together with the aforementioned ATP synthesis reaction.

Therefore, if only ADP is supplied to epidermal cells, ATP synthesized from ADP does not bind to the P2 receptor and may be immediately subjected to the above degradation. Therefore, in order to make ATP converted from the supplied ADP work efficiently, it is necessary to suppress the ATP decomposition reaction.
In the above-described series of degradation reactions, the present inventors show that a xanthine derivative (Tsuzuki and Newburgh, 1975) known to inhibit 5′-nucleotidase inhibits the expression of NTPPDase involved in ATP degradation. I found.

  That is, when a xanthine derivative is supplied to epidermal cells in the presence of ADP, ATP is synthesized by ADP by an extracellular ATP synthase, and inhibition of ATP degradation by the xanthine derivative simultaneously functions. By these actions, the ATP concentration in the epidermal cells is remarkably higher than that in the normal state, the binding of ATP to the P2Y receptor is activated, and the proliferation of the epidermal cells is promoted.

In particular, at the epidermal cell level, when the concentration of xanthine derivative is 0.0005 to 5% in the presence of 0.01 to 100 mM ADP, the extracellular release amount of ATP increases synergistically.
Even if the xanthine derivative is applied to epidermal cells without the addition of ADP, a sufficient effect cannot be obtained in improving ATP production.
When the concentration of xanthine derivative in the epidermal cells is adjusted to the above-mentioned suitable level by application of the external preparation for skin according to the present invention, 0.0005 to 5% by mass of the xanthine derivative is preferably added to the composition. Preferably it is 0.0005-0.5 mass%.
Examples of xanthine derivatives that can be used in the present invention include xanthine, aminophylline, theophylline, choline theophylline, caffeine, theobromine, 1,7-dimethylxanthine, oxtrifin, diprofylline, proxyphylline, and the like. One kind or a combination of two or more kinds can be used. In the present invention, caffeine and / or theophylline are particularly suitable.
The compounding ratio of the xanthine derivative to ADP is preferably 1: 1 to 5: 1 as a mass ratio.

  In addition to the above, as a means for inhibiting NTPDase, it is known to expose epidermal cells to a low pH environment, and it has been used as a general chemical peeling agent. That is, glycolic acid or the like, which is a main component of a conventional chemical peeling agent, has an effect of releasing ATP from cells through stimulation of the VR1 receptor and inhibiting the degradation of ATP. On the other hand, in the present invention, by blending ADP and xanthine derivative, a high epidermal cell proliferation effect can be achieved without giving acid stimulation.

  The skin external preparation composition of the present invention is not only the above-mentioned essential components but also other components usually used in skin external preparations such as cosmetics and pharmaceuticals, for example, powder components, liquid fats, solid fats, waxes, hydrocarbons, higher fatty acids. Higher alcohols, esters, silicones, anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants, water-soluble polymers, thickeners, film agents, UV absorbers, sequestering agents, Lower alcohols, polyhydric alcohols, sugars, amino acids, organic amines, polymer emulsions, pH adjusters, skin nutrients, vitamins, antioxidants, antioxidant aids, fragrances, water, etc. are blended as necessary, It can be produced by a conventional method according to the intended dosage form.

  Other ingredients that can be blended include, for example, preservatives (ethyl paraben, butyl paraben, etc.); anti-inflammatory agents (eg, glycyrrhizic acid derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, zinc oxide, allantoin, etc.); whitening agents ( For example, placenta extract, saxifrage extract, arbutin, etc.); various extracts (eg, buckwheat, auren, shikon, peonies, assembly, birch, sage, loquat, carrot, aloe, mallow, iris, grape, yokuinin, loofah, Lily, saffron, nematode, ginger, hypericum, onionis, garlic, red pepper, chimpi, red snapper, seaweed, etc.), activator (eg, royal jelly, photosensitizer, cholesterol derivative, etc.); nicotine Benzyl ester, nicotinic acid β-butoxyethyl ester, capsaicin, gingerone, cantalis tincture, ictamol, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclandrate, cinnarizine, trazoline, acetylcholine, verapamil, cephalanthin , Γ-oryzanol, etc.); antiseborrheic agents (eg, sulfur, thianthol, etc.); anti-inflammatory agents (eg, tranexamic acid, thiotaurine, hypotaurine, etc.) and the like.

The dosage form of the external preparation for skin of the present invention is arbitrary, solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, gel, mist, spray, Any dosage form such as mousse, roll-on, stick, etc. may be used. Alternatively, a preparation impregnated or coated on a sheet such as a nonwoven fabric is also possible. Further, the product form of the external preparation for skin of the present invention is also arbitrary, facial cosmetics such as lotion, emulsion, cream and pack; makeup cosmetics such as foundation, lipstick and eye shadow; sunscreen cosmetics (sunscreen agent) ); Body cosmetics; aromatic cosmetics; skin cleansing agents such as make-up removers, body shampoos, etc .; hair cosmetics such as hair liquids, hair tonics, hair conditioners, shampoos, rinses, hair restorers; ointments and the like. In particular, the present invention can be suitably used as a leave-on type cosmetic that can be used on a daily basis and does not require washing immediately after application.
Examples of the present invention are specifically shown below, but the present invention is not limited to these. In the following examples, all component concentrations are based on cultured cells.

<Selection of ATP analog>
Test Method Commercially available normal human epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were cultured according to the protocol in KGM-2 medium (manufactured by Kurabo Industries Co., Ltd.). Cells were seeded in a 96-well black plate at 1 × 10 ⁴ per well, cultured for 24 hours, washed with PBS (−), and allowed to stand for 1 hour.
Thereafter, ADP, UTP, GTP, adenosine, AMP, and UDP were each added to the medium of each well so as to be 10 μM, and treated with an ATP assay kit (ATPLite, manufactured by Perkin Elmer) for 5 minutes at room temperature. The whole plate was placed in a luminescent plate reader and measured.
The amount of extracellular ATP released by each ATP analog addition obtained from this test is shown in FIG.

As shown in FIG. 1, when adenosine, AMP, and UDP were added, the release of ATP to the outside of the cells was hardly observed as in the case of the ATP analog-free (medium) sample. On the other hand, when ADP, UTP, and GTP were added, a clear increase in ATP release was observed.
From the above, it is clear that the addition of ADP, UTP, and GTP enhances ATP production of skin cells.

<Influence of ATP analog on cell proliferation>
About the ADP, UTP, and GTP in which the increase in ATP production was recognized in the said test, the effect with respect to cell proliferation was evaluated using the alamar blue reagent.
Test Method Commercially available normal human epidermal keratinocytes (NHEK, manufactured by Kurabo Industries Co., Ltd.) were cultured according to the protocol in KGM-2 medium (manufactured by Kurabo Industries Co., Ltd.). The cells were seeded in a 96-well black plate at 3 × 10 ³ per well and cultured for 4 days. On the fifth day of culture, ADP, UTP, and GTP were added to the medium of each well at a concentration of 0.01 to 250 μM, respectively, and the alamar blue reagent (manufactured by BIOSOURCE) was added the next day. After further 24 hours, the fluorescence intensity at 590 nm was measured with a fluorescence plate reader.
The fluorescence intensity in an untreated culture system to which no ATP analog was added was defined as 100%, and the relative absorbance of each sample to which ADP, UTP, and GTP were added at a concentration of 0.01 to 250 μM was calculated, and this was calculated as the cell. The growth rate was used. The results are shown in FIG.

As shown in FIG. 2, in the culture system to which GTP was added, a slight increase in the cell growth rate was observed in the low concentration range, but started to decrease from 1.95 μM to the peak, and at 250 μM, it was almost the same as that in the untreated case. It became. When UTP was added, the cell proliferation rate increased with increasing UTP concentration. When ADP was added, the cell growth rate was excellent at all concentrations. In particular, the growth rate tended to increase from the addition of 62.5 μM or more.
From the above results, it is particularly preferable to add ADP in the proliferation of skin cells.

Further, MTT assays were performed in cell lines supplemented with 0.03-250 μM of ATP, ADP, and adenosine, respectively. The calculation of the test method and cell growth rate was as described above. The results are shown in FIG.
As shown in FIG. 3, an increase in cell proliferation rate was observed with the addition of ATP and ADP, but higher cell proliferation was observed with the sample to which ADP was added. When adenosine was added, the cell growth rate decreased significantly as the concentration increased.
From the above test results, it is clear that ADP is particularly suitable as a substance that increases extracellular ATP release in epidermal cells and promotes cell proliferation.

<ADP concentration>
Test Method Commercially available normal human epidermal keratinocytes (NHEK, Kurabo Industries) were cultured in KGM-2 medium (Kurabo) according to the protocol. The cells were seeded in a 96-well black plate at 1 × 10 ⁴ per well, cultured for 24 hours, washed with PBS (−), and allowed to stand for 1 hour.
Thereafter, ADP was added to the culture medium of each well so as to be 0, 0.01, 0.1, 1, 10, 100 μM, respectively, and at room temperature using an ATP assay kit (ATPLite, manufactured by Perkin Elmer). The sample was processed for a minute and placed in a luminescent plate reader for measurement.
The amount of extracellular ATP released by each concentration of ADP obtained from this test is shown in FIG.

As shown in FIG. 4, in the sample in which the concentration of ADP was higher than 1 μM, a significant increase in ATP release was observed. On the other hand, the increase in the amount of extracellular ATP released hardly increased until the ADP concentration reached 1 μM.
From the above, it has been clarified that the ADP concentration is preferably 10 μM (0.01 mM) or more in order to obtain the effect of promoting the extracellular ATP release amount.

<ATP-degrading enzyme inhibitory ability of xanthine derivatives>
Test Method Commercially available normal human epidermal keratinocytes (NHEK, Kurabo Industries) were cultured in KGM-2 medium (Kurabo) according to the operation manual. The cells were seeded in a 96-well black plate at 1 × 10 ⁴ per well, cultured for 24 hours, washed with PBS (−), and allowed to stand for 1 hour.
Thereafter, caffeine was added to the medium of each well so as to have a concentration of 0%, 0.0005%, and 0.005%, and cultured at 37 ° C. for 24 hours.
After culturing, total RNA was extracted from cultured cells in each well using ISOGEN (manufactured by Nippon Gene) according to the protocol, cDNA was prepared by a conventional method, and RT-PCR was performed. The PCR product was subjected to agarose gel electrophoresis, and the expression of NTPDases 1 to 4 was confirmed. The results are shown in FIG.
(Primers and probes)
NTPDase1
5'- ATGCAGGGTTAAAGGTCCTGGAATCTC
5'-TGACTGAATTTGCCCAGCAGATAGTTG
NTPDase2
5'-CTACCGAGTCTACACCCACAGCTTC
5'-TCCACAGTGTAGAAGAAGGCAGAGA
NTPDase3
5'- AAAGCTACTTCAAGTCCCAGCCCTTTG
5'-CAGGAGCATTGCCAGAAACTTCTTCTC
NTPDase4
5'- CGAGTATTTGTTTACTGCTGGCCAAGG
5'-GGGATATCGGTCAGAAGGTCTTCCAG

As shown in FIG. 5, NTPDases 2 to 4, which are ATP-degrading enzymes, were expressed in cultured human epidermal cells. As previously reported, NTPDase1 was not expressed in epidermal cells (Georgiou JG et al,: Human epidermal and monocyte-derived langerhans
cells express functional P2X7 receptors. J Invest Dermatol: 125: 482-490, 2005).
In all of the three types of degrading enzymes (NTPDases 2 to 4) in which expression was observed, suppression of expression due to addition of caffeine was observed. This enzyme inhibitory effect was observed from a very low caffeine concentration, and the enzyme expression was suppressed as the concentration increased.

Further, HaCaT cells (human keratinocytes) were seeded in a 96-well plate at 1 × 10 ⁴ per well, and 24 hours later, BSS (NaCl 150 mM; KCl 5 mM; CaCl 2 1.8 mM; D-glucose 10 mM; After washing with 25 mM HEPES and adjusting the pH to 7.4 with NaOH, the mixture was allowed to stand for 30 minutes.
Thereafter, ATP 10 μM, ATP 10 μM + caffeine 0.0005% mixed solution, ATP 10 μM + theophylline 10 μM mixed solution were added to the medium of each well, respectively. Absorbance at 620 nm was measured with a reader.
The absorbance in the system (unstimulated) cultured for 24 hours without addition of ATP and xanthine derivative was taken as 100%, and the relative value of the absorbance of each sample was calculated, and this was taken as the amount of free phosphoric acid. The results are shown in FIG.

As shown in FIG. 6, when ATP was added to epidermal cells, the amount of free phosphate increased compared to normal (unstimulated). This is probably because the addition of ATP activated the homeostasis maintenance function of the cell and activated the ATP decomposition reaction accompanied by the release of phosphate. When a mixed solution of ATP and caffeine or theophylline was added to the culture system, the amount of free phosphate decreased. This is presumably because caffeine and theophylline reduced the amount of free phosphate by inhibiting the ATP degradation reaction.
From the above results, it is clear that the xanthine derivative inhibits the expression of NTPDase, and the decomposition reaction of free ATP in epidermal cells is suppressed.

<ATP release amount by ADP + xanthine derivative>
Test Method Commercially available normal human epidermal keratinocytes (NHEK, Kurabo Industries) were cultured in KGM-2 medium (Kurabo) according to the operation manual. The cells were seeded in a 96-well black plate at 1 × 10 ⁴ per well, cultured for 24 hours, washed with PBS (−), and allowed to stand for 1 hour.
Thereafter, only the culture solution was added to the medium of each well so that ADP was 10 μM and GTP was 10 μM, and caffeine was added to each of the wells so that the concentrations were 0, 0.0005, and 0.005%. Samples were prepared, treated for 5 minutes at room temperature using an ATP assay kit (ATPLite, manufactured by Perkin Elmer), and placed on a luminescent plate reader for measurement. The results are shown in FIG. In addition, FIG. 8 shows the result of comparison between the ADP-free medium (control) and the ADP 10 μM-added medium with and without 0.0005% caffeine.

In the case of the medium only, the increase in ATP release was hardly observed even when the caffeine concentration was increased. In the case of a medium supplemented with GTP, the amount of ATP released was significantly increased by adding GTP alone as compared with the case of using only the culture solution, but almost no effect on the amount of ATP released by the addition of caffeine was observed.
On the other hand, in the case of the medium added with ADP, the caffeine-free sample did not reach the amount of extracellular ATP released by adding GTP, but when caffeine was added, the amount of extracellular ATP released increased dramatically, and GTP was added. Better ATP release (approximately 300 nM).
In addition, according to FIG. 8, when ADP is not added, even if an appropriate amount of caffeine is added, an increase in the amount of ATP released is hardly observed (control), whereas in the ADP compounding system, caffeine is added. It can be seen that a significant increase in ATP release occurred.
This remarkable increase in ATP is thought to be due to the enhancement of ATP synthesis based on the addition of ADP as an ATP substrate and the suppression of ATP degradation based on the addition of caffeine. Furthermore, ADP and caffeine act synergistically with respect to the increase in the ATP release amount from the comparison between the increase in the ATP release amount by the addition of caffeine in the control of FIG. 8 and the increase range in the ADP-added medium. It was guessed.
From the above, it is clear that the amount of extracellular ATP released from epidermal cells is remarkably promoted particularly by coexisting ADP with a xanthine derivative.
Furthermore, when further examination was performed, when the ADP: xanthine derivative concentration was 1: 1 to 5: 1 by mass ratio, a particularly remarkable increase in the amount of extracellular ATP released was observed.

<Effect of ADP + xanthine derivative on cell proliferation>
ADP and xanthine derivatives were applied to the test skin and changes in cell proliferation activity were measured.
The test skin is a three-dimensional skin model consisting of an epidermis layer consisting of human-derived epidermal keratinocytes (epidermal keratinocytes, one of the epidermal cells) and a collagen gel embedded with dermal fibroblasts. It has a thickness closer to that of human skin, making it easier to measure information-transmitting substances while culturing. A stratum corneum is also formed on the epidermis, and it is possible to apply and remove the drug.

Test Method Test skin (LSE-011A, manufactured by Toyobo Co., Ltd.) was cultured according to the protocol. The attached silicon ring is mounted on the skin, and water (control), ADP 3 mM solution, ADP 10 mM solution, ADP 30 mM solution, caffeine 0.1% solution, ADP and caffeine solution (ADP concentration 10 mM, caffeine concentration 0.1%) ) Was placed in the center of the ring, and after culturing for 24 hours, the culture solution and test skin were collected. BrdU (5-bromo-2′-deoxy-uridine) was added to the culture solution (final concentration 50 μM) 2 hours before collection to label the DNA of the replicating cells.
The test skin was sampled to have a width of 2 mm so as to include the central part and the length of the entire model diameter, and was embedded in a paraffin block according to a conventional method to prepare a sliced slice. This section was immunostained with an anti-BrdU antibody, and the number of cell nuclei (BrdU positive cells) labeled with BrdU was counted as an index of cell proliferation activity. BrdU is incorporated into replicating DNA as an analog of thymidine and is used to identify proliferating cells. When the tissue is collected 1-2 hours after adding BrdU and stained with an antibody against BrdU, the nuclei of proliferating cells are stained. The more nuclei, the higher the proliferation activity.
FIG. 9 shows the anti-BrdU stained image of each section and the number of BrdU positive cells per section.

As shown in the left image of FIG. 9, the number of cells that incorporated BrdU and stained was increased by the addition of ADP. In particular, a large number of BrdU positive cells were observed in the sample to which caffeine was added in addition to ADP. In each image of FIG. 9, the upper band-like portion is the epidermis and the lower portion is the portion corresponding to the dermis, and the stained cells appear as dark dots.
In addition, as shown in the right graph of FIG. 9, an increase in the number of BrdU positive cells was also observed in the addition of ADP solution or caffeine solution compared to water, but particularly with a solution containing both ADP and caffeine, A very high increase in the number of positive cells was shown. This increase in the number of cells is more significant than the increase due to ADP alone and the increase due to caffeine alone, and it is considered that the combined use of ADP and caffeine has a synergistic cell growth effect.

<Comparison with chemical peeling agent>
The following comparative test was conducted on skin cells to which ADP and xanthine derivatives were added and skin cells that had been treated with glycolic acid (GA), which is a general chemical peeling agent component.
Test Method Commercially available normal human epidermal keratinocytes (NHEK, Kurabo Industries) were cultured in KGM-2 medium (Kurabo) according to the operation manual. The cells were seeded in a 96-well black plate at 1 × 10 ⁴ per well, cultured for 24 hours, washed with PBS (−), and allowed to stand for 1 hour.
Thereafter, each well cell was stimulated by exposing it to a glycolic acid solution having a different concentration for 5 to 10 seconds. The same cells were stimulated twice, and after washing the acid with the culture solution, the culture solution was added and cultured at 37 ° C. for 24 hours. In addition, cells in another well were added with 10 μM ADP and 0.0005% caffeine and cultured in the same manner. After incubation, the plate was treated with an ATP assay kit (ATPLite, manufactured by Perkin Elmer) for 5 minutes at room temperature, and the plate was placed in a luminescent plate reader and measured.

As shown in FIG. 10, the increase in the amount of ATP released by glycolic acid (GA) appears most prominently when the GA concentration is 0.59%, and the pH of the GA solution at that time is very high at 3.7. showed that. On the other hand, the sample containing ADP 10 μM and caffeine 0.0005% showed an ATP release amount (about 270 nM) equivalent to that treated with a GA concentration of 0.59%. The culture solution pH at that time was 7.6, which was close to physiological pH.
According to Japanese Patent Application Laid-Open No. 2006-262806, treatment with 40% glycolic acid (pH 2.8) is performed on a skin model for 3 minutes, and after 10 minutes, free ATP in the culture solution is about 300 nm, It has been suggested that such ATP release can promote epidermal cell proliferation. This is almost equal to the ATP release amount in the presence of ADP and caffeine shown in FIG. Considering these, it can be considered that an extracellular ATP release amount of about 300 nM can serve as an indicator of promoting the proliferation of epidermal cells.
That is, in order to obtain a cell proliferation action (chemical peeling effect) through ATP release by stimulation of VR1 receptor by GA, it is necessary to apply a strong acid to the skin and immediately wash it off. On the other hand, in the present invention, it is considered that by adding ADP and caffeine, cell proliferation action equivalent to the chemical peeling effect can be obtained without giving strong stimulation to the epidermal cells by acid.
In addition, since the blending system of ADP and caffeine is close to physiological pH and there is almost no irritation, it can be used for a long time without being washed off immediately after application to the skin.

Although the example of formulation of the skin external preparation composition concerning this invention is shown below, these do not limit this invention.
<Prescription Example 1: Cream>
(Ingredient) (mass%)
ADP 1
Caffeine 0.5
Squalane 10
Cetostearyl alcohol 3.5
Beeswax 3
Reduced lanolin 5
Polyoxyethylene (20) sorbitan monopalmitate 2
Stearic acid monoglyceride 2
1,3-butylene glycol 0.1
Glycerin 4
Preservative 0.3
Fragrance 0.05
pH adjuster Suitable amount Purified water Residual

<Formulation example 2: lotion>
(Ingredient) (mass%)
ADP 0.5
Caffeine 0.5
Ethanol 5
1,3-butylene glycol 4
Polyoxyethylene (18) oleyl alcohol ether 0.5
Glycerin 4
Preservative 0.2
Fragrance 0.05
pH adjuster Suitable amount Purified water Residual

<Prescription Example 3: Latex>
(Ingredient) (mass%)
ADP 0.5
Caffeine 0.5
Stearic acid 1.5
Cetyl alcohol 0.5
Beeswax 2
Polyoxyethylene (10) monooleate 1
Glycerin 7
1,3-butylene glycol 0.5
Preservative 0.3
Perfume 0.03
pH adjuster Suitable amount Purified water Residual

<Formulation example 4: lotion>
(Ingredient) (mass%)
Glycerin 2.0
Dipropylene glycol 2.0
1,3-butylene glycol 3.0
Polyoxyethylene polyoxypropylene dimethyl ether 3.0
Sodium hyaluronate 0.2
PPG-13 decyltetradeces-24 0.5
Sodium 4-methoxysalicylate 0.5
Water-soluble collagen 0.1
ADP 0.0005
Theophylline 0.0005
Ethanol 10.0
Citric acid Appropriate amount Sodium citrate Appropriate amount EDTA Appropriate amount Phenoxyethanol Appropriate perfume Appropriate amount Purified water Residue

<Prescription Example 5: Latex>
(Ingredient) (mass%)
Glycerin 10.0
Dipropylene glycol 5.0
Polyethylene glycol 20000 0.5
Polyoxyethylene polyoxypropylene dimethyl ether 5.0
Hydrogenated polydecene 4.0
Vaseline 2.0
Dimethicone 3.5
Batyl alcohol 1.0
Pentaerythrityl tetraethylhexanoate 2.0
Cetyl ethylhexanoate 4.0
Polyoxyethylene glyceryl isostearate 2.0
Polyoxyethylene glyceryl distearate 2.0
(Acryloyldimethyltaurate ammonium / VP) copolymer 1.5
Ethanol 5.0
ADP 0.1
Caffeine 0.05
Caustic potash appropriate amount EDTA appropriate amount phenoxyethanol appropriate amount methylparaben appropriate amount perfume appropriate amount purified water remainder

<Prescription Example 6: Essence>
(Ingredient) (mass%)
Glycerin 10.0
1,3-butylene glycol 5.0
Polyoxyethylene polyoxypropylene dimethyl ether 5.0
Erythritol 0.5
Cetyl octanoate 1.0
Decamethylcyclopentasiloxane 2.0
Squalane 2.0
Tetraoctanoic acid pentaerythrit 1.0
Polyacrylamide 0.1
Ethanol 5.0
(Na-acrylate / acryloyldimethyltaurine) copolymer 0.1
(C10-C30) acrylic acid / methacrylic acid copolymer 0.1
ADP 0.5
Theoferrin 0.5
Tranexamic acid methylamide hydrochloride 3.0
Dipotassium glycyrrhizinate 0.05
Tocopherol acetate 0.5
Yeast extract 1.0
Silica 2.0
Sodium metaphosphate Appropriate potassium hydroxide Appropriate citric acid Appropriate sodium citrate Appropriate yellow iron oxide Appropriate phenoxyethanol Appropriate fragrance Appropriate purified water Residue

<Prescription Example 7: Cream>
(Ingredient) (mass%)
Glycerin 12.0
Dipropylene glycol 8.0
Hydrogenated polydecene 2.0
Vaseline 4.0
Decamethylcyclopentasiloxane 5.0
Cetyl alcohol 1.0
Pentaerythrityl tetraethylhexanoate 2.0
Polyoxyethylene glyceryl distearate 2.0
Polyoxyethylene phytosterol 0.5
Ethanol 3.0
Agar 1.0
Succinoglucan 1.0
(Dimethylacrylamide / acryloyldimethyltaurine Na) cross polymer 0.5
ADP 5.0
Theophylline 1.0
Tranexamic acid 3.0
Vitamin C ethyl 0.5
Rose extract 0.5
Citric acid Appropriate amount Sodium citrate Appropriate amount Sodium metaphosphate Appropriate amount Phenoxyethanol Appropriate fragrance Appropriate amount Purified water Residue

<Prescription Example 8: Daytime serum>
(Ingredient) (mass%)
Glycerin 5.0
Acetylated sodium hyaluronate 0.001
Decamethylcyclopentasiloxane 1.0
Caprylyl Methicone 2.0
Ethylhexyl ethylhexanoate 2.0
Isostearic acid 1.5
PEG-10 Dimethicone 1.0
Polyoxyethylene hydrogenated castor oil 0.5
Ethanol 10.0
Octocrylene 8.0
Octyl methoxycinnamate 7.0
Titanium oxide 0.3
Zinc oxide 0.2
Succinoglucan 0.5
Polyacrylamide 0.5
(Na-acrylate / acryloyldimethyltaurine) copolymer 0.2
Ascorbic acid glucoside 3.0
Dipotassium glycyrrhizinate 0.01
ADP 0.0008
Caffeine 0.0006
Citric acid Appropriate amount Sodium citrate Appropriate amount Sodium metaphosphate Appropriate amount Phenoxyethanol Appropriate fragrance Appropriate amount Purified water Residue

  The skin external preparation compositions of Formulation Examples 1 to 3 were all hypoallergenic and exhibited a high epidermal cell proliferation effect.

It is a graph which shows the amount of extracellular ATP released by addition of an ATP analog in cultured epidermal cells. It is a graph which shows the cell growth effect | action by addition of ATP analog in a cultured epidermal cell. It is a graph which shows the cell growth effect | action by addition of ATP analog in a cultured epidermal cell. It is a graph which shows the amount of extracellular ATP released by the concentration of ADP in cultured epidermal cells. It is an image which shows the expression suppression of ATP-degrading enzyme by caffeine in a cultured epidermal cell. It is a graph which shows the effect of a xanthine derivative with respect to the free phosphate amount which arises by decomposition | disassembly by addition of ATP in a cultured epidermal cell. It is a graph which shows the amount of extracellular ATP release by a caffeine density | concentration in a cultured epidermal cell. It is a graph which shows the change of the amount of extracellular ATP released by addition of ADP and caffeine in cultured epidermal cells. It is a graph which shows the cell growth by addition of ADP and caffeine in a skin model, and the change of the number of BrdU positive cells. It is a graph which shows the change of the amount of extracellular ATP by GA of each density | concentration, or ADP and caffeine treatment in a cultured epidermal cell.

Claims (8)

  A skin external preparation composition comprising ADP and a xanthine derivative.   2. The skin external preparation composition according to claim 1, comprising 0.01 to 100 mM of ADP and 0.0005 to 5% by mass of a xanthine derivative.   The skin external preparation composition according to claim 1 or 2, wherein the xanthine derivative is caffeine and / or theophylline.   It is an epidermal cell growth promoter, The skin external preparation composition in any one of Claims 1-3 characterized by the above-mentioned.   The external preparation composition for skin according to any one of claims 1 to 4, wherein the mixing ratio of ADP: xanthine derivative is 1: 1 to 5: 1 by mass ratio.   A leave-on type cosmetic comprising the skin external preparation composition according to any one of claims 1 to 5.   A method for promoting the proliferation of epidermal cells, comprising applying an external preparation composition for skin containing ADP and a xanthine derivative to the skin surface.   The method for promoting epidermal cell proliferation according to claim 7, wherein the skin external preparation composition contains 0.01 to 100 mM of ADP and 0.0005 to 5 mass% of a xanthine derivative.