Anti-aging and anti-inflammatory agents

Abstract

The objective of this invention is to provide a novel active ingredient that can suppress aging-related inflammation. [Solution] Azulene derivatives have the effect of suppressing the production of SASP factors, and are therefore useful as active ingredients in anti-aging and anti-inflammatory agents.

Description

Technical Field

[0001] The present invention relates to an anti-aging inflammation inhibitor.

Background Art

[0002] Most normal somatic cells have a determined number of divisions and can no longer divide once a certain number of divisions is reached. This state where division reaches its limit is called cellular senescence, and cells that have divided up to the limit number are called senescent cells. It has been found that senescent cells cause chronic inflammation through the secretion of inflammatory cytokines via the senescence-associated secretory phenotype (SASP).

[0003] Examples of SASP factors include inflammatory cytokines (interferon (IL)-1α / β, IL-6, IL-8, TNF-α), chemokines (CCL, CXCL), extracellular matrix degrading enzymes (MMPs), growth factors (EGF, VEGF, PGDF), extracellular matrix (fibronectin, collagen), etc. (Non-Patent Document 1).

[0004] CCL2, which is an example of a C-C motif chemokine receptor (CCL), is an important regulatory factor in periodontal disease and a therapeutic target. Specifically, it has been reported that the administration of bindarit, a CCL2 inhibitor, to a periodontal disease model was effective in reducing osteoclasts (Non-Patent Document 2).

[0005] Furthermore, it has been reported that the expression localization of CCL2 changes during the progression of periodontal disease. Specifically, the localization of CCL2 changes in the order of (1) giant cells on the surface of the alveolar bone, (2) giant cells (the number of CCL2-positive giant cells further increases), immune cells infiltrating the periodontal tissue, intravascular cells, and gingival cells, (3) furthermore, the periodontal ligament, (4) the surface of the alveolar bone and the root surface (the number of CCL2-positive periodontal ligament cells decreases) (Non-Patent Document 3).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] While some compounds useful as SASP factor inhibitors, such as CCL2 inhibitors, are already in practical use as pharmaceuticals, they require a doctor's prescription and careful handling. On the other hand, considering that periodontal disease and other conditions caused by SASP in senescent cells in periodontal tissue are common, it is desirable to be able to select a more versatile active ingredient that can suppress the production of SASP factors.

[0008] Therefore, the purpose of this disclosure is to provide a new active ingredient that can suppress aging inflammation. [Means for solving the problem]

[0009] The inventors of this invention conducted diligent research to solve the aforementioned problems and discovered that azulene derivatives have the effect of suppressing the production of CCL2, a SASP factor. The present invention was completed by further research based on this finding.

[0010] In other words, this disclosure provides inventions in the following embodiments. Item 1. Anti-aging and anti-inflammatory agents containing azulene derivatives. Item 2. An anti-aging inflammation agent as described in Item 1, used to suppress aging inflammation in periodontal tissue. Item 3. An anti-aging and anti-inflammatory agent according to item 1 or 2, used as an oral composition. Item 4. An anti-aging and anti-inflammatory agent according to any one of items 1 to 3, for use in application to CCL2 expression sites in periodontal tissue. Item 5. An anti-aging and anti-inflammatory agent described in any of items 1 to 4, used for the prevention or improvement of periodontal disease. Item 6. A method for suppressing aging inflammation, comprising administering an effective amount of azulene derivative to a subject requiring suppression of aging inflammation. Item 7. Use of azulene derivatives for the manufacture of compositions for suppressing aging and inflammation. Item 8. Azulene derivatives for use in the prevention or treatment of diseases associated with aging inflammation. Item 9. Non-therapeutic use of azulene derivatives to suppress aging inflammation. [Effects of the Invention]

[0011] According to this disclosure, aging inflammation can be suppressed by using azulene derivatives as novel active ingredients. [Brief explanation of the drawing]

[0012] [Figure 1] This disclosure demonstrates the inhibitory effect of the anti-aging and anti-inflammatory agent (sodium azulene sulfonate) on the production of SASP factor (CCL2) in senescent cells. [Figure 2] This study demonstrates the effect of a common anti-inflammatory component (glycyrrhetinic acid) on SASP factor (CCL2) production in senescent cells. [Figure 3] This study demonstrates the effect of a common anti-inflammatory component (allantoin) on SASP factor (CCL2) production in senescent cells. [Figure 4] This study shows the effect of a common anti-inflammatory component (alcloxa) on SASP factor (CCL2) production in senescent cells. [Figure 5] This study shows the effect of a common anti-inflammatory component (tranexamic acid) on SASP factor (CCL2) production in senescent cells. [Figure 6] This study demonstrates the effect of a common anti-inflammatory component (tocopherol acetate) on SASP factor (CCL2) production in senescent cells. [Figure 7]Shows the effect of a general anti-inflammatory component (ε-aminocaproic acid) on the production of SASP factor (CCL2) in senescent cells.

Mode for Carrying Out the Invention

[0013] Hereinafter, the senescence inflammation inhibitor of the present disclosure will be described in detail. In this specification, a numerical range indicated by two numerical values and "~" includes the two numerical values as the lower limit value and the upper limit value. For example, the notation of 2 to 15% by weight means 2% by weight or more and 15% by weight or less.

[0014] Active ingredients The senescence inflammation inhibitor of the present disclosure contains an azulene derivative as an active ingredient. The azulene derivative used in the present disclosure is a compound in which one or more substituents selected from the group consisting of an acidic functional group and an alkyl group are bonded to azulene, and a salt thereof. Examples of the acidic functional group include a sulfo group, a carboxyl group, and the like. Examples of the alkyl group include an alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 or 1 to 2 carbon atoms.

[0015] Specific examples of the azulene derivative include azulene sulfonic acid (guaiazulene sulfonic acid), dimethylisopropylazulene (guaiazulene), 1,4-dimethyl-7-ethylazulene (camaiazulene), 1,4-dimethyl-7-ethylazulene-3-sulfonic acid (camaiazulene sulfonic acid), and the like.

[0016] When azulene derivatives are in the form of salts, the type of salt is not particularly limited, as long as it is pharmaceutically or physiologically acceptable. Examples include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; other metal salts such as aluminum salts; ammonium salts; carboxylate salts such as acetate, trifluoroacetate, butyrate, palmitate, stearate, fumarate, maleate, succinate, malonate, lactate, tartrate, and citrate; organic sulfonates such as methanesulfonate, toluenesulfonate, and tosylate; organic amine salts such as methylamine salt, triethylamine salt, triethanolamine salt, morpholine salt, piperazine salt, pyrrolidine salt, tripyridine salt, and picoline salt; and inorganic salts such as hydrochloride, sulfate, nitrate, hydrobromide, and phosphate.

[0017] These azulene derivatives may be used individually or in combination of two or more.

[0018] Among these azulene derivatives, from the viewpoint of further enhancing the anti-aging and anti-inflammatory effects, compounds in which at least one or more acidic functional groups are bonded to the azulene skeleton and salts thereof are preferred, more preferably compounds in which at least one or more sulfo groups are bonded to the azulene skeleton and salts thereof are preferred, even more preferably azulene sulfonic acid and salts thereof are preferred, even more preferably alkali metal salts of azulene sulfonate are preferred, and particularly preferably sodium azulene sulfonate is preferred.

[0019] In the anti-aging inflammation inhibitors of this disclosure, the content of the azulene derivative is not particularly limited and can be appropriately set according to the type of azulene derivative used, the degree to which the anti-aging inflammation inhibitory effect should be imparted, and the formulation form and use of the anti-aging inflammation inhibitor, for example, 0.001 to 10% by weight is a possible range. From the viewpoint of further enhancing the anti-aging inflammation inhibitory effect, the content of the azulene derivative is preferably 0.004 to 10% by weight, more preferably 0.008 to 10% by weight, even more preferably 0.014 to 10% by weight, even more preferably 0.018 to 10% by weight, 0.018 to 5% by weight, 0.018 to 1% by weight, 0.018 to 0.5% by weight, or 0.018 to 0.1% by weight.

[0020] Other ingredients The anti-aging and anti-inflammatory agents disclosed herein may or may not contain other pharmacological components in addition to the active ingredients described above, as needed. Examples of such pharmacological components, whether present or absent, include antihistamines, local anesthetics (dibucaine hydrochloride, ethyl aminobenzoate, etc.), bactericides (isopropylmethylphenol, cetylpyridinium chloride, benzethonium chloride, chlorhexidine hydrochloride, chlorhexidine hydrochloride gluconate, triclosan, lauroyl sarcosine salt, hinokitiol, etc.), astringents (sodium chloride, Phellodendron amurense extract, Angelica acutiloba extract, Paeonia lactiflora extract, allantoin, etc.). Examples include cloxamine, other anti-inflammatory agents (tranexamic acid, dipotassium glycyrrhizate, β-glycyrrhetinic acid, ε-aminocaproic acid, lysozyme chloride, methyl salicylate, cineole, etc.), antioxidants (pycnogenol, ascorbic acid, ascorbic acid glucoside), cell activators (allantoin, etc.), skin protectants, blood circulation promoting ingredients (tocopherol acetate, tocopherol nicotinate, panthenol, etc.), hemostatic agents (carbazochrome, etc.), and mucopolysaccharides.

[0021] Furthermore, the anti-aging and anti-inflammatory agents of this disclosure may or may not contain base materials and / or additives other than the components described above, as necessary, in order to obtain the desired formulation form. Such base materials and additives, whether present or absent, are not particularly limited to the extent that they are pharmaceutically or physiologically acceptable, but examples include water, lower (1-5 carbon) monohydric alcohols, polyhydric alcohols, natural oils and fats, hydrocarbon oils, ester oils, fatty acid alkyl esters, fatty acids, fatty acid esters, higher (6 or more carbon) monohydric alcohols, cholesterol, glyceryl tri-2-ethylhexanoate, cetyl 2-ethylhexanoate, silicone oil; emulsifiers; cleansing components (surfactants, sodium bicarbonate, etc.), cooling agents, preservatives, fragrances, colorants, viscosity modifiers, pH adjusters, wetting agents, abrasives, stabilizers, antioxidants, UV absorbers, chelating agents, adhesives, buffers, solubilizers, preservatives, etc.

[0022] Dosage form, formulation, and product classification The dosage form of the anti-aging inflammation agent of this disclosure is not particularly limited and may be liquid, semi-solid, solid, or any other form. The anti-aging inflammation agent of this disclosure may be a non-emulsified composition or an emulsified composition, but a non-emulsified composition is preferred, and a non-emulsified aqueous composition is preferred.

[0023] The anti-aging and anti-inflammatory agents disclosed herein can be formulated in the form of gels, creams, lotions, emulsions, liquids, ointments, pastes, jellies, aerosols, powders, granules, capsules, candies, drops, lozenges, tablets, chewables, gummies, edible films, etc. These formulations can be prepared by compounding them using additives appropriate to the formulation, in accordance with known methods described in the General Provisions for Formulations of the Seventeenth Revised Japanese Pharmacopoeia, etc.

[0024] Furthermore, product classifications of anti-aging and anti-inflammatory agents in this disclosure include topical preparations and oral preparations, more specifically, topical pharmaceuticals, topical quasi-drugs, oral pharmaceuticals, and oral quasi-drugs. Preferably, product classifications of anti-aging and anti-inflammatory agents in this disclosure include mucosal preparations. Mucosal preparations include oral preparations and other mucosal preparations (e.g., suppositories and vaginal preparations), with oral preparations being preferred. Specifically, these include mouthwash, gargles, dental creams, oral ointments, toothpastes (paste, gel toothpaste, liquid toothpaste (dental rinse)), oral tablets (buccal tablets, gums, lozenges, adhesive tablets, etc.), oral capsules (for oral spray), oral sprays, etc.

[0025] Purpose The anti-inflammatory agents of this disclosure are used to suppress inflammation caused by cells that have divided to their limit, i.e., senescent cells. Preferably, the anti-inflammatory agents of this disclosure are used to suppress inflammation based on CC motif chemokine receptor 2 (CCL2).

[0026] The anti-aging inflammation inhibitors of this disclosure are preferably used to suppress anti-aging inflammation in the oral cavity, more preferably in periodontal tissue, and even more preferably applied to CCL2 expression sites in periodontal tissue.

[0027] The anti-aging and anti-inflammatory agents of this disclosure are preferably used for the prevention or improvement of periodontal disease. The periodontal disease may be either gingivitis or periodontitis. The stage of periodontal disease is not particularly limited and may be stage I, stage II, stage III, or stage IV. The grade of periodontal disease is also not particularly limited and may be grade A, grade B, or grade C. The progression stage of periodontal disease based on the localization of CCL2 is also not particularly limited and may be any of the following: (1) a stage in which CCL2 is localized in giant cells on the surface of the alveolar bone; (2) a stage in which the number of CCL2-positive giant cells increases compared to stage (1), and CCL2 is also localized in immune cells, endovascular cells, and gingival cells infiltrating the periodontal tissue; (3) a stage in which CCL2 is also localized in the periodontal ligament; or (4) a stage in which the number of CCL2-positive periodontal ligament cells decreases compared to stage (3), and CCL2 is localized on the surface of the alveolar bone and the root surface. If the anti-aging and anti-inflammatory agent of this disclosure is a topical agent, it may be applied at either the prevention stage or the progression stage of periodontal disease, but it is preferable to apply it at the stage described in (2) and / or (3) above, where CCL2 is present at a relatively shallow location from the surface of the periodontal tissue (including the inner surface of the periodontal pocket).

[0028] Since senescent cells exist regardless of age or sex, inflammation caused by senescent cells, i.e., senescent inflammation, can also occur regardless of age or sex. Therefore, the target population for the senescent inflammation inhibitors disclosed herein is not particularly limited in terms of age or sex. When the senescent inflammation inhibitors disclosed herein are used for the prevention or improvement of periodontal disease, target populations include men or women in late infancy, childhood, adolescence, adulthood, middle age, or old age, and among these, men or women in middle age or old age with a high accumulation of senescent cells are preferred. When the senescent inflammation inhibitors disclosed herein are used for the prevention or improvement of periodontal disease, target populations among women further include women in pregnancy, childbirth, ovulation, luteal phase, menopause, and post-menopausal periods, as well as women taking hormonal drugs such as oral contraceptives. [Examples]

[0029] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0030] Test Example 1 (1) Test method 1. Cellular senescence was induced in cultured human gingival fibroblasts (HGnF cells) by treating them with Doxorubicin (100 ng / mL) for 13-14 days to produce senescent HGnF cells (hereinafter referred to as "senescent cells"). For each test substance, cells with the same viability and proliferation rate were used as senescent cells. Furthermore, cells that showed CCL2 expression more than twice the amount of non-senescent cells (described later) after senescence induction were selected as senescent cells within a reasonably reproducible range. 2. Separately, HGnF cells that had not undergone senescence induction (hereinafter referred to as "non-senescent cells") were prepared. 3. Senescent cells were cultured for 24 hours after being added to the test substances, sodium azulene sulfonate, glycyrrhetinic acid, allantoin, alcloxa, tranexamic acid, tocopherol acetate, or ε-aminocaproic acid, at the concentrations shown in Figures 1 to 7. In this test system, it was confirmed that the test substances were non-cytotoxic at all tested concentrations. Furthermore, it was confirmed that sodium azulene sulfonate did not cause the removal of senescent cells during the tested culture time. 4. After culturing, RNA was extracted from all cells using the Relia Prep RNA Cell Miniprep System (Promega) according to the product protocol. 5. After extraction, 1 μl of Random Primer (Takara Bio) was added to 1 μg of RNA, and the sample was adjusted to 12 μl with RN-ase Free Water. The sample was then heat-treated at 65°C for 5 minutes using a T-Personal Thermocyler (Biometra) to denature the RNA, and then rapidly cooled on ice. 6. Add the reverse transcription reaction mixture (4 μl of 5×RT Buffer, 2 μl of 10 mM dNTPs, 1 μl of RNase Inhibitor, 0.5 μl of ReverTraAce, 0.5 μl of RNase-Free H2O (all reagents except H2O are from TOYOBO)) to the RNA-denatured sample, and adjust to a final volume of 20 μl. The resulting sample was reverse transcribed to cDNA using a T-Personal Thermocyler (conditions: 30°C for 10 minutes → 42°C for 60 minutes → 85°C for 5 minutes → 4°C Hold). 7. Quantitative real-time PCR was performed using the SYBR Green Kit (Qiagen) and CCL2-specific primers in a QuantStudio (Thermofisher Scientific) system. 8. The relative expression levels of the CCL2 gene were determined by a comparative quantitative method (ΔΔCt method). The same procedure as above was followed for non-senescent or senescent cells, except that the test substance was not added.

[0031] (2) Results As shown in Figure 1, senescent cells (without the addition of sodium azulene sulfonate) showed a significantly increased expression level of CCL2 compared to non-senescent cells. On the other hand, when sodium azulene sulfonate was added to senescent cells, the expression level of CCL2 decreased in a concentration-dependent manner. In other words, it was found that sodium azulene sulfonate can suppress senescent inflammation by inhibiting the production of CCL2, a SASP factor in senescent cells.

[0032] On the other hand, as shown in Figures 2 to 7, glycyrrhetinic acid, allantoin, alcloxa, tranexamic acid, tocopherol acetate, and ε-aminocaproic acid, which are common anti-inflammatory ingredients used in oral compositions, were all unable to suppress the production of CCL2, a SASP factor in senescent cells; in other words, no inhibitory effect on aging inflammation was observed.

Claims

1. An anti-aging and anti-inflammatory agent containing azulene derivatives.

2. An anti-aging inflammation agent according to claim 1, used to suppress aging inflammation in periodontal tissue.

3. An anti-aging and anti-inflammatory agent according to claim 1, used as an oral composition.

4. An anti-aging and anti-inflammatory agent according to claim 1, for use in application to CCL2 expression sites in periodontal tissue.

5. An anti-aging inflammation agent according to claim 1, used for the prevention or improvement of periodontal disease.

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