Pharmaceutical composition for hair growth, scalp or skin modification, wound healing, bone formation promotion or hair modification

Abstract

The present invention provides a pharmaceutical composition and a method for hair growth, scalp or skin modification, wound healing, bone formation promotion or hair modification, the pharmaceutical composition comprising a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Description

[0001] This application is a divisional application of an invention patent application with an application date of December 26, 2018, application number 201880050803.4 (international application number PCT / JP2018 / 047736), and invention name “Pharmaceutical composition for hair growth, scalp or skin modification, wound healing, bone formation promotion or hair modification”. Technical Field

[0002] The present invention relates to pharmaceutical compositions and methods for hair enhancement, scalp or skin modification, wound healing, bone formation promotion or hair modification. Background Art

[0003] Mammalian milk (particularly colostrum) contains various substances including nutrients, such as immune substances, intracellular transmitters, exosomes, mRNA, cytokines, etc. When an infant orally ingests these various substances, they are taken into the body and contribute to the development of the infant.

[0004] By comparing the presence or absence of adipocytes, it was reported that adipocytes contribute to the niche of skin stem cells and drive the hair cycle (Non-Patent Document 1). In Non-Patent Document 1, it is suggested that the appearance of CD34-positive cells, the increase of PPARγ-positive adipocytes, and the maturation of adipocytes are related to the elongation of hair in the growth phase. In addition, in Non-Patent Document 2, it is reported that an increase in tissue-resident M2-like macrophages is observed when tissue regeneration occurs in tissues.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Festa E et al., Cell, 146: 761-71, 2011

[0008] Non-patent document 2: Satoh T. et al., Nature, 495: 524-528, 2013 Summary of the invention

[0009] The present invention provides a topical pharmaceutical composition for improving the scalp or skin. In addition, the present invention provides a topical pharmaceutical composition for treating wounds. In addition, the present invention provides a topical pharmaceutical composition for promoting hair growth or improving the hair.

[0010] The present inventors have found that a composition comprising milk, a centrifuged supernatant fraction of milk, and soybean oil, and fatty acids such as the main fatty acids contained therein are useful for improving the quality of the scalp or skin, healing wounds, promoting bone formation, and improving the quality of hair. The present invention is based on these findings.

[0011] According to the present invention, the following inventions are provided.

[0012] (1) A pharmaceutical composition, which is (i) a pharmaceutical composition for topical administration for improving the scalp or skin, (ii) a pharmaceutical composition for topical administration for treating wounds, or (iii) a pharmaceutical composition for topical administration for promoting hair growth or for improving the hair, comprising a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof.

[0013] (2) The pharmaceutical composition according to (1) above, which is used for improving the scalp or skin.

[0014] (3) The pharmaceutical composition according to (1) above, which is used for treating wounds.

[0015] (4) The pharmaceutical composition according to (1) above, which is used for promoting hair growth or improving hair quality.

[0016] (5) The pharmaceutical composition according to (1) above, which is used for promoting bone formation.

[0017] (6) The pharmaceutical composition according to the above (6), wherein the saturated fatty acid is in a form coated on a biocompatible film.

[0018] (7) The pharmaceutical composition according to any one of (1) to (6) above, wherein the saturated fatty acid is one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid.

[0019] (8) The pharmaceutical composition according to any one of (1) to (7) above, further comprising an unsaturated fatty acid.

[0020] (9) The pharmaceutical composition according to any one of (1) to (6) above, comprising soybean oil or milk, or an extract or processed product thereof, containing a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof.

[0021] (10) The pharmaceutical composition according to (9) above, wherein the milk is sterilized.

[0022] (11) The pharmaceutical composition according to (10) above, wherein the milk is sterilized at high temperature.

[0023] The present invention is advantageous in providing a pharmaceutical composition or a therapeutic method that can be used for the above-mentioned various pharmaceutical uses and can continue to exert its effects even after the treatment is completed. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are photographs showing the effect of emulsion administration on the epidermis of hair.

[0025] Figure 2A This is a partial photograph showing the effect of emulsion administration on reducing gray hair.

[0026] Figure 2B This is a photograph of the side of the head showing the effect of emulsion administration on reducing gray hair.

[0027] Figure 3 This is a graph showing the relationship between emulsion administration and the thickness of the fat layer of the skin. #1 to #6 represent 1 to 6 months after treatment, respectively.

[0028] Figure 4A Histological staining of type I collagen and type III collagen in skin tissue before and after treatment is shown.

[0029] Figure 4B Histological staining of type I collagen and type III collagen in skin tissue before and after treatment is shown.

[0030] Figure 5 This is a graph showing the effect of milk administration on the size of fat cells.

[0031] Fig. 6A This is a graph showing the effect of milk administration on wound healing.

[0032] Figure 6B This is a graph showing the effect of lactoferrin administration on wound healing.

[0033] Figure 6C This is a graph showing the effect of administration of saturated fatty acids or unsaturated fatty acids on wound healing.

[0034] Fig.6D This is a graph showing the effect of soybean oil administration on wound healing.

[0035] Figure 7 The results of immunohistological staining (staining of Ki67-positive adipocytes and CD68-positive macrophages) of the dorsal subcutaneous fat layer tissue of Wistar rats administered with milk are shown.

[0036] Fig. 8A The results of immunohistological staining (CD31 staining and CD34 staining) of scalp fat layer tissue based on emulsion administration are shown.

[0037] Figure 8B The results of immunohistological staining of scalp fat layer tissue (staining of CD68-positive macrophages, staining of CD163-positive tissue-resident M2-like macrophages, and staining of PPARγ-positive cells) based on emulsion administration are shown.

[0038] Fig. 9 The graph shows the results of immunohistological staining (staining of CD68 and PPARγ-positive cells) of the dorsal subcutaneous fat layer tissue of rats to which lactoferrin was orally administered.

[0039] Fig. 10A The results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after administration of physiological saline are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0040] Fig. 10B The results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after palmitic acid administration are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0041] Fig. 10C The results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after stearic acid administration are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0042] Fig. 10D The results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after administration of myristic acid are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0043] Fig.10E The results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after oleic acid administration are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0044] Fig.10F The results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after administration of α-linolenic acid are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0045] Figure 10GThe results of staining for CD68 and PPARγ positive cells in tissues 4 and 8 weeks after linoleic acid administration are shown. The arrows in the figure indicate the sites where positive cells are present, and the numbers in parentheses at the lower right of each staining image indicate the number of positive cells in the visual field.

[0046] Fig.11A The total of the length × the number of hairs for each hair length based on emulsion administration is shown.

[0047] Fig. 11B It is shown in the form of a bar graph Fig.11A Result graph. Fig. 11B In the figure, "1.4 mm or more" refers to the total length of hairs with a length of 1.4 mm or more, and "all" refers to the total length of hairs with a length of 0.9 mm or more.

[0048] Fig.12 The images were obtained by placing a biocompatible membrane coated with palmitic acid or a biocompatible membrane not coated with palmitic acid on a bone resection site (8.8 mm in diameter) and observing the subsequent bone formation using 3D-CT. For tissue sampling, a 3 mm diameter hole was artificially opened in a portion of the bone after bone formation.

[0049] Fig.13 Figure 2 shows hematoxylin-eosin stained images of bone sections obtained by tissue sampling after bone formation. The dotted ellipse represents the newly formed bone at the bone resection site. The numerical values ​​represent the area of ​​each bone formed (number of pixels and mm 2 ).

[0050] Fig.14 The graph shows changes in gene expression of bone morphogenetic protein-4 (Bmp4) and bone morphogenetic protein-7 (Bmp7) in subjects administered with palmitic acid or milk.

[0051] Fig.15 The changes in gene expression relative to the control caused by palmitic acid administration or milk administration are shown. DETAILED DESCRIPTION

[0052] In the present specification, "subject" refers to a mammal. Examples of mammals include humans (male and female).

[0053] In the present specification, "milk" refers to milk derived from mammals. Examples of mammals include humans and cows.

[0054] In the present specification, "whey" refers to an aqueous solution obtained by removing milk fat components, casein, etc. from milk. Whey can be obtained, for example, by subjecting milk to an operation such as centrifugation and removing the precipitate.

[0055] In this specification, "scalp" refers to the area of ​​the skin on the head including the top of the head, excluding the face, jaw and neck (including the ears). In this specification, "skin" is used to include the scalp and skin other than the scalp. The human scalp has a diameter of about 700 mm. 2 ~About 800mm 2 area.

[0056] In this specification, "modification" means: improving its texture; improving its texture; enhancing its texture; or making the texture better. In this specification, "improvement" is used to include making it better than the current situation and making a bad part better.

[0057] In this specification, "hair" refers to hair growing on the skin. In this specification, "hair" refers to hair growing on the scalp.

[0058] In the present specification, "white hair" refers to hair, particularly hair that lacks pigments such as eumelanin (or true melanin) and / or pheomelanin and appears white. In the present specification, "black hair" refers to hair that contains eumelanin and appears black.

[0059] In this specification, the "cuticle layer" is a structure covering the surface of the hair and is a structure existing in the outermost layer of the hair. The cuticle layer plays a role in protecting the hair from external stimuli and preventing the loss of water or components from the inner cortex layer to the outside. The cuticle layer covers the hair in a scale-like manner from the root to the tip of the hair.

[0060] In this specification, "trauma" refers to physical damage to tissues. Trauma includes wounds on the body surface.

[0061] According to the present invention, there is provided:

[0062] (A) A pharmaceutical composition for topical administration for improving the scalp or skin, comprising a therapeutically effective amount of a fatty acid (particularly a saturated fatty acid) or a pharmaceutically acceptable salt thereof;

[0063] (B) A pharmaceutical composition for topical administration for treating wounds, comprising a therapeutically effective amount of a fatty acid (particularly a saturated fatty acid) or a pharmaceutically acceptable salt thereof;

[0064] (C) a pharmaceutical composition for topical administration for promoting hair growth or for modifying hair quality, comprising a therapeutically effective amount of a fatty acid (especially a saturated fatty acid) or a pharmaceutically acceptable salt thereof; and

[0065] (D) A pharmaceutical composition for promoting bone formation, comprising a therapeutically effective amount of a fatty acid (especially a saturated fatty acid) or a pharmaceutically acceptable salt thereof

[0066] (Hereinafter, the pharmaceutical compositions described in (A) to (D) above may be collectively referred to as "the pharmaceutical composition of the present invention").

[0067] In one embodiment of the present invention, as a pharmaceutical composition containing a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof, milk, milk extracts (e.g., whey) and processed milk (e.g., dairy products, such as whey of cheese, whey of yogurt, etc.) and mixtures thereof can be cited. Milk, milk extracts or processed milk products can be sterilized, for example, low-temperature sterilization (e.g., heating sterilization at 62°C to 68°C) or high-temperature sterilization (heating sterilization at 120°C or above). The sterilization of milk is generally divided into low-temperature sterilization at 62°C to 68°C for about 30 minutes (e.g., 30 minutes at 65°C) and high-temperature sterilization at 120°C to 150°C for 1 second to 4 seconds (e.g., 2 seconds to 3 seconds at 120°C to 130°C). Therefore, in one embodiment of the present invention, the pharmaceutical composition of the present invention contains milk as an active ingredient.

[0068] In one embodiment of the present invention, the pharmaceutical composition containing a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof may include animal oils, vegetable oils, extracts or processed products of these oils (e.g., soybean oil, soybean oil extracts or soybean oil processed products), or mixtures thereof.

[0069] In the present specification, the extract derived from milk or soybean oil is a fraction extracted from milk or soybean oil, sometimes referred to as a milk extract or a soybean oil extract, respectively, and contains a therapeutically effective amount of saturated fatty acids or pharmaceutically acceptable salts thereof.

[0070] In the present specification, processed products of milk or soybean oil are obtained by processing milk or soybean oil, and are sometimes referred to as processed milk products or processed soybean oil products, respectively, and each contains a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof.

[0071] As the composition containing a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof, there can be mentioned solutions containing milk and whey, and freeze-dried preparations thereof.

[0072] The fatty acid contained in the pharmaceutical composition for topical administration of the present invention may be a fatty acid contained as a component of milk.

[0073] In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for topical administration of the present invention is one or more saturated fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid.

[0074] In one embodiment of the present invention, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention may be a saturated fatty acid, for example, one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid.

[0075] In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for topical administration of the present invention is palmitic acid.

[0076] In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for topical administration of the present invention is stearic acid.

[0077] In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for topical administration of the present invention is myristic acid.

[0078] In one embodiment of the present invention, the pharmaceutical composition for topical administration of the present invention may further contain an unsaturated fatty acid. The unsaturated fatty acid may contain, for example, one or more unsaturated fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid.

[0079] In one embodiment of the present invention, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is oleic acid.

[0080] The fatty acid contained in the pharmaceutical composition for topical administration of the present invention is α-linolenic acid.

[0081] The fatty acid contained in the pharmaceutical composition for topical administration of the present invention is linoleic acid.

[0082] In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention may be a mixture of at least one saturated fatty acid and at least one unsaturated fatty acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is palmitic acid and linoleic acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is palmitic acid and oleic acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is stearic acid and linoleic acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is stearic acid and oleic acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is palmitic acid, stearic acid and linoleic acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is palmitic acid, stearic acid and oleic acid. In a certain embodiment, the fatty acid contained in the pharmaceutical composition for topical administration of the present invention is myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and α-linolenic acid.

[0083] In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, may contain, for example, one or more fatty acids selected from saturated fatty acids {herein, "may contain" may include the case where it is naturally contained in milk and the case where it is added to milk}. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, may further contain unsaturated fatty acids. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, may contain, for example, one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, may contain, for example, one or more unsaturated fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, may contain, for example, one or more saturated fatty acids and one or more unsaturated fatty acids. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, can contain one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid, and one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, can contain myristic acid, palmitic acid and oleic acid. In a certain embodiment, the milk, milk extract or milk processed product that can be used in the present invention, or a mixture thereof, can contain myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and α-linolenic acid. In a certain embodiment of the present invention, the above-mentioned one or more fatty acids can be added to the pharmaceutical composition in the form of milk, milk extract or milk processed product, or a mixture thereof.

[0084] In a certain embodiment of the present invention, the content of one or more fatty acids selected from saturated fatty acids and unsaturated fatty acids can be enhanced in milk, milk extract or milk processed products or their mixtures. Regarding enhancement, as long as the fatty acid content after enhancement is increased compared to the fatty acid content in milk, milk extract or milk processed products or their mixtures before enhancement. Enhancement can be performed by adding one or more fatty acids selected from saturated fatty acids and unsaturated fatty acids to milk, milk extract or milk processed products or their mixtures. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more saturated fatty acids and one or more unsaturated fatty acids. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid, and the enhancement of the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In one embodiment, the enhancement may be an enhancement of the content of each of myristic acid, palmitic acid, and oleic acid. In one embodiment, the enhancement may be an enhancement of the content of each of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, and α-linolenic acid.

[0085] In one embodiment of the present invention, the lactoferrin content in milk, milk extract, processed milk, or a mixture thereof can be reduced. The lactoferrin content can be reduced by methods such as high temperature sterilization.

[0086] In one embodiment of the present invention, milk, a milk extract or a processed milk product, or a mixture thereof may have an enhanced fatty acid content and a reduced lactoferrin content.

[0087] In a certain embodiment of the present invention, animal oil, vegetable oil, extract or processed product of these oils or mixture thereof may contain one or more fatty acids selected from saturated fatty acids. In a certain embodiment, animal oil, vegetable oil, extract or processed product of these oils or mixture thereof may further contain unsaturated fatty acids. In a certain embodiment, animal oil, vegetable oil, extract or processed product of these oils or mixture thereof that can be used in the present invention may contain one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid. In a certain embodiment, animal oil, vegetable oil, extract or processed product of these oils or mixture thereof that can be used in the present invention may contain one or more unsaturated fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, animal oil, vegetable oil, extract or processed product of these oils or mixture thereof that can be used in the present invention may contain one or more saturated fatty acids and one or more unsaturated fatty acids. In a certain embodiment, the animal oil, vegetable oil, extract or processed product of these oils or mixtures thereof that can be used in the present invention may contain one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid, and one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, the animal oil, vegetable oil, extract or processed product of these oils that can be used in the present invention may contain myristic acid, palmitic acid and oleic acid. In a certain embodiment, the animal oil, vegetable oil, extract or processed product of these oils or mixtures thereof that can be used in the present invention may contain myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and α-linolenic acid.

[0088] In a certain embodiment of the present invention, the content of one or more fatty acids selected from saturated fatty acids and unsaturated fatty acids can be enhanced in animal oil, vegetable oil, extracts or processed products of these oils or mixtures thereof. Regarding enhancement, as long as the fatty acid content after enhancement is increased compared with the fatty acid content in the animal oil, vegetable oil, extracts or processed products of these oils or mixtures thereof before enhancement. Enhancement can be carried out, for example, by adding one or more fatty acids selected from saturated fatty acids and unsaturated fatty acids to animal oil, vegetable oil, extracts or processed products of these oils or mixtures thereof. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, enhancement can be, for example, the enhancement of the content of one or more saturated fatty acids and one or more unsaturated fatty acids. In a certain embodiment, the enhancement may be, for example, an enhancement of the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid, and an enhancement of the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linoleic acid. In a certain embodiment, the enhancement may be an enhancement of the content of each of myristic acid, palmitic acid and oleic acid. In a certain embodiment, the enhancement may be an enhancement of the content of each of myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and α-linolenic acid.

[0089] In a certain embodiment, the pharmaceutical composition for topical administration of the present invention contains the fatty acid in a solubilized form. In a certain embodiment, the pharmaceutical composition for topical administration of the present invention contains the fatty acid in the form of a pharmaceutically acceptable salt. In a certain embodiment, the pharmaceutical composition for topical administration of the present invention contains the fatty acid in the form of an emulsion. In a certain embodiment, the fatty acid in the pharmaceutical composition for topical administration of the present invention is not in the form of an ester or a glyceride.

[0090] Fatty acids are soluble in dimethyl sulfoxide, ethanol, chloroform, and ether. When fatty acids form pharmaceutically acceptable salts, such as sodium salts, their water solubility increases. Fatty acid salts can form micelles and dissolve in water.

[0091] In a certain embodiment, the pharmaceutical composition for topical administration of the present invention can be a preparation for injection. In a certain embodiment of the present invention, the pharmaceutical composition for topical administration of the present invention can be provided in the form of a kit comprising a freeze-dried preparation of a solution containing a fatty acid and water for injection, and the freeze-dried preparation can be prepared using water for injection when necessary. Water for injection can be heated for use.

[0092] The pharmaceutical composition for promoting bone formation of the present invention can be provided in a state of being coated on a biocompatible film. Therefore, in the present invention, a drug containing a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof coated on a biocompatible film can be provided. As a biocompatible film, for example, a film having a surface of calcium phosphate (e.g., a film of calcium phosphate), a film having a surface of polylactic acid (e.g., a film of polylactic acid), a film having a surface of polyglycolic acid (e.g., a film of polyglycolic acid), a film having a surface of a copolymer or block copolymer of lactic acid and polyglycolic acid (e.g., a film of a copolymer or block copolymer of lactic acid and polyglycolic acid), and a film having a surface of hydroxyapatite (e.g., a film of hydroxyapatite). The coating of the active ingredient on the biocompatible film can be carried out, for example, by dripping a solution in which the active ingredient is dissolved onto the biocompatible film and drying it. For example, about 10 μg to about 50 μg of the active ingredient of the drug can be applied to the biocompatible film for use.

[0093] The pharmaceutical composition for topical administration for improving the scalp or skin, which contains a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof, improves the scalp starting from the vicinity of the administration site after administration, and the effect gradually spreads to the surrounding area over time. Therefore, it can be spread over the scalp or skin so that the effect of the invention spreads to the surrounding area over time, or it can be densely distributed so that the time required for the spread is shortened. In one embodiment of the present invention, the pharmaceutical composition of the present invention can be applied to, for example, 1 cm 2 ~10cm 2 , 1cm 2 ~5cm 2 , 1cm 2 ~4cm 2 , 1cm 2 ~3cm 2 , 1cm 2 ~2cm 2 、0.5cm 2 ~2cm 2 、0.7cm 2 ~1.5cm 2 In one embodiment of the present invention, the pharmaceutical composition of the present invention can be, for example, at 0.5 cm 2 The middle is less than one place, within 0.6cm 2 The middle is less than one place, within 0.7cm 2 The middle is less than one place, within 0.8cm 2 The middle is less than one place, within 0.9cm 2 Less than one spot or within 1 cm 2In one embodiment of the present invention, the pharmaceutical composition of the present invention can be topically administered, for example, just below the subcutaneous layer, or from the dermis to the upper layer of the fat layer. Administration can be performed, for example, by injection.

[0094] Alternatively, a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof may be administered in a solution of, for example, 10 μL to 50 μL, 15 μL to 30 μL, 15 μL to 25 μL, or about 20 μL per site.

[0095] The administration site can be appropriately selected, and for example, the drug may be administered to the entire scalp, or to a portion of the entire scalp that is poor compared to other portions.

[0096] In addition, surprisingly, the inventors have found that when the drug is administered to a relatively good part of the entire scalp, the effect is easily spread to the surrounding area. The good part is highly responsive to the pharmaceutical composition of the present invention, and the effect spreads and also spreads to the surrounding area of ​​the administration site. The improvement effect brought about by the spread is strong in places closer to the administration site, and can spread to a range of several centimeters (for example, 1cm to 4cm) from the administration site. Therefore, the drug can be administered to a relatively good part of the scalp. In addition, since the improvement effect spreads from the good part to the relatively poor part, the treatment effect on the poor part can also be obtained by only administering the drug to the relatively good part.

[0097] Therefore, the drug can be administered to a relatively good part of the scalp, and the improvement of the poor part can be attempted by the propagation effect. In addition, if the improvement is achieved in the relatively poor part, the responsiveness to the pharmaceutical composition of the present invention is improved. Therefore, if the drug can be administered to a relatively good part of the scalp and the poor part can be improved by the propagation effect, the pharmaceutical composition of the present invention can be administered to the part that was once poor.

[0098] It is believed that each time the pharmaceutical composition of the present invention is administered, tissue stimulation at the administration site, macrophage activity or immature adipocyte activity increases, and repair function is enhanced. Administration is preferably performed after diagnosing the patient's symptoms (condition) and the degree of improvement.

[0099] In one embodiment, the topical administration can be set to 150 to 250 locations (e.g., about 200 locations) or 250 to 800 locations on the scalp of each adult, and the number of administration sites can be determined as needed. Here, the term "about" means a numerical range including ±10% or ±5% of the numerical value following the term.

[0100] The pharmaceutical composition of the present invention can be administered at a dosing interval of, for example, twice a month to once every six months, twice a month to once every five months, twice a month to once every four months, twice a month to once every three months, twice a month to once every two months, once 1.5 times a month to once every 1.5 months, or once 1.2 times a month to once every 1.2 months, for example, about once a month, but is not particularly limited.

[0101] The pharmaceutical composition of the present invention has a scalp and skin improvement effect even if it is only administered once, and the effect is sustainable. Therefore, the administration can be once or multiple times. The pharmaceutical composition of the present invention can also be continuously administered until the treatment is completed, or until the patient is satisfied. In particular, according to the present invention, the improvement effect of the scalp and skin begins to appear in the part with good condition, and slowly appears in the part with poor condition. Therefore, the treatment period and treatment interval can be changed according to the condition of the scalp and skin. The treatment period of the pharmaceutical composition of the present invention can be set, for example, to a minimum of one treatment for 3 years, a minimum of one treatment for 2 years, a minimum of one treatment for 1.5 years, a minimum of one treatment for 1 year, a minimum of one treatment for 8 months, a minimum of one treatment for 6 months, or a minimum of one treatment for 4 months. In addition, the pharmaceutical composition of the present invention can be administered before the changes caused by aging are observed in the hair, scalp, and skin, and can also be administered when the above changes are observed or after the above changes are observed. Furthermore, the pharmaceutical composition of the present invention can be administered again to a subject treated by administration of the pharmaceutical composition before, when, or after changes due to aging are observed in the hair, scalp, or skin.

[0102] In one embodiment of the present invention, the pharmaceutical composition described in (A) above may be a pharmaceutical composition for rejuvenation or promotion of rejuvenation of the scalp or skin. In one embodiment of the present invention, the pharmaceutical composition described in (A) above may be a pharmaceutical composition for regeneration or promotion of regeneration of the scalp or skin. In one embodiment of the present invention, the pharmaceutical composition described in (A) above may be a pharmaceutical composition for activating the scalp or skin. In one embodiment of the present invention, the pharmaceutical composition described in (A) above may be used in combination with a hair thickening agent or a hair growth agent.

[0103] In one embodiment of the present invention, the pharmaceutical composition described in (B) above can be used to treat wounds. In the present invention, the treatment of wounds can be healing of wounds, promotion of healing of wounds, or advancement of healing of wounds.

[0104] By improving the scalp, the effect also reaches the hair itself. And, as shown in the examples described later, as the improvement effect of hair, there can be listed the reduction of white hair, the reduction of the weight of the white hair part, the reduction of the proportion of white hair, the reduction of the proportion of white hair, the improvement of the roughness of the epidermis, the improvement of the thickness and extension speed of hair. Therefore, in a certain embodiment of the present invention, the pharmaceutical composition described in (C) above can be a pharmaceutical composition for reducing white hair. As the reduction of white hair, there can be listed the reduction of the number of white hair roots, the reduction of the weight of the white hair part, and the reduction of the proportion of white hair. The reduction of white hair can be accompanied by an increase in the number of hair roots colored by various melanins, an increase in the weight of such hair parts, and an increase in the proportion of such hair. In a certain embodiment of the present invention, the pharmaceutical composition described in (C) above can be a pharmaceutical composition for improving the roughness of the epidermis of hair. As the improvement of the roughness of the epidermis of hair, there can be listed the improvement of the degree of arrangement of the epidermis and the reduction of the disordered area of ​​the epidermis. In one embodiment of the present invention, the pharmaceutical composition described in (C) above may be a pharmaceutical composition for improving the thickness or extension speed of hair. Examples of the improvement of the thickness of hair include making the hair thicker, increasing the number of thick hairs, and increasing the proportion of thick hairs. Examples of the improvement of the extension speed include, for example, increasing the number or proportion of hairs having an extension speed of 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, 15 mm or more, 16 mm or more, 17 mm or more, 18 mm or more, 19 mm or more, 20 mm or more, or 21 mm or more per month.

[0105] In one aspect of the present invention, there is provided:

[0106] (a) A method for modifying the scalp or skin in a subject in need thereof (or a site in need thereof), comprising topically administering to the scalp or skin a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof;

[0107] (b) a method for treating a wound in a subject in need thereof, comprising topically administering to the scalp or skin a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof;

[0108] (c) a method of promoting hair growth in a subject in need thereof, or a method of modifying hair quality in a subject in need thereof, comprising topically administering to the scalp or skin a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof; and

[0109] (d) A method for promoting bone formation in a subject in need thereof, comprising administering a therapeutically effective amount of a fatty acid (especially a saturated fatty acid) or a pharmaceutically acceptable salt thereof to a site in need of bone formation (e.g., a bone damage site) {herein, the therapeutically effective amount of a fatty acid (especially a saturated fatty acid) or a pharmaceutically acceptable salt thereof can be administered in a state coated on a biocompatible film}.

[0110] In one aspect of the present invention, there is provided:

[0111] (a) use of a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a topical pharmaceutical preparation for improving the scalp or skin;

[0112] (β) Use of a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical preparation for topical administration for treating wounds;

[0113] (γ) use of a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a topical pharmaceutical preparation for promoting hair growth or a topical pharmaceutical preparation for improving hair quality; and

[0114] (δ) Use of a therapeutically effective amount of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition or pharmaceutical preparation for promoting bone formation {herein, the pharmaceutical preparation can be provided in a state where a therapeutically effective amount of a fatty acid (especially a saturated fatty acid) or a pharmaceutically acceptable salt thereof is coated on a biocompatible film}.

[0115] In a certain embodiment of the present invention, the pharmaceutical composition may include an excipient (e.g., a solvent, a cosolvent, a solubilizer, a wetting agent, a suspending agent, a thickening agent, an emulsifier, a chelating agent, a buffer, a pH regulator, an antioxidant, a reducing agent, an antibacterial agent, a preservative, a filler, a protective agent, or an isotonic agent) in addition to a therapeutically effective amount of a fatty acid or a pharmaceutically acceptable salt thereof. In a certain embodiment of the present invention, the pharmaceutical composition may be in the form of an injection. In a certain embodiment of the present invention, the pharmaceutical composition may be administered topically, such as intradermally or subcutaneously.

[0116] Example

[0117] Example 1: Preparation and administration of milk

[0118] (1) Milk was prepared as follows.

[0119] As milk for administration to humans, so-called colostrum (milk collected within 3 weeks after giving birth) was obtained from the breasts of 34-year-old women and 40-year-old women using a breast pump.

[0120] As milk for administration to rats, hand-expressed cow colostrum or commercially available cow milk was used.

[0121] (2) The milk was administered as follows.

[0122] The milk was injected into the right or left half of the human scalp or the back skin of the rat in an amount of 20 μL per place. In the case of humans, for each patient, a total of 2 mL of colostrum was injected into half of the patient's head (i.e., 100 places per person). In the case of rats, three areas were set from the head to the tail on the back of 12-week-old Wistar rats (male), and each area was further divided into two areas on the left and right of the vertebrae, thereby setting a total of 6 areas on the back. 20 μL / place of milk was injected into each of the two places near the center of each area. It should be noted that the distance between the above two places was set to 1 cm.

[0123] Example 2: Changes in hair quality

[0124] In this example, it was examined whether improvements were observed in the cuticle layer of the hair before and after the treatment.

[0125] As milk, human colostrum described in Example 1 is used. The average extension rate of hair is about 10 mm per month. One hair is collected from each of the administration site and non-administration site, and considering the extension rate of the patient's hair, a portion of hair corresponding to 3 months before treatment and a portion of hair corresponding to 3.5 months after the start of treatment are used as samples. On the day of hair collection or the next day, the hair at the inspection site is fixed to a fixing plate with resin glue. Carbon fixation is performed on the second or third day, and on the same day, the state of the epidermis of the hair is observed using a scanning electron microscope according to a conventional method. As a negative object, the hair of the untreated part (non-administration site) of the same patient is used. The newly extended part of the hair (root side) is compared with the existing part (front end side). Representative examples are shown in Figure 1 . Figure 1 This is an electron microscope image of hair from the same patient.

[0126] As a result, in the treated hair, the cuticle layer was rough at the tip side, while the cuticle layer was neat at the newly extended part, i.e., the root side, and the difference in hair quality was significantly improved. In contrast, in the untreated part of the hair of the negative subject, the cuticle layer was rough at both the root and the tip side, and no improvement effect of hair quality was confirmed in the untreated part.

[0127] The same evaluation was performed on more patients. The evaluation was performed according to the following scoring based on the electron microscope images. The evaluation was performed by 7 medical practitioners, including doctors. The average of the scores was calculated.

[0128] Hair quality evaluation score sheet

[0129] 5 points: The epidermis is neat and no roughness is observed

[0130] 4 points: The epidermis is smoother than average, but slightly rough.

[0131] 3 points: The neatness of the epidermis is average

[0132] 2 points: The epidermis is rougher than average, and peeling is slightly observed

[0133] 1 point: The epidermis is rough as a whole, and peeling is observed as a whole

[0134] The results are shown in Table 1.

[0135] [Table 1]

[0136] Table 1: Improvement in hair quality evaluation due to treatment

[0137]

[0138] As shown in Table 1, the difference in the score between the tip and the root of the same hair was found, and the result showed that the root was rated higher on both the treated and untreated sides. Specifically, the average score of the electron microscope image of the root on the treated side was 3.52, while the average score of the tip on the treated side was 2.79, and the difference was statistically significant (p=0.017). More specifically, the degree of improvement (score of the root - score of the tip) was 0.73 on the treated side and 0.39 on the untreated side, and the degree of improvement on the treated side was high (n=4, 33-56 years old, 4 females, average age 45.8 years old). In addition, as shown in Table 1, the average score of the root on the treated side was 3.52, while the average score of the root on the untreated side was 2.93, and it was confirmed that the difference in the score between the roots on the treated side and the untreated side was statistically significant (p=0.036). This shows that the hair quality improvement effect is shown in the root part of the treated side, that is, the newly grown hair part after treatment, through the administration of the emulsion.

[0139] Example 3: Changes in hair quality

[0140] In this example, the improvement effect before and after treatment was examined with attention paid to the color of hair (gray hair).

[0141] Specifically, two points where the lines connecting the two ears and the top of the head are extended from the corners of the eyes of the patient to the head and intersect are tattooed with ink, and a 2 cm range is shaved with the mark as the center. Three days after shaving, 2 mL of human colostrum described in Example 1 is injected into one part of the scalp at 100 places, and observation is performed one month later. A trichogram is taken with each mark as the center (Canon Power Shot A520, Tokyo, Japan), and a circle with a diameter of 11 mm (area 95 mm) with the tattoo as the center included in the shooting range is taken. 2 ) were photographed and the hair was measured visually.

[0142] As a result, in the shaved area, among the three patients who had white hair before treatment, all of the three patients had less white hair after a single injection 6 months after treatment, with an average reduction of 7.7 hairs / 95mm in each of the above-mentioned shooting ranges. 2 The number of white hairs on the treated side was reduced to 92.9% before and after treatment. The white hair reduction rate when the number before treatment on the untreated side was set as 100 was 28.2% lower than when the number before treatment on the treated side was set as 100, and the number of white hairs on the treated side was significantly reduced (n=3).

[0143] A representative example of the hair of a patient (48-year-old female) six months after shaving her head and a single injection of colostrum is shown in Figure 2A .like Figure 2A As shown in the figure, no reduction in white hair was observed in the non-administered area, but the number of white hairs was significantly reduced in the administered area. Specifically, the number of white hairs in the non-administered area was 49 before treatment and increased to 63 after 6 months, while the number of white hairs in the administered area was 85 before treatment and decreased to 66 after 6 months.

[0144] Furthermore, for a patient (50-year-old male) who had received 4 injections of 20 μL of human colostrum described in Example 1 at each site on the scalp, the temporal part 13 months after the start of treatment was compared with the temporal part on the non-treated side. Figure 2B As shown. Figure 2B As shown, the area of ​​the white hair area on the non-treated side of the temple is 833cm 2 On the other hand, the area of ​​white hair on the temporal part of the treated side was 294cm 2 . Thus, it can be seen that the administration of colostrum reduces the amount of gray hair. Figure 2B In the above, a grid was superimposed on the photograph of the temporal region, and a mark "○" was placed in each square of the grid that was visually judged to be white. It can be seen that the number of marks "○" was reduced on the treated side compared to the non-treated side.

[0145] Example 4: Verification of the scalp improvement effect

[0146] The modification of hair is as described above, and it was verified what kind of modification this is based on the scalp.

[0147] As the milk, human colostrum described in the Examples was used. 20 μL of the milk was injected into 100 sites on the scalp of the patient (six patients were injected once, and one patient was injected four times once a month).

[0148] By using an echo test with a 10Mz probe, the change in the thickness of the fat layer of the subcutaneous tissue of the scalp after a single dose of the drug was observed. In this test, the thickness of the area from the sebaceous gland area to the lower part was measured, and the thickness of the area roughly up to the dermis was measured (however, the dermis was not included in the measured value). The results are as follows Figure 3 as shown in . Figure 3 In the data, "#1 to #6" show the data obtained after 1 to 6 months of treatment, respectively. Figure 3 As shown, 6 months after the start of treatment, a slight increase in the thickness of the fat layer was observed, but no significant change was observed.

[0149] Example 5: Scalp tissue image

[0150] In the above examples, the effects of improving the epidermis and reducing gray hair were confirmed. In this example, in order to examine whether these effects are caused by the improvement of the scalp, the amount of collagen in the scalp was examined.

[0151] 20 μL of milk was administered subcutaneously to each site of the scalp of a human patient. The administration was performed once a month. As the milk, the human colostrum described in Example 1 was used.

[0152] (1) Changes in collagen profile

[0153] First, the increase and decrease of type I collagen and type III collagen were observed. Specifically, sections of scalp tissue (the surface from the epidermis toward the skull) were prepared, and type I collagen and type III collagen were stained using a Sirius Red staining kit reagent (Polysciences, Inc., Cat#: 24901-250) according to the manufacturer's instructions. Type I collagen was stained yellow, and type III collagen was stained green. In this system, collagens other than type III collagen were stained red and yellow.

[0154] A polarizing filter was installed on a microscope (Nikon Co., Ltd., OLYMPUS Co., Ltd.), and the stained image was observed and photographed with a camera attached to the microscope (DP-22 OLYMPUS Co., Ltd., DS-Fi3 NIKONINSTECH Co., Ltd.). A digital image was created in grayscale and stored in a computer, and the amount of collagen was estimated from the brightness of each pixel. Figure 4A The scalp sample of a female patient (49 years old) 5 months after a single administration is shown. Figure 4B Shown is a scalp sample of a male patient (50 years old) who received 4 monthly dosings, 15 months after the initial treatment.

[0155] As a result, if Figure 4A and 4B As shown, the presence of type I collagen and type III collagen was observed on both the pre-treatment and untreated sides.

[0156] Figure 4A In the table, the number of green pixels (equivalent to the amount of type III collagen), the number of yellow pixels (equivalent to the amount of type I collagen), and the number of red and yellow pixels (equivalent to the total amount of collagen other than type III collagen) are summarized.

[0157]

[0158] As shown in Table 2, in patients 5 months after a single dose, the proportion of type III collagen decreased from 49.6% to 19.6%, and the proportion of type I collagen increased from 30.1% to 62.3%. It can be seen that the total amount of collagen other than type III collagen also increased from 50.4% to 80.4%, and it is believed that this increase is mainly caused by the increase in type I collagen.

[0159] Figure 4B In the table, the number of green pixels (equivalent to the amount of type III collagen), the number of yellow pixels (equivalent to the amount of type I collagen), and the number of red and yellow pixels (equivalent to the total amount of collagen other than type III collagen) are summarized.

[0160]

[0161] In addition, as shown in Table 3, for patients 15 months after the start of treatment, the following was observed in the comparison between the untreated side and the treated side: Type III collagen decreased significantly from 26.3% to 2.8%, and Type I collagen increased from 24.7% to 56.3% (reference Figure 4B ). It can be seen that the total amount of collagen other than type III collagen also increased from 73.7% to 97.2%, and it is believed that this increase is mainly caused by the increase of type I collagen.

[0162] The same tendency was observed in the comparison of the treated side before and after, and the comparison of the untreated side and the treated side. It can be seen that: after milk administration, type III collagen decreased and type I collagen increased. In addition, the same observation was made in human samples (n=3), and the following situation was observed in any sample: after milk administration, type III collagen decreased and type I collagen increased. It should be noted that regarding this result, the same tendency as the increase of type I collagen stained with yellow was also confirmed in the collagen stained with red and yellow (except type III collagen).

[0163] The decrease of type III collagen and the increase of type I collagen are phenomena that occur during tissue repair, especially wound healing. The results of this example suggest that the administration of the milk induces tissue repair of the scalp and skin.

[0164] (2) Rejuvenation of fat cells

[0165] Therefore, rejuvenation of adipocytes was observed.

[0166] As fat cells grow, their cytoplasm almost all turns into fat droplets, and fat droplets accumulate in the cytoplasm and hypertrophy occurs. Therefore, large cells mean mature old cells, and small cells are immature new cells (i.e., immature fat cells). In this example, it was confirmed whether small and immature cells increase by administering milk. More specifically, human scalp tissue sections administered with 20 μL of milk were observed to confirm changes in the size of fat cells in the fat layer. The image of the fat cells was approximated as a circle, and they were classified as S, M, or L based on their diameter. Cells with a diameter of 51 μm or less were recorded as "S", cells with a diameter of more than 51 μm and less than 63 μm were recorded as "M", and cells with a diameter of more than 63 μm were recorded as "L", and the sum was calculated. Regarding patients, the study was conducted on a patient who had been administered a single dose for 6 months (hereinafter referred to as "Patient 1"), and a patient who had been administered 4 doses once a month and had been treated for 15 months (hereinafter referred to as "Patient 2"). The results are as follows Figure 5 shown.

[0167] like Figure 5 As shown, in patients 6 months after treatment (patient 1, 51-year-old male) and 15 months after treatment (patient 2, 50-year-old male), the proportion of "S" fat cells increased significantly by milk administration. In addition, when comparing patient 1 with patient 2, the increase in the proportion of "S" fat cells was more significant in patient 2. This shows that the increase in the proportion of "S" fat cells was greater in 4 treatments compared to 1 treatment.

[0168] These results suggest that the administration of milk increases immature cells and rejuvenates tissues. In addition, it is suggested that the rejuvenation of tissues may be related to the enhancement of tissue repair ability.

[0169] Example 6: Effect on wound healing

[0170] Therefore, in this example, it was confirmed whether the administration of milk has the effect of accelerating the healing of skin wounds.

[0171] Wound preparation: The backs of 12-week-old Wistar rats were shaved with a hair clipper, and a circular full-thickness skin defect with a diameter of 8 mm was made on the left and right sides of the head and tail. Four full-thickness skin defects were made in each rat.

[0172] Administration of milk: 20 μL of milk was subcutaneously injected at four locations 4 mm from the wound edge, top, bottom, left, and right. As milk, commercially available cow's milk sterilized by heat (130°C, 2 seconds and 65°C, 30 minutes) as described in Example 1 was used.

[0173] Administration of fatty acids: Sterile pure water (administration solution) containing palmitic acid, stearic acid, myristic acid, linolenic acid, α-linolenic acid and oleic acid as fatty acids contained in milk was administered, saturated fatty acids were administered after heating, and unsaturated fatty acids were administered at room temperature. Regarding administration, 20 μL of the administration solution (containing 1 mg of fatty acids) was administered subcutaneously at 4 locations 4 mm above, below, left and right from the wound edge.

[0174] Trauma healing score

[0175] 5 points: Cured

[0176] 4 points: The area of ​​the wound has been reduced by more than 70%

[0177] 3 points: The reduction rate of the wound area is 50% or more and less than 70%

[0178] 2 points: The reduction rate of the wound area is 20% or more and less than 50%

[0179] 1 point: The reduction rate of the wound area is less than 20%

[0180] The results were expressed as a ratio of the above scores divided by the scores of saline. Fig. 6A As shown, the effect of promoting wound healing was observed in the group administered with milk.

[0181] 8 mL x 2 bottles of commercially available low-temperature sterilized milk (65 degrees, 30 minutes) were centrifuged using a centrifuge (2600 g x 6 minutes), and 2 mL of the supernatant, which was equivalent to one-fourth of the supernatant after removing the foaming part, was collected from each bottle, and the amount of the two bottles was combined into 4 mL, which was stirred and used in the experiment (hereinafter the obtained sample is referred to as the "centrifuged sample"). The obtained centrifuged sample was administered to the wound site in the same manner as above at a dosage of 20 μL per site. The results are as follows: Fig. 6A The “centrifuged sample” is shown in FIG.

[0182] like Fig. 6A As shown, the centrifuged sample exhibited an improvement effect on wound healing equal to or greater than that of milk. This suggests that the wound healing effect of milk is mainly due to the effect of the components contained in the components that were not precipitated by centrifugation. In addition, it can be seen that natural healing of wounds also occurred after administration of physiological saline, but both milk administration and centrifuged sample administration promoted healing more than natural healing after administration of physiological saline ( Fig. 6A ).

[0183] Next, the effect of promoting wound healing produced by lactoferrin was studied. Lactoferrin purchased from Lion (product name: NICE 62971) was used, and 20 μg or 60 μg was administered at one site using the same method as described above. Specifically, the lactoferrin was pulverized and fully dissolved in pure water. The lactoferrin concentration was adjusted in such a way that the above amount was contained in the injection volume of 20 μL. As for the results, the values ​​obtained by dividing the scores after 7 days and 10 days by the scores of the saline administration group were obtained. The results are as follows. Figure 6B As shown. Figure 6B As shown in Figure 2, lactoferrin did not show a significant promoting effect on wound healing. This suggests that components other than lactoferrin have a promoting effect on wound healing.

[0184] Normal raw milk contains about 3 mg of lactoferrin (also called "LF") per 1L. About 0.06 μg of lactoferrin is contained in 20 μL of raw milk. In addition, lactoferrin is not heat-resistant and is believed to be basically disappeared from the cow's milk sterilized at high temperature used in the above examples (refer to Noro Mirai et al., Electrophoresis 59: 21-24, 2015). Therefore, the promotion effect of wound healing was observed when the high-temperature sterilized cow's milk was administered, and the promotion effect of wound healing was also observed when the centrifuged sample was administered. On the other hand, the promotion effect of wound healing was almost not observed when lactoferrin was administered, which is a result consistent with the conclusion that lactoferrin is not an effective ingredient.

[0185] The above examples show that administration of milk or a supernatant of milk centrifugation promotes wound healing. The above examples also suggest the possibility of activating skin tissue regeneration by administration of milk or a supernatant of milk centrifugation.

[0186] Next, the effective ingredients in the milk were identified. Specifically, the fatty acids contained in the milk were administered to the wound site as described above to examine whether there was an effect of promoting wound healing. Figure 6C As shown. Figure 6C As shown, palmitic acid, stearic acid and myristic acid have a high promoting effect on wound healing. This indicates that these fatty acids may contribute to the promoting effect of wound healing in emulsion administration. In addition, oleic acid, α-linolenic acid and linoleic acid have a weak promoting effect on wound healing.

[0187] In addition, edible soybean oil (manufactured by Riken Agrochemicals Co., Ltd., Fukuoka Plant), which is known to be rich in palmitic acid, stearic acid, linoleic acid, and α-linolenic acid, was administered to the wound site to investigate whether it has an effect of promoting wound healing. Fig.6D As shown. Fig.6D As shown, soybean oil exhibits a high promoting effect on wound healing.

[0188] In addition, typical fatty acids contained in 1g of general cow's milk or 1g of soybean oil and their contents are as follows.

[0189]

[0190] Example 7: Tissue-resident M2-like macrophages and PPARγ-positive adipocytes in scalp tissue after emulsion administration Cell detection

[0191] It has been reported that when tissue regeneration occurs in peripheral tissues (particularly adipose tissue), an increase in tissue-resident M2-like macrophages occurs (Satoh T. et al., Nature, 495: 524-528, 2013), the appearance of CD34-positive cells, or an increase in PPARγ-positive adipocytes occurs (Festa E et al., Cell, 146: 761-71, 2011). Therefore, in this example, an increase in tissue-resident M2-like macrophages or an increase in PPARγ-positive cells was investigated in the scalp tissue after administration of 20 μL of milk. In addition, an increase in Ki67-positive cells, which is an activation marker for cell proliferation, was confirmed in the back skin of 12-week-old Wistar rats. Tissue-resident M2-like macrophages were detected as CD68-positive cells or CD163-positive cells. In this example, human colostrum was administered to humans as milk, and bovine colostrum was administered to rats.

[0192] Utilize conventional method to the tissue section of the back skin of the milk administration group (single administration) of multiple rats is fixed, and is provided for immunohistological staining.For rat, respectively, the two positions of the administration site and the position (distant position) 10mm away from the administration site are sampled after 5 weeks and 8 weeks of administration.For patients, half of the head is treated, and the remaining half is set as untreated. The section including the 3mm diameter punching from the scalp surface of the patient to the top of the skull periosteum or to the top of the temporal fascia is fixed.In addition, the primary antibody and secondary antibody used in the dyeing in this embodiment are as follows.

[0193] [Table 5]

[0194] Table 5: Primary and secondary antibodies used in immunohistological staining of rat samples

[0195]

[0196] The results are as follows Figure 7 As shown. Figure 7 As shown, the number of Ki67-positive adipocytes and CD68-positive macrophages increased after 5 weeks of administration, and each decreased after 8 weeks of administration. In contrast, in the distal part, no positive cells were observed after 5 weeks of administration, but the number of Ki67-positive cells and CD68-positive macrophages were observed after 8 weeks of administration.

[0197] This shows that, through the administration of milk, Ki67-positive adipocytes increase, that is, adipocytes are activated and their division is activated; and macrophages in the tissue increase. According to the microscope images, Ki67-positive adipocytes are mainly adipocytes and adipose stem cells. However, after 8 weeks of administration, the number of Ki67-positive adipocytes and macrophages decreased. On the other hand, surprisingly, an increase in the number of Ki67-positive adipocytes and CD68-positive macrophages was also observed at a distant site later than the administration site. This suggests that the effect of milk administration spreads in the tissue.

[0198] The same experiment was performed on humans. In addition to staining for CD31, staining for CD34, CD68, CD163, and PPARγ was also performed on human tissues.

[0199] [Table 6]

[0200] Table 6: Primary and secondary antibodies used in immunohistological staining of human samples

[0201]

[0202] in addition, Fig. 8ACD31 and CD34 staining are shown. CD31 positive cells were observed only in the vascular endothelium. On the other hand, CD34 positive cells were observed in areas other than blood vessels. This suggests that CD31 negative CD34 positive cells increase at the administration site by milk administration. Fig. 8A As shown, an increase in CD34-positive cells per unit area was confirmed before treatment and in the fifth month after a single treatment (female, 49 years old). In addition, regarding CD34-positive cells, the average number of 3mm punched slices on the untreated side was 106.7 (n=3), while the average number on the treated side was 129.7 (n=3), and an increase in CD34-positive cells was observed on the treated side in any patient. It should be noted that CD34-positive cells are known to be adipose stem cell markers, and an increase in CD34-positive cells suggests an increase in adipose stem cells. Therefore, this result shows that the adipose tissue at the site of milk administration is modified to produce adipose stem cells.

[0203] The results of staining for CD68-positive macrophages and PPARγ-positive cells are shown in Figure 8B As shown. Figure 8B As shown, in the case of humans, CD68-positive macrophages, CD163-positive macrophages, and PPARγ-positive cells per unit area were also increased by the administration of 20 μL of milk (female, 49 years old). In addition, for male patients (4 doses, 50 years old) 15 months after the start of treatment, it was also observed that CD68-positive macrophages, CD163-positive macrophages, and PPARγ-positive cells per unit area increased on the treated side under the action of milk.

[0204] Thus, at the milk administration site, tissue-resident M2-like macrophages increased, indicating the involvement of immature adipocytes, which are PPARγ-positive cells.

[0205] It should be noted that Fig. 8A and 8B In the diagram, the symbol "H" represents hair follicles, the symbol "V" represents blood vessels, and the symbol "S" represents sebaceous glands.

[0206] Example 8: Study on tissue changes caused by lactoferrin (large volume forced oral ingestion)

[0207] As a comparative test, lactoferrin was administered instead of milk to confirm whether an increase in CD68-positive cells and PPARγ-positive cells occurred. Regarding lactoferrin, 3 tablets of NICE ESSENCE (LION, 62971) and 300 mg of effective lactoferrin (daily intake for humans) were orally administered to 12-week-old Wistar rats (n=3). The administration was performed once a day, injected into the stomach using an oral probe (forced ingestion), and paraffin-embedded sections of the back skin were prepared using conventional methods 8 weeks after administration for immunohistological staining. The results are as follows: Fig. 9 shown.

[0208] like Fig. 9 As shown in the results, no increase in CD68-positive cells and PPARγ-positive cells was observed when lactoferrin was administered. This indicates that, although lactoferrin is claimed to have a hair thickening effect, it does not have a skin improvement effect, unlike milk. Fig. 9 As shown, when the normal tissue (back) of 18-week-old Wistar rats was stained with anti-CD68 antibody and PPARγ antibody, no significant difference was found between the normal tissue and the 8th week (20 weeks old) of lactoferrin administration. It has been reported that lactoferrin inhibits the activity of PPARγ while inhibiting the adipose differentiation of preadipocytes (reference M. Yag, et al., Journal of Oral Science, 50: 419-425, 2008), and the above results are consistent with the report. Components other than lactoferrin in milk are considered to be the main cause of the rejuvenation of adipocytes and the increase of preadipocytes caused by milk administration.

[0209] Example 9: Tissue changes and PPARγ activity induced by representative fatty acids contained in milk

[0210] Next, representative fatty acids contained in the milk were administered to multiple sites on the back skin of 12-week-old Wistar rats. The administration sites were as described in Example 1 (2) above, and the tissue changes caused by the administration were observed by immunohistological staining. More specifically, the changes in CD68-positive macrophage staining and PPARγ-positive cell images produced by the administration of 20 μL / site of physiological saline and 20 μL / site of a dosing solution containing 1 mg of fatty acids were observed. For the samples of Wistar rats, samples were collected at the administration sites (4 weeks after administration and 8 weeks after administration). The results are as follows. Figures 10A to 10G shown. Fig. 10A The results of the physiological saline administration group (negative control) are shown.

[0211] like Figures 10B to 10D As shown in Figure 2, when palmitic acid, stearic acid or myristic acid was administered, an increase in PPARγ positive cells and CD68 positive cells was observed at the administration site 4 weeks after administration. In addition, after 8 weeks of administration of any of the above fatty acids, the increase in PPARγ positive cells and CD68 positive cells at the administration site was still maintained.

[0212] It should be noted that many CD68-positive cells were found to be infiltrated at the saturated fatty acid injection site, and fibrosis of normal tissue was confirmed. Figures 10B to 10DIn the experiment, changes in adipose tissue in the area closest to the administration site where normal tissue was maintained (non-fibrotic area) were observed.

[0213] like Figures 10E to 10G As shown in respectively, in the case of oleic acid, α-linolenic acid or linoleic acid administration, after 4 weeks of administration, no significant increase of PPARγ positive cells and CD68 positive cells was confirmed compared with the control, but an increase of PPARγ positive cells was confirmed after 8 weeks of administration. On the other hand, in the case of oleic acid, α-linolenic acid or linoleic acid administration, no significant increase of CD68 positive cells was confirmed (a weaker effect was confirmed overall). CD68 positive cells differentiate into tissue resident M2-like macrophages under the action of PPARγ, promoting the maintenance of tissue homeostasis. It is believed that in the case of saturated fatty acids such as palmitic acid, stearic acid or myristic acid, an increase in CD68 positive cells and an increase in PPARγ positive cells are produced, so in the tissues administered with fatty acids, tissue resident M2-like macrophages increase. On the other hand, it is believed that in the case of unsaturated fatty acids, PPARγ positive cells increase, but no significant increase of CD68 positive cells is confirmed, and it is believed that the induction effect of tissue resident M2-like macrophages is limited. In addition, this result is consistent with the result in the above-mentioned Example 6, that is, saturated fatty acids exert a high wound healing promoting effect, while unsaturated fatty acids have a limited wound healing promoting effect. It has been reported that the PPARγ activity of macrophages is closely related to the function of adipocytes (reference Odegaard JI, et al., Nature, 447: 1116-1121, 2007). Therefore, the increase of CD68 positive cells and PPARγ positive cells in adipose tissue caused by saturated fatty acid administration is also a result of the investigation that supports the improvement of skin quality by saturated fatty acid administration. It should be noted that the increase of PPARγ positive adipocytes was confirmed based on the increase of PPARγ positive cells with fat droplets in the cells, and the result suggests the activation of PPARγ positive adipose stem cells produced by saturated fatty acid administration.

[0214] Palmitic acid, stearic acid or myristic acid are saturated fatty acids, while oleic acid, α-linolenic acid or linoleic acid are unsaturated fatty acids. Figures 10B to 10GThe results showed that saturated fatty acids have an effect of inducing the expression of PPARγ positive cells (including immature adipocytes) in tissues at an early stage, and unsaturated fatty acids have an effect of inducing the expression of PPARγ positive cells (immature adipocytes) later than saturated fatty acids. From this result, it is believed that saturated fatty acids induce strong tissue inflammation and aggregate macrophages, but at the same time as macrophages aggregate to tissues, PPARγ positive cells increase, thereby suppressing induced inflammation. On the other hand, it is believed that unsaturated fatty acids have a weak effect of aggregate macrophages to tissues, and the inflammation suppression effect caused by PPARγ is dominant.

[0215] Fatty acids are known to promote the secretion of inflammatory cytokines (reference Hotamisligil GS, Nature, 542: 177-185, 2017). In the results of this embodiment, saturated fatty acids induce a strong inflammatory response, on the other hand, by increasing PPARγ positive cells to reduce inflammation and promote tissue regeneration. This suggests the following possibility: PPARγ positive cells increased in tissues by saturated fatty acid administration induce adipose stem cells, immature adipocytes, induce M2 macrophages, and cause wound healing reactions and tissue regeneration. It should be noted that it is confirmed that the expression of PPARγ and its effect on macrophages cause the activation of immune cells (particularly the induction of M1 macrophages to M2 macrophages) (reference Croadell A. et al., PPAR Research, ID 549691, 2015).

[0216] Example 10: Relationship between scalp improvement, hair quality improvement and hair thickening effect

[0217] The above Examples 1 to 9 show that the administration of the emulsion leads to the regeneration and improvement of the scalp and skin, thereby producing an effect of improving the hair quality.

[0218] In this example, based on the results of the above examples, the effect of emulsion administration was evaluated by focusing on hair that grows faster. Specifically, hair that grows more than 0.9 mm in 3 days was extracted from the scalp image, and the total length of the growth was calculated. It should be noted that it is known that adult hair grows at a rate of about 10 mm per month (i.e., about 1 mm in 3 days).

[0219] For this purpose, a portion of the scalp of a plurality of adults (6 treated with only one dose and 1 treated once a month for 4 months) who were administered with 20 μL / site of emulsion was shaved, and images of the shaved site were taken 3 days later and image analysis was performed. In addition, the same site was shaved again one year after administration, and images of the shaved site were taken 3 days later and image analysis was performed. After shaving, the hair had an average length of 0.4 mm. Hereinafter, for hair having a length of 1.4 mm or more 3 days after shaving, the total length of extension was calculated.

[0220] The counting result of the length of the hair part of this patient obtained by using image analysis software is shown in FIG. Fig.11A and 11B Here, as a representative measurement example, the treatment course comparison counting results of patient A (50 years old / male, once a month treatment for 4 months, one year after the start of treatment) are shown in Fig.11A The results of comparative measurements of the untreated side and the treated side of two patients (patient A, patient B (47 years old / female, treatment with only one dose, one year after treatment)) are shown in Fig. 11B .

[0221] Fig.11A In this study, measurements of patient A were compared before treatment and one year after the start of treatment. Fig. 10A , the sum of the lengths of hairs having a length of 1.4 mm or more 3 days after shaving (1.4 mm or more) and the sum of the lengths of all hairs including hairs having a length of 0.9 mm or more and less than 1.4 mm 3 days after shaving (total) are shown respectively. In the case of patient A, the sum of the lengths of hairs having a length of 1.4 mm or more 3 days after treatment was 311.9 before treatment, but was 337.9 one year after the start of treatment. The sum of the lengths of all hairs 3 days after shaving was 405.7 before treatment, but was 419.2 one year after the start of treatment.

[0222] Fig. 11B The results of the untreated and treated sides of patients A and B were compared one year after the start of treatment. Fig. 10B , the sum of the lengths of hairs longer than 1.4 mm 3 days after shaving (1.4 mm or longer) and the sum of the lengths of all hairs including hairs longer than 0.9 mm and shorter than 1.4 mm 3 days after shaving (total) are shown respectively. In the case of patient A, the sum of the lengths of hairs longer than 1.4 mm 3 days after shaving was 199.4 on the untreated side, but 337.9 on the treated side. In the case of patient B, the sum of the lengths of hairs longer than 1.4 mm 3 days after shaving was 114.3 on the untreated side, but 300.7 on the treated side.

[0223] Similarly, in the case of patient A, the total length of hair longer than 0.9 mm 3 days after shaving was 357.8 on the untreated side, but 419.2 on the treated side. In the case of patient B, the total length of all hair 3 days after shaving was 261.5 on the untreated side, but 378.1 on the treated side.

[0224] As a negative control, saline was injected into one half of the head. The changes in hair (the number of hair roots longer than 1.4 mm) caused by the injection of saline into one side of the head were observed monthly for 6 months. The change rate between before treatment and the sixth month of treatment was -0.84% ​​to +1.24%, with an average of 1.03%. In addition, the changes in the sixth month of treatment between the treated side injected with saline and the untreated side were -0.99% to +1.25%, with an average of 1.06% (n=4). The changes in hair (the sum of the length of hair longer than 1.4 mm) caused by the injection of saline into one side of the head were observed monthly for 6 months. The changes between before treatment and the sixth month of treatment were -0.79% to +1.33%, with an average of 1.05%. In addition, the changes in the sixth month of treatment between the treated side injected with saline and the untreated side were -0.95% to +1.28%, with an average of 1.07% (n=4). Thus, in the group administered with physiological saline as a negative control, no significant increase in hair of 1.4 mm or more was observed on the third day after shaving.

[0225] Thus, it was found that the administration of the emulsion (20 μL / one site) showed improvement in scalp quality and the resulting improvement in the rate of hair growth.

[0226] In addition, when the hair with a length of 1.4 mm or more was examined after 3 days, it was found through image analysis that the hair with a length of 1.4 mm or more was thicker than the hair with a slow elongation rate (hair with a length of less than 1.4 mm after 3 days). Specifically, the average thickness of the hair with a length of 2.1 to 2.2 mm after shaving was 97.42 μm, while the average thickness of the hair with an elongation of less than 1.4 mm was 75.39 μm.

[0227] This result shows that the administration of emulsion (20 μL / one site) increases the number of hairs that grow faster and thicker.

[0228] By administering the emulsion, improvements in the tension and elasticity of the hair were observed (the tension and elasticity of the hair increased, and an increase in the volume of the hair was observed), which is a result consistent with an increase in thick hair.

[0229] Thus, hair growth was observed in the subjects who received the emulsion administration, and the modification of the scalp due to the emulsion administration resulted in an improvement in the rate of hair extension and an increase in thick, elastic, and tensile high-quality hair.

[0230] Example A11: Bone formation promoting effect

[0231] In this example, the effect of palmitic acid administration on bone formation was confirmed.

[0232] Creation of bone defect: The heads of 12-week-old (4 rats) and 18-week-old (1 rat) Wistar rats (male) were shaved with a hair clipper, and a tangent line was drawn diagonally above the eye and in front of the opposite ear, and a skull defect with a diameter of 8.8 mm was created behind the intersection. The procedure was performed in the order of 1. general anesthesia, 2. skin incision, 3. periosteal stripping, and 4. skull removal (diameter 8.8 mm). The skull defect was created while slowly cutting the surrounding bones using a trephine and a micro drill, and finally the bone was lifted to create the skull defect.

[0233] Observation of bone formation: A hydroxyapatite film was prepared according to the size of the bone defect and placed on the bone defect. At this time, 0.02 mg (20 μg) of palmitic acid was applied on the film. Specifically, 0.02 mL (20 μL) of a solution prepared by dissolving 1 mg of palmitic acid in 1 mL was dropped onto the film, dried and placed on the bone defect.

[0234] After 8 weeks of feeding, the animals were slaughtered after tissue collection, perfused and fixed, and the cross-section of the skull was stained with hematoxylin-eosin and observed under a microscope. Then, the area of ​​the part where bone formation was confirmed in the case of using a film coated with palmitic acid and in the case of using a film not coated was compared. Image J was used for analysis.

[0235] The 3D-CT image of the skull was acquired using the following system.

[0236] Device name ScanXmate-E090 manufactured by Comscan

[0237] Shooting conditions: X-ray tube voltage 88 kV

[0238] X-ray tube current 89 μA

[0239] Resolution: 30.649 μm / pixel

[0240] The three-dimensional construction was performed using VGSTUDIO MAX (Volume Graphics Corporation).

[0241] The results are as follows Fig.12 As shown. Fig.12 As shown in , when a film coated with palmitic acid was used, the skull was almost completely closed by the formation of new bone, whereas when a film not coated with palmitic acid was used, the formation of new bone was insufficient.

[0242] Then, hematoxylin-eosin stained sections were Fig.13 As shown. Fig.13 As shown in FIG. 1 , when the palmitic acid-coated film is placed, a large amount of red-stained bone is formed (see the dotted line), whereas when the film is placed, a small amount of red-stained bone is formed (see the dotted line). The stained area (number of pixels and mm) of each bone formation in the tissue fragment is calculated. 2 ) for comparison, the film coated with palmitic acid has 12132 pixels (0.984 mm 2 ), while the comparison is only 1025 pixels (0.083mm 2 ). The formed bone was significantly thicker in the films coated with palmitic acid than in the control films.

[0243] This indicates that palmitic acid has the effect of promoting bone formation.

[0244] Bone tissue slices (3 mm in diameter) were collected at slaughter.

[0245] After 8 weeks of feeding, tissues were collected and fixed by perfusion. Bone tissue was collected using a trephine (3 mm in diameter) when bone formation was sufficient, and bone rongeurs (rongeurs) were used to collect the original bone and regenerated bone from the open bone margins for samples with insufficient bone formation (rats).

[0246] The collected tissue pieces were immersed in RNA stabilizing solution (Thermo Fisher Scientific, Lithuania), frozen with liquid nitrogen, and stored at -80°C. Total RNA was purified and extracted from the stored tissue pieces as follows: 25 mg of tissue pieces were sealed in a sample tube together with 5 stainless steel beads (3 mm in diameter), and the tissue pieces were homogenized using the RNeasy Mini kit (QIAGEN GmbH, Hilden, Germany) and Tissue Lyser II (QIAGEN GmbH, Hilden, Germany). After that, the total RNA was purified and extracted using a centrifugal column according to the operating procedures attached to the kit. For gene expression analysis using quantitative PCR, Integrated DNA Technologies, Inc (Skoki, IL, USA) was commissioned to design and synthesize gene-specific primers and probes. Using PrimeTime Gene Expression Premix (IntegratedDNA Technologies, Inc, Skoki, IL, USA) and Rotor-Gene Q PCR device (QIAGEN GmbH, Hilden, Germany), PCR analysis was performed twice for each gene in each sample to analyze the expression level of the target gene. The information of seven rat genes used in the gene expression analysis by quantitative PCR (qPCR; ΔΔCt method) and the base sequences of the synthesized probe and primer sets are as follows.

[0247] 1.) Ribosomal protein lateral stalk subunit P0 (RplP0), Rattus norvegicus: mRNA, 1,093 bp, NM_022402.2, GI: 310616731

[0248] 6-FAM / CCT GTC TTC / ZEN / CCT GGG CAT CAC G / IABkFQ

[0249] CAA TCC CTG ACG CAC CG

[0250] TGT CTG CTC CCA CAA TGA AG

[0251] 2.) CXC motif chemokine ligand 12 (Cxcl12), transcript variant 1, Rattus norvegicus: mRNA 1,880 bp NM_022177.3 GI: 7649650

[0252] 56-FAM / TCA ACA CTC / ZEN / CAA ACT GTG CCC TTC A / 3IABkFQ

[0253] GAG CCA ACG TCA AAC ATC TG

[0254] GGC TTT GTC CAG GTA CTC TTG

[0255] 3.) CXC motif chemokine receptor 4 (Cxcr4), Rattus norvegicus: mRNA 1,726 bp NM_022205.3 GI: 82617587

[0256] 56-FAM / CAA TGC TCG / ZEN / CTC TCC AGC CCT / 3IABkFQ

[0257] CGT TTG GTG CTC CGG TAG

[0258] TCT CCA GAC CCT ACT TCT TCG

[0259] 4.) Integrin subunit alpha 4 (Itga4), Rattus norvegicus: mRNA 3,736 bp NM_001107737.1 GI: 157819948

[0260] 56-FAM / CGA AGG ACG / ZEN / TCA AAT CAG CCA GTC A / 3IABkFQ

[0261] GCA TCT CCT CTA CAT ACT CAC AG

[0262] CAC CAA CCG CTA CAT CAA CA

[0263] 5.)CD34, Rattus norvegicus: CD34 molecule (Cd34), mRNA 1,161 bp NM_001107202.2 GI: 169790781

[0264] 56-FAM / TCC CTG GAA / ZEN / GTA CCA GCC ACT ACT / 3IABkFQ

[0265] GGA GTA TTT CCA CCA GTT CCT AC

[0266] GAT GGC TGG TGT GGT CTT ATT

[0267] 6.) Bone morphogenetic protein-4 (BMP4), Rattus norvegicus: mRNA 1,559 bp NM_012827.2 GI: 148747215

[0268] 56-FAM / CCT TGT TTT / ZEN / CTG TCA AGA CAC CAT GAT TCC / 3IABkFQ

[0269] ATA AAA CGA CCA TCA GCA TTC G

[0270] GCC TTT CCA GCA AGT TTG TTC

[0271] 7.) Bone morphogenetic protein-7 (BMP7), Rattus norvegicus: mRNA 2,448 bp NM_001191856.2 GI: 683523995

[0272] 56-FAM / TGC GAT GAT / ZEN / CCA GTC CTG CCA G / 3IABkFQ

[0273] CGT TCA TGT AGG AGT TCA GAG G

[0274] CTG TAT GTT AGC TTC CGA GAC C

[0275] The difference in expression level (Ct value) between the mRNA of ribosomal phosphoprotein large subunit P0 (RplP0) and the target gene as a housekeeping gene (average of two samples, one at 12 weeks and one at 18 weeks of age) and the difference in expression level between RplP0 and the target gene in the control (12 weeks of age, no palmitic acid administration) were compared, and the difference was converted into a gene expression ratio (fold), and the fold was further log2-converted and shown in the figure (reference Fig.14 ).

[0276] like Fig.14 As shown, compared with the case where only the film was embedded, increases in BMP4 and BMP7, which are important for bone formation, were confirmed.

[0277] Example A12: Changes in gene expression in tissues after subcutaneous administration

[0278] Then, the changes of gene expression in the tissue were confirmed. Three regions were set from the head to the tail of the back of 12-week-old Wistar rats (male), and each region was divided into two regions on the left and right of the vertebrae, thus setting a total of 6 regions on the back. Near the center of each region, 20 μL / a portion of milk or palmitic acid was injected into each 2 places. It should be noted that the distance between the above 2 places was set to 1 cm.

[0279] The collected tissue slices were frozen with liquid nitrogen and stored at -80°C. Total RNA was purified and extracted from the stored tissue slices as follows: 25 mg of tissue slices were sealed in a sample tube together with 5 stainless steel beads (3 mm in diameter), and the tissue slices were homogenized using the RNeasy Mini kit (QIAGEN GmbH, Hilden, Germany) and Tissue Lyser II (QIAGEN GmbH, Hilden, Germany). After that, the total RNA was purified and extracted using a centrifugal column according to the operating procedures attached to the kit. For gene expression analysis using quantitative PCR, Integrated DNA Technologies, Inc (Skoki, IL, USA) was commissioned to design and synthesize gene-specific primers and probes. Using PrimeTime Gene Expression Premix (Integrated DNA Technologies, Inc, Skoki, IL, USA) and Rotor-Gene Q PCR device (QIAGEN GmbH, Hilden, Germany), PCR analysis was performed twice for each gene in each sample to analyze the expression level of the target gene. The difference in expression level (Ct value) between the ribosomal phosphoprotein large subunit P0 (RplP0) as a housekeeping gene and the mRNA of the target gene (average of 3 animals tested twice for each sample) and the difference in expression level between RplP0 and the mRNA of the target gene in the control (no administration of palmitic acid or milk) were compared, and their differences were converted into gene expression ratios (multipliers), and the multipliers were further log2-converted and shown in the graph.

[0280] The results are as follows Fig.15 As shown. Fig.15As shown, in both the palmitic acid administration and milk administration, the expression levels of Cxcl12, Cxcr4, and Itga4 in the tissues increased significantly. Itga4 and Cxcr4 are markers confirmed in bone marrow cells, and their expression in tissues indicates that cells derived from the bone marrow are recruited to the tissues. The expression of Cxcl12 in the tissues also indicates that the mechanism for accepting these cells derived from the bone marrow is also prepared. In addition, Fig.15 As shown, an increase in CD34 was also observed. It is considered that adipocytes may be induced in the presence of bone marrow progenitor cells and macrophages.

[0281] Reference List

[0282] Noro Mirai et al., Electrophoresis, 59: 21-24, 2015

[0283] Satoh T. et al., Nature: 495, 524-528, 2013

[0284] Festa E et al., Cell, 146: 761-71, 2011

[0285] M. Yag, et al., Journal of Oral Science, 50: 419-425, 2008

[0286] Odegaard JI, et al., Nature, 447: 1116-1121, 2007

[0287] Hotamisligil GS, Nature, 542: 177-185, 2017

[0288] Croadell A. et al., PPAR Research, ID 549691, 2015

Claims

1. Use of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for topical administration for treating wounds, wherein the saturated fatty acid comprises a saturated fatty acid consisting of stearic acid and / or myristic acid.

2. Use of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for topical administration for treating wounds, wherein the saturated fatty acid consists of stearic acid and myristic acid.

3. Use of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for topical administration for treating wounds, wherein the saturated fatty acid consists of stearic acid.

4. Use of a saturated fatty acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for topical administration for treating wounds, wherein the saturated fatty acid consists of myristic acid.

5. Use of palmitic acid or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for promoting bone formation.

6. The use according to claim 5, wherein: The saturated fatty acid in the pharmaceutical composition is in the form of being coated on a biocompatible film.

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