JP5582516B2 - Fibroblast growth factor regulatory peptide - Google Patents

Description

  The present invention relates to a peptide having at least fibroblast growth factor 5 (hereinafter referred to as FGF5) -like activity, an FGF5-like drug containing the peptide as an active ingredient, an FGF5 activity promoter, a therapeutic agent for diseases based on FGF5 deficiency, The present invention relates to a hair growth inhibitor, a hair removal agent, a whitening agent, an sunscreen agent, or the like.

  It is known that various polypeptide growth factors such as fibroblast growth factor (hereinafter referred to as FGF) are expressed in skin tissue. In mice and humans, FGF is encoded by 22 different genes, and in particular, FGF5 has been reported to be involved in hair growth (see Non-Patent Document 1). Therefore, an FGF5 antagonist that inhibits the hair growth inhibitory action by FGF5 is expected as an inhibitor against hair loss (see Non-Patent Document 2), and a polypeptide comprising a part of FGF5 is known as an FGF5 antagonist (patent) References 1 and 2 and Non-Patent Reference 3).

On the other hand, as a physiological function of FGF5, in addition to an inhibitory effect on hair growth, a proliferating action of fibroblasts, a nutritional or functional regulating action of the cranial nervous system, a whitening action, an ultraviolet protective action and the like are known (Non-Patent Document 4). 5, Patent Documents 3 and 4), FGF5-like active substance or FGF5-like activity promoting substance has hair growth inhibitory action, hair loss action, and fibroblast proliferation action in addition to these actions. It is expected to be used for various drugs based on the brain or nervous system nutrition or function regulating action, whitening action and UV protection action. Under such circumstances, the present inventors have found that an FGF5 agonist having a partial amino acid sequence of FGF5 has a tyrosinase inhibitory action and is useful as a whitening agent (see Patent Document 5).
Japanese Patent Laid-Open No. 2002-293720 JP 2005-298371 A specification JP 09-316096 specification JP 2000-344796 specification JP 2007-99677 specification Hebert, JM., Rosenquist, T., Gotz, J. and Martin, GR., 1994.FGF-5 as a regulator of the hair growth cycle: Evidence from targeted and spontaneous mutations.Cell 78: 1017-1025. Suzuki, S., Ota Y., Ozawa K. and Imamura T. 2000. Dual-mode regulation of hair growth cycle by two Fgf-5 gene products. J. Invest. Dearmatol. 114: 456-463. Ito, C., Saitoh, Y., Fujita, Y., Yamazaki, Y., Imamura, T., Oka, S. and Suzuki, S. 2003. Decapeptide with fibroblast growth factor (FGF) -5 partial sequence inhibits hair growth suppressing activity of FGF-5. J. Cell. Physiol. 197: 272-283. Zhan, X., Bates, B., Hu, X., and Goldfarb, M., 1988. The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol. Cell Biol. 8: 3487-3495. Haub, O., Drucker, B., Goldfarb, M., 1990. Expression of the murine fibroblast growth factor-5 gene in the adult central nervous system.Proc. Natl. Acad. Sci. USA. 87: 8022-8026.

  An object of the present invention is to provide a novel FGF5 agonist having a structure different from that of conventional FGF5 agonist peptides as described in the above-mentioned patent documents, simple peptide synthesis, low cost, and excellent transdermal absorbability. The object is to find a peptide and to provide a pharmaceutical composition, quasi-drug composition or cosmetic useful as a hair growth inhibitor, a hair removal agent or a whitening agent using the peptide.

  In order to solve the above problems, the present inventors synthesized various peptides with reference to the amino acid sequence of FGF5, and examined the FGF5-like activity of these peptides in detail. The inventors have found a substance having an extremely low molecular weight and effective FGF5-like activity in these peptides, and further enhancing and promoting the activity of FGF5, thereby completing the present invention. That is, the present invention is as follows.

(1) The amino acid sequence has a sequence of Gly-Ser-Leu-Tyr, Ser-Leu-Tyr, Gly-Ser-Leu, Leu-Tyr, or Leu-Tyr-Phe-Arg, and at least fibroblasts A peptide having a cell growth factor 5-like activity or a salt thereof.
(2) The peptide or salt thereof according to (1), which is represented by the amino acid sequence according to any one of (I) to (V) below.
(I) Gly-Ser-Leu-Tyr
(II) Ser-Leu-Tyr
(III) Gly-Ser-Leu
(IV) Leu-Tyr
(V) Leu-Tyr-Phe-Arg
(3) A drug having fibroblast growth factor 5-like activity, comprising the peptide according to (1) or (2) as an active ingredient.
(4) A fibroblast growth factor 5 activity promoter comprising the peptide according to (1) or (2) as an active ingredient.
(5) A therapeutic agent for a disease caused by FGF5 deficiency, comprising the peptide according to (1) or (2) as an active ingredient.
(6) A hair growth inhibitor or hair removal agent comprising the peptide according to (1) or (2) as an active ingredient.
(7) A tyrosinase inhibitor comprising the peptide according to (1) or (2) as an active ingredient.
(8) A whitening or sunscreen agent comprising the peptide according to (1) or (2) as an active ingredient.

The peptide of the present invention has a very short sequence as an FGF5 agonist, has a very simple peptide synthesis, is inexpensive and has excellent transdermal absorbability. In particular, the peptide of the above amino acid sequence (IV) has a molecular weight of 1 / in comparison with the peptide consisting of 6 or 13 amino acids described in Patent Document 5, which has been conventionally known as an FGF5 agonist peptide. Very small, 3 or 1/6.
Therefore, according to the present invention, it is possible to provide a drug having a lower molecular weight and an inexpensive FGF5-like action or an FGF5-like activity promoting action, and the FGF5-like activity or the promoting action can be effectively utilized. As a skin external preparation, it can greatly contribute to the development of hair growth inhibitors and hair removal agents, tyrosinase inhibitors, whitening agents, sunscreen agents and the like. That is, the present invention provides a pharmaceutical composition, quasi-drug composition or cosmetic which contains an FGF5 agonist peptide as an active ingredient and is extremely useful as a skin external preparation such as a hair growth inhibitor, a hair removal agent, and a whitening agent. It is to provide.

The polypeptide of the present invention has the sequence described in the above (1) and is included in the present invention as long as it has at least FGF5-like cell proliferation activity. The length of the amino acid sequence is not particularly limited, but a peptide having 6 or less amino acids is desirable in terms of ease of synthesis and drug absorbability.
Specific examples of the peptide in the present invention include those having the following amino acid sequences.
(I) Gly-Ser-Leu-Tyr (SEQ ID NO: 1)
(II) Ser-Leu-Tyr
(III) Gly-Ser-Leu
(IV) Leu-Tyr
(V) Leu-Tyr-Phe-Arg (SEQ ID NO: 2)

These peptides of the present invention may have one or several amino acids at the N-terminus of each peptide. On the other hand, when one or several amino acids are added to the C-terminal, they often have an FGF5 inhibitory action (Japanese Patent Laid-Open No. 2005-298371).
The peptide of the present invention has at least an FGF5-like action and also has an action of further enhancing and promoting the activity of FGF5. Therefore, the peptide of the present invention has hair growth inhibitory action, fibroblast proliferation action, brain nervous system nutrition or function regulating action, hair growth inhibitory action, tyrosinase inhibitory action, whitening action, UV protection action, etc. Various symptoms and diseases caused by deficiency can be improved or treated.

  The peptide of the present invention can be synthesized very easily by a chemical method such as a solid phase method and a liquid phase method usually used for peptide synthesis. In addition, according to a biological technique such as normal enzyme treatment, a plant or animal-derived protein is hydrolyzed by enzyme treatment or the like, and a peptide having the sequence shown in (I) to (V) above is separated from the hydrolyzate. It can also be obtained by purification and screening using FGF5-like cell proliferation activity or FGF5-like cell proliferation activity promoting action as an index. Furthermore, plants and foods containing a large amount of proteolytic enzymes may contain them as peptides, and desired peptides can be separated and purified from these plants and foods.

  When the peptide of the present invention is used as a hair growth inhibitor, a hair removal agent, a whitening agent, or the like, one or more peptides selected from the above peptides or physiologically acceptable salts thereof are directly used in the form of, for example, an aqueous solution. It can also be used, and can also be produced and administered as a pharmaceutical form containing one or more of the above peptides as an active ingredient using one or more pharmaceutically acceptable formulation additives. . Examples of such a preparation form include external preparations such as creams, sprays, coatings, and patches, but may be administered directly to the affected area as injections, for example. Further, the polypeptide may be blended in shampoo or rinse, or may be prepared by encapsulating in a liposome, for example.

As will be described later, the peptide of the present invention has an activity of promoting FGF5-like cell growth. For example, FGFR1 (III) c, which is one of FGF receptors, is forcibly expressed on the cell surface by gene expression manipulation. When the Ba / F3 cell line (R1c / Ba / F3 cell), which is a mouse IL-3-dependent proB cell, was used, the FGF5-like cell proliferation activity under the conditions described in detail in Example 2 was determined based on the amino acid sequence ( 115% at the time of addition of 1 mM peptide of I), 156% of the peptide of amino acid sequence (II), 156% of the peptide of amino acid sequence (III), 153% of the peptide of amino acid sequence (IV), amino acid sequence (V ) Shows a high cell proliferation activity of 159%.
Furthermore, as a result of the simultaneous addition of FGF5 and these peptides, it has been confirmed that each peptide promotes cell proliferation activity by FGF5. In addition, none of the peptides acts on the cell proliferation of the cells by Interleukin-3, and the peptide of the present invention can serve as an FGF5 activity promoter having high specificity for FGF5.

  As described above, the peptide of the present invention has an FGF5-like cell proliferative activity, and further has an action of promoting the cell proliferative activity of FGF5, and is useful as a hair growth inhibitor, a hair removal agent, or a whitening agent. It provides external or cosmetic products. Hereinafter, examples will be shown to describe the present invention in more detail, but the present invention is not limited to these examples.

Example 1 (1) below shows the use of R1c / Ba / F3 cells having the FGF receptor (FGFR1 (III) c) on the cell surface, which was used to measure the cell proliferation activity of FGF5 of the polypeptide of the present invention. Preparation examples are shown.
In Example 1 (2), the peptide of the amino acid sequences (I) to (V) of the present invention shows FGF5-like cell growth activity alone against R1c / Ba / F3 cells grown by FGF5. It is. These peptides were also shown to further promote the growth of R1c / Ba / F3 cells by FGF5 and have an FGF5 activity promoting action.
In Example 2, the in vivo action of the peptide of the amino acid sequence (IV) of the present invention was tested by applying it to C3H / He mice in the resting period of the hair cycle. It is shown that there is an effect of.

[Example 1]
(1) R1c / Ba / F3 cells were prepared according to a method known per se described in Reference Document 1 below. That is, human FGFR-1 (III) c plasmid (reference document 2) was inserted into Ba / F3 cells by the electroporation method. Stable R1c / Ba transformed by subculture in RPMI medium containing 10% FBS, 500 μg / ml anti-antibiotic G-418 Sulfate (Promega), 10 ng / ml FGF1 and 10 μg / ml heparin / F3 cells were obtained.
Reference 1: Yoneda, A., Asada, M., Oda, Y., Suzuki, M. and Imamura, T., 2000. Engineering of an FGF-proteoglycan fusion protein with heparin-independent, mitogenic activity. Nat. Biotec 18 (6): 641-644.
Reference 2; Ornitz, DM., Xu, J., Colvin, JS., McEwen, DG., MacArthur, CA., Coulier, F., Gao, G. and Goldfarb, M., 1996. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271 (25): 15292-15297.

(2) Various concentrations of the peptides of the present invention ((I) to (I)) were added to a 96-well cell culture plate containing RPMI1640 medium (measuring medium) containing 10% FBS and 500 μg / ml Antibiotic G-418 Sulfate. (V)), Heparin (5 μg / ml), FGF5 (1.54 × 10 ⁻⁹ M: manufactured by Wako Pure Chemical Industries, Ltd.), and the above R1c / Ba / F3 cells suspended in the medium for measurement (5 per well) x 10 ⁴ pieces) was added and cultured in 100 μl of medium per well for 72 hours. The proliferative activity of R1c / Ba / F3 cells was determined by adding 5 μl of WST-8 (Wako Pure Chemical Industries, Ltd.) solution to each well and further culturing for 3 hours to develop a 450 nm color associated with the production of WST-8 formazan. Determined by measurement. The results are shown in FIGS. 1a and b and FIGS. 3a and b.

FIG. 1a shows the result of adding peptides (final concentration 1 mM) of the amino acid sequences (I) to (IV) of the present invention to the culture medium without adding FGF5. These peptides are all FGF5-like. It was shown to have cell proliferation activity. According to FIG. 1a, the FGF5-like cell growth activity is 115% when the peptide of amino acid sequence (I) is added and 100% of the peptide of amino acid sequence (II) when the cell growth activity without addition of peptide is 100%. It had a cell growth activity of 156%, the peptide of amino acid sequence (III) was 156%, and the peptide of amino acid sequence (IV) was 153%. Regarding the cell growth activity of each peptide, when the addition concentration of each peptide was lowered, the cell growth activity decreased depending on the addition concentration.
On the other hand, FIG. 1b is a graph showing the action of further promoting the cell proliferation activity by FGF5 (FGF5 activity promoting action) for the peptides (I) to (IV) of the present invention (final concentration 1 mM). According to FIG. 1b, the FGF5 activity-promoting action of the peptide of the present invention is as follows. For the peptide of amino acid sequence (IV), cell growth by adding peptide (IV) (1 mM) alone without adding FGF5 is 450 nm. Although the amount was 0.018, the amount of color development increased by 0.057 when FGF5 and the peptide were added, greatly exceeding the cell proliferation activity induced by the addition of the peptide alone. This result showed that the peptide had an FGF5 activity promoting action to enhance FGF5 activity. The activity enhancing / promoting action of FGF5 was shown for all the peptides (I) to (IV) of the present invention.

FIG. 3a shows the result of adding the peptide (final concentration 1 mM) of the amino acid sequence (V) of the present invention to the culture medium without adding FGF5, and this peptide has FGF5-like cell proliferation activity. Indicated. According to FIG. 3a, FGF5-like cell growth activity has 159% cell growth activity when peptide (V) (final concentration 1 mM) is added, assuming that cell growth activity when no peptide is added is 100%. It was. The cell proliferation activity of peptide (V) decreased depending on the addition concentration when the addition concentration of peptide was lowered.
On the other hand, FIG. 3b is a graph showing the action of further promoting cell proliferation activity by FGF5 (FGF5 activity promoting action) for the peptide of amino acid sequence (V) of the present invention (final concentrations 1 mM and 0.1 mM). According to FIG. 3b, the FGF5 activity promoting action of the peptide of the present invention is as follows. In the peptide of the amino acid sequence (V), FGF5 was not added, and the cell proliferation by addition of peptide (V) (1 mM) alone was 450 nm. Although the amount of color development increased by 0.029, the amount of color development increased by 0.061 when peptide (V) (1 mM) was added in the presence of FGF5, significantly exceeding the cell proliferation activity induced by addition of the peptide alone. When the addition concentration of the peptide was further reduced, the increase in color development amount was 0.009 when peptide (V) (final concentration 0.1 mM) was added in the presence of FGF5, and the FGF5 activity promoting action decreased depending on the addition concentration. . This result showed that the peptide had an FGF5 activity promoting action to enhance FGF5 activity.

(3) In order to examine the nonspecific cell growth activity promoting action of the peptide, the same procedure as in (2) above was used, except that a medium containing 50 pg / ml mouse IL-3 was used instead of FGF5 and heparin. The color development based on the promoting activity of R1c / Ba / F3 cell proliferation activity was measured. As a result, each of the peptides (I) to (V) hardly promoted the proliferation of R1c / Ba / F3 cells by IL-3 in a solution having a final concentration of 1 mM. From this, it was shown that all the peptides of amino acid sequences (I) to (V) are FGF5 activity promoting agents with high specificity.

[Example 2]
In order to see the in vivo effects of FGF5-like active substances, they were tested in C3H / He mice in the rest period of the hair cycle.
Peptide (IV) was dissolved in physiological phosphate buffer (PBS (-)) and the same amount of ethanol was added to prepare a 50% ethanol solution of peptide (IV). Thereafter, glycerol was added to 1% to prepare a test solution. The peptide (IV) solution thus obtained (containing 49.5% ethanol and 1% glycerol) was applied to the back skin of 7-week-old C3H / He male mice in the rest period of the hair cycle. The mice were anesthetized and the back hair was gently shaved with a clipper. After shaving, 12 mM peptide (IV) solution (containing 49.5% ethanol and 1% glycerol) as a test solution, 2% minoxidil solution as a positive control, water-ethanol-glycerol solution (49.5% water, 49.5% as a negative control) % Ethanol and 1% glycerol) and 200 μl each were applied to the back skin of 5 mice per group (day 0). Similarly, it was applied daily for 11 days (excluding the 5th and 6th days) and the hair growth on the back of the mice on the 7th, 10th, 14th, 17th, 22nd and 24th day was performed. The state was observed visually, and photography was performed, and a hair growth score was obtained. The hair growth score is 1) skin blackening: 1 point, 2) short hair level: 2 points, 3) normal hair level: 3 points, and the area of each hair growth state occupies the total shaved area The ratio was expressed in% and was calculated by the following formula. According to this calculation method, when the total shaved area is restored to the normal level, the score is 100 points.
Hair growth score = (ratio of skin darkening area (%) × 1 + (percentage of short hair area (%) × 2 + normal hair area (%) × 3) / 3)
As a result of observing the hair growth on the back of the mouse on the 7th day, 10th day, 14th day, 17th day, 22nd day and 24th day after the first application, as shown in FIG. 2, the peptide (IV) solution was applied. The group had a lower hair growth score compared to the negative control group (water-ethanol-glycerol solution (containing 49.5% water, 49.5% ethanol, 1% glycerol) application group), and hair growth was significantly suppressed.

Example 3
A hair shampoo was produced according to the following formulation.
(Prescription)
Component Weight%
1. 12 mM peptide (IV) solution 0.1
2. Sodium lauryl ether sulfate ethanol 20
3. Sodium lauryl sulfate 10
4. 1,3 Butylene glycol 1
5. Perfume appropriate amount 6. Purified water remaining (100 in total)
The prescription ingredients were heated to 80 ° C., mixed with stirring, then cooled with stirring to produce the hair shampoo of the present invention.

Example 4
A hair liquid was produced according to the following formulation.
(Prescription)
Component Weight%
1. 12 mM peptide (IV) solution 0.1
2. Ethanol 40
3. Glycerin 1
4). Perfume appropriate amount 5. Purified water remaining (100 in total)
After adding the said formulation component and stirring and dissolving, purified water was added and the hair liquid was manufactured.

Example 5
This example shows the formulation and production method of a hair cream using the peptide of the present invention.
A hair cream was prepared according to the following formulation.
(Prescription)
Component Weight%
1. 12 mM peptide (IV) solution 0.1
2. Liquid paraffin 40
3. Vaseline 1
4). Cetostearyl alcohol 1
5. Methyl polysiloxane 1
6). Methyl paraoxybenzoate 0.2
7). Propylene glycol 5
8). Perfume appropriate amount 9. Purified water remaining (100 in total)
The said prescription component was stirred and mixed and the hair cream of this invention was manufactured.

Graph (a) showing the results of measuring the FGF5-like R1c / Ba / F3 cell proliferation activity of peptides (1 mM) of amino acid sequences (I) to (IV) of the present invention, and R1c / Ba of FGF5 by each peptide It is a graph (b) which shows the result of having measured the effect | action which promotes / F3 cell proliferation activity. For the peptide of amino acid sequence (IV) of the present invention, in order to see the action in vivo, the results of the test applied to the back skin of C3H / He mice in the resting period of the hair cycle for 11 days are shown by the hair growth score. It is a graph. FGF5-like R1c / Ba / F3 cell proliferation activity of the peptide (1 mM) of the amino acid sequence (V) of the present invention is shown in graph (a), and R1c / Ba / of FGF5 by peptide (V). It is a graph (b) which shows the result of having measured the effect | action which promotes F3 cell proliferation activity.

Claims (5)

The peptide or its salt which has a fibroblast growth factor 5-like activity shown by the amino acid sequence as described in the following (I) or (V).
(I) Gly-Ser-Leu-Tyr
(V) Leu-Tyr-Phe-Arg Characterized in that it contains a peptide represented by the amino acid sequence described in the following (I) or (V) as an active ingredient, drug agents having fibroblast growth factor 5 like activity.
(I) Gly-Ser-Leu- Tyr
(V) Leu-Tyr-Phe-Arg An activity promoter for fibroblast growth factor 5, comprising a peptide represented by the amino acid sequence described in (V) below as an active ingredient .
(V) Leu-Tyr-Phe-Arg A therapeutic agent for a disease caused by FGF5 deficiency, comprising as an active ingredient a peptide represented by the amino acid sequence described in (I) or (V) below:
(I) Gly-Ser-Leu- Tyr
(V) Leu-Tyr-Phe-Arg A hair growth inhibitor or hair removal agent comprising a peptide represented by the amino acid sequence described in (IV) below as an active ingredient (except when applied to cosmetics).
(IV) Leu- Tyr

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