JP2008031094A - Scf binding inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an SCF (stem cell factor) binding inhibitor and a bleaching agent, inhibiting SCF binding to a c-kit receptor, useful as pharmaceuticals or cosmetics. <P>SOLUTION: The invention relates to the SCF binding inhibitor comprising compounds represented by following (1)-(3) and a compound selectd from phosphorylated polysacchrides selected from such as phosphorylated mannan oligosaccharide (originated from Y1892), phosphorylated galactans (originated from Y6493) and phosphorylated galactans (originated from Y6502), or salt of the same, as active component. Wherein (1); 7, 8-dihydrofolic acid (1a) and 7, 8-dihydropteroic acid (1b), (2); dGMP(2a) and 1-(5'-phosphoribosyl)-5-amino-4-imidazole carboxamide (2b), (3); phosphoenolpyruvic acid (3a), 2-isopropylmalic acid (3b), and 2-oxobutyric acid (3c). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a stem cell factor (hereinafter referred to as “SCF”) binding inhibitor and a whitening agent.

  Pigmentation after sunburn, spots and freckles generally result from increased production of melanin by activation of pigment cells (melanocytes) in the skin due to stimulation by ultraviolet exposure to the skin, hormonal abnormalities or genetic factors It is believed that.

  SCF is a ligand for the c-kit receptor expressed on the surface of hematopoietic stem cells, and is known as a membrane-bound growth factor that promotes the proliferation and differentiation of hematopoietic cells. In addition, it has become clear that it is also expressed on the surface of mast cells, melanocytes, and germ cells (Non-patent Document 1), and research on the in vivo effects of SCF has progressed, including involvement in immediate allergy. It has been. Recently, in a tonlasgenic mouse that expresses SCF only in the skin, melanin synthesis is enhanced by induction of mast cells and proliferation of melanocytes (Non-patent Document 2), and phosphorylation of c-kit on melanocytes. It has been reported that melanogenesis is enhanced (Non-patent Document 3), and SCF is thought to be deeply involved in melanin overproduction.

  Therefore, if the binding between the c-kit receptor on melanocytes and SCF can be specifically inhibited, excessive production of melanin can be suppressed, and skin browning and occurrence of spots and freckles can be prevented or improved. It becomes possible to do.

On the other hand, 7,8-dihydrofolic acid is known as the first product in the folic acid biosynthesis system (Non-patent Document 4), and 7,8-dihydropteroyl acid is synthesized in vivo by dihydropteroic acid pyrophosphorylase. (Non-Patent Document 5), dGMP is known as a constituent deoxynucleotide of DNA (Non-Patent Document 6), and 1- (5′-phosphoribosyl) -5-amino-4-imidazolecarboxamide has AMPK activation. It is known that there is an action (Non-Patent Document 7), phosphoenolpyruvate is known to have a hair-growing action (Patent Document 1), and 2-isopropylmalic acid is known as an intermediate of leucine biosynthesis ( Non-patent document 8), 2-oxobutyric acid is known to be produced in vivo by threonine undergoing a dehydratase reaction (non-patent document 9). Although the oxidized polysaccharide has been reported to have protein adsorption inhibiting effect (Patent Document 2), it is not known at all that there is SCF binding inhibition activity to these compounds.
JP 2004-168728 A JP 2002-29948 A J.Exp.Med., 183,2681-2686,1996 J.Exp.Med., 187,1565-1573,1998 Molecular Bioloy of the Cell 3, 197-209 1992 The Great Dictionary of Chemistry 1989 Tokyo Chemical Co., Ltd. Biochemical Dictionary (2nd edition) 1990 Tokyo Chemical Co., Ltd. The Great Dictionary of Chemistry 1989 Tokyo Chemical Co., Ltd. Obesity Research 11, 79-81, 2005 Biochemical Dictionary (2nd edition) 1990 Tokyo Chemical Co., Ltd. Biochemical Dictionary (2nd edition) 1990 Tokyo Chemical Co., Ltd.

  The present invention relates to providing an SCF binding inhibitor and a whitening agent that inhibits the binding of SCF to a c-kit receptor and is useful as a medicine or cosmetic.

  The present inventors searched for compounds that specifically inhibit the binding of SCF to the c-kit receptor on the cell surface. As a result, the following specific folic acid (1), purine-related substance (2), C3- The present inventors have found that C4 unit (3) and phosphorylated polysaccharide have SCF binding inhibitory activity and are effective in preventing or improving pigmentation and stains and freckles due to excessive production of melanin.

  That is, the present invention relates to an SCF binding inhibitor and a whitening agent comprising as an active ingredient a compound selected from compounds represented by the following general formulas (1) to (3) and phosphorylated polysaccharides or salts thereof. .

  According to the SCF binding inhibitor or the whitening agent of the present invention, excessive production of melanin in the skin can be suppressed, and pigmentation and spot / sobacus after sunburn can be prevented or improved.

  The SCF binding inhibitor of the present invention specifically inhibits the binding of SCF to the c-kit receptor on the cell surface, particularly on the melanocyte surface, and causes browning of the skin and generation of spots and freckles due to excessive production of melanin. It has prevention or improvement effect.

  In the present invention, the compounds represented by the general formula (1) are 7,8-dihydrofolic acid (1a) and 7,8-dihydropteroic acid (1b) as shown below, and the general formula (1) The compounds represented by the formulas are dGMP (2a) and 1- (5′-phosphoribosyl) -5-amino-4-imidazolecarboxamide (2b), and the compound represented by the general formula (3) is phosphoenol pyruvin. Acid (3a), 2-isopropylmalic acid (3b) and 2-oxobutyric acid (3c).

  Examples of salts of these compounds include alkali metal salts such as lithium, sodium and potassium, alkaline earth metals such as beryllium, magnesium and calcium, primary to tertiary amines, quaternary ammonium salts, arginine, lysine and the like. And basic salts with amino acids and the like, mineral acids such as hydrochloric acid, iodic acid and sulfuric acid, and acidic salts with organic acids such as acetic acid, citric acid and p-toluenesulfonic acid.

  The above compounds are all known compounds, and can be obtained from known organic chemical synthesis, or from natural products or microbial metabolites (for example, Bacillus subtilis metabolites) containing these compounds, using a known method. In addition, there are commercially available products, and these can be used. Examples of such commercially available products include 7,8-dihydrofolic acid (Schircks Laboratories 16.206), 7,8-dihydropteroyl acid (Schircks Laboratories 16.114), dGMP (SIGMA D-9625), 1- (5′-phosphoribosyl). ) -5-amino-4-imidazolecarboxamide (SIGMA A-1393), phosphoenolpyruvate (Wako 325-20641), 2-isopropylmalic acid (ALDRICH 333115), 2-oxobutyric acid (ALDRICH K401) and the like. .

The phosphorylated polysaccharide of the present invention refers to a polysaccharide (including oligosaccharides) having at least one, preferably two or more phosphate groups in the molecule. Examples of the constituent polysaccharide include mannan and galactan. Preferably, plant-derived mannan, plant-derived galactan, microbial-derived mannan, and microbial-derived galactan are used. Of these, phosphorylated polysaccharides derived from yeast are preferred, and phosphorylated mannan oligosaccharides derived from H. capsulata NRRL Y-1842, phosphorylated galactans derived from Sporobolomyces NRRL Y-6493, and phosphorylated galactans derived from Sporobolomyces NRRL Y-6502 are particularly preferred. .
Moreover, as these salts, sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt etc. are mentioned.

  These phosphorylated polysaccharides can be obtained from the culture solution of the microorganism by, for example, the method described in Slodki ME et al., J. Bacteriol, 82; 269-274 (1961).

  Since the compound of the present invention or a salt thereof has an activity of inhibiting the binding of SCF to c-kit, as shown in the examples described later, it has an effect of overproduction of melanin caused by the activation of c-Kit on melanocytes. It can be suppressed (Reference: The Journal of Biological Chemistry 275, 33321-33328, 2000). Therefore, the compound of the present invention or a salt thereof can be used as an SCF binding inhibitor or a whitening agent, and can be used for producing an SCF binding inhibitor or a whitening agent. The SCF binding inhibitor and whitening agent can be used as pharmaceuticals, quasi-drugs, cosmetics and the like for preventing or improving the browning of skin and the occurrence of spots and freckles. In addition, the SCF binding inhibitor or whitening agent can be used as a quasi-drug or cosmetic that displays SCF binding inhibition or whitening as a concept and displays that effect as necessary.

  Examples of the dosage form when the SCF binding inhibitor or whitening agent of the present invention is used as a pharmaceutical agent include oral administration by tablet, capsule, granule, powder, syrup, etc., or injection, external preparation, suppository, transdermal Any of parenteral administration using an absorbent or the like may be used. In order to prepare the pharmaceutical preparation, the plant of the present invention or an extract thereof alone or other pharmaceutically acceptable excipient, binder, extender, disintegrant, surfactant, lubricant , Dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents and the like can be used in appropriate combinations. The content of the compound of the present invention or a salt thereof in the preparation is preferably 0.1 to 20% by mass, particularly preferably 0.5 to 10% by mass. When the SCF binding inhibitor or whitening agent of the present invention is used as a pharmaceutical, the daily dose per adult is, for example, 0.001 to 1000 mg, particularly 0.01 as the compound of the present invention or a salt thereof. It is preferably ˜100 mg.

In addition, when the SCF binding inhibitor or whitening agent of the present invention is used as a quasi-drug or cosmetic, it can be used as a skin external preparation, a cleaning agent, or a makeup cosmetic. Depending on the method of use, a lotion, It can be provided in various dosage forms such as emulsion, gel, cream, ointment, powder, granule and the like. Such quasi-drugs and cosmetics of various dosage forms include oily ingredients, moisturizing agents, which are blended with the plant of the present invention or an extract thereof alone or in quasi-drugs, skin cosmetics and cleaning agents. Prepared by appropriately combining agents, powders, pigments, emulsifiers, solubilizers, detergents, UV absorbers, thickeners, medicinal ingredients, fragrances, resins, antibacterial and antifungal agents, plant extracts, alcohols, etc. can do. Examples of the medicinal component include other whitening ingredients such as hormone agents, kojic acid, arbutin, placenta extract, chamomile extract, and lucinol.
The content of the compound of the present invention in the quasi drug or cosmetic is preferably 0.01 to 100% by mass, particularly preferably 0.05 to 70% by mass.

<Preparation of evaluation sample>
Each sample consists of 7,8-dihydrofolic acid (Schircks Laboratories 16.206), 7,8-dihydropteroylic acid (Schircks Laboratories 16.114), dGMP (SIGMA D-9625), 1- (5′-phosphoribosyl) -5-amino. -4-imidazole carboxamide (SIGMA A-1393), phosphoenolpyruvate (Wako 325-20641), 2-isopropylmalic acid (ALDRICH 333115), 2-oxobutyric acid (ALDRICH K401), phosphorylation derived from H.capsulata NRRL Y1842 Mannan oligosaccharide, Sporobolomyces NRRL Y6493-derived phosphorylated galactan, and Sporobolomyces NRRL Y6502-derived phosphorylated galactan were each dissolved in distilled water to a concentration of 1 mM.

Example 1 SCF binding inhibitory activity Recombinant human c-kit (Soluble c-kit; s-kit (R & D systems)) dissolved in PBS at 200 μg / ml was added to a 96-well plate at 50 μl / well, Left at 4 ° C. overnight. The next day, the wells were washed twice with PBS, then 5% BSA (Bovine Serum Albumin; Wako Pure Chemical Industries, Ltd.)-PBST (0.1% Tween20-PBS) was added until the wells were full, and at room temperature for over 1 hour I left it alone. After the wells were washed twice with PBST, the evaluation sample (final concentration 5% (v / v)), final concentration 100 pM SCF (“Recombinant Human SCF” (Peprotech)), final concentration 0.5% BSA, PBS And a total of 100 μl / well was added, followed by shaking at room temperature for 2 hours or more. After the wells were washed 5 times with PBST, anti-SCF antibody (“Rabbit polyclonal to SCF” (abcam)) with a final concentration of 0.5 μg / ml, BSA with a final concentration of 0.5%, and PBS were added to give a total of 100 μl / well. And then shaken at room temperature for more than 2 hours or left at 4 ° C. overnight. The wells were washed 5 times with PBST, and then 100 μl / well of HRP-labeled anti-rabbit IgG antibody (ECL Anti-rabbit IgG (Amersham Biosciences)) diluted 1000-fold with 0.5% BSA-PBST was added. Shake for more than an hour. After the wells were washed 10 times with PBST, 100 μl / well of TMB substrate solution (“TMB Microwell Peroxidase Substrate System” (KPL)) was added, and the mixture was allowed to stand at 10 ° C. for 20 minutes at room temperature with light shielding. Furthermore, 100 μl / well of TMB reaction stop solution (“TMB Stop Solution” (KPL)) was added to stop the reaction, the absorbance at 450 nm was measured with a plate reader, and the amount of SCF bound to s-kit was measured. As a control, a well to which the same amount of the solvent was added instead of the evaluation sample was prepared. In addition, a well in which the solvent was added instead of the evaluation sample was prepared without immobilizing s-kit, and this absorbance was used as a blank (nonspecific binding amount). The obtained absorbance was obtained by subtracting the blank value to obtain the specific binding amount and expressed as a relative value with the control value being 100.

  As shown in Table 1, it was confirmed that the compounds of the present invention have SCF binding inhibitory activity against c-kit.

Claims (2)

An SCF binding inhibitor comprising as an active ingredient a compound selected from compounds represented by the following general formulas (1) to (3) and a phosphorylated polysaccharide or a salt thereof.

A whitening agent comprising as an active ingredient a compound selected from compounds represented by the following general formulas (1) to (3) and a phosphorylated polysaccharide or a salt thereof.