Uses of saturated fatty acids or their pharmaceutically permissible salts

Abstract

The present invention provides a pharmaceutical composition and method comprising a therapeutically effective amount of saturated fatty acid or a pharmaceutically permissible salt thereof as an active ingredient for use in improving hair, scalp or skin, wound healing, promoting bone formation, or improving hair quality.

Description

Technical Field

[0001] This invention relates to pharmaceutical compositions and methods for improving hair, scalp or skin, wound healing, promoting bone formation, or improving hair quality. Prior Technology

[0002] Mammal milk (especially colostrum) contains a wide variety of nutrients, such as immune substances or intracellular transport substances, extracellular bodies, mRNA, and cytokines. If an infant ingests these substances orally, they are absorbed into the body and contribute to the infant's development.

[0003] Through comparative analysis of the presence and absence of adipocytes, it has been reported that adipocytes contribute to the microenvironment of skin stem cells and drive hair growth (Non-Patent Literature 1). Non-Patent Literature 1 has suggested that the presence of CD34-positive cells, the increase of PPARγ-positive adipocytes, and the maturation of adipocytes are associated with hair growth during the anagen phase. Furthermore, Non-Patent Literature 2 has reported an increase in tissue-native M2-like macrophages observed during tissue regeneration. [Previous Technical Documents] [Non-patent literature]

[0004] [Non-Patent Literature 1] Festa E et al., Cell, 146: 761-71, 2011 [Non-Patent Literature 2] Satoh T. et al., Nature, 495: 524-528, 2013 Summary of the Invention

[0005] This invention provides a topical pharmaceutical composition for modifying the scalp or skin. It further provides a topical pharmaceutical composition for treating wounds, and also provides a topical pharmaceutical composition for promoting hair growth or modifying hair.

[0006] The inventors have discovered that compositions comprising milk, the supernatant of milk after centrifugation, soybean oil, and the main fatty acids contained therein, etc., have the potential to improve the scalp or skin, heal wounds, promote bone formation, and improve hair quality. This invention is based on these findings.

[0007] According to the present invention, the following invention is provided. (1) A pharmaceutical composition which is (i) a topical pharmaceutical composition for use in modifying the scalp or skin, (ii) a topical pharmaceutical composition for use in treating wounds, or (iii) a topical pharmaceutical composition for use in promoting hair growth or for use in modifying hair, comprising a therapeutically effective amount of saturated fatty acid or a pharmaceutically permissible salt thereof. (2) The pharmaceutical composition described in (1) above is used to modify the scalp or skin. (3) The pharmaceutical ingredients described in (1) above are used to treat wounds. (4) The pharmaceutical ingredients described in (1) above are used to promote hair growth or to improve hair quality. (5) The pharmaceutical ingredients described in (1) above are used to promote bone formation. (6) The pharmaceutical composition described in (6) above is in the form of saturated fatty acids coated on a biocompatible membrane. (7) The pharmaceutical composition described in any of (1) to (6) above, wherein the aforementioned saturated fatty acid is one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid. (8) The pharmaceutical composition described in any of (1) to (7) above further includes unsaturated fatty acids. (9) The pharmaceutical composition described in any of (1) to (6) above contains soybean oil or emulsion or such extract or processed product, wherein the soybean oil or emulsion or such extract or processed product contains a therapeutically effective amount of saturated fatty acid or its pharmaceutically permissible salt. (10) The pharmaceutical composition described in (9) above, wherein the milk is sterilized. (11) The pharmaceutical composition described in (10) above, wherein the milk is sterilized at high temperature.

[0008] According to the present invention, it is advantageous to provide a pharmaceutical composition or treatment method that can be used for the various pharmaceutical applications described above, and which can continue to exert its effects even after the treatment has ended. Simple Explanation of the Diagram

[0009] [Figure 1] Figure 1 is a photograph showing the effect of milk application on the cuticle of hair. [Figure 2A] Figure 2A is a partial photograph showing the effect of breast milk application on the reduction of gray hair. [Figure 2B] Figure 2B is a photograph of the head from the side, showing the effect of breast milk administration on the reduction of gray hair. [Figure 3] Figure 3 shows the relationship between milk delivery and the thickness of the skin's fat layer. #1 to #6 represent treatment results 1 to 6 months later. [Figure 4] Figure 4 shows tissue staining of type I and type III collagen in skin tissue before and after treatment. [Figure 5] Figure 5 shows the effect of milk administration on the size of fat cells. [Figure 6A] Figure 6A is a diagram showing the effect of breast milk on wound healing. [Figure 6B] Figure 6B shows the effect of lactoferrin on wound healing. [Figure 6C] Figure 6C is a diagram showing the effect of saturated or unsaturated fatty acids on wound healing. [Figure 6D] Figure 6D is a diagram showing the effect of soybean oil on wound healing. [Figure 7] Figure 7 shows the results of immunohistochemical staining (staining of Ki67-positive adipocytes and CD68-positive macrophages) of the subcutaneous adipose tissue on the back of Wistar rats induced by milk administration. [Figure 8A] Figure 8A shows the results of immunohistochemical staining (CD31 staining and CD34 staining) of the scalp fat layer tissue induced by milk administration. [Figure 8B] Figure 8B shows the results of immunohistochemical staining of scalp fat tissue induced by milk administration (staining of CD68-positive macrophages, staining of CD163-positive tissue-normal M2-like macrophages, and staining of PPARγ-positive cells). [Figure 9] Figure 9 shows the results of immunohistochemical staining (staining of CD68 and PPARγ positive cells) of the subcutaneous adipose tissue on the back of rats induced by oral administration of lactoferrin. [Figure 10A] Figure 10A shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after administration of physiological saline. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 10B] Figure 10B shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after palmitic acid administration. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 10C] Figure 10C shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after stearic acid administration. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 10D] Figure 10D shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after myristic acid administration. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 10E] Figure 10E shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after oleic acid administration. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 10F] Figure 10F shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after α-linolenic acid administration. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 10G] Figure 10G shows the staining results of CD68 and PPARγ positive cells in tissues 4 and 8 weeks after linoleic acid administration. The arrows in the figure indicate the locations of positive cells, and the numbers in parentheses at the bottom right of each stained image indicate the number of positive cells in the field of view. [Figure 11A] Figure 11A shows the total length of hair per hair × number of hairs induced by milk. [Figure 11B] Figure 11B is a bar chart showing the results of Figure 11A. In Figure 11B, "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. [Figure 12] Figure 12 shows the images obtained by 3D-CT observation of subsequent bone formation after placing the bone resection site (8.8 mm in diameter) on a biofit membrane coated with palmitic acid or an uncoated biofit membrane. For the bone after bone formation, a 3 mm diameter hole was artificially perforated in part of the bone for tissue sampling. [Figure 13] Figure 13 shows hematoxylin-eosin stained images of bone sections taken from tissue samples after bone formation. The dashed ellipses represent newly formed bone at the bone resection site. The values ​​represent the area (number of pixels and mm²) of each formed bone. [Figure 14] Figure 14 shows the changes in gene expression of Bone morphogenetic protein-4 (Bmp4) and Bone morphogenetic protein-7 (Bmp7) in subjects given palmitic acid or milk. [Figure 15] Figure 15 shows the changes in gene expression induced by palmitic acid administration or milk administration relative to the control. Implementation

[0010] In this specification, the term "object" refers to mammals. Examples of mammals include humans (male and female).

[0011] In this instruction manual, the term "milk" means milk derived from mammals. Examples of mammals include humans and cows.

[0012] In this specification, "whey" refers to an aqueous solution obtained by removing milk fat or casein from milk. Whey can be obtained by removing precipitates, for example, by centrifuging the milk.

[0013] In this specification, "scalp" refers to the skin of the head, excluding the top of the head, face, jaw, and neck (including the ears). In this specification, "skin" is used to include the scalp and all skin outside the scalp. The human scalp has an area of ​​approximately 700 mm² to approximately 800 mm².

[0014] In this specification, the term "improvement" means to modify, enhance, upgrade, or optimize the quality. The term "improvement" is used in the sense of being better than the current state, including improvements to previously poor aspects.

[0015] In this instruction manual, the term "hair" refers to hair growing on the skin. The term "hair head" refers to hair growing on the scalp.

[0016] In this specification, "white hair" means hair that is white due to a lack of pigments such as eumelanin (or true melanin) and / or pheomelanin, particularly scalp hair. In this specification, "black hair" means hair that contains eumelanin and appears black.

[0017] In this specification, the term "keratin" refers to the surface structure covering the hair, which is the outermost layer of the hair. The keratin plays a role in protecting the hair from external stimuli and preventing the loss of moisture or components from the inner cortex to the outside. The keratin covers the hair in a scaly manner from the root to the hair tip.

[0018] In this specification, the term "trauma" means physical damage to tissues. Trauma includes injuries to the body surface.

[0019] According to the present invention, it is provided (A) A topical pharmaceutical composition intended for the modification of the scalp or skin, comprising a therapeutically effective amount of fatty acids (particularly saturated fatty acids) or their pharmaceutically permissible salts; (B) A topical pharmaceutical composition for the treatment of wounds, comprising a therapeutically effective amount of fatty acids (particularly saturated fatty acids) or their pharmaceutically permissible salts; and (C) Topical pharmaceutical formulations for promoting hair growth or for modifying hair quality, comprising a therapeutically effective amount of fatty acids (particularly saturated fatty acids) or pharmaceutically permissible salts thereof; and (D) Pharmaceutical formulations used to promote bone formation, which contain therapeutically effective amounts of fatty acids (especially saturated fatty acids) or their pharmaceutically permissible salts. (Hereinafter, the pharmaceutical ingredients described in (A) to (D) above will sometimes be referred to as "the pharmaceutical ingredients of the present invention").

[0020] In one embodiment of the present invention, examples of substances containing a therapeutically effective amount of saturated fatty acids or their pharmaceutically permissible salts include milk, milk extracts (e.g., whey), and processed milk products (e.g., dairy products, such as whey from cheese, whey from yogurt, etc.), as well as mixtures thereof. Milk, milk extracts, or processed milk products may also be sterilized, for example, by low-temperature sterilization (e.g., heat sterilization at 62°C to 68°C) or high-temperature sterilization (heat sterilization at 120°C or above). Sterilization of milk is generally broadly divided into low-temperature sterilization at 62°C to 68°C for about 30 minutes (e.g., at 65°C for 30 minutes) and high-temperature sterilization at 120°C to 150°C for 1 second to 4 seconds (e.g., at 120°C to 130°C for 2 seconds to 3 seconds). Thus, in one embodiment of the present invention, the pharmaceutical composition of the present invention includes milk as an active ingredient.

[0021] In one embodiment of the present invention, as a substance containing a therapeutically effective amount of saturated fatty acids or their pharmaceutically permissible salts, examples include animal oils, vegetable oils, such oil extracts or processed products (e.g., soybean oil, soybean oil extract or soybean oil processed products), or mixtures thereof.

[0022] In this specification, extracts from milk or soybean oil are portions extracted from milk or soybean oil, sometimes referred to as milk extracts or soybean oil extracts respectively, each containing therapeutically effective amounts of saturated fatty acids or their pharmaceutically permissible salts.

[0023] In this specification, "milk or soybean oil processed product" refers to the product obtained by processing milk or soybean oil, sometimes referred to as milk processed product or soybean oil processed product respectively, each containing a therapeutically effective amount of saturated fatty acid or its pharmaceutically permissible salt.

[0024] Examples of compositions containing therapeutically effective amounts of saturated fatty acids or their pharmaceutically permissible salts include solutions containing milk and whey, as well as freeze-dried preparations of the like.

[0025] The fatty acids contained in the topical pharmaceutical composition of the present invention may be fatty acids contained as components of milk.

[0026] In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for partial application of the present invention is one or more saturated fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid.

[0027] In one embodiment of the present invention, the fatty acids contained in the pharmaceutical composition for local application of the present invention may be saturated fatty acids, such as one or more fatty acids selected from the group consisting of palmitic acid, stearic acid and myristic acid.

[0028] In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for local application of the present invention is palmitic acid. In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for local application of the present invention is stearic acid. In one embodiment of the present invention, the saturated fatty acid contained in the pharmaceutical composition for local application of the present invention is myristic acid.

[0029] In one embodiment of the present invention, the pharmaceutical composition for local application of the present invention may further include unsaturated fatty acids. As unsaturated fatty acids, they may include one or more unsaturated fatty acids selected from the group consisting of oleic acid, α-linolenic acid and linolenic acid. In one embodiment of the present invention, the fatty acid contained in the pharmaceutical composition for partial application of the present invention is oleic acid. The fatty acid contained in the topical pharmaceutical composition of the present invention is α-linolenic acid. The fatty acid contained in the topical pharmaceutical composition of the present invention is linoleic acid.

[0030] In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention may be a mixture of at least one saturated fatty acid and at least one unsaturated fatty acid. In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention are palmitic acid and linoleic acid. In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention are palmitic acid and oleic acid. In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention are stearic acid and linoleic acid. In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention are stearic acid and oleic acid. In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention are palmitic acid, stearic acid, and oleic acid. In one embodiment, the fatty acids contained in the pharmaceutical composition for partial administration of the present invention are palmitic acid, stearic acid, and oleic acid. In one particular embodiment, the fatty acids contained in the pharmaceutical composition for local application of the present invention are myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, and α-linolenic acid.

[0031] In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may contain, for example, one or more fatty acids selected from saturated fatty acids {here, "may contain" can include those naturally present in milk and those added to milk}. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may further contain unsaturated fatty acids. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may contain, for example, one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may contain, for example, one or more unsaturated fatty acids selected from the group consisting of oleic acid, α-linolenic acid, and linolenic acid. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may contain, for example, one or more saturated fatty acids and one or more unsaturated fatty acids. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this 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 linolenic acid. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may contain myristic acid, palmitic acid, and oleic acid. In one embodiment, the milk, milk extract, or milk processed product, or mixture thereof, which can be used in this invention, may contain myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, and α-linolenic acid. In one embodiment of the present invention, the above-mentioned one or more fatty acids may be added to a pharmaceutical composition in the form of milk, milk extract or milk processed product or a mixture thereof.

[0032] In one embodiment of the present invention, the content of one or more fatty acids selected from saturated and unsaturated fatty acids may be increased in milk, milk extract, milk processed product, or mixtures thereof. During enhancement, the fatty acid content after enhancement simply needs to be increased compared to the fatty acid content in the milk, milk extract, milk processed product, or mixtures thereof before enhancement. Enhancement may be performed, for example, by adding one or more fatty acids selected from saturated and unsaturated fatty acids to the milk, milk extract, milk processed product, or mixtures thereof. In one embodiment, enhancement may, for example, be an increase in the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid. In one embodiment, enhancement may, for example, be an increase in the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid, and linolenic acid. In one embodiment, enhancement may, for example, be an increase in the content of one or more saturated fatty acids and one or more unsaturated fatty acids. In a given sample, enhancement may, for example, be an increase in the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid, or an increase in the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid, and linolenic acid. In a given sample, enhancement may be an increase in the content of each of myristic acid, palmitic acid, and oleic acid. In a given sample, enhancement may be an increase in the content of each of myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, and α-linolenic acid.

[0033] In one embodiment of the present invention, the lactoferrin content may be reduced in milk, milk extract, milk processed product, or mixtures thereof. The reduction in lactoferrin content may also be achieved by methods such as high-temperature sterilization. In one embodiment of the present invention, the fatty acid content of milk, milk extract, milk processed product, or mixture thereof may be increased and the lactoferrin content reduced.

[0034] In one embodiment of the present invention, animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof may contain one or more fatty acids selected from saturated fatty acids. In one embodiment, animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof may further contain unsaturated fatty acids. In one embodiment, animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof that can be used in the present invention may contain one or more fatty acids, for example, selected from the group consisting of palmitic acid, stearic acid, and myristic acid. In one embodiment, animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof that can be used in the present invention may contain one or more unsaturated fatty acids, for example, selected from the group consisting of oleic acid, α-linolenic acid, and linolenic acid. In one embodiment, animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof that can be used in the present invention may contain, for example, one or more saturated fatty acids and one or more unsaturated fatty acids. In one embodiment, the animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof used in this 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 linolenic acid. In one embodiment, the animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof used in this invention may contain myristic acid, palmitic acid, and oleic acid. In one embodiment, the animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof used in this invention may contain myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, and α-linolenic acid.

[0035] In one embodiment of the present invention, the content of one or more fatty acids selected from saturated and unsaturated fatty acids may be increased in animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof. During enhancement, the fatty acid content after enhancement simply needs to be increased compared to the fatty acid content in the animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof before enhancement. Enhancement may be performed, for example, by adding one or more fatty acids selected from saturated and unsaturated fatty acids to animal oils, vegetable oils, such oil extracts or processed products, or mixtures thereof. In one embodiment, enhancement may, for example, be an increase in the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid. In one embodiment, enhancement may, for example, be an increase in the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid, and linolenic acid. In one embodiment, enhancement may, for example, be an increase in the content of one or more saturated fatty acids and one or more unsaturated fatty acids. In a given sample, enhancement may, for example, be an increase in the content of one or more fatty acids selected from the group consisting of palmitic acid, stearic acid, and myristic acid, or an increase in the content of one or more fatty acids selected from the group consisting of oleic acid, α-linolenic acid, and linolenic acid. In a given sample, enhancement may be an increase in the content of each of myristic acid, palmitic acid, and oleic acid. In a given sample, enhancement may be an increase in the content of each of myristic acid, palmitic acid, stearic acid, oleic acid, linolenic acid, and α-linolenic acid.

[0036] In one embodiment, the pharmaceutical composition for partial application of the present invention contains fatty acids in a soluble form. In another embodiment, the pharmaceutical composition for partial application of the present invention contains fatty acids in the form of a pharmaceutically permissible salt. In another embodiment, the pharmaceutical composition for partial application of the present invention contains fatty acids in the form of an emulsion. In another embodiment, the pharmaceutical composition for partial application of the present invention contains fatty acids that are not in the form of esters or glycerides.

[0037] Fatty acids are soluble in dimethyl sulfoxide, ethanol, chloroform, and diethyl ether. Their water solubility increases when fatty acids are formulated into pharmaceutically acceptable salts, such as sodium salts. Alternatively, fatty acid salts can be made into microparticles that dissolve in water.

[0038] The topical pharmaceutical composition of the present invention, in a certain state, can be an injectable formulation. In one state of the present invention, the topical pharmaceutical composition of the present invention can be provided in a kit form, the kit comprising: a freeze-dried formulation containing a solution of fatty acids and water for injection, wherein the freeze-dried agent can be prepared with water for injection as needed. The water for injection can also be heated for use. The pharmaceutical composition of the present invention for promoting bone formation can also be provided in a state of being coated onto a biofit membrane. Thus, in the present invention, a medicine comprising a therapeutically effective amount of saturated fatty acid or its pharmaceutically permissible salt coated onto a biofit membrane can be provided. Examples of biofit membranes include films having a calcium phosphate surface (e.g., a calcium phosphate film), films having a polylactic acid surface (e.g., a polylactic acid film), films having a polyglycolic acid surface (e.g., a polyglycolic acid film), films having a surface of a copolymer or block copolymer of lactic acid and polyglycolic acid (e.g., a copolymer or block copolymer of lactic acid and polyglycolic acid), and films having a surface of hydroxyapatite (e.g., a hydroxyapatite film). Coating the active ingredient onto the biofit membrane can be performed, for example, by dripping a solution obtained by dissolving the active ingredient onto the biofit membrane and then drying it. For example, approximately 10-50 μg of the pharmaceutical active ingredient can be coated onto the biofit membrane for use.

[0039] A topical pharmaceutical composition containing a therapeutically effective amount of saturated fatty acids or their pharmaceutically permissible salts, used to modify the scalp or skin, provides improvement to the scalp from the area of ​​application after application, with the effect spreading slowly to the surrounding area over time. Therefore, it can be dispersed on the scalp or skin to allow the effect of the invention to spread over time, or it can be concentrated to shorten the time required for spread. In one embodiment of the invention, the pharmaceutical composition is not particularly limited, and for example, a single application site can be defined as 1 cm²~10 cm², 1 cm²~5 cm², 1 cm²~4 cm², 1 cm²~3 cm², 1 cm²~2 cm², 0.5 cm²~2 cm², or 0.7 cm²~1.5 cm². In one embodiment of the present invention, the pharmaceutical composition of the present invention may be administered, for example, at a dosage density of 0.5 cm² or less, 0.6 cm² or less, 0.7 cm² or less, 0.8 cm² or less, 0.9 cm² or less, or 1 cm² or less. In another embodiment of the present invention, the pharmaceutical composition of the present invention may be administered, for example, locally directly below the subcutaneous layer or from the dermis to the upper fat layer. Administration may be performed, for example, by injection.

[0040] In addition, therapeutically effective amounts of saturated fatty acids or their pharmaceutically permissible salts may be administered, for example, in solutions of 10 μL to 50 μL, 15 μL to 30 μL, 15 μL to 25 μL, or about 20 μL per dose.

[0041] The application site can be chosen appropriately; for example, it can be applied to the entire scalp, or to a less desirable part of the scalp compared to other areas.

[0042] Furthermore, surprisingly, the inventors discovered that when applied to a relatively healthy area of ​​the scalp, the effect readily spreads to the surrounding area. The healthy area exhibits a higher responsiveness to the pharmaceutical composition of this invention, and its effect spreads to the periphery of the application site. The improvement effect resulting from this spread is strongly manifested in locations closer to the application site, reaching a range of several centimeters (e.g., 1cm to 4cm) from the application site. Therefore, it is possible to apply the medicine to healthy areas of the scalp. Furthermore, since the improvement effect spreads from the healthy area to less healthy areas, even if only the healthy area is treated, the less healthy area can also achieve a therapeutic effect. Therefore, the product can also be applied to better areas of the scalp, attempting to improve undesirable areas through the spreading effect. Furthermore, if improvement can be achieved in undesirable areas, the responsiveness to the pharmaceutical composition of the present invention is enhanced. Thus, if the product can be applied to better areas of the scalp, improving undesirable areas through the spreading effect, the pharmaceutical composition of the present invention can then be applied to previously undesirable areas subsequently. It is generally believed that each administration of the pharmaceutical composition of this invention will increase tissue stimulation, macrophage activity, or the activity of young adipocytes at the administration site, thereby enhancing repair function. Administration is intended to be based on observation of the patient's symptoms (condition) and the degree of improvement.

[0043] In a given scenario, the aforementioned local application can be set to 150 to 250 (e.g., approximately 200) or 250 to 800 scalp spots per adult, with the number of application spots determined as needed. Here, the term "approximately" means a range of values ​​including ±10% or ±5% of the values ​​following this term.

[0044] The pharmaceutical composition of the present invention is not particularly limited, and can be administered at intervals such as twice a month to once every 6 months, twice a month to once every 5 months, twice a month to once every 4 months, twice a month to once every 3 months, twice a month to once every 2 months, 1.5 times a month to once every 1.5 months, or 1.2 times a month to once every 1.2 months, for example, about once a month.

[0045] The pharmaceutical composition of this invention has a skin-improving effect on the scalp or skin even with a single application, and this effect is sustainable. Therefore, the application can be single or multiple times. The pharmaceutical composition of this invention can also be applied continuously until the treatment is completed or until the patient is satisfied. Specifically, according to this invention, the skin-improving effect on the scalp or skin begins to appear in areas of good condition and appears later in areas of poor condition. Therefore, the treatment period and treatment interval can be varied according to the condition of the scalp or skin. The treatment period using the pharmaceutical composition of this invention can be set, for example, 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. Furthermore, the pharmaceutical composition of this invention can be applied before, during, or after the observation of age-related changes in hair, scalp, or skin. Furthermore, the pharmaceutical composition of the present invention can also be administered again to subjects who have been treated with the pharmaceutical composition, before, during, or after the observation of changes in hair, scalp, or skin caused by aging.

[0046] In one embodiment of the present invention, the pharmaceutical composition described in (A) above may be a pharmaceutical composition for the rejuvenation or rejuvenation promotion 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 the regeneration or regeneration promotion 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 the activation of 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 growth agent or hair thickening agent.

[0047] 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 the healing of wounds, the promotion of the healing of wounds, or the early healing of wounds.

[0048] By improving the scalp, the effect also reaches the hair itself. Furthermore, as shown in the embodiments described later, the hair improvement effects include a reduction in gray hair, a reduction in the amount of gray hair, a reduction in the proportion of gray hair, an improvement in the roughness of the cuticle, and an improvement in the thickness and length of the hair. Therefore, in one embodiment of the present invention, the pharmaceutical composition described in (C) above can be a pharmaceutical composition used to reduce gray hair. As a reduction in gray hair, the reduction in the number of gray hairs, a reduction in the amount of gray hair, and a reduction in the proportion of gray hair can be included. The reduction in gray hair may be accompanied by an increase in the number of hairs obtained by coloring with various melanin, an increase in the amount of such hair, and an increase in the proportion of such hair. In one embodiment of the present invention, the pharmaceutical composition described in (C) above can be a pharmaceutical composition used to improve the roughness of the hair cuticle. To improve the roughness of the stratum corneum in hair, examples include increasing the neatness of the stratum corneum arrangement and reducing disordered areas of the stratum corneum. In one embodiment of the present invention, the pharmaceutical composition described in (C) above may be a pharmaceutical composition used to improve the thickness or elongation speed of hair. As for improving the thickness of hair, examples include thickening the hair, increasing the number of thicker hairs, and increasing the proportion of thicker hairs. As for improving the elongation speed, examples include increasing the number or proportion of hairs with an elongation 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.

[0049] In one aspect of the present invention, it is provided (a) A method for modifying the scalp or skin in a desired object (or desired site), comprising topically administering a therapeutically effective amount of saturated fatty acids or their pharmaceutically permissible salts to the scalp or skin; (b) A method of treating trauma in a person in need, comprising administering a therapeutically effective amount of saturated fatty acid or a pharmaceutically permissible salt thereof to the scalp or skin; (c) A method for promoting hair growth in subjects in need, or a method for modifying hair in subjects in need, comprising topical administration of a therapeutically effective amount of saturated fatty acids or their pharmaceutically permissible salts to the scalp or skin; and (d) A method for promoting bone formation in a subject in need, comprising administering a therapeutically effective amount of fatty acids (especially saturated fatty acids) or their pharmaceutically permissible salts to the site of bone formation (e.g., the site of bone injury) {here, the therapeutically effective amount of fatty acids (especially saturated fatty acids) or their pharmaceutically permissible salts may also be administered in a state of coating the biocompatible membrane}.

[0050] In one aspect of the present invention, it is provided (α) Therapeutic use of saturated fatty acids or their pharmaceutically permissible salts in the manufacture of topical pharmaceutical preparations for the modification of the scalp or skin; (β) The use of therapeutically effective amounts of saturated fatty acids or their pharmaceutically permissible salts in the manufacture of topical pharmaceutical preparations for the treatment of wounds; (γ) The use of therapeutically effective amounts of saturated fatty acids or their pharmaceutically permissible salts, in the manufacture of topical pharmaceutical preparations for promoting hair growth or for modifying hair; and (δ) The use of therapeutically effective amounts of saturated fatty acids or their pharmaceutically permissible salts in the manufacture of pharmaceutical compositions or preparations for promoting bone formation {here, the pharmaceutical preparations may provide therapeutically effective amounts of fatty acids (especially saturated fatty acids) or their pharmaceutically permissible salts coated onto the body's adaptive membrane}.

[0051] In one embodiment of the present invention, the pharmaceutical composition may, in addition to a therapeutically effective amount of fatty acids or their pharmaceutically permissible salts, also include excipients (e.g., solvents, co-solvents, soluble agents, wetting agents, suspending agents, thickeners, emulsifiers, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antibacterial agents, preservatives, fillers, protective agents, or isotropic agents). In one embodiment of the present invention, the pharmaceutical composition may be in the form of an injectable preparation. In one embodiment of the present invention, the pharmaceutical composition may be administered topically, such as intradermal or subcutaneously. [Example]

[0052] <Example 1: Milk Preparation and Administration> (1) The milk is prepared as follows. Colostrum, or milk collected within three weeks after childbirth, is obtained from the breasts of women aged 34 and 40 using a breast pump. As for the milk to be administered to rats, in the case of cow's milk, it is the colostrum obtained by hand-squeezing or commercially available cow's milk. (2) Milk is fed as follows. Colostrum was injected at a rate of 20 μL per site into the right or left half of the human scalp or the dorsal skin of rats. In humans, a total of 2 mL of colostrum was injected into the half of the head of each patient (i.e., 100 sites per patient). In rats, three regions were defined on the back of 12-week-old male Weiss rats from head to tail, and each region was further divided into two regions along the left and right sides of the spine, resulting in a total of six regions on the back. Two sites near the center of each region were injected with 20 μL of colostrum per site. The distance between the two sites was set at 1 cm.

[0053] <Example 2: Changes in Hair Texture> In this embodiment, the investigation focused on whether improvements were visible in the stratum corneum of the hair before and after treatment.

[0054] As the milk, human colostrum as described in Example 1 was used. The average hair growth rate was approximately 10 mm per month. One hair follicle was collected from both the treated and untreated areas. Considering the patient's hair growth rate, samples were taken from the hair follicles corresponding to the period 3 months prior to treatment and the hair follicles corresponding to the period 3.5 months after the start of treatment. On the day of hair collection or the following day, the hair follicles from the examination area were fixed to a fixation plate with resin. Carbon fixation was performed on the following day or the day after that, and the state of the hair cuticle was observed by scanning electron microscopy on the same day using the standard method. Untreated hair follicles (untreated areas) from the same patient were used as negative samples. The newly grown portion (root side) and the existing portion (tip side) of the hair were compared. A representative example is shown in Figure 1. Figure 1 shows an electron microscopic image of hair from the same patient.

[0055] As a result, in the treated hair, a rough cuticle was observed on the tip side, while the cuticle on the root side of the newly grown portion was neat, indicating a significant improvement in hair quality. In contrast, in the untreated hair of the negative subjects, a rough cuticle was observed on both the root and tip sides, and no improvement in quality was observed in the untreated portion. []

[0056] The same assessment was performed on a larger number of patients. The assessment was based on electron microscopy images and the following scoring system. The assessment was performed by seven physicians and other medical professionals. The average score was calculated.

[0057] <Hair Quality Assessment Scoring Sheet> 5 points: The stratum corneum is neat and there is no roughness. 4 points: The stratum corneum is relatively even compared to the average, but some roughness can be seen. 3 points: The uniformity of the stratum corneum is average. 2 points: Compared to the average, the stratum corneum is rough, and some peeling can be seen. 1 point: The stratum corneum is rough overall, and peeling is visible throughout.

[0058] The results are shown in Table 1.

[0059]

[0060] As shown in Table 1, the differences in scores between the tip and root of the same hair were calculated. The results showed that the scores for the root were higher on both the treated and untreated sides. Specifically, the average score of the root electroradiography on the treated side was 3.52, while the average score for the tip was 2.79, a statistically significant difference (p=0.017). More specifically, the degree of improvement (root score - tip score) was 0.73 on the treated side and 0.39 on the untreated side, indicating a greater degree of improvement on the treated side (n=4, 33–56 years old, 4 females, mean age 45.8 years). Furthermore, as shown in Table 1, the average score of the root on the treated side was 3.52, while the average score on the untreated side was 2.93, confirming a statistically significant difference in root scores between the treated and untreated sides (p=0.036). Therefore, it is clear that by applying breast milk, the hair quality is improved in the root area of ​​the treated side, that is, in the newly grown hair after treatment.

[0061] <Example 3: Changes in Hair Texture> In this embodiment, we focus on hair color (gray hair) and investigate the improvement effect before and after treatment. Specifically, the patient's inner corners of the eyes were extended to the sides of the head, and two points were tattooed with ink at the intersection of the lines connecting the ears and the top of the head. A 2cm area centered on these marks was then shaved. Three days after shaving, 20μL of human colostrum, as described in Example 1, was injected into one scalp. A total of 2mL was injected into 100 locations across half of the head, and observations were conducted one month later. Hair trichograms were taken at each point centered on the marks (Canon Power Shot A520, Tokyo, Japan). Hair within a 11mm diameter circle (95mm²) centered on the tattoo was photographed and visually measured. As a result, in the shaving section mentioned above, among the three patients with gray hair before treatment, all three experienced a reduction in gray hair 6 months after a single injection, with an average reduction of 7.7 gray hairs per 95mm² per photographed area (n=3). Furthermore, the number of gray hairs on the treated side decreased to 92.9% after treatment. The gray hair reduction rate when the untreated side's pre-treatment threshold was set to 100 was 28.2% lower than when the treated side's threshold was set to 100, indicating a significant reduction in gray hair on the treated side (n=3).

[0062] Figure 2A shows a representative example of a patient's (48-year-old female) hair six months after a single injection of colostrum following shaving. As shown in Figure 2A, no reduction in gray hair was observed in the non-injection area, while a significant reduction in gray hair was observed in the injection area. Specifically, in the non-injection area, the number of gray hairs was 49 before treatment and increased to 63 after 6 months; in contrast, in the injection area, the number decreased from 85 before treatment to 66.

[0063] Furthermore, the scalp of a 50-year-old male patient who received 20 μL of human colostrum as described in Example 1 four times at each scalp treatment site was compared with the untreated scalp 13 months after the start of treatment. The results are shown in Figure 2B. As shown in Figure 2B, the area of ​​gray hair on the untreated scalp was 833 cm², compared to 294 cm² on the treated scalp. Thus, it is clearly evident that colostrum administration reduces gray hair. In Figure 2B, a grid was overlaid on the photograph of the contralateral scalp, and squares that were visually white were marked with "○". It is clearly evident that the number of "○" marks was reduced on the treated side compared to the untreated side.

[0064] <Example 4: Verification of the scalp-improving effect> The changes in hair texture shown above have verified what kind of changes in the scalp they are based on.

[0065] As the milk, it is the human colostrum described in the usage examples. 20 μL of milk was injected into each scalp of the patient, for a total of 100 injections (6 patients per injection, 4 injections per patient per month).

[0066] The thickness of the subcutaneous fat layer of the scalp was observed after a single treatment using echocardiography with a 10 MHz probe. This examination generally measures the thickness of the area from the sebaceous glands down to the dermis (however, the dermis is not included in the measured values). The results are shown in Figure 3. In Figure 3, "#1~#6" represent data obtained 1 to 6 months after treatment. As shown in Figure 3, although a slight increase in fat layer thickness was observed 6 months after the start of treatment, no significant change was observed.

[0067] <Example 5: Histological Imaging of the Scalp> In the above embodiments, the effects of improving the stratum corneum or reducing gray hair can be confirmed. In this embodiment, in order to investigate whether these effects are caused by improvements in the scalp, the amount of collagen in the scalp was investigated.

[0068] 20 μL of milk was administered subcutaneously to each scalp of a human patient. This administration was performed monthly. The milk used was human colostrum as described in Example 1.

[0069] (1) Changes in collagen imaging First, the increase or decrease of type I and type III collagen was observed. Specifically, tissue sections of the scalp (from the epidermis towards the skull) were prepared, and type I and type III collagen were stained using the Picrosirius Red Stain Kit (Polysciences, Inc., Cat#: 24901-250) according to the manufacturer's instructions. Type I collagen stained yellow, and type III collagen stained green. In this system, collagen other than type III stained reddish-yellow.

[0070] The stained images were observed using a microscope (Nikon Corporation, OLYMPUS Corporation) with a polarizing filter, and photographed using a camera attached to the microscope (DP-22 OLYMPUS Corporation, DS-Fi3 Corporation, Nikon Instech). Digital images were created from grayscale and stored on a computer, and the amount of collagen was inferred from the brightness of each pixel. Figure 4A shows a scalp sample from a female patient (49 years old) 5 months after a single treatment, and Figure 4B shows a scalp sample from a male patient (50 years old) who received four monthly treatments 15 months after the initial treatment.

[0071] As shown in Figures 4A and 4B, the presence of type I and type III collagen can be observed on both the pre-treatment and untreated sides.

[0072] The number of green pixels (equivalent to the amount of type III collagen), yellow pixels (equivalent to the amount of type I collagen), and red-yellow pixels (equivalent to the total amount of collagen other than type III) in Figure 4A are summarized in the table.

[0073]

[0074] As shown in Table 2, in patients 5 months after a single administration, the proportion of type III collagen decreased from 49.6% to 19.6%, while the proportion of type I collagen increased from 30.1% to 62.3%. Although the total amount of collagen other than type III also increased from 50.4% to 80.4%, it is generally believed that this increase was mainly due to the increase in type I collagen.

[0075] The number of green pixels (equivalent to the amount of type III collagen), yellow pixels (equivalent to the amount of type I collagen), and red-yellow pixels (equivalent to the total amount of collagen other than type III) in Figure 4B are summarized in the table.

[0076]

[0077] Furthermore, as shown in Table 3, in patients 15 months after the start of treatment, comparing the untreated and treated sides, it was observed that type III collagen decreased significantly from 26.3% to 2.8%, while type I collagen increased from 24.7% to 56.3% (see Figure 4B). Although the total amount of collagen other than type III also increased from 73.7% to 97.2%, this increase is generally considered to be mainly due to the increase in type I collagen.

[0078] The same trend was observed in before-and-after comparisons of the treated side and in left-and-right comparisons of the untreated and treated sides, clearly demonstrating that breast milk administration decreased type III collagen and increased type I collagen. Furthermore, the same observation was performed on human samples (n=3), and the results showed that in all samples, breast milk administration resulted in a decrease in type III collagen and an increase in type I collagen. This result was also observed in collagen stained with red-yellow chromatograms (other than type III), showing the same trend as the increase in type I collagen stained with yellow chromatograms. The decrease in type III collagen and the increase in type I collagen are phenomena that occur during tissue repair, particularly wound healing. The results of this example suggest that the administration of breast milk induces tissue repair in the scalp and skin.

[0079] (2) Rejuvenation of fat cells Therefore, we observed the rejuvenation of adipocytes. As adipocytes grow, most of their cytoplasm transforms into lipid droplets, which accumulate within the cytoplasm, causing hypertrophy. Thus, larger cell lines represent mature, older cells, while smaller cells are considered young, new cells (i.e., young adipocytes). In this embodiment, we investigated whether the administration of breast milk increased the number of smaller, young cells. More specifically, we observed changes in the size of adipocytes in the fat layer by examining human scalp tissue sections that had been administered 20 μL of breast milk. Adipocytes are approximately circular in shape and are classified as S, M, or L based on their diameter. Cells with a diameter of 51 μm or less were designated "S," cells larger than 51 μm but smaller than 63 μm were designated "M," and cells larger than 63 μm were designated "L," and these were summed. The treatment was administered to patients who had received a single administration 6 months prior (hereinafter referred to as "Patient 1") and patients who received four monthly administrations for 15 months from the start of treatment (hereinafter referred to as "Patient 2"). The results are shown in Figure 5.

[0080] As shown in Figure 5, at 6 months post-treatment (Patient 1, a 51-year-old male) and 15 months post-treatment (Patient 2, a 50-year-old male), the proportion of adipocytes in the "S" region significantly increased with the administration of breast milk. Furthermore, comparing Patient 1 and Patient 2, the increase in the proportion of adipocytes in the "S" region was more significant in Patient 2. Therefore, it is clear that the increase in the proportion of adipocytes in the "S" region was greater in the group receiving four treatments than in the group receiving one treatment.

[0081] These results demonstrate that the administration of breast milk leads to an increase in young cells and tissue rejuvenation. Furthermore, they suggest that tissue rejuvenation may involve enhanced tissue repair capabilities.

[0082] <Example 6: Effect on wound healing> Therefore, in this embodiment, it was determined whether the administration of breast milk had the effect of accelerating the healing of skin injuries.

[0083] Induction of trauma: The backs of 12-week-old Wesleyan rats were shaved with clippers, and one circular full-thickness skin defect with a diameter of 8 mm was created on each side of the head and tail. Four full-thickness skin defects were created in each individual. Milk administration: 20 μL of milk was injected subcutaneously at four points (top, bottom, left, and right) 4 mm from the edge of the wound. The milk used was commercially available cow's milk that had been heat-sterilized (130 degrees for 2 seconds and 65 degrees for 30 minutes) as described in Example 1. Fatty acid administration: As fatty acids contained in the milk, a sterile purified water (administration solution) containing palmitic acid, stearic acid, myristic acid, linolenic acid, α-linolenic acid, and oleic acid was administered. Saturated fatty acids were heated, while unsaturated fatty acids were administered at room temperature. 20 μL of the administration solution (containing 1 mg of fatty acids) was administered subcutaneously at four points approximately 4 mm from the wound edge.

[0084] <Trauma Healing Rating> [] 5 points: Cured 4 points: The area of ​​the wound site was reduced by more than 70%. 3 points: The reduction rate of the wound area is more than 50% but less than 70%. 2 points: The reduction rate of the wound area is more than 20% but less than 50%. 1 point: The reduction rate of the wound area is less than 20%.

[0085] The results were obtained by dividing the above scores by the scores of physiological saline and expressing the results as a ratio. As shown in Figure 6A, the group given breast milk showed an effect of promoting wound healing.

[0086] Two 8mL bottles of commercially available low-temperature sterilized milk (65°C, 30 minutes) were centrifuged (2600g, 6 minutes). Two-quarters (2mL) of the supernatant (after removing foamy material) were collected from each bottle. These two portions were combined to make a total of 4mL. This 4mL was stirred and used in the experiment (hereinafter, the obtained sample is referred to as the "centrifuged sample"). 20μL of the obtained centrifuged sample was administered to each wound site in the same manner as described above. The results are shown in Figure 6A, "Centrifuged Sample".

[0087] As shown in Figure 6A, the centrifuged sample exhibited an effect on wound healing equal to or greater than that of breast milk. This suggests that the wound healing effect induced by breast milk is primarily due to the components contained within the milk that do not precipitate during centrifugation. Furthermore, it was shown that although natural wound healing also occurs after the administration of physiological saline, both the administration of breast milk and the centrifuged sample promoted healing compared to the natural healing following the administration of physiological saline (Figure 6A).

[0088] Secondly, the effect of lactoferrin on promoting wound healing was examined. Lactoferrin was purchased from Lion Pharmaceuticals (product name: Nice Rim Essence 62971), and administered at a dose of 20 μg or 60 μg using the same method as described above. Specifically, the lactoferrin was thoroughly dissolved in purified water after being pulverized. The lactoferrin concentration was adjusted to include the above amount in the 20 μL injection volume. The results were obtained by dividing the scores at 7 days and 10 days by the scores of the saline-treated group. The results are shown in Figure 6B. As shown in Figure 6B, lactoferrin did not show a significant promoting effect on wound healing. This suggests that components other than lactoferrin may have a promoting effect on wound healing.

[0089] In typical raw milk, lactoferrin (also known as "LF") is considered to contain approximately 3 mg per liter. 20 μL of raw milk should contain approximately 0.06 μg of lactoferrin. Furthermore, lactoferrin is heat-sensitive and is generally considered to have almost completely disappeared from the high-temperature sterilized milk used in the above examples (see Noro et al., Electrophoresis, 59:21-24, 2015). Therefore, while high-temperature sterilized milk showed a wound-healing-promoting effect, and centrifuged samples also showed a wound-healing-promoting effect, almost no wound-healing-promoting effect was observed with lactoferrin, consistent with the conclusion that lactoferrin is not an effective component.

[0090] The above embodiments clearly demonstrate that administering breast milk or the supernatant of breast milk promotes wound healing. Furthermore, the above embodiments suggest the possibility of activating skin tissue regeneration through the administration of breast milk or the supernatant of breast milk.

[0091] Secondly, the effective components in breast milk were identified. Specifically, the fatty acids contained in breast milk were applied to the wound as described above to investigate their effect on promoting wound healing. The results are shown in Figure 6C. As shown in Figure 6C, palmitic acid, stearic acid, and myristic acid clearly demonstrated a high promoting effect on wound healing. Therefore, it is clear that these fatty acids are likely beneficial in promoting wound healing through breast milk application. Furthermore, oleic acid, α-linolenic acid, and linolenic acid showed a weaker effect on promoting wound healing.

[0092] Furthermore, edible soybean oil (manufactured at Riken Agrochemical Co., Ltd., Fukuoka Plant), known to be rich in palmitic acid, stearic acid, linolenic acid, and α-linolenic acid, was applied to the wound to investigate its effect on promoting wound healing. The results are shown in Figure 6D. As shown in Figure 6D, soybean oil exhibited a high promoting effect on wound healing.

[0093] In addition, the representative fatty acids and their contents contained in 1g of ordinary milk or 1g of soybean oil are shown below.

[0094] <Example 7: Detection of tissue-normal M2-like macrophages and PPARγ-positive adipocytes in scalp tissue after milk administration> It has been reported that during tissue regeneration in peripheral tissues (particularly adipose tissue), an increase in tissue-normal M2-like macrophages (Satoh T. et al., Nature, 495: 524-528, 2013), the appearance of CD34-positive cells, or an increase in PPARγ-positive adipocytes (Festa E et al., Cell, 146: 761–71, 2011) occurs. Therefore, in this example, we investigated whether an increase in tissue-normal M2-like macrophages or PPARγ-positive cells occurred in scalp tissue after administration of 20 μL of milk. Furthermore, we confirmed the presence of Ki67-positive cells as an activity marker for cell proliferation in the dorsal skin of 12-week-old Wesleyan rats. Tissue-normal M2-like macrophage lines were detected as CD68-positive or CD163-positive cells. In this example, the milk used was human colostrum in humans and bovine colostrum in rats.

[0095] Tissue sections of the dorsal skin from multiple rats given milk (single administration) were fixed using standard methods for immunohistochemical staining. In rats, samples were taken at two locations: 5 weeks and 8 weeks after administration, at the administration site and at a location 10 mm distal to the administration site. The treated half of the head was considered the patient, while the remaining half was considered untreated. Sections with a diameter of 3 mm, encompassing the scalp surface of the patient down to the periosteum of the skull or the lateral head muscle membrane, were fixed. Furthermore, in this embodiment, the primary and secondary antibody systems used for staining are as follows.

[0096]

[0097] The results are shown in Figure 7. As shown in Figure 7, the number of Ki67-positive adipocytes and CD68-positive macrophages increased after 5 weeks of treatment and decreased after 8 weeks of treatment. In contrast, in distant sites, no positive cells were observed after 5 weeks of treatment, but Ki67-positive cells and CD68-positive macrophages became observable after 8 weeks of treatment. Therefore, it is clear that breast milk administration increases the number of Ki67-positive adipocytes, i.e., adipocytes become activated and proliferate, and also increases the number of macrophages within the tissue. Microscopic images generally suggest that Ki67-positive adipocytes are primarily adipocytes and adipose-derived stem cells. However, after 8 weeks of administration, the number of Ki67-positive adipocytes and macrophages decreased. Surprisingly, an increase in the number of Ki67-positive adipocytes and CD68-positive macrophages was observed later at distal sites than at the administration site. This suggests that the effects of breast milk administration spread throughout the tissue.

[0098] The same experiments were conducted on humans. In addition to CD31, human tissues were also stained with CD34, CD68, CD163, and PPARγ.

[0099]

[0100] Furthermore, Figure 8A shows CD31 and CD34 staining. 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, through breast milk administration, an increase in CD31-negative CD34-positive cells was observed at the administration site. As shown in Figure 8A, an increase in CD34-positive cells per unit area was observed before treatment and at 5 months after a single treatment (female, 49 years old). Furthermore, regarding CD34-positive cells, the overall average number in a 3mm punch section was 106.7 (n=3) on the untreated side, compared to an average of 129.7 (n=3) on the treated side. An increase in CD34-positive cells was observed on the treated side in all patients. Additionally, CD34-positive cells are known markers for adipose-derived stem cells, and an increase in CD34-positive cells suggests an increase in adipose-derived stem cells. Therefore, these results clearly indicate that the adipose tissue at the breast milk administration site is modified, leading to the production of adipose-derived stem cells.

[0101] The staining results for CD68-positive macrophages and PPARγ-positive cells are shown in Figure 8B. As shown in Figure 8B, in humans, administration of 20 μL of breast milk increased the number of CD68-positive macrophages, CD163-positive macrophages, and PPARγ-positive cells per unit area (female, 49 years old). Furthermore, in male patients (4 administrations, 50 years old) who had undergone treatment for 15 months, an increase in CD68-positive macrophages, CD163-positive macrophages, and PPARγ-positive cells per unit area induced by breast milk was also observed on the treated side. Thus, at the site of milk delivery, the tissue shows an increase in M2-like macrophages, indicating the involvement of young adipocytes belonging to PPARγ-positive cells. Additionally, in Figures 8A and 8B, the symbol "H" represents a hair follicle, the symbol "V" represents a blood vessel, and the symbol "S" represents a sebaceous gland.

[0102] <Example 8: An Examination of Tissue Changes Through Lactoferrin (High-Volume Forced Oral Intake)> As a comparative experiment, lactoferrin was administered instead of breast milk to confirm whether an increase in CD68-positive and PPARγ-positive cells occurred. Lactoferrin was administered orally to 12-week-old Wesleyan rats (n=3) using three tablets of Nice Rim Essence (Lion, 62971) and 300 mg of effective lactoferrin (human daily intake). Administration was once daily via oral feeding tube (forced ingestion). After 8 weeks of administration, paraffin-embedded sections of the dorsal skin were prepared using standard methods for immunohistochemical staining. The results are shown in Figure 9.

[0103] As shown in Figure 9, no increase in CD68-positive cells or PPARγ-positive cells was observed after lactoferrin administration. This clearly indicates that although lactoferrin administration emphasizes hair growth, unlike milk, it does not have a skin-modifying effect. Furthermore, as shown in Figure 9, when normal tissue (back) of 18-week-old Wesleyan rats was stained with anti-CD68 antibody and PPARγ antibody respectively, no significant difference was found between normal tissue and lactoferrin administration at week 8 (20 weeks old). It has been reported that lactoferrin inhibits adipocyte differentiation while suppressing PPARγ activity (see M. Yag, et al., Journal of Oral Science, 50: 419-425, 2008), and the above results are consistent with this report. It is generally believed that components other than lactoferrin in milk are the key factors contributing to the rejuvenation of adipocytes and the increase in adipocyte precursor cells induced by milk administration.

[0104] Example 9: Tissue changes and PPARγ activity induced by representative fatty acids contained in milk. Secondly, as in Example 1(2) above, representative fatty acids contained in milk were administered to multiple sites on the back skin of 12-week-old Wess rats, and the tissue changes induced by the administration were observed by immunohistochemical staining. More specifically, changes in CD68-positive macrophage staining and PPARγ-positive cell images were observed after administration of 20 μL / L of physiological saline and 20 μL / L of an administration solution containing 1 mg of fatty acid. Samples from Wess rats were taken from the administration sites (4 weeks and 8 weeks after administration). The results are shown in Figures 10A-10G. Figure 10A shows the results in the group administered physiological saline (negative control).

[0105] As shown in Figures 10B-10D, after 4 weeks of administration of palmitic acid, stearic acid, or myristic acid, an increase in PPARγ-positive cells or CD68-positive cells was observed at the administration site. Furthermore, even after 8 weeks of administration of all the aforementioned fatty acids, the increase in PPARγ-positive cells and CD68-positive cells at the administration site was maintained. Furthermore, infiltration of numerous CD68-positive cells was observed at the saturated fatty acid injection site, indicating fibrosis of normal tissue. Therefore, in Figures 10B-10D, changes in adipose tissue were observed at the point where normal tissue closest to the injection site was maintained (non-fibrotic area). As shown in Figures 10E-10G, after 4 weeks of administration of oleic acid, α-linolenic acid, or linolenic acid, no significant increase in PPARγ-positive or CD68-positive cells was observed compared to the control. An increase in PPARγ-positive cells was observed after 8 weeks of administration. On the other hand, no significant increase in CD68-positive cells was observed after administration of oleic acid, α-linolenic acid, or linolenic acid (overall, a weak effect was observed). CD68-positive cells differentiate into tissue-resident M2-like macrophages via PPARγ, promoting tissue homeostasis. Since an increase in CD68-positive and PPARγ-positive cells occurs after administration of saturated fatty acids such as palmitic acid, stearic acid, or myristic acid, it is generally believed that tissue-resident M2-like macrophages increase in tissues treated with fatty acids. On the other hand, while PPARγ-positive cells increased in unsaturated fatty acids, no significant increase in CD68-positive cells was observed, suggesting that the induction effect of tissue-resident M2-like macrophages is limited. Furthermore, this result is consistent with the following: in Example 6 above, saturated fatty acids exhibited a higher effect in promoting wound healing, while the effect of unsaturated fatty acids in promoting wound healing was limited. It has been reported that macrophage PPARγ activity is highly correlated with adipocyte function (see Odegaard JI, et al., Nature, 447:1116-1121, 2007). Therefore, the increase in CD68-positive cells and PPARγ-positive cells in adipose tissue induced by saturated fatty acid administration also supports the findings of the study on skin modification induced by saturated fatty acid administration. In addition, the increase in PPARγ-positive cells with intracellular lipid droplets confirms the increase in PPARγ-positive adipocytes, suggesting the activation of PPARγ-positive adipose stem cells induced by saturated fatty acid administration.

[0106] Palmitic acid, stearic acid, or myristic acid are saturated fatty acids, while oleic acid, α-linolenic acid, or linolenic acid are unsaturated fatty acids. The results in Figures 10B-10G show that saturated fatty acids induce PPARγ-positive cell expression (including young adipocytes) in tissues at an earlier stage, while unsaturated fatty acids induce PPARγ-positive cell expression (in young adipocytes) at a relatively later stage. Therefore, it is generally believed that saturated fatty acids induce stronger tissue inflammation and macrophage aggregation, but while inducing macrophage aggregation in tissues, they also increase the number of PPARγ-positive cells, suppressing the induced inflammation. On the other hand, it is generally believed that unsaturated fatty acids are less effective at inducing macrophage aggregation in tissues, and that PPARγ-induced anti-inflammatory effects are superior.

[0107] It is known that fatty acids promote the secretion of inflammatory cytokines (see Hotamisligil GS, Nature, 542:177-185, 2017). In the results of this example, saturated fatty acids induced a strong inflammatory response, but on the other hand, the inflammation was reduced by the increase of PPARγ-positive cells, promoting tissue regeneration. This suggests the possibility that the administration of saturated fatty acids can induce the formation of adipose-derived stem cells or young adipocytes, and induce the formation of M2 macrophages, thereby triggering a wound healing response and tissue regeneration. In addition, it can be confirmed that the expression of PPARγ and its effect on macrophages can induce the activation of immune cells (especially the induction of M1 macrophages into M2 macrophages) (see Crodell A. et al., PPAR Research, ID 549691, 2015).

[0108] <Example 10: The Relationship Between Scalp Improvement, Hair Quality Improvement, and Hair Growth Enhancement> According to Examples 1-9 above, it is clear that applying breast milk can cause scalp or skin regeneration and improvement, thereby producing the effect of improving hair quality. In this embodiment, based on the results of the above embodiments, the effect of milk administration is evaluated focusing on hair with a faster growth rate. Specifically, hairs that have grown by more than 0.9 mm in 3 days are extracted from scalp images, and the total length of growth is calculated. In addition, it is known that in adult hair, growth occurs at a rate of approximately 10 mm per month (i.e., approximately 1 mm per 3 days). For this purpose, a portion of the scalp of several adults (6 treated with a single dose and 1 treated monthly for 4 months) who received 20 μL / L of breast milk were shaved. Images of the shaved area were taken 3 days later for image analysis. Furthermore, the same area was shaved again 1 year after treatment, and images of the shaved area were taken 3 days later for image analysis. On average, the hairs were 0.4 mm long after shaving. The total length increase was calculated for hairs that were 1.4 mm or longer 3 days after shaving.

[0109] The results of the patient's hair length count, obtained by using image analysis software, are shown in Figures 11A and 11B. Here, as a representative measurement example, the treatment progress of patient A (50 years old / male, treated once a month for 4 months, 1 year after the start of treatment) is compared and the results are shown in Figure 11A. The results of the comparison between the untreated side and the treated side of two patients (patient A and patient B (47 years old / female, treated only once, 1 year after treatment)) are shown in Figure 11B.

[0110] Figure 11A compares the measurement results of patient A before treatment and one year after treatment began. Figure 10A shows the total length of hairs with a length of 1.4 mm or more three days after shaving (≥1.4 mm), and the total length of all hairs including those with a length of 0.9 mm or more but less than 1.4 mm three days after shaving (all hairs). In patient A, the total length of hairs with a length of 1.4 mm or more three days after treatment was 311.9 before treatment and 337.9 one year after treatment began. The total length of all hairs three days after shaving was 405.7 before treatment and 419.2 one year after treatment began.

[0111] Figure 11B compares the measurement results of the untreated and treated sides of patients A and B one year after the start of treatment. Figure 10B shows the total length of hairs with a length of 1.4 mm or more three days after shaving (≥1.4 mm), and the total length of all hairs including those with a length of 0.9 mm or more but less than 1.4 mm three days after shaving (total). In patient A, the total length of hairs with a length of 1.4 mm or more three days after shaving is 199.4 on the untreated side and 337.9 on the treated side. In patient B, the total length of hairs with a length of 1.4 mm or more three days after shaving is 114.3 on the untreated side and 300.7 on the treated side. Similarly, in patient A, the total length of hairs longer than 0.9 mm three days after shaving was 357.8 on the untreated side and 419.2 on the treated side. In patient B, the total length of all hairs three days after shaving was 261.5 on the untreated side and 378.1 on the treated side.

[0112] A negative control was prepared by injecting physiological saline solution into one side of the scalp monthly for six months. Changes in hair length (number of hairs longer than 1.4 mm) induced by monthly injections of physiological saline solution into one side of the scalp were observed. The change rate before treatment and at the sixth month of treatment ranged from -0.84% ​​to +1.24%, with an average of 1.03%. Furthermore, between the treated and untreated sides, the change at the sixth month of treatment ranged from -0.99% to +1.25%, with an average of 1.06% (n=4). Changes in hair length (total length of hairs longer than 1.4 mm) induced by monthly injections of physiological saline solution into one side of the scalp monthly for six months were also observed. The change before treatment and at the sixth month of treatment ranged from -0.79% to +1.33%, with an average of 1.05%. Furthermore, between the treated and untreated sides, the change at the sixth month of treatment ranged from -0.95% to +1.28%, with an average of 1.07% (n=4). Thus, as a negative control, no significant increase in hairs larger than 1.4 mm was observed in the group given physiological saline on the 3rd day after shaving.

[0113] Thus, it is clearly known that administration of breast milk (20 μL / L) can show scalp improvement and the resulting increase in hair elongation rate.

[0114] Furthermore, if hairs longer than 1.4 mm were examined 3 days later, image analysis clearly showed that they were thicker than hairs with slower growth rates (hair shorter than 1.4 mm after 3 days). Specifically, the average thickness of hairs 2.1–2.2 mm long 3 days after shaving was 97.42 μm, compared to an average thickness of 75.39 μm for hairs shorter than 1.4 mm. These results clearly demonstrate that the administration of breast milk (20 μL / L) resulted in faster hair growth and an increase in thicker hairs. During milk administration, an improvement in the tightness or resilience of the hair can be observed (an increase in the tightness or resilience of the hair, and an increase in the amount of hair), which is consistent with the result of an increase in coarser hair. Thus, an increase in hair can be observed in subjects who have been given milk, and the scalp modification caused by milk administration involves an improvement in hair elongation rate and an increase in thicker, more resilient, and firmer quality hair.

[0115] <Example A11: Bone Formation Promotion Effect> In this embodiment, the effect of palmitic acid administration on bone formation was confirmed.

[0116] Creation of the bone defect: The heads of 12-week-old (4 individuals) and 18-week-old (1 individual) male Wess rats were shaved with clippers. A diagonal tangent was drawn above the eyes and in front of the opposite ear, and an 8.8 mm diameter skull defect was created behind the intersection of these tangents. The procedure was performed sequentially: 1. General anesthesia; 2. Skin incision; 3. Periosteal stripping; 4. Skull bone removal (8.8 mm diameter). The skull defect was created by slowly cutting away the surrounding bone using a trephine bur and a microdrill, and finally, the bone was lifted to create the defect. Observation of bone formation: A hydroxyapatite film, prepared to match the size of the bone defect, was loaded onto the bone defect. At this time, 0.02 mg (20 μg) of palmitic acid was coated onto the film. Specifically, 0.02 mL (20 μL) of the mixture obtained by dissolving 1 mg in 1 mL was dropped onto the film, dried, and then loaded onto the bone defect.

[0117] Eight weeks after rearing, the skulls were slaughtered and perfused for fixation after tissue collection. Cross-sections of the skulls were stained with hematoxylin and eosin for microscopic observation. The area of ​​visible bone formation was then compared between the use of a palmitic acid-coated film and an uncoated film. ImageJ was used for analysis.

[0118] The 3D-CT images of the skull were obtained using the following system. Device name: Comscantecno, manufacturer: ScanXmate-E090 Filming conditions: X-ray tube voltage 88kV X-ray tube current 89μA Resolution 30.649μm / pixel The three-dimensional construction system was implemented using VGSTUDIO MAX (VOLUME GRAPHICS).

[0119] The results are shown in Figure 12. As shown in Figure 12, when using a film coated with palmitic acid, the skull is almost completely closed due to the formation of new bone. In contrast, when using an uncoated film, the formation of new bone is not sufficient.

[0120] Secondly, the sections stained with hematoxylin and eosin are shown in Figure 13. As shown in Figure 13, when a palmitic acid-coated film is placed on the tissue section, a large amount of red-stained bone (refer to the dotted line) is formed. In contrast, when a film is placed on the tissue section, less red-stained bone (refer to the dotted line) is formed. If the stained area (pixels and mm²) of the bone formation in the tissue section is compared, it is 12,132 pixels (0.984 mm²) in the palmitic acid-coated film, compared to only 1,025 pixels (0.083 mm²) in the control. The formed bone is significantly thicker in the palmitic acid-coated film than in the control film.

[0121] Therefore, it is clearly known that palmitic acid has a bone formation promoting effect. Bone tissue sections (3 mm in diameter) were collected at slaughter. After 8 weeks of feeding, tissue was collected and perfused for fixation. Bone tissue was collected using a bone bur (3 mm in diameter) when bone formation was insufficient. For specimens (rats) with insufficient bone formation, bone forceps (Luer forceps) were used to collect existing bone and regenerated bone from the open bone margin.

[0122] Tissue slides were immersed in RNA Stabilization Solution (Thermo Fisher Scientific, Lithuania), frozen in liquid nitrogen, and stored at -80°C. For the purification and extraction of total RNA from the stored tissue slides, 25 mg of tissue slides, along with five stainless steel beads (3 mm in diameter), were sealed in a sample tube. The tissue slides were homogenized using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) and TissueLyser II (QIAGEN GmbH, Hilden, Germany), and then the experimental procedures included in the kit were performed using a spin column. Gene expression analysis via quantitative PCR involved designing and synthesizing gene-specific primers and probes, outsourced to Integrated DNA Technologies, Inc. (Skokie, IL, USA). PrimeTime Gene Expression Master Mix reagent (Integrated DNA Technologies, Inc., Skokie, IL, USA) and a Rotor-Gene QPCR device (QIAGEN GmbH, Hilden, Germany) were used. Two PCR analyses were performed on each gene from each sample to determine the expression level of the target gene. Information on the seven rat genes used in the gene expression analysis via quantitative PCR (qPCR) and the base sequences of the synthesized probes and primers are as follows.

[0123] The difference in expression levels (Ct values) between Ribosomal protein lateral stalk subunit P0 (RplP0), belonging to Housekeeping Genes, and the target gene mRNA was compared (average of two tests, one individual at 12 weeks of age and one at 18 weeks of age) with the difference in expression levels between RplP0 and the target gene mRNA in the control group (12 weeks of age, without palmitic acid administration). These differences were converted into gene expression ratios (fold change), and the fold change was further log2 converted and plotted on a graph (see Figure 14).

[0124] As shown in Figure 14, compared to simply embedding a membrane, an increase in BMP4 and BMP7, which are important for bone formation, can be observed.

[0125] <Example A12: Changes in gene expression in tissues after subcutaneous administration> Secondly, changes in gene expression within the tissue were identified. Three regions were defined on the back of 12-week-old male Weiss rats, from head to tail. Each region was further divided into two subregions along the left and right sides of the spine, resulting in a total of six regions on the back. Two injections of 20 μL of latex or palmitic acid were administered near the center of each region. The distance between the two injection sites was set at 1 cm. Tissue slides were frozen in liquid nitrogen and stored at -80°C. For total RNA extraction from the stored tissue slides, 25 mg of tissue slides, along with five stainless steel beads (3 mm in diameter), were sealed in sample tubes. The slides were homogenized using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany) and TissueLyser II (QIAGEN GmbH, Hilden, Germany), and then performed using a rotary column according to the included experimental procedure. For gene expression analysis via quantitative PCR, the design and synthesis of gene-specific primers and probes were outsourced to Integrated DNA Technologies, Inc. (Skokie, IL, USA). PrimeTime Gene Expression Master Mix reagent (Integrated DNA Technologies, Inc., Skokie, IL, USA) and a Rotor-Gene Q PCR device (QIAGEN GmbH, Hilden, Germany) were used. Two PCR analyses were performed on each sample, one gene, to determine the expression level of the target gene. The difference in expression level (Ct value) between Ribosomal protein lateral stalk subunit P0 (RplP0) belonging to Housekeeping Genes and the target gene mRNA (average of 3 individuals in 2 tests for each specimen) was compared with the difference in expression level between RplP0 and the target gene mRNA in the control group (without palmitic acid or milk). These differences were converted into gene expression ratios (fold change), and the fold change was further log2 converted and displayed on a graph.

[0126] The results are shown in Figure 15. As shown in Figure 15, it is clear that the expression levels of Cxcl12, Cxcr4, and Itga4 in tissues were significantly increased during palmitic acid and milk administration. Itga4 and Cxcr4 are markers visible in bone marrow cells, and their expression in tissues indicates the recruitment of bone marrow-derived cells. The expression of Cxcl12 in tissues also indicates that the mechanism for receiving these bone marrow-derived cells has been simultaneously perfected. Furthermore, as shown in Figure 15, an increase in CD34 is also observed. The possibility of adipocyte induction in the presence of bone marrow precursor cells and macrophages can be considered.

[0127] <List of References>

[0128]

Claims

1. Use of a saturated fatty acid or a pharmaceutically permissible salt thereof for the preparation of a pharmaceutical composition coated on a biocompatible membrane, the pharmaceutical composition being used to increase M2-like macrophages, promote bone formation, and regenerate new bone by administration to bone defects, the saturated fatty acid comprising palmitic acid or stearic acid.

2. Use of a soybean oil or emulsion or such extract or processed product for preparing a pharmaceutical composition coated on a biocompatible membrane, the pharmaceutical composition being used to increase M2-like macrophages, promote bone formation, and regenerate new bone by administration to bone defects, the soybean oil or emulsion or such extract or processed product comprising palmitic acid or stearic acid.

3. As claimed in claim 1 or 2, wherein the aforementioned pharmaceutical composition further comprises unsaturated fatty acids.

4. As requested in item 2, wherein the milk is sterilized.

5. As requested in item 2, wherein the milk is sterilized at high temperature.

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