JP4742365B2 - Tyrosinase inhibitor, whitening agent and whitening topical agent - Google Patents

Description

  The present invention relates to a tyrosinase inhibitor, a whitening agent, and a whitening external preparation.

Among fibroblast growth factors, it has been reported that fibroblast growth factor 5 (hereinafter referred to as “FGF-5”) is involved in hair growth (non-patent literature). 1, Patent Document 1). In addition, FGF-5 antagonists such as fibroblast growth factor 5S (hereinafter referred to as “FGS-5S”) are known to inhibit the effect of suppressing hair growth by FGF-5, and are described as hair restorers. (Non-Patent Document 2). Furthermore, a polypeptide comprising a part of FGF-5, which inhibits the inhibitory action of FGF-5 on hair growth, has been reported as an FGF-5 antagonist (Patent Document 2, Non-Patent Document 3).
Thus, FGF-5 is known to be greatly involved in hair growth and inhibition thereof.

On the other hand, skin spots and buckwheat are activated by melanocytes due to irritation caused by exposure to ultraviolet rays of sunlight, and tyrosinase in melanocytes activates tyrosine to produce a large amount of melanin pigment in the skin, resulting in abnormal deposition. Caused by. Kojic acid and placenta extract are widely used for the prevention and treatment of such spots and freckles.
However, for cosmetics using kojic acid, carcinogenicity due to kojic acid has been reported, and at present, production and import of cosmetics using kojic acid have been discontinued. In addition, the placental extract still has problems in terms of safety.
That is, a whitening agent having a new type of whitening effect is desired.

JP 2002-293720 A JP 2000-344796 J. et al. Cell 78: 1017-1025. J. et al. Invest. Dearmatol. 114: 456-463. J. et al. Cell. Physiol. 197,272-283.

  An object of the present invention is to solve the above-mentioned problems, and to provide a tyrosinase inhibitor and the like.

As a result of intensive studies by the inventor under the above-mentioned problems, the inventors have found that peptides having a specific amino acid sequence derived from FGF-5 or FGF-5S have a tyrosinase inhibitory action, and have completed the present invention. Specifically, it was achieved by the following means.
(1) A tyrosinase inhibitor comprising a peptide comprising all or part of fibroblast growth factor 5 or fibroblast growth factor 5S, a modified peptide of the peptide having tyrosinase inhibitory activity, or a salt thereof .
(2) A tyrosinase inhibitor comprising a peptide comprising any of the following amino acid sequences, a modified peptide of the peptide having a tyrosinase inhibitory activity similar to that of the peptide, or a salt thereof.
(A) an amino acid sequence described in SEQ ID NO: 1 or 2; or (b) an amino acid sequence in which one to several amino acids are deleted, substituted and / or inserted in the amino acid sequence described in SEQ ID NO: 1 or 2. An amino acid sequence having tyrosinase inhibitory activity.
(3) A tyrosinase inhibitor comprising a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2, a modified peptide of the peptide having a tyrosinase inhibitory activity similar to that of the peptide, or a salt thereof.
(4) A tyrosinase inhibitor comprising a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, a modified peptide of the peptide having a tyrosinase inhibitory activity similar to that of the peptide, or a salt thereof.
(5) A whitening agent containing the tyrosinase inhibitor according to any one of (1) to (4).
(6) A whitening external preparation containing the tyrosinase inhibitor according to any one of (1) to (4).

The tyrosinase inhibitor of the present invention can inhibit the action of tyrosinase. Therefore, it has become possible to suppress the production of melanin pigments produced from tyrosine. For this reason, it became possible to use as a whitening agent which suppresses the production | generation of a melanin pigment. Specifically, it can be used as cosmetics, pharmaceuticals, and quasi drugs for the purpose of preventing or treating spots and freckles due to sunburn and the like.
Furthermore, since the peptide used in the present invention also has a hair removal effect, it can be used as a whitening agent that also has a hair removal effect.

  Hereinafter, the contents of the present invention will be described in detail. In the present specification, “to” is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.

The tyrosinase inhibitor in the present invention inhibits the synthesis of melanin pigment in melanocytes by inhibiting the action of tyrosinase.
The tyrosinase inhibitor of the present invention contains a specific peptide.
The peptide used in the present invention is, for example, a peptide consisting of all or part of FGF-5 or FGF-5S and having a tyrosinase inhibitory action. Here, the peptide referred to in the present invention refers to a peptide in which amino acids are peptide-bonded, and is intended to include polypeptides and proteins without departing from the spirit of the present invention.
The peptide used in the present invention is preferably a peptide containing any one of the following amino acid sequences, or a modified peptide of the peptide, which is a modified peptide having the same tyrosinase inhibitory activity as the peptide.
(A) amino acid sequence described in SEQ ID NO: 1 (Leu Ser Gln Val His Arg) or SEQ ID NO: 2 (Pro Asp Gly Lys Val Asn Gly Ser His Glu Ala Asn Met): or (b) described in SEQ ID NO: 1 or 2 An amino acid sequence in which 1 to several amino acids are deleted, substituted and / or inserted, and has tyrosinase inhibitory activity.
Here, the tyrosinase inhibitory activator of the present invention only needs to contain any one of the above amino acid sequences as a part of the amino acid sequence of the peptide used in the present invention. As long as it exhibits tyrosinase inhibitory activity, the above (a ) Or (b), an additional amino acid sequence may be added to the N-terminus and / or C-terminus of the amino acid sequence. However, as one embodiment of the present invention, a peptide comprising only the amino acid sequence described in the above (a) or (b) is provided.

  A modified peptide of the above-described peptide used in the present invention and having the same tyrosinase inhibitory activity as the above-described peptide used in the present invention is also within the scope of the present invention. In the modified peptides described above, the term “modification” should be interpreted in the broadest sense including chemical and biological modifications. Examples of modifications include functional group conversion such as alkylation, esterification, halogenation, or amination, oxidation, reduction, addition, or elimination, sugar compounds (monosaccharides, disaccharides, Oligosaccharides or polysaccharides) or lipid compounds and the like, phosphorylation, biotinylation, and the like, but are not limited thereto.

Further, the range of the above “1 to several” is not particularly limited, but for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably about 1 to 3. means.
In the peptide used in the present invention, the type of amino acid to be inserted or substituted is not particularly limited, but is preferably an L-amino acid. The number and position of amino acids to be inserted or substituted are not particularly limited.
Thus, in order for a peptide obtained by substitution with an amino acid to have the same tyrosinase inhibitory activity as the peptide before substitution, it may be preferable that the substituted amino acids are amino acids having similar properties to each other. . Specifically, substitution is made between amino acids belonging to hydroxy amino acids, amino acids belonging to aliphatic amino acids, amino acids belonging to acidic amino acids, and amino acids belonging to basic amino acids. Of course, other amino acids can be substituted as long as they have tyrosinase inhibitory activity.
The number of amino acid residues of the peptide used in the present invention is not particularly defined as long as it has tyrosinase inhibitory activity, but it is preferably 5 to 20, more preferably 6 to 15.

  Whether (b) a peptide comprising an amino acid sequence in which one to several amino acids are deleted, substituted and / or inserted in the amino acid sequence described in SEQ ID NO: 1 or 2, or a modified peptide has tyrosinase inhibitory activity Those skilled in the art can easily confirm by the test methods described in detail and specifically in the examples of the present specification, or by appropriately modifying or modifying the test methods.

  When the peptide used in the present invention is chemically synthesized, it can be synthesized by conventional means of peptide synthesis. Examples thereof include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. That is, the amino acid that can constitute the peptide used in the present invention is condensed with the remaining portion, and when the product has a protecting group, the protecting peptide can be removed to synthesize the target peptide. Any known method may be used as the condensation method or the removal of the protecting group.

After the reaction, the peptide used in the present invention can be purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. In the peptide used in the present invention, the C-terminus is usually a carboxyl group (COOH) or carboxylate (COO ⁻ ), but the C-terminus may be an amide (—CONH ₂ ) or an ester (—COOR). Furthermore, as a peptide used in the present invention, a peptide whose N-terminal amino group is protected with a protecting group, or a complex peptide such as a glycopeptide to which a sugar chain is bonded can be employed.

As another method for producing the peptide used in the present invention, a gene encoding the amino acid sequence of the peptide used in the present invention is used, and a recombinant protein ( Peptide). The obtained crude peptide can be purified using means commonly used for protein or peptide purification, such as gel filtration, reverse phase HPLC, or ion exchange column purification.
In particular, since the amino acid sequence of FGF-5 and the base sequence of the DNA encoding them are clear, they can be prepared using genetic recombination techniques based on these sequences. FGF-5 or FGF-5S is not particularly defined as long as it does not depart from the spirit of the present invention, and examples thereof include FGF-5 or FGF-5S derived from human, mouse and rat. Among these, human-derived FGF-5 or FGF-5S is preferable.

DNA encoding FGF-5 derived from human, mouse or rat has a known nucleotide sequence (Zhan X. et al., Mol. Cell. Biol 8: 3487-3495, 1988; Haubo. Et al., Proc. Natl). Acad. Sci. USA 87: 8022-8026, 1990; Hattori Y. et al., Biochim. Biophys. Acta 1306: 31-33, 1996), well known to those skilled in the art such as PCR or hybridization. The obtained gene can be obtained from a chromosomal DNA or cDNA library by a cloning method. FGF-5 is encoded by the FGF-5 gene.
On the other hand, DNA encoding human or mouse-derived FGF-5S can be obtained by PCR or hybridization based on a known base sequence (Ozawa K et al., J. Biel. Chem. 273: 29262-29271, 1998). The gene can be obtained from a chromosomal DNA or cDNA library by gene cloning methods well known to those skilled in the art. FGF-5S is encoded by the exon 1 and exon 3 genes of the FGF-5 gene.
More specifically, the peptide contained in the tyrosinase inhibitor of the present invention can be synthesized, for example, referring to the descriptions in paragraph numbers 0016 to 0018 of JP-A-2002-29372.

Since the tyrosinase inhibitor in the present invention inhibits the synthesis of melanin pigment, it can be used as a whitening agent. When used as a whitening agent, the tyrosinase inhibitor of the present invention may be administered, but it can be preferably administered as a whitening agent containing other components that can be produced by methods well known to those skilled in the art.
For example, it can be produced by adding pharmacologically and pharmaceutically acceptable additives. Specifically, excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizing agents or solubilizing agents, isotonic agents, pH adjusting agents, Stabilizers, propellants, and pressure-sensitive adhesives can be mentioned. Furthermore, you may mix | blend 1 type (s) or 2 or more types of other whitening agents with the whitening agent of this invention within the range which does not deviate from the meaning of this invention.
The dosage of the whitening agent of the present invention is not particularly limited, and can be appropriately selected according to the type of active ingredient, and further various weights that should normally be considered such as the user's weight, age, type, symptom, and administration route. Depending on the factor, it can be appropriately increased or decreased.
The whitening agent may be a whitening internal preparation or a whitening external preparation, but is preferably a whitening external preparation.

The whitening internal preparation may be administered as an injection, an infusion, or the like by intravenous injection or orally. In the case of a whitening internal preparation, it can be used in the range of, for example, 0.0001 to 10000 mg, preferably 0.001 to 1000 mg per adult day.
Specifically, it can be used as pharmaceuticals, quasi drugs, foods / beverages for the purpose of whitening. In this case, the amount of the tyrosinase inhibitor of the present invention is preferably contained in the range of 0.0000000001 to 10.0% by weight in terms of solid content with respect to the total amount of the whitening internal preparation. The content is more preferably within the range of 0.000000001 to 5.0% by weight, and even more preferably within the range of 0.00000001 to 1.0% by weight.

As a whitening external preparation, it can be used as a spray, a coating agent or a patch. When used as an external preparation, it can be used, for example, in the range of 0.0001 to 10000 mg, preferably 0.001 to 1000 mg per adult day.
Specifically, it can be used as pharmaceuticals, quasi drugs, cosmetics, and the like. In this case, the amount of the tyrosinase inhibitor of the present invention is preferably contained within the range of 0.0000000001 wt% to 10.0 wt% in terms of solid content with respect to the total amount of the whitening external preparation. The content is more preferably within the range of 0.000000001% to 5.0% by weight, and even more preferably within the range of 0.00001% to 1.0% by weight.

  In particular, the whitening agent of the present invention is preferably used in cosmetics. When using the whitening agent of this invention as cosmetics, the component used for normal cosmetics etc. can be mix | blended suitably as needed. Examples of such components include those described above, for example, water, ethanol, oily components, moisturizers, thickeners, preservatives, emulsifiers, medicinal components, powders, ultraviolet absorbers, fragrances, and emulsion stabilizers. Can be mentioned. Moreover, the dosage form can also be selected arbitrarily and a cream, an ointment, an emulsion, a lotion, a solution, a gel etc. are illustrated. It can also be in the form of a pack, powder, stick or the like.

  The present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.

Preparation of Recombinant FGF-5 Protein Recombinant FGF-5 protein (hereinafter referred to as “recombinant FGF-5 protein”) comprising the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing is described in the literature J. Biol. Chem. 273, 29262-29271.
Specifically, the brain was extracted from an ICR mouse 6-week-old female individual (manufactured by CLEA Japan), and RNA was extracted therefrom. A cDNA mixture was obtained from this RNA using M-MLV reverse transcriptase (Moloney Murine Leukemia Virus, manufactured by Invitrogen, 2825-013) using random hexaoligonucleotide as a primer. Next, the target gene was amplified from the obtained cDNA mixture by PCR reaction. As a result, a DNA fragment having a size corresponding to the recombinant FGF-5 protein was obtained. This was separated by gel electrophoresis and cut out from the gel. A plasmid was constructed by incorporating the obtained DNA fragment into its cloning site using pET-3 as a cloning vector. Using this plasmid as a vector, a transformant was obtained using Escherichia coli BL21 (DE3) pLysS strain (Calbiobiochem / Novabiochem Corporation, 69451-3) as a host. The transformant was inoculated into 40 ml of Luria-Bertani medium (LB medium), cultured at 37 ° C. for 16 hours, and then transplanted to a new LB medium and further cultured. As an inducer, 1 mM isopropyl thiogalactoside (IPTG) was added and further cultured for 3 hours. After culture, the cells were centrifuged at 6000 rpm for 15 minutes to obtain bacterial cells. The cells were taken in a test tube and sonicated for 2 minutes in an ice bath. Thereafter, the mixture was centrifuged at 12,000 rpm for 1 hour, and a supernatant containing the recombinant FGF-5 protein was collected.

Next, the obtained recombinant FGF-5 protein was purified as follows.
Heparin sepharose was suspended in 10 mM Tris-HCl buffer (pH 7.4) and washed with the same solution. To this suspension, NaCl was added to a final concentration of 0.5 M, and the supernatant containing the recombinant FGF-5 protein was added, followed by gentle stirring at 4 ° C. for 3 hours. After washing with 10 mM Tris-HCl buffer (pH 7.4), the column was packed, washed with 10 mM Tris-HCl buffer (pH 7.4) containing 0.7 M NaCl, and then containing 2 M NaCl as an eluent. The desired recombinant FGF-5 protein was eluted using 10 mM Tris-HCl buffer (pH 7.4). From the culture solution of E. coli 4L, 300 μg of a partially purified product of recombinant FGF-5 protein could be obtained. The purified sample was separated by SDS-PAGE, tested for purity, and stored at -80 ° C.

Tyrosinase inhibitory activity Mouse B16 melanoma cells (Dainippon Pharmaceutical Co., Ltd., 09-6322) were mixed with 10% by mass fetal bovine serum (FBS) (Cancer International, CC3008-504) and 0.5 mM theophylline (Wako Pure Chemical ( (Strain), 201-09931) and suspended in Dulbecco's modified Eagle medium (DMEM medium), 2 × 10 ³ cells per well were added to a 96-well cell culture plate, and a carbon dioxide incubator (5%, CO ₂ , 37 days). Two days later, the medium was replaced with a DMEM medium not containing theophylline, and the partially purified product of the recombinant FGF-5 protein was added and further cultured for 3 days. After culturing, the medium was removed by aspiration, and each well contained 1% Triton X-100 (Triton X-100) (ICN Biomedicals, 194854) (phosphate buffered saline (PBS) (- )) Was added, and the well was vigorously shaken, and then the absorbance at 475 nm was measured (the final concentration of the partially purified product of the recombinant FGF-5 protein in this sample was 1.5 × 10 ⁻⁹ M). ). After the measurement, 10 mM L-dihydroxyphenylalanine (L-DOPA) was added and reacted for 1 hour. Similarly, the absorbance at 475 nm was measured, and the tyrosinase inhibitory activity was quantified from the amount of change in absorbance (1).
The cytotoxicity of the partially purified product of the recombinant FGF-5 protein to melanoma cells was determined by adding WST-8 solution (Wako Pure Chemical Industries, Ltd.) to each well after adding the partially purified product of the recombinant FGF-5 protein and culturing for 3 days. 5 μl was added, and the mixture was further cultured for 3 hours, and the color development at 450 nm associated with the production amount of WST-8 formazan was measured (2).
The tyrosinase inhibitory activity of the partially purified product of the recombinant FGF-5 protein is divided by the cytotoxicity value of the test sample obtained by the measurement of (2) with respect to the tyrosinase inhibitory activity obtained by the measurement of (1). Was calculated by correcting the cytotoxicity of the test sample.

Peptide 1 and peptide 2 shown in SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized by a chemical synthesis method using a peptide synthesizer and purified by high performance liquid chromatography. The amino acid sequence of the peptide was analyzed with a protein sequencer to obtain a preparation with a final purity of 80% or more.
The tyrosinase activity was similarly measured for these peptides and kojic acid (Sigma, K3125-5G). The final concentrations in these samples for measuring tyrosinase inhibitory activity were 1 mM, 1 mM, and 4.4 mM in the order of peptide 1, peptide 2, and kojic acid.

The results of tyrosinase inhibitory activity are shown in FIG. As is clear from FIG. 1, the partially purified product of the recombinant FGF-5 protein (denoted as FGF-5 in FIG. 1) was 26% despite being measured at a concentration of 1.5 × 10 ⁻⁹ M. Inhibitory activity was shown. In addition, peptide 1 measured at a concentration of 1 mM showed 75% inhibitory activity in peptide 2 measured at a concentration of 1% and 75%, respectively.
On the other hand, kojic acid measured at a concentration of 4.4 mM inhibited 53% activity.
That is, it has been clarified that the peptide of the present invention exhibits high tyrosinase inhibitory activity even at a significantly lower concentration than kojic acid.

Measurement of DNA synthesis promoting activity Balb / c3T3 cells (American Type Culture Collection (ATCC), CCL-163) ⁴ × 10 ⁴ cells / 500 μl were seeded in a 48-well culture plate, and in the presence of 10% by mass fetal bovine serum (FBS), The cells were cultured in DMEM medium for 24 hours. Next, after washing the cells with the same medium, a DMEM medium containing 0.3% by mass of activated charcoal-treated bovine serum (CS) was added and cultured for another 48 hours to synchronize the cells. After adding heparin 5 μg / ml (final concentration) to the reaction solution, add a test solution containing FGF-5, incubate for 16 hours, and then add ³ HTdR ( ³ H-methyl thymidine) (from Moravek Biochemicals). Then, the cells were further cultured for 4 hours at 37 ° C. in a CO ₂ incubator. After aspirating the culture supernatant and washing the cells with PBS, 200 μl of 0.025% Trypsin-0.02% EDTA2Na-PBS is added per well and reacted at 30 ° C. for 15-20 minutes. Cells were peeled off. Cells were collected on a filter paper with a cell harvester, the filter paper was dried, 2 ml of toluene-based scintillator / section was added, and the uptake of tritium thymidine into the cells was measured with a liquid scintillation counter (the final concentration of FGF-5 was 2). × 10 ⁻⁷ mM).
Moreover, FGF-5 was changed similarly to heparin (5 μg / M), peptide 1 (1.0 mM) or peptide 2 (1.0 mM).
As a result, as shown in FIG. 2, FGF-5, peptide 1 or peptide 2 each promoted DNA synthesis.
As a result, it was revealed that peptide 1 or peptide 2 has FGF-5-like activity. On the other hand, it is shown by the said nonpatent literature 3 (J. Cell. Physiol. 197,272-283) etc. that FGF-5 controls a hair cycle and inhibits hair growth by administering to a mouse | mouth subcutaneously. Therefore, it has been clarified that the tyrosinase inhibitor in the present invention is a whitening agent having a hair removal effect.

It is a figure which shows the tyrosinase activity inhibition rate of the peptide used by this invention. It is a figure which shows the uptake | capture rate of the tritium thymidine of the peptide used by this invention.

Claims (3)

A tyrosinase inhibitor comprising fibroblast growth factor 5, or a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2, or a salt thereof. A whitening agent comprising the tyrosinase inhibitor according to claim 1. A whitening external preparation containing the tyrosinase inhibitor according to claim 1.

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