WO2022107866A1 - 育毛剤 - Google Patents
育毛剤 Download PDFInfo
- Publication number
- WO2022107866A1 WO2022107866A1 PCT/JP2021/042505 JP2021042505W WO2022107866A1 WO 2022107866 A1 WO2022107866 A1 WO 2022107866A1 JP 2021042505 W JP2021042505 W JP 2021042505W WO 2022107866 A1 WO2022107866 A1 WO 2022107866A1
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- WO
- WIPO (PCT)
- Prior art keywords
- hair
- growth
- phytosphingosine
- present
- skin
- Prior art date
Links
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/41—Amines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
Definitions
- the present invention relates to a hair restorer. More specifically, the present invention relates to a hair growth agent which is an external preparation containing phytosphingosine.
- the hair-growth agent which is an external preparation containing chiro-inositol described in Patent Document 4
- the hair-growth agent is only supported by the hair-growth effect in subjects who are not insulin resistant, and the number of administration subjects is limited. Therefore, it does not fully meet the demands of a wide range of consumers who desire a hair growth effect and a hair type / quality improvement effect.
- Phytosphingosine is known as a component of cosmetic raw materials (see Patent Document 5). However, there are no reports on the hair-growth effect of phytosphingosine.
- An object of the present invention is to provide a hair-growth agent having an excellent hair-growth effect.
- the first means of the present invention for solving the above-mentioned problems is a hair-growth agent which is an external preparation containing phytosphingosine.
- the second means of the present invention for solving the above-mentioned problems is the hair growth agent according to the first means of the present invention, wherein the content of phytosphingosine is 0.001 to 20% by weight based on the whole. be.
- the third means of the present invention for solving the above-mentioned problems is the first means or the second means of the present invention in which the content of phytosphingosine is 0.005 to 10% by weight based on the whole.
- the described hair restorer is the first means or the second means of the present invention in which the content of phytosphingosine is 0.005 to 10% by weight based on the whole.
- the fourth means of the present invention for solving the above-mentioned problems is the hair growth agent according to any one of the first to third means of the present invention, which is used for promoting hair stem growth or hair growth. Is.
- the fifth means of the present invention for solving the above-mentioned problems is the hair growth agent according to any one of the first to fourth means of the present invention, which is used for improving the hair stem elongation rate. Is.
- the sixth means of the present invention for solving the above-mentioned problems is the hair growth agent according to any one of the first to fourth means of the present invention, which is used to improve the maximum hair shaft length. Is.
- the seventh means of the present invention for solving the above-mentioned problems is the hair growth agent according to any one of the first to fourth means of the present invention, which is used for increasing the hair shaft diameter. be.
- the eighth means of the present invention for solving the above-mentioned problems is the hair growth agent according to any one of the first to fourth means of the present invention, which is used for increasing the number of hairs. ..
- the ninth means of the present invention for solving the above-mentioned problems is a hair-growth agent according to any one of the first to eighth means of the present invention, which is a solution.
- the tenth means of the present invention for solving the above-mentioned problems is the hair growth according to any one of the first to ninth means of the present invention for hair, hair, eyebrows and / or eyelashes. It is an agent.
- the eleventh means of the present invention for solving the above-mentioned problems is a hair-growth method including administration of the hair-growth agent according to any one of the first to tenth means of the present invention to a subject. ..
- Another means of the present invention for solving the above-mentioned problems is a scalp care agent which is an external preparation containing phytosphingosine.
- Another means of the present invention for solving the above-mentioned problems is a method for improving scalp symptoms, which comprises administering a scalp care agent, which is an external preparation containing phytosphingosine, to a subject.
- phytosphingocin as an active ingredient of a hair restorer which is an external preparation
- hair stem growth promotion hair shaft elongation rate improvement
- maximum hair shaft length in hair, hair, eyebrows and / or eyelashes are achieved.
- An excellent hair restorer and scalp care agent that have an effect of improving hair stalks, an effect of increasing the diameter of the hair shaft, and an scalp care effect will be provided.
- FIG. 1 is a plot showing changes in hair shaft length at a drug-applied site in mice after application of a 60% ethanol aqueous solution.
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- the first hair cycle shows standard data applied with a 60% ethanol aqueous solution containing no drug.
- FIG. 2 is a plot showing changes in hair shaft length at the drug application site in mice after application of a 60% ethanol aqueous solution containing minoxidil (5%).
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- the first hair cycle shows standard data applied with a 60% ethanol aqueous solution containing no drug.
- FIG. 1 is a plot showing changes in hair shaft length at a drug-applied site in mice after application of a 60% ethanol aqueous solution.
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- FIG. 3 is a plot showing changes in hair shaft length at the drug application site in mice after application of a 60% ethanol aqueous solution containing minoxidil (3%).
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- the first hair cycle shows standard data applied with a 60% ethanol aqueous solution containing no drug.
- FIG. 4 is a plot showing changes in hair shaft length at the drug application site in mice after application of a 60% ethanol aqueous solution containing minoxidil (1%).
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- the first hair cycle shows standard data applied with a 60% ethanol aqueous solution containing no drug.
- FIG. 5 is a plot showing changes in hair shaft length at the drug application site in mice after application of a 60% ethanol aqueous solution containing phytosphingosine (3%).
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- the first hair cycle shows standard data applied with a 60% ethanol aqueous solution containing no drug.
- FIG. 6 is a plot showing changes in hair shaft length at the drug application site in mice after application of a 60% ethanol aqueous solution containing phytosphingosine (1%).
- the vertical axis represents the hair shaft length (mm), and the horizontal axis represents the number of days.
- the first hair cycle shows standard data applied with a 60% ethanol aqueous solution containing no drug.
- FIG. 7 is a graph showing changes in VEGF gene expression level in human dermal papilla cells when stimulated with tosphingocin for 72 hours.
- FIG. 8 shows the evaluation of cell division activity by phytosphingosine stimulation. Cells in which BrdU uptake can be confirmed are indicated by upward triangles ( ⁇ or ⁇ ).
- FIG. 9 shows the evaluation of cell division activity by phytosphingosine stimulation.
- the active ingredient of the hair restorer and the scalp care agent which are external agents according to the present invention, consists of phytosphingosine.
- the concentration of phytosphingosine which is an active ingredient in the hair restorer and scalp care agent of the present invention, is 0.001 to 20% by weight based on the total amount of the hair restorer and scalp care agent. More specifically, it is 0.005 to 10% by weight.
- the hair restorer and scalp care agent of the present invention include pharmaceuticals, non-pharmaceutical products, hair, hair, eyebrows and / or cosmetics including eyelid cosmetics and scalp cosmetics, and ointments, paps, liniments, lotions, and external liquid preparations. , Creams, gels, emulsions, hair tonics, hair sprays, microneedles and the like, but are not limited to these.
- ingredients such as additives that are permissible may be blended.
- Ingredients such as this additive include, for example, excipients, stabilizers, odorants, bases, dispersants, diluents, anionic surfactants, amphoteric surfactants, nonionic surfactants, and cationic surfactants.
- Activators anionic polymers, nonionic polymers, ethylene oxide / propylene oxide block copolymers, alcohols, emulsifiers, transdermal absorption promoters, pH adjusters, preservatives, colorants, fats and oils, mineral oils, etc. Oils, moisturizers, thickeners, polymers, film-forming agents, UV absorbers, cell activators, moisturizers, inorganic salts, functional beads and capsules, silicones, metal chelating agents, antioxidants, preservatives, Cooling agents, deodorants, pigments, dyes, fragrances, sugars, amino acids, vitamins, organic acids, organic amines, plant extracts, viscosity modifiers such as clay minerals and various polymers, but are limited to these. It is not something that is done.
- the hair growth agent and scalp care agent of the present invention may contain known components having effects such as hair growth, hair growth, and hair growth.
- the dose of the active ingredient per administration of the hair restorer and the scalp care agent in the means of the present invention can be adjusted so that the effects of the hair restorer and the scalp care agent of the present invention are exhibited.
- the dose thereof can be, for example, 0.005 to 200 mg, specifically 0.05 to 100 mg, and more specifically 0.5 to 10 mg.
- the number of administrations of the hair restorer and scalp care agent of the present invention can be one or more so that the effects of the hair restorer and scalp care agent of the present invention can be exhibited.
- the number of administrations of the hair restorer and the scalp care agent of the present invention can be, for example, 1 to 6 times per day. Then, specifically, it can be 1 to 3 times a day, and more specifically, 1 to 2 times a day.
- the hair restorer and scalp care agent of the present invention are related to hair shaft growth promotion, hair growth and hair loss prevention, and are preferably related to hair shaft growth promotion and hair growth.
- promoting hair shaft growth means improving the hair shaft elongation rate, improving the maximum hair shaft length, and / or increasing the hair shaft diameter.
- hair growth means that hair growth has stopped or the ability to grow hair has decreased in areas where hair is not growing (hair shafts do not appear outside the epidermis) or where the number of hairs is small. It means promoting the growth of new hair from the opened pores and increasing the number of hairs, specifically, shortening the resting period in the hair cycle and / or resuming the stopped hair cycle. ..
- having a hair shaft growth promoting effect means having an advantageous action on hair stem growth promoting effect, and a characteristic showing a hair shaft growth promoting effect is referred to as "hair shaft growth promoting activity”. Further, “having a hair growth effect” means having an advantageous action on hair growth, and a characteristic showing a hair growth effect is referred to as "hair growth promoting activity”.
- hair loss means a phenomenon in which hair stems are shed from pores, and more specifically, it means an increase in inhibitory cytokines and the like that inhibit cell proliferation and cell death thereof.
- the characteristic showing the hair loss prevention effect is called “hair loss prevention activity”.
- “having a hair loss preventing effect” means that the number of hair follicles shed from the pores is reduced through inhibition or reduction of inhibitory cytokines and suppression of cell death, and promotes and develops hair follicles. It is a physiological phenomenon that is different from the characteristics that show the hair effect.
- the "scalp symptom” means a symptom such as dandruff, rough skin of the scalp, dryness of the scalp, erythema, itch, and pimples.
- "improvement of scalp symptom” means suppression or improvement of dandruff, rough skin of scalp, dryness of scalp, erythema, itch, pimple and the like.
- the hair restorer of the present invention can be used to improve the hair shaft elongation rate or the hair shaft maximum length.
- the hair shaft elongation rate can be improved by, for example, up to 110%, specifically by about 25 to 110%, as compared with the hair stem elongation rate in the standard data of the hair cycle. More specifically, it can be improved by about 33 to 110%.
- the maximum hair shaft length can be improved by, for example, up to 49%, specifically by about 1 to 49%, as compared with the maximum hair shaft length in the standard data of the hair cycle. More specifically, it can be improved by about 2 to 49%.
- the hair restorer of the present invention can be used to increase the diameter of the hair shaft.
- new hair grows from pores where hair growth is stopped or hair growth ability is reduced at a site where hair is not growing (hair shaft does not come out from the epidermis) or the number of hair is small. It can be used to promote this and increase the number of hairs, and more specifically to shorten the resting period in the hair cycle and / or to resume the stopped hair cycle.
- the hair restorer and scalp care agent of the present invention can be used not only for humans but also for animals such as livestock and pets.
- a method for hair growth and a method for improving scalp symptoms which comprises administering an external preparation containing phytosphingosine to a subject including humans and animals such as livestock and pets.
- the planned back body hair skin collection site of 7 to 8 week old C57BL / 6N mice was removed and bred for 12 to 14 days. Then, after euthanizing the hair-extracted C57BL / 6N mouse by cervical spine dislocation, an appropriate amount of back body hair skin was collected from the planned back body hair skin collection site.
- DMEM10 DMEM medium
- the collected skin was immersed in DMEM medium (hereinafter referred to as "DMEM10") containing 10 mM HEPES, 10% fetal bovine serum, and 1% penicillin / streptomycin solution.
- DMEM10 DMEM medium
- the collected back hair skin is pinched with bent tweezers and soaked in a sterilizing solution for 10 seconds for treatment. Sterilization was performed using a fresh solution in the order of povidone iodine 7% solution treatment twice, PBS ( ⁇ ) treatment three times, and DMEM10 treatment twice. After sterilization, it was immersed in clean DMEM10.
- the transparent connective tissue attached to the cutaneous muscle layer of the skin was excised using curved scissors, and the hair group was cut into rectangular strips along the hair flow. At that time, the hair follicles were adjusted to have 5 rows on the short axis, and the hair follicles on the long axis were cut into 6 rows and blocked.
- the skin test piece derived from the back body hair skin prepared above was transplanted into 4 to 6 week old Balb / c nu / nu mice.
- mice were anesthetized with isoflurane according to a conventional method. Then, the back of the mouse was disinfected with a povidone iodine 7% solution, and then the mouse was placed in a natural lying position. Then, the back of the mouse was punctured with a Manny Offsalmic Knife (Manny Co., Ltd., Japan) to form a transplant wound extending from the epidermal layer of the skin to the lower layer of the dermis layer.
- a Manny Offsalmic Knife Manny Co., Ltd., Japan
- a skin test piece derived from the back body hair skin was inserted into the formed transplant wound so that the hair group faces the body surface side of the transplant wound.
- the transplantation depth of the skin test piece was adjusted so that the upper end of the hair group was exposed at the upper end of the transplant wound.
- the transplanted wound into which the skin test piece derived from the back body hair skin was transplanted was covered with Nurse Van (registered trademark) (Sample Planet Co., Ltd., Japan) and surgical tape (3M Japan Ltd., Japan) as protective tapes. Protected the transplant wound.
- the hair shaft diameter was measured using the hair after application of the drug.
- the hair shaft diameter was 15.89 ⁇ m when a control 60% ethanol aqueous solution was applied.
- the hair shaft diameter when the minoxidil 5% solution was applied was 18.28 ⁇ m.
- the rate of increase from the control hair shaft diameter when minoxidil was applied was 115%.
- the hair shaft diameter was 20.15 ⁇ m.
- the rate of increase from the control hair shaft diameter when phytosphingosine was applied was 127%.
- Human dermal papilla cells and medium Purchase human dermal papilla cells (catalog number: CA602t05a, white race, derived from 29-year-old male, Toyo Spinning Co., Ltd. (Japan)) and make them listed in the protocol. The cells were maintained and cultured for test evaluation.
- Test method Human dermal papilla cells were seeded on a 24-well plate so as to have 6 ⁇ 10 3 cells / well. After culturing in a CO 2 incubator (5% CO 2 , 37 ° C.) for 1 day, the cells were replaced with a medium containing each test drug. The cell plates were then returned to the CO 2 incubator and cultured for an additional 72 hours. After culturing, total RNA was extracted from each well, recovered, and reverse transcribed into cDNA. The expression of the VEGF gene was measured by the real-time PCR method using the prepared cDNA. The GAPDH gene was used as an internal standard, and the expression level of the VEGF gene was calculated as a relative value to the negative control group.
- 300 ⁇ L of lysis buffer RL was added per well, and the cells were lysed by pipetting. 300 ⁇ L of 70% ethanol was added to the cytolysate and mixed by pipetting. The sample solution was added to FastGene RNA binding volume and centrifuged at 10,000 g for 1 minute at room temperature. The filtrate that passed through the column was discarded from the collection tube, the FastGene RNA binding volume was returned to the original collection tube, 600 ⁇ L of the washing buffer RW1 was added to the FastGene RNA binding volume, and the mixture was centrifuged at 10000 g for 1 minute at room temperature.
- the FastGene RNA binding collection was transferred to a new collection tube and set, 700 ⁇ L of the wash buffer RW2 was added to the FastGene RNA binding collection, and the mixture was centrifuged at 10000 g for 1 minute at room temperature.
- the FastGene RNA binding collection was transferred to a new collection tube, set, and centrifuged at 15000 rpm for 1 minute at room temperature.
- the FastGene RNA binding volume was transferred to a new collection tube and set, 50 ⁇ L of elution buffer RE was added to the center of the membrane of the FastGene RNA binding volume, and the mixture was centrifuged at 10000 g for 1 minute at room temperature to recover the purified RNA.
- the concentration of the recovered RNA was measured by NanoDrop Lite (catalog number: ND-LITE, Thermo Fisher Scientific Co., Ltd.) and stored at ⁇ 80 ° C. until the next cDNA conversion operation.
- FastGene scriptaseII cDNAsynthesis 5 x Ready Mix (catalog number: NE-LS64, Japan Genetics Co., Ltd. (Japan)) was used for the synthesis of cDNA.
- the total RNA produced in the new tube was diluted with RNase Free Water so that the concentration was 20 ng / mL, and 4 ⁇ L of FastGene scriptaseII cDNAsynthesis 5 ⁇ Ready Mix was added to 16 ⁇ L of this sample solution, and the mixture was stirred with vortex.
- a MiniAmp thermal cycler Thermo Fisher Scientific Co., Ltd.
- the cDNA was synthesized by incubating at 25 ° C. for 10 minutes, 42 ° C. for 60 minutes, and 85 ° C. for 5 minutes.
- the cDNA synthesized by the above method was used for real-time PCR.
- Each cDNA gene diluent is added to a predetermined well of a 96-well plate, and a primer is added and mixed with THUNDERBIRD SYBR qPCR Mix (catalog number: QPS-201, Toyo Spinning Co., Ltd. (Japan)), and QuantStudio 7 Flex Real. -Gene expression was analyzed by Time PCR System (catalog number: 4485693, Thermo Fisher Scientific Co., Ltd.).
- As a PCR reaction 40 cycles of 95 ° C. for 5 seconds and 60 ° C. for 30 seconds were performed.
- Primer for detecting VEGF gene expression Forward: aggccagcacataggagaga (SEQ ID NO: 1) Reverse direction: acgcgagtctgtgttttgc (SEQ ID NO: 2)
- Primer for detecting GAPDH gene expression Forward: catccctgcctctactggcgctgcc (SEQ ID NO: 3)
- Reverse direction ccaggatgcccttgagggggccctc (SEQ ID NO: 4)
- the relative expression level of each gene was calculated as follows.
- the Ct value (number of PCR cycles) was calculated from the intersection of the amplification curve of each gene and the threshold line.
- the relative expression level is the value obtained by dividing the Ct value of the target gene by the Ct value of the internal standard GAPDH gene.
- Section preparation A block of paraffinized skin tissue is sectioned with a microtome (Leica Microsystems GmbH, Germany), and the section is attached to a platinum-coated slide glass (Platinum Pro (Matsunami Glass Industry Co., Ltd., Japan)). It was allowed to stand in warm steam at ° C for 8 to 10 hours to dry.
- a microtome Leica Microsystems GmbH, Germany
- Platinum-coated slide glass Platinum Pro (Matsunami Glass Industry Co., Ltd., Japan)
- phytosphingosine which is an active ingredient of the means of the present invention
- At 3% at least the cell division activity in hair follicles was improved, and the results suggesting that the active ingredient of the means of the present invention has hair growth activity.
- the application of phytosphingosine, which is the active ingredient of the means of the present invention enhances the cell division activity in the basal layer of the epidermis, and that the active ingredient of the means of the present invention provides a scalp care effect.
- phytosphingosine which is the active ingredient of the means of the present invention, exhibits excellent hair-growth activity and at the same time exhibits an excellent scalp care effect at the applied portion. It became.
- the hair stem growth promoting effect and hair shaft in hair such as hair, hair, eyebrows and / or eyelashes. It is possible to provide new hair growth agents and scalp care agents that have an effect of improving the elongation rate, an effect of improving the maximum length of the hair shaft, an effect of increasing the diameter of the hair shaft, and an effect of scalp care.
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Abstract
Description
(1)実験動物
C57BL/6Nマウス(オス)およびBalb/c nu/nuマウス(メス)を日本エスエルシー株式会社(日本)より購入し、飼育の後に以下の実験に供した。なお、動物の飼育および試験は、関連法規、省令、および指針を遵守し、理化学研究所実験倫理審査会の承認のもとに実施した。
以下の試薬をそれぞれ用意した。
比較例1:60%エタノール水溶液
比較例2:ミノキシジル 5%溶液
比較例3:ミノキシジル 3%溶液
比較例4:ミノキシジル 1%溶液
実施例1:フィトスフィンゴシン 3%溶液
実施例2:フィトスフィンゴシン 1%溶液
毛周期の1周期目は、60%エタノール水溶液をプラセボとして塗布する。上記の皮膚試験片を移植したBalb/c nu/nuマウスに、マイクロピペットを用いて25μLの60%エタノールを、皮膚試験片の生着させた左右の背部にそれぞれ塗布した。その後、ドライヤーを用いて冷風をあててエタノールを素早く乾燥させた。この作業を、マウス左右背部に各4回ずつ繰り返した。
Balb/c nu/nuマウスの皮膚試験片の移植部位から3領域をそれぞれ選択し、それら領域からそれぞれ5本の毛を選択して、発毛の状態を確認し記録した。観察と記録は、目視およびデジタルマイクロスコープ(株式会社キーエンス、日本)により行った。
(1)実験動物
上記試験例1と同様の手順にて、7~8週齢のC57BL/6Nマウス(SLC)より背部体毛皮膚を採取し、作製した背部体毛皮膚由来の毛群を4~6週齢のBalb/c nu/nuマウス(SLC)に移植した後、薬剤を塗布した。動物の飼育および実験は、関連法規、省令、および指針を遵守し、理化学研究所実験倫理審査会の承認のもとに実施した。
以下の3つの溶液を試験に使用した。
60%エタノール水溶液
ミノキシジル 5%溶液
フィトスフィンゴシン 1%溶液
(3)太毛化の計測方法
試験例1において3周期目終了した成長した毛幹を用いて、太毛化の計測を行った。採取した毛幹のうち、Zigzag毛の2本を用いた。その中心部分の太くなっている範囲を1辺 100μmの正方形で3か所を選択した。選択した範囲の5か所を計測した。
薬剤塗布後の毛を用い、毛幹径を計測した。
対照となる60%エタノール水溶液を塗布した場合の毛幹径は15.89μmであった。一方、ミノキシジル 5%溶液を塗布した場合の毛幹径は18.28μmであった。ミノキシジルを塗布した場合の対照となる毛幹径からの増加率は115%であった。これに対し、フィトスフィンゴシン 1%溶液を塗布した場合には、毛幹径は20.15μmであった。フィトスフィンゴシンを塗布した場合の対照となる毛幹径からの増加率は127%であった。
(1)ヒト毛乳頭細胞及び培地について
ヒト毛乳頭細胞(カタログ番号:CA602t05a、白色人種、29歳男性由来、東洋紡株式会社(日本))を購入し、プロトコールに記載されるようにして細胞を維持・培養して試験評価を行った。
試験用薬剤として、以下の各濃度(終濃度)の薬剤溶液を調製し、使用した。
比較例5:ミノキシジル 30μM
比較例6:アデノシン 100μM
実施例3:フィトスフィンゴシン 0.3125μM
実施例4:フィトスフィンゴシン 1.25μM
ヒト毛乳頭細胞を6×103個/ウェルとなるように、24ウェルプレートに播種した。CO2インキュベーター(5%CO2、37℃)内で、1日間培養後、各試験用薬剤を含む培地に置換した。その後、細胞プレートをCO2インキュベーターに戻し、さらに72時間培養した。培養後、各ウェルより全RNAを抽出、回収して、それをcDNAに逆転写した。調製したcDNAを用いてリアルタイムPCR法にてVEGF遺伝子の発現を測定した。内部標準としてGAPDH遺伝子を用い、陰性対照群との相対値としてVEGF遺伝子の発現量を算出した。
VEGF遺伝子発現検出用プライマー
順方向:aggccagcacataggagaga(配列番号1)
逆方向:acgcgagtctgtgtttttgc(配列番号2)
GAPDH遺伝子発現検出用プライマー
順方向:catccctgcctctactggcgctgcc(配列番号3)
逆方向:ccaggatgcccttgagggggccctc(配列番号4)
各遺伝子の増幅曲線と閾値線との交点より、Ct値(PCRサイクル数)を算出した。目的遺伝子のCt値より内部標準GAPDH遺伝子のCt値で除した値が相対発現量となる。
ヒト毛乳頭細胞にフィトスフィンゴシンを72時間作用させた後のVEGF遺伝子の発現量の変化を測定し、その結果を、図7に示した。
7~8週齢のBALB/c-nu/nuマウス(日本エスエルシー株式会社、日本)より背部体毛皮膚を採取した。動物の飼育および実験は、関連法規、省令、および指針を遵守し、理化学研究所実験倫理審査会の承認のもとに実施した。
以下の薬剤をそれぞれ用意した。
実施例:フィトスフィンゴシン3%溶液
比較例:60%エタノール水溶液
BALB/c-nu/nuマウスの左右背部に、マイクロピペットを用いて25μLの薬剤を塗布した。その後、塗布部位にドライヤーを用いて冷風をあてて薬剤を素早く乾燥させた。この作業を左右背部に各4回ずつ繰り返した。この薬剤塗布作業を抜毛した翌日から7日間行った。
7日間薬剤を塗布したBALB/c-nu/nuマウスから背部体毛皮膚を採取する24時間および48時間前に、各マウスの後足部分の腹部に10mg/mLのBrdU/生理食塩水0.5mL、注射器(テルモ株式会社、日本)を用いて投与した。
8日目に投与した時間と同じ時間にBALB/c-nu/nuマウスを頸椎脱臼により安楽死させた後に、毛球部を傷つけないように皮膚組織を採取した。採取した皮膚組織をSuperFix(東洋紡株式会社、日本)に16~24時間固定液に浸した後、1×PBSで4回洗い、新しい1×PBSに入れ保管した。その皮膚組織を必要な部分を切り出しし、パラフィン液交換機(Leica Microsystems GmbH、ドイツ)にてパラフィンブロックを作製した。
パラフィン化した皮膚組織のブロックをミクロトーム(Leica Microsystems GmbH、ドイツ)で切片化し、プラチナコートしたスライドガラス(プラチナプロ(松浪硝子工業株式会社、日本))に切片を張り付けて、40℃のウォームスチームで8~10時間放置し、乾燥させた。
脱パラフィン処理を施すため、サンプルの乗ったスライドガラスを、キシレン、キシレン、100%エタノール、95%エタノール、70%エタノールに、順に3分間ずつ浸した。脱パラフィン処理を施したスライドガラスを、室温にて2M HClに30分間浸した。スライドガラスをベンタディスカバリーULTRA(自動染色装置)にセットし、プログラムを起動した。1次抗体Anti-BrdU antibody-Proliferation Maker(abcam、英国)を希釈溶液(1%BSA、0.1%TX100/PBS)にて160倍希釈し、1次抗体を加えるタイミングで100μL/サンプル数添加した。2次抗体Donkey Anti-Sheep IgG H&L(Alexa Flour 594)(Thermo Fisher Scientific、米国)およびへキスト(Hoechst33342)を希釈溶液(1%BSA、0.1%TX100/PBS)にて500倍希釈し、100μL/サンプル数添加した。プログラム終了後、0.1%TX100/PBSにて流しながら洗浄し、1×PBSに浸した。水溶性封入剤(0.5%没食子酸、90%グリセロール/PBS)を80μL/サンプル数滴下した後、カバーガラスを乗せ、黄色チップで空気を押し出し、アスピレーターでカバーガラスの位置を修正しながら余分な封入剤を除き、トップコート(マニュキュア用)を使ってカバーガラスの4辺に封をした。トップコートが乾いていることを確認し、AxioScanで画像をスキャンし、解析した。
BrdU数は表皮層、真皮層の100μm2を3か所、毛包は同程度の大きさのものを3つ計測した。
本試験例では、BALB/c-nu/nuマウスを用いて、60%エタノールへ溶解した濃度3%のフィトスフィンゴシンを試験用の薬剤液とし、連日塗布を施した。陰性対照として60%を塗布し施した。これら塗布は、1回/日で総塗布量100μLとなるようにして7日間行った。また、6、7日目の同じ時間にBrdUを腹部から投与し、その翌日に皮膚組織を採取した。採取した皮膚組織をパラフィン化し切片を作製し、BrdUの免疫染色を行い、BrdU数の計測により細胞活性を観察した。結果を図8及び図9に示す。
Claims (11)
- フィトスフィンゴシンを含む外用剤である育毛剤。
- フィトスフィンゴシンの含有量が全体に対して0.001~20重量%である、請求項1に記載の育毛剤。
- フィトスフィンゴシンの含有量が全体に対して0.005~10重量%である、請求項1または請求項2に記載の育毛剤。
- 毛幹成長促進または発毛に用いるための、請求項1~請求項3のいずれか1項に記載の育毛剤。
- 毛幹伸長速度を向上させるために使用する、請求項1~請求項4のいずれか1項に記載の育毛剤。
- 毛幹最大長を向上させるために使用する、請求項1~請求項4のいずれか1項に記載の育毛剤。
- 毛幹径を増大させるために使用する、請求項1~請求項4のいずれか1項に記載の育毛剤。
- 毛数を増加させるために使用する、請求項1~請求項4のいずれか1項に記載の育毛剤。
- 溶液である、請求項1~請求項8のいずれか1項に記載の育毛剤。
- 頭髪、須毛、眉毛および/または睫毛用の、請求項1~請求項9のいずれか1項に記載の育毛剤。
- 請求項1~請求項10のいずれか1項に記載の育毛剤を対象に投与することを含む育毛方法。
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JP2022563834A JPWO2022107866A1 (ja) | 2020-11-19 | 2021-11-18 | |
US18/037,943 US20240099951A1 (en) | 2020-11-19 | 2021-11-18 | Hair growth stimulant |
TW110143483A TW202237080A (zh) | 2020-11-19 | 2021-11-19 | 育毛劑 |
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JP2020192152 | 2020-11-19 | ||
JP2020-192152 | 2020-11-19 | ||
JP2021-163773 | 2021-10-04 | ||
JP2021163773 | 2021-10-04 |
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WO2022107866A1 true WO2022107866A1 (ja) | 2022-05-27 |
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PCT/JP2021/042505 WO2022107866A1 (ja) | 2020-11-19 | 2021-11-18 | 育毛剤 |
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US (1) | US20240099951A1 (ja) |
JP (1) | JPWO2022107866A1 (ja) |
TW (1) | TW202237080A (ja) |
WO (1) | WO2022107866A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022265053A1 (ja) * | 2021-06-19 | 2022-12-22 | 株式会社アジュバンホールディングス | 育毛剤 |
WO2022265054A1 (ja) * | 2021-06-19 | 2022-12-22 | 株式会社アジュバンホールディングス | 育毛剤 |
WO2022265052A1 (ja) * | 2021-06-19 | 2022-12-22 | 株式会社アジュバンホールディングス | 育毛剤 |
Citations (5)
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JPH0525023A (ja) * | 1991-07-18 | 1993-02-02 | Pola Chem Ind Inc | 養毛料 |
JPH09227343A (ja) * | 1996-02-15 | 1997-09-02 | L'oreal Sa | 2−アミノ−1,3−アルカンジオールを含有する、髪抜け遅延および/または髪成長誘発および刺激剤 |
JP2000281519A (ja) * | 1999-03-25 | 2000-10-10 | Kao Corp | 毛成長促進剤 |
KR20170114317A (ko) * | 2016-04-04 | 2017-10-16 | (주) 셀티스바이오 | 파이토스핑고신과 효모추출물을 유효성분으로 포함하는 탈모방지 및 발모촉진용 조성물 |
KR20200129009A (ko) * | 2019-05-07 | 2020-11-17 | 주식회사 닥터스칼프 | 피토스핑고신을 이용한 두피 재생 및 모발성장을 촉진하는 화장품 |
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2021
- 2021-11-18 JP JP2022563834A patent/JPWO2022107866A1/ja active Pending
- 2021-11-18 WO PCT/JP2021/042505 patent/WO2022107866A1/ja active Application Filing
- 2021-11-18 US US18/037,943 patent/US20240099951A1/en active Pending
- 2021-11-19 TW TW110143483A patent/TW202237080A/zh unknown
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JPH0525023A (ja) * | 1991-07-18 | 1993-02-02 | Pola Chem Ind Inc | 養毛料 |
JPH09227343A (ja) * | 1996-02-15 | 1997-09-02 | L'oreal Sa | 2−アミノ−1,3−アルカンジオールを含有する、髪抜け遅延および/または髪成長誘発および刺激剤 |
JP2000281519A (ja) * | 1999-03-25 | 2000-10-10 | Kao Corp | 毛成長促進剤 |
KR20170114317A (ko) * | 2016-04-04 | 2017-10-16 | (주) 셀티스바이오 | 파이토스핑고신과 효모추출물을 유효성분으로 포함하는 탈모방지 및 발모촉진용 조성물 |
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DATABASE GNPD MINTEL; 13 December 2011 (2011-12-13), "Root Power Restorative Hair Tonic", XP055931579, Database accession no. 1692824 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2022265053A1 (ja) * | 2021-06-19 | 2022-12-22 | 株式会社アジュバンホールディングス | 育毛剤 |
WO2022265054A1 (ja) * | 2021-06-19 | 2022-12-22 | 株式会社アジュバンホールディングス | 育毛剤 |
WO2022265052A1 (ja) * | 2021-06-19 | 2022-12-22 | 株式会社アジュバンホールディングス | 育毛剤 |
Also Published As
Publication number | Publication date |
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US20240099951A1 (en) | 2024-03-28 |
TW202237080A (zh) | 2022-10-01 |
JPWO2022107866A1 (ja) | 2022-05-27 |
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