JP2010006734A - Skin care preparation for external use comprising growth factor, and bioproduct and its preservation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preservation method permitting preservation for a long period of time of a growth factor, which is an effective ingredient in the cosmetic field, the biological field and the pharmaceutical field, as a skin care preparation for external use, a biological preparation or a pharmaceutical. <P>SOLUTION: The growth factor, which is an effective ingredient in the cosmetic field, the biological field and the pharmaceutical field, for example, a fibroblast growth factor or an epithelial cell growth factor, is separated from an aqueous solution and is preserved in a dry solid state, and they are mixed when used. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a topical skin preparation or pharmaceutical preparation containing a growth factor in the cosmetics field, biotechnology field and pharmaceutical field, and a method for storing the same. More specifically, the present invention relates to a growth factor-containing external skin preparation or biopharmaceutical or pharmaceutical preparation and a storage method thereof, in which a growth factor and an aqueous solution are separated and stored and mixed during use.

Growth factors are also called growth factors or cell growth factors, and are a general term for endogenous proteins that promote proliferation and differentiation of specific cells in an animal body. It acts to regulate various cytological and physiological processes, and can act as a signal substance between cells by specifically binding to a receptor protein on the surface of a target cell.
Epidermal growth factor (EGF) is a protein that mainly promotes cell proliferation of epithelial cells. Cohen, who received the Nobel Prize in 1986, continued to study growth factors and injected newborn mice with components obtained from the mouse submandibular gland. It was found that the development of the plant occurred early. Cohen isolated this heat-stable protein from the mouse submandibular gland and confirmed the effect of this protein on the mouse microscopically. The effect on the eyelids and teeth was the result of proliferation of epithelial cells. Apparently, it was named EGF. That is, by using EGF as a cosmetic ingredient as well as a pharmaceutical or bio product, the epithelial layer of the skin is actively renewed and is expected to be modified to a youthful skin.

  On the other hand, fibroblast growth (proliferation) factor (FGF) is a protein that promotes cell proliferation of fibroblasts. That is, it is a protein consisting of 150 to 300 amino acids, and was originally purified from brain extracts. Two types of FGF were initially discovered and named as aFGF (acidic FGF or FGF-1 or FGF1) and bFGF (basic FGF or FGF-2 or FGF2). At present, 22 types of FGF have been identified in humans, including FGF-1, FGF-2, and FGF-7 (KGF). With the progress of functional analysis of these FGFs, FGF is not just a fibroblast growth factor, but a morphogenic factor having cell proliferation / differentiation activity against various cells, a tissue repair factor that works in the case of tissue damage, and living body homeostasis. It has been shown that it acts as a metabolic regulator to maintain sex and as an angiogenic factor that builds new blood vessels to send nutrients to cells. Therefore, FGF is attracting attention in the fields of developmental biology and regenerative medicine as a pharmaceutical and biopharmaceutical. Furthermore, as an application of FGF-1 as a cosmetic ingredient, it is expected to act on the dermis and the like to promote cell renewal and rejuvenate the skin.

FGF-7 is also called KGF, and KGF is also translated as keratinocyte growth factor, keratinocyte growth factor, and keratinocyte growth factor. KGF receptors are found in epithelial cells of many tissues such as tongue, oral cavity, mucous membrane, esophagus, stomach, intestine, salivary gland, lung, liver, pancreas, kidney, bladder, mammary gland, skin (hair follicle and sebaceous gland), and eye lens. It is known to exist and binds to this receptor tyrosine kinase to send a signal into the cell.
As an application of KGF pharmaceuticals, biopharmaceuticals, and cosmetic ingredients, hair growth has attracted attention. This is because KGF is produced from hair papilla cells and acts on the hair matrix cells, and is interpreted as causing hair growth by promoting proliferation and division of the hair matrix cells.

While the above cell growth factors are expected as ingredients for external preparations for skin, biopharmaceuticals and pharmaceuticals, the instability of growth factors has become a problem.
Growth factors are very unstable at room temperature, particularly in the presence of moisture (Patent Document 1). For example, it has been reported that the biological activity of an FGF-1 aqueous solution decreases to 0.1% of the initial value after 12 hours under the condition of 37 degrees (Non-patent Document 1). Further, it has been reported that the biological activity of an EGF aqueous solution decreases to 62% of the initial value after 48 days under the condition of 37 degrees (Patent Document 2).

Various methods have been developed so far to stabilize growth factors. As a method for stabilizing EGF, it has been reported that an EGF preparation is produced by containing a carboxyvinyl polymer, which is an acidic polymer, in an EGF aqueous solution, and is usefully used as an external skin preparation and cosmetics (Patent Literature). 1). Furthermore, there has been reported an attempt to maintain the EGF bioactivity in the EGF aqueous solution for a long period by containing cellulose derivatives such as hydroxypropylcellulose and methylcellulose in the EGF aqueous solution (Patent Document 2).
It has also been reported that a metal cation such as zinc is added to an EGF aqueous solution to prevent EGF biodegradation in the aqueous solution and stabilize EGF by ionic bond between EGF and zinc ( Patent Document 3).

  However, in any case, when the growth factor is stored in an aqueous solution, a decrease in biological activity over time cannot be avoided. Even if the stabilizer is contained in the EGF aqueous solution, the stability of the EGF aqueous solution is usually about 2 months when stored at 4 ° C. (Patent Document 1).

  As a method for stabilizing FGF-1, a genetically modified FGF-1 in which the type of amino acid is changed from one to several in the amino acid sequence of FGF-1 is used to stabilize FGF-1. Has been reported (Non-Patent Document 1 and Non-Patent Document 2). However, from the viewpoint of safety when used for humans, there remains room for doubt about the use of genetically modified FGF-1 as pharmaceuticals for humans, topical skin preparations, and cosmetics.

  It has been reported that the stability of FGF-1 increases when heparin is contained in the FGF-1 aqueous solution. In a normal FGF-1 aqueous solution, the biological activity of FGF-1 decreases to 0.1% of the initial value after 12 hours, but in the presence of heparin, it is around 20% even after 96 hours. It has been reported that the biological activity value is maintained (Non-patent Document 1). However, it has been difficult to keep the FGF-1 aqueous solution in a state where the activity is maintained for one month or longer.

If it is possible to maintain the biological activity of the growth factor in the long term, it will be possible to preserve the skin preparations and bioproducts containing the growth factor in the long term, and the growth factor is acting effectively. Develop skin preparations and bio products. Furthermore, the use of an external skin preparation that maintains EGF and FGF-1 activity for humans is expected to promote skin renewal, rejuvenation, and hair growth. In addition, the growth factor-containing cell culture medium can be stored for a long period of time, and it is expected to greatly contribute to stem cell research or clinical application of stem cells.
Japanese Patent Publication No. 2003-523399

U.S. Pat. No. 4,717,717

US Pat. No. 5,130,298

Zakrzewska M, Krowarsch D, Wiedlocha A, Olsnes S., Otlewski J., J. Mol. Bio., 352, 860-875 (2005)

Zakrzewska M, Krowarsch D, Wiedlocha A, Otlewski J., Protein Eng. Des. Sel., 17, 603-611 (2004)

It becomes possible to easily produce an external preparation for skin, a biopharmaceutical or a pharmaceutical containing a growth factor that can be stored for a long time. It is possible to provide a highly active growth factor-containing external skin preparation, biopharmaceutical or pharmaceutical product to society. The biologically active growth factor contributes to skin whitening, renewal and rejuvenation, anti-aging effect, and hair growth effect. Ideal for manufacturing and storage of pharmaceuticals and biopharmaceuticals.

It is intended to provide an external preparation for skin and a bioproduct containing a growth factor that can be stored for a long period of time by storing the growth factor in a solid state without dissolving it in an aqueous solution and mixing it in the aqueous solution at the time of use, and a method for storing the same .

  In view of the above points, the present inventors have conducted extensive research, and as a result, the growth factor is stored in a solid form without being dissolved in an aqueous solution, and mixed in the aqueous solution at the time of use. It was found that the activity can be maintained for a long time.

  Growth factors according to the present invention include (1) EGF, which is an epidermal growth factor, (2) fibroblast growth factors, FGF-1 (acidic FGF: aFGF), FGF-2 (basic FGF: bFGF), FGF-3 (int-2), FGF-4 (hst-1 / kaposi-FGF), FGF-5, FGF-6 (hst-2), FGF-7 (KGF), FGF-8, FGF-9, FGF-10 and other FGF, (3) vascular endothelial growth factor (VEGF), (4) hepatocyte growth factor (HGF), (5) platelet-derived growth factor (PDGF), and the like.

  When a growth factor is dissolved in an aqueous solution, the biological activity of the growth factor decreases with time. Therefore, even after purchasing a growth factor-containing skin preparation or biopharmaceutical, if stored for a certain period of time before use, the biological activity of the growth factor was usually significantly reduced at the time of use. . The best way to prevent this is to separate and store the growth factor and the aqueous solution in which it is dissolved before use, and to mix the growth factor and the aqueous solution for the first time during use. In this case, the solid of the growth factor is poured into one container and stored in a sealed state, and the aqueous solution is poured into another container, and (1) the aqueous solution is poured into the growth factor-containing container at the time of use. A method of injecting a solid growth factor into the aqueous solution-containing container is conceivable. Furthermore, it is also effective to isolate the growth factor and the aqueous solution in the same container using a film, break the film during use, and dissolve the growth factor in the aqueous solution.

Injecting a growth factor stabilizer into the above-mentioned growth factor-containing container, or in an aqueous solution, or in both containers is ideal for long-term storage of skin preparations and bioproducts containing additional growth factors It is. Possible stabilizers include (1) negatively charged polymers and polypeptides, (2) proteins, (3) sugars and polysaccharides, (4) hydrophilic polymers, (5) lipids, and the like. (1) As a negatively charged polymer and polypeptide, polyacrylic acid, carbomer, polyitaconic acid or a copolymer containing them, polyalginic acid, and the like are useful as growth factor stabilizers. (2) As proteins, albumin, lysozyme and the like are useful as growth factor stabilizers. 3) As sugars and polysaccharides, glucose, galactose, mannitol, sucrose, hyaluronic acid, heparin, heparan sulfate and the like are useful as growth factor stabilizers. It is effective to include the stabilizer in an amount of about 0.1 to 1000 times the weight of the growth factor.

      EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

  100 μg of epidermal growth factor (EGF, purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted in a dry state into a 100 ml glass tube with a lid. Furthermore, 50 ml of pure water (purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted into another glass tube with a lid. Six glass tubes each containing dry EGF and pure water were stored at 37 ° C. for 1 to 6 months. After each month, pure water stored in a glass tube was injected into a glass tube containing EGF, and then the EGF content was measured using a Quantikine EGF ELISA kit (R & D, U.S.A.).

  The results are shown in Table 1 (No. 1). In addition, the numerical value in Table 1 has shown the percentage compared with the initial concentration of EGF.

100 μg of epidermal growth factor (EGF, purchased from Sigma-Aldrich Japan Co., Ltd.) and 10 mg of mannitol were inserted into a 100 ml glass tube with a lid in a dry state. Furthermore, 50 ml of pure water (purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted into another glass tube with a lid. Six glass tubes each containing dry EGF and pure water were stored at 37 ° C. for 1 to 6 months. After each month, pure water stored in a glass tube was poured into a glass tube containing EGF, and then the EGF content was measured using a Quantikine EGF ELISA kit (R & D, USA).
The results are shown in Table 1 (No. 2). EGF activity can be maintained higher when stored in a solid state together with a stabilizer such as mannitol (No. 2 in Table 1) than when no mannitol is added (No. 1 in Table 1). Became clear.

100 μg of epidermal growth factor (EGF, purchased from Sigma Aldrich Japan) and 50 ml of pure water were inserted into a 100 ml glass tube with a lid. Six glass tubes containing EGF aqueous solutions were stored at 37 ° C. for 1 to 6 months. Every month, the EGF content in the EGF aqueous solution stored in the glass tube was measured using Quantikine EGF ELISA kit (R & D, USA).
The results are shown in Table 1 (No. 3). It was revealed that EGF activity can be maintained higher when stored in a solid state (No. 1 in Table 1) than when stored as an EGF aqueous solution.

100 μg of epidermal growth factor (EGF, purchased from Sigma-Aldrich Japan), 10 mg of mannitol and 50 ml of pure water were inserted into a 100 ml glass tube with a lid. Six glass tubes containing EGF aqueous solutions were stored at 37 ° C. for 1 to 6 months. Every month, the EGF content in the EGF aqueous solution stored in the glass tube was measured using Quantikine EGF ELISA kit (R & D, USA).
The results are shown in Table 1 (No. 4). It was clarified that EGF activity can be maintained higher when stored in a solid state (No. 2 in Table 1) than when stored as an EGF aqueous solution.

  100 μg of epidermal growth factor (EGF, purchased from Sigma-Aldrich Japan) and 10 mg of hyaluronic acid (purchased from Sigma-Aldrich Japan) were inserted in a dry state into a 100 ml glass tube with a lid. Furthermore, 50 ml of pure water (purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted into another glass tube with a lid. The dried EGF and 6 glass tubes containing pure water were each stored at 37 ° C. for 1 to 6 months. After each month, pure water stored in a glass tube was injected into a glass tube containing EGF, and then the EGF content was measured using a Quantikine EGF ELISA kit (R & D, U.S.A.). The results are shown in Table 1 (No. 5). When EGF is stored in a solid state together with a stabilizer such as hyaluronic acid (No. 5 in Table 1), EGF activity is higher than when no hyaluronic acid is added (No. 1 in Table 1). It became clear that it could be retained.

100 μg of fibroblast growth factor (FGF-1, purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted in a dry state into a 100 ml glass tube with a lid. Furthermore, 50 ml of pure water (purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted into another glass tube with a lid. The above-mentioned dried FGF and pure water-containing glass tubes were each stored at 25 ° C. for 6 months. Six months later, pure water stored in a glass tube was injected into a glass tube containing FGF-1, and the amount of FGF-1 bioactivity was determined in the literature (M. Zakrzewska et al., J. Nol. Bio., 352, 860 ( 2005)). The results are shown in Table 2 (No. 6). The FGF-1 bioactivity (ED50) measurement method is described below.
NIH 3T3 cells (ATCC) were cultured in DMEM medium containing 20% fetal calf serum to grow the cells. The cells were cultured for 24 hours in DMEM medium containing 2.5 μg / ml insulin and 2.5 μg / ml transferrin. Thereafter, the FGF-1 aqueous solution described above diluted to various concentrations was added to the medium, and the cells were further cultured for 12 hours. The cells were detached from the flask by trypsin treatment, and after nuclear staining using propylene iodide, the amount of DNA was measured by flow cytometry measurement. The percentage of the number of cells corresponding to S phase, M phase, and G2 phase (FGF-1 biological activity) was measured for each FGF-1 concentration. A similar experiment was conducted using fresh FGF-1 stored frozen. From the above, the concentration of FGF aqueous solution (ED50) showing 50% of the FGF bioactivity measured at a concentration of 100 ng / ml FGF-1 (fresh refrigerated FGF-1) in the medium was determined for each FGF. -1 Measurement was performed on an aqueous solution.

100 μg of fibroblast growth factor (FGF-1, purchased from Sigma-Aldrich Japan) and 50 ml of pure water were inserted into a 100 ml glass tube with a lid. Six glass tubes each containing this FGF-1 aqueous solution were stored at 25 ° C. for 6 months. Six months later, a method for measuring FGF bioactivity (ED50) in an FGF aqueous solution stored in a glass tube was performed.
The results are shown in Table 2 (No. 7). It was revealed that FGF-1 activity can be maintained higher when FGF is stored in a solid state (No. 6 in Table 1) than when stored as an FGF-1 aqueous solution.

  100 μg of fibroblast growth factor (FGF-1, purchased from Sigma-Aldrich Japan Co., Ltd.) and 10 mg of hyaluronic acid (purchased from Sigma-Aldrich Japan Co., Ltd.) are inserted into a 100 ml glass tube with a lid in a dry state. did. Furthermore, 50 ml of pure water (purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted into another glass tube with a lid. The above-mentioned dried FGF-1 and 6 glass tubes containing pure water were each stored at 25 ° C. for 6 months. Six months later, the FGF-1 bioactivity (ED50) measurement method in the FGF-1 aqueous solution stored in the glass tube was performed. The results are shown in Table 2 (No. 8). It is clear that FGF-1 can be maintained at a higher level when FGF-1 is stored in a solid state together with an FGF-1 stabilizer such as hyaluronic acid than when no stabilizer is added (No. 6). became.

  100 μg of fibroblast growth factor (FGF-1, purchased from Sigma-Aldrich Japan) and 2 mg of heparin sodium (purchased from Sigma-Aldrich Japan) are inserted into a 100 ml lidded glass tube in a dry state. did. Furthermore, 50 ml of pure water (purchased from Sigma-Aldrich Japan Co., Ltd.) was inserted into another glass tube with a lid. The dried FGF-1 and 6 glass tubes containing pure water were each stored at 25 ° C. for 6 months. Six months later, a method for measuring the amount of FGF bioactivity (ED50) in an FGF aqueous solution stored in a glass tube was performed. The results are shown in Table 2 (No. 9). It has been clarified that FGF-1 can be maintained at a higher level when FGF-1 is stored in a solid state together with heparin which is a stabilizer, compared to the case where no stabilizer is added (No. 6 in Table 2). It was.

  As shown in FIG. 1, it is separated into two layers with a film in one bottle, 20 ml of pure water is inserted into the A layer, and epithelial cell growth factor (EGF, Sigma-Aldrich Japan Co., Ltd.) is inserted into the B layer. Purchased) and 10 mg of mannitol was inserted in a dry state. The dried EGF and 6 glass tubes containing pure water were each stored at 25 ° C. for 1 to 6 months. By depressing the piston indicated by C in FIG. 1 every one month, pure water injected into the glass tube and EGF were mixed. After stirring well by hand, EGF content was measured using Quantikine EGF ELISA kit (R & D, U.S.A.). The results are shown in Table 1 (No. 10). It was revealed that EGF activity can be maintained higher when EGF is stored in a solid state than when it is stored in an aqueous solution (Table 1 No. 4).

  As shown in FIG. 1, the film is separated into two layers in one bottle, and 100 μg of fibroblast growth factor (FGF-1, purchased from Sigma-Aldrich Japan Co., Ltd.) is dried in the A layer. Inserted. Further, 20 ml of pure water was inserted into the B layer. The dried FGF-1 and 6 glass tubes containing pure water were each stored at 25 ° C. for 6 months. After 6 months, the piston indicated by C in FIG. 1 was pushed down to mix the pure water injected into the glass tube with FGF-1. After stirring well by hand, the amount of FGF-1 bioactivity (ED50) in the FGF-1 aqueous solution stored in the glass tube was measured. The results are shown in Table 2 (No. 11). It was clarified that FGF-1 activity can be maintained higher when FGF-1 is stored in a solid state than when it is stored in an aqueous solution (No. 7 in Table 2).

  As shown in FIG. 1, it is separated into two layers with a film in one bottle. In layer A, 100 μg of fibroblast growth factor (FGF-1, purchased from Sigma-Aldrich Japan) and 2 mg of heparin Sodium (purchased from Sigma-Aldrich Japan) was inserted in a dry state. Further, 20 ml of pure water was inserted into the B layer. The dried FGF-1 and 6 glass tubes containing pure water were each stored at 25 ° C. for 6 months. After 6 months, the piston indicated by C in FIG. 1 was pushed down to mix the pure water injected into the glass tube with FGF-1. After stirring well by hand, the amount of FGF-1 bioactivity (ED50) in the FGF-1 aqueous solution stored in the glass tube was measured. The results are shown in Table 2 (No. 12). It was revealed that FGF-1 activity can be maintained higher when FGF-1 is stored in a solid state together with heparin, a stabilizer, as compared to the case where no stabilizer is added.

It is a figure which shows the 2 layer isolation | separation type growth factor preservation | save bottle which concerns on Example 10, 11, 12. FIG. In the figure, A and B contain an aqueous solution or a solid growth factor, and the aqueous phase and the solid growth factor are separated by a film D. By pushing down the piston C and damaging the film D, the aqueous solution layer and the solid growth factor can be mixed. In addition, after the film D is broken, the aqueous growth factor solution can be taken out from the tube E.

Claims (9)

A skin external preparation and a bio product produced by storing a solid growth factor in a container and injecting an aqueous solution into the container at the time of use. The skin external preparation and biopharmaceutical according to claim 1, wherein the growth factor is a fibroblast growth factor. The external preparation for skin and biopharmaceutical according to claim 1, wherein the growth factor is an epidermal growth factor. Skin external preparation is cosmetics, lotion, hair preparation, hair growth promoter, hair water, hair cream, nutrition cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, body lotion The skin external preparation according to claim 1, which is a body cream, body oil, body essence, makeup cup base, foundation, shampoo, rinse or body soap. The external preparation for skin and biopharmaceutical according to claim 1, wherein the aqueous solution contains a stabilizer for growth factor. The external preparation for skin and biopharmaceutical according to claim 1, wherein the growth factor stabilizer and a solid state growth factor are hermetically stored in a container. The skin external preparation according to claim 1, wherein the preparation is prepared by using a storage bottle in which a growth factor and an aqueous solution are separated from each other with a film, damaging the film during use, and mixing the growth factor and the aqueous solution. And preservation method of bio product The preservation method according to claim 7, wherein the growth factor is a fibroblast growth factor. The preservation method according to claim 7, wherein the growth factor is an epidermal growth factor.

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