JP2023053658A - Intracellular NAD+ production promoter - Google Patents

Abstract

To provide a novel intracellular NAD+ production promoter.SOLUTION: An intracellular NAD+ production promoter contains hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients.SELECTED DRAWING: Figure 1

Description

The present invention relates to intracellular NAD ⁺ production promoters.

Nicotinamide adenine dinucleotide (NAD ⁺ ) is a coenzyme that mediates redox reactions in vivo and plays a central role in energy metabolism. There are two biosynthetic pathways for NAD ⁺ : the de novo pathway using tryptophan as a starting material, and the salvage pathway that recycles the nicotinamide ring of nicotinamide (NAM) and nicotinamide riboside (NR).

NAD ⁺ is also deeply involved in post-translational modifications of proteins (e.g., deacetylation, ADP-ribosylation, etc.), and is known to regulate various intracellular functions such as differentiation and proliferation. (Patent Document 1). For example, poly ADP-ribose polymerase (PARP), an enzyme involved in DNA single-strand damage repair, utilizes NAD ⁺ for ADP-ribosylation. In addition, sirtuins (Sirtuins 1 to 7), which play important roles in the aging control mechanism, are all NAD ⁺ -dependent deacetylases, and sirtuin activity is also controlled by the concentration of NAD ⁺ , which accepts acetyl groups. It is It is generally known that increased sirtuin activity causes changes such as increased expression of antioxidant enzyme genes and suppressed expression of inflammatory protein genes.

On the other hand, it has been found that intracellular NAD ⁺ levels decrease with aging and cause various aging symptoms and related diseases. For example, a decrease in NAD ⁺ level is known to affect metabolic dysfunction, DNA repair disorder, inflammation, cellular senescence, reduced resistance to oxidative stress, neurodegeneration, etc. (Non-Patent Document 1). On the other hand, it has been reported that age-related diseases can be treated by promoting NAD ⁺ biosynthesis or inhibiting NAD ⁺ degradation. For example, it has been reported in animal models that increasing intracellular NAD ⁺ levels delays aging, restores muscle function, and provides protection against metabolic diseases (Non-Patent Document 2).

Patent Document 2 reports the use of nicotinic acid riboside or nicotinamide riboside derivatives and their reduced derivatives as precursors for increasing NAD ⁺ .

In recent years, infusion therapy and supplements of nicotinamide mononucleotide (NMN) known as an intermediate in the biosynthetic pathway of NAD ⁺ have been used for anti-aging to increase intracellular NAD ⁺ levels. One of its effects is to suppress deterioration of the skin caused by aging such as wrinkles, sagging, spots, and dullness. In addition, NMN-containing cosmetics activate sirtuins by increasing NAD ⁺ in epidermal and subcutaneous cells, promotes the production of natural moisturizing factors (NMF), and prevents skin aging. (Patent Documents 3 and 4).

Japanese Patent Publication No. 2018-530614 Japanese Patent Publication No. 2019-514874 WO2019/054485 WO2019/078177

NAD+ metabolism and its roles in cellular processes during aging, Anthony J.; Covarrubias et al. , Nature Reviews Molecular Cell Biology vol. 22, pp. 119-141, 2021 De novo NAD+ synthesis enhances mitochondrial function and improves health, Elena Katsyuba et al. , Nature vol. 563, pp. 354-359, 2018

An object of the present invention is to provide a novel intracellular NAD ⁺ production promoter.

As a result of intensive studies to achieve the above object, the present inventors have found that hinokitiol or a salt thereof and nicotinic acid or a salt thereof have an intracellular NAD ⁺ production-promoting effect, and completed the present invention. reached.

Nicotinic acid or a salt thereof administered simultaneously with hinokitiol or a salt thereof and an intracellular NAD ⁺ production promoter containing nicotinic acid or a salt thereof as active ingredients, which is an embodiment of the present invention, and hinokitiol or a salt thereof as an active ingredient, and an intracellular NAD ⁺ production promoter containing hinokitiol or a salt thereof as an active ingredient, which is administered simultaneously with nicotinic acid or ^a salt thereof ^. It has the effect of increasing concentration. Since functions such as DNA repair and antioxidant stress can be restored by increasing the intracellular NAD ⁺ concentration in the epidermis and subcutaneous skin, the intracellular NAD ⁺ production promoter is a preventive agent for skin aging symptoms. Alternatively, it is also useful as an improver. In addition, a cosmetic composition or a composition for external use containing the intracellular NAD ⁺ production promoter promotes NAD ⁺ production in the epidermal and subcutaneous cells of the skin, and is suitable for aging care. A topical composition can be provided.
As used herein, the term "administration at the same time" means that one is acting while the other is being administered to cause both to act together. The administrations can be done together or at different times. Alternatively, a single agent containing both may be administered, or two agents containing each may be administered. Similarly, "adding simultaneously" means working together by adding the other while one is working. The additions can be made together or at different times. Moreover, it may be added as a single agent containing both, or may be added as two agents containing each.

That is, embodiments of the present invention include the following [1] to [13].
[1] An intracellular NAD ⁺ production promoter containing hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients.
[2] An intracellular NAD ⁺ production promoter containing nicotinic acid or a salt thereof as an active ingredient, which is administered simultaneously with hinokitiol or a salt thereof.
[3] An intracellular NAD ⁺ production promoter containing hinokitiol or a salt thereof as an active ingredient, which is administered simultaneously with nicotinic acid or a salt thereof.
[4] The intracellular NAD ⁺ production promoter according to [1] to [3], wherein the molar ratio of hinokitiol or its salt to nicotinic acid or its salt to be administered is 1-3:1-60.
[5] The intracellular NAD ⁺ production promoter according to [1] to [4], which is an agent for preventing or improving skin aging symptoms.
[6] The intracellular NAD ⁺ production promoter of [5], wherein the skin aging symptoms are wrinkles, sagging, spots, freckles, rough skin, or dry skin.
[7] A cosmetic composition containing the intracellular NAD ⁺ production promoter according to [1] to [6] as an active ingredient.
[8] A cosmetic containing the cosmetic composition according to [7] as an active ingredient.
[9] A composition for external use, containing the intracellular NAD ⁺ production promoter according to [1] to [6] as an active ingredient.
[10] An external preparation containing the composition for external use according to [9] as an active ingredient.
[11] A method for preventing or ameliorating skin aging symptoms, which comprises administering the intracellular NAD ⁺ production inhibitor according to [1] to [6] to a human.
[12] The intracellular NAD ⁺ production inhibitor according to [1] to [6], which is used for preventing or improving skin aging symptoms.
[13] Use of the intracellular NAD ⁺ production inhibitor according to [1] to [6] for producing an agent for preventing or improving skin aging symptoms.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel intracellular NAD ⁺ production promoter.

1 is a bar graph showing the amount of intracellular NAD ⁺ when each compound that is a precursor for NAD ⁺ biosynthesis was added alone or together with hinokitiol in one example of the present invention.

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the objects, features, advantages, and ideas of the present invention are apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. can. DETAILED DESCRIPTION OF THE INVENTION The embodiments, specific examples, and the like set forth below are indicative of preferred embodiments of the invention, and are presented by way of illustration or description, without regard to the invention. It is not limited. Based on this description, it will be apparent to those skilled in the art that various alterations and modifications can be made within the spirit and scope of the invention disclosed herein.
MR Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2012 ); as described in standard protocol collections such as FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. or a modified or modified method thereof can be used. When using a commercially available reagent kit or measuring device, the attached protocol is used unless otherwise specified.

In one embodiment of the present invention, nicotinic acid or a salt thereof administered simultaneously with hinokitiol or a salt thereof and an intracellular NAD ⁺ production promoter containing nicotinic acid or a salt thereof as active ingredients, hinokitiol or a salt thereof It is an intracellular NAD ⁺ production-promoting agent containing as an active ingredient, or an intracellular NAD ⁺ production-promoting agent containing hinokitiol or a salt thereof as an active ingredient, which is administered simultaneously with nicotinic acid or a salt thereof.

<Intracellular NAD ⁺ production promoter>
Hinokitiol is a known compound and is a monocyclic monoterpene with an unsaturated seven-membered ring.
Nicotinic acid (NA) is a known compound named 3-pyridinecarboxylic acid in the IUPAC nomenclature.

The salt of hinokitiol is not particularly limited as long as it is pharmaceutically, pharmacologically or physiologically acceptable. Examples of such salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; organic base salts such as ammonium salts and trialkylamine salts;
The intracellular NAD ⁺ production promoter containing hinokitiol or salts thereof may contain one type of hinokitiol or salts thereof, or may contain two or more types of hinokitiols or salts thereof. In particular, hinokitiol and hinokitiol sodium are preferably included as hinokitiol or a salt thereof.

The salt of nicotinic acid is not particularly limited as long as it is pharmaceutically, pharmacologically or physiologically acceptable. Examples of such salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; organic base salts such as ammonium salts and trialkylamine salts;
The intracellular NAD ⁺ production-enhancing agent containing nicotinic acid or a salt thereof may contain one type of nicotinic acid or a salt thereof, or may contain two or more types of nicotinic acid or salts thereof. In particular, nicotinic acid and sodium nicotinate are preferably included as nicotinic acid or a salt thereof.

Hinokitiol and its salts used for the production of intracellular NAD ⁺ production promoters may be extracted from natural products, chemically synthesized, or obtained as commercial products. Examples of raw material plants for extracts of hinokitiol and salts thereof include Taiwan cypress, Aomori hiba, North American western red cedar, and the like. The purity of the extract is not particularly limited, and it may be a highly purified product or a crudely purified product that does not inhibit the effect of promoting intracellular NAD ⁺ production. As the crudely purified hinokitiol, for example, an intermediate substance in the purification process of extracting and purifying hinokitiol and its salt from a raw material plant may be used.

Nicotinic acid and its salts used for the production of intracellular NAD ⁺ production-enhancing agents may be synthesized by known methods or obtained as commercial products. Nicotinic acid and its salt may be used singly or in combination of two or more compounds.

When the intracellular NAD ⁺ production promoter containing nicotinic acid or a salt thereof is a liquid formulation, the concentration of nicotinic acid or a salt thereof is not particularly limited, and the concentration of hinokitiol or a salt thereof used in combination, the formulation form, the method of use, etc. It is set accordingly. In addition, when the intracellular NAD ⁺ production promoter containing hinokitiol or its salt is a liquid formulation, the concentration of hinokitiol or its salt is not particularly limited, and the concentration, formulation form, method of use, etc. of nicotinic acid or its salt used in combination are not particularly limited. is set as appropriate.

The molar ratio of hinokitiol or a salt thereof and nicotinic acid or a salt thereof contained in the intracellular NAD ⁺ production promoting agent containing both hinokitiol or a salt thereof and nicotinic acid or a salt thereof is not particularly limited. It is appropriately set according to the type of hinokitiol or its salt and nicotinic acid or its salt, formulation form, method of use, and the like. For example, the molar ratio of each active ingredient is preferably hinokitiol or its salt: nicotinic acid or its salt = 1 to 3: 1 to 60, more preferably 1 to 3: 2 to 30. -3:6-20 is more preferred.

The intracellular NAD ⁺ production-promoting agent according to the present embodiment is ^, for example, an excipient, diluent, moisturizer, pH adjuster, emulsifier, solubilizer, Dispersants, thickeners, surfactants, preservatives, antioxidants, oils and fats, hydrocarbons, fatty acids, alcohols, fragrances, powders, pigments, and the like may be contained.

The dosage form of the intracellular NAD ⁺ production promoter according to the present embodiment is not particularly limited and can be arbitrarily selected according to the purpose of use. form, solution form, powder form, powder form, pack form, gel form, and the like.

Intracellular NAD ⁺ production promoters can promote DNA repair by PARP and suppress the expression of inflammatory protein genes by increasing the concentration of intracellular NAD ⁺ (Nature Reviews Molecular Cell Biology Vol.22, pp.119-141, February 2021). That is, by increasing the intracellular NAD ⁺ concentration, it is possible to improve the metabolic function in the skin, promote DNA repair, suppress inflammation, suppress or improve cellular aging, and enhance oxidative stress resistance. (Nature vol.563, pp.354-359, 2018; International Publication No. 2019/078177). Therefore, the intracellular NAD ⁺ production promoter according to the present embodiment is a cell aging preventive agent or ameliorating agent (especially a skin aging preventive agent or ameliorating agent), a DNA repair promoter, an anti-inflammatory agent, and an oxidative stress resistance enhancer. It can be suitably used as an agent. Symptoms of skin aging include, for example, wrinkles, sagging, spots, freckles, rough skin, and dry skin.

<Composition>
The intracellular NAD ⁺ production promoter according to this embodiment can be incorporated into cosmetic compositions, food compositions, reagent compositions and pharmaceutical compositions.

The cosmetic composition containing the intracellular NAD ⁺ production promoter according to one embodiment of the present invention is, for example, a cosmetic composition for improving metabolic function of skin, a cosmetic composition for promoting DNA repair, a cosmetic for suppressing or improving cell aging. It can be used as a composition, a cosmetic composition for enhancing resistance to oxidative stress, and the like.
The forms of cosmetics containing these cosmetic compositions as active ingredients are not particularly limited, and examples include lotions, creams, milky lotions, ointments, lotions, oils, all-in-one gels, packs, serums, foundations, cleansers, and facial cleansers. , cleansers, shampoo rinses, eye shadows, eyeliners and the like. These cosmetics are preferably applied to the skin as a coating agent.

A food composition containing an intracellular NAD ⁺ production promoter according to one embodiment of the present invention includes, for example, a food composition for improving metabolic function, a food composition for promoting DNA repair, and a food composition for suppressing or improving cell aging. It can be used as a food composition, a food composition for enhancing resistance to oxidative stress, and the like.
The type of food containing the food composition as an active ingredient is not particularly limited. etc.), confectionery (gums, jellies, candies, etc.), health foods (tablets, capsules, tablets, etc.), nutritional supplements (energy drinks, etc.), supplements, nutrient functional foods, food with function claims, health food, specified health food, pet food, animal feed, and the like.

A reagent composition containing an intracellular NAD ⁺ production-enhancing agent according to one embodiment of the present invention can be used in vitro as a reagent. When this reagent is administered to a culture such as a cultured cell, a cultured tissue, or a cultured organ, the culture promotes intracellular NAD ⁺ production, suppresses an increase in the concentration of intracellular reactive oxygen species, and improves metabolic function. , promotion of DNA repair, inhibition or improvement of cell senescence, enhancement of resistance to oxidative stress, and the like. The method of using the reagent is not particularly limited, and for example, hinokitiol or its salt and nicotinic acid or its salt may be added simultaneously to the medium.

A pharmaceutical composition containing an intracellular NAD ⁺ production promoter according to one embodiment of the present invention is, for example, a pharmaceutical composition for improving metabolic function, a pharmaceutical composition for promoting DNA repair, or a pharmaceutical composition for suppressing or improving cell senescence. , a pharmaceutical composition for enhancing oxidative stress resistance, and the like.
A medicament containing the pharmaceutical composition as an active ingredient can be administered to a human or non-human animal individual. The mode of administration is not particularly limited, and may be oral administration or parenteral administration, for example. The dosage form for oral administration is not particularly limited, and examples thereof include tablets, powders, granules, fine granules, capsules, liquids, emulsions, syrups, suspensions, lozenges and the like. The dosage form for parenteral administration is not particularly limited, and examples include injections (intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, etc.), drips, external preparations (ointments, powders, granules, liquids, gels, emulsions, creams, lotions, mousses, jellies, sheets, sprays, etc.), suppositories and the like, but external preparations are preferred.

^As described above, the intracellular NAD ⁺ production promoter according to the present embodiment can increase the concentration of NAD ⁺ in cells, particularly in the epidermis and subcutaneous. It is useful as a preventive agent and an improving agent for aging symptoms. In addition, it is possible to provide cosmetic compositions and topical compositions containing the same.

EXAMPLES The present invention will now be described more specifically based on examples. However, the present invention is not limited by the following examples.

[Test Example 1: Evaluation of promoting action of NAD ⁺ production in cells]
nicotinic acid (NA), nicotinic acid mononucleotide (NAMN), nicotinic acid riboside (NAR), vitamin E nicotinate (NAVE), nicotinamide (NAM), nicotinamide, a group of compounds that are precursors for NAD ⁺ biosynthesis NAD ⁺ production in cells when mononucleotide (NMN) and nicotinamide riboside (NR) were added alone or in combination with hinokitiol to human normal epidermal keratinocytes (NHEK: Normal Human Epidermal Keratinocyte) was evaluated by the following method.

NHEK (manufactured by Kurashiki Boseki Co., Ltd.) was seeded in a 96-well plate at 1.0×10 ⁵ cells/well and cultured at 37° C. and 5% CO ₂ . One day later, NA, NAMN, NAR, NAVE, NAM, NMN, and NR compounds were added alone or in combination with hinokitiol to the respective wells at final concentrations shown in Table 1 below. After culturing for 6 hours at 37° C. and 5% CO ₂ , the cells were harvested and the relative amount of intracellular NAD ⁺ was measured using Promega's NAD/NADH-Glo™ Assay. A Synergy HTX multiplate reader (BioTek) was used to measure luminescence. In FIG. 1, the relative amount of intracellular NAD ⁺ in the control NT (no compound or hinokitiol added) was set to 100%, and the relative intracellular NAD ⁺ amount was shown in a bar graph on the vertical axis. In the graph, except for NT, the white bar graph shows the relative NAD ⁺ amount when each compound was added alone, and the black bar graph shows the relative NAD ⁺ amount when hinokitiol was added in addition to each compound. is shown. In NT, the white bar graph indicates the reference NAD ⁺ amount when nothing is added, and the black bar graph indicates the relative NAD ⁺ amount when only hinokitiol is added.

As shown in FIG. 1, when only hinokitiol was added without adding any compound, there was no significant difference in the amount of intracellular NAD ⁺ compared to the control to which nothing was added. When hinokitiol was added, the amount of intracellular NAD ⁺ increased compared to when NA was added alone, and a synergistic effect between NA and hinokitiol was observed. However, synergistic effects with hinokitiol were not observed with other nicotinic acid analogues.
Thus, NA and hinokitiol synergistically promote intracellular NAD ⁺ production.

Claims (10)

An intracellular NAD ⁺ production promoter containing hinokitiol or a salt thereof and nicotinic acid or a salt thereof as active ingredients. An intracellular NAD ⁺ production promoter containing nicotinic acid or a salt thereof as an active ingredient, which is administered simultaneously with hinokitiol or a salt thereof. An intracellular NAD ⁺ production promoter containing hinokitiol or a salt thereof as an active ingredient, which is administered simultaneously with nicotinic acid or a salt thereof. The intracellular NAD ⁺ production promoter according to any one of claims 1 to 3, wherein the molar ratio of hinokitiol or its salt to nicotinic acid or its salt to be administered is 1-3:1-60. The intracellular NAD ⁺ production promoter according to any one of claims 1 to 4, which is an agent for preventing or improving skin aging symptoms. 6. The agent for promoting intracellular NAD ⁺ production according to claim 5, wherein the skin aging symptoms are wrinkles, sagging, spots, freckles, rough skin, or dry skin. A cosmetic composition comprising the intracellular NAD ⁺ production promoter according to any one of claims 1 to 6 as an active ingredient. A cosmetic containing the cosmetic composition according to claim 7 as an active ingredient A composition for external use, comprising the intracellular NAD ⁺ production-enhancing agent according to any one of claims 1 to 6 as an active ingredient. An external preparation containing the composition for external use according to claim 9 as an active ingredient.

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