JP7748722B2 - PDE4 inhibitors - Google Patents

Description

The present invention relates to PDE (phosphodiesterase) 4 inhibitors, and more specifically to PDE4 inhibitors containing propolis.

Phosphodiesterase (PDE) is a general term for enzymes that hydrolyze phosphate diesters to phosphate monoesters, and there are 11 types of PDE (PDE1 to PDE11).

PDE breaks down and inactivates substances (second messengers) that play a role in transmitting information within cells. Preventing the breakdown of these second messengers is thought to be effective in treating a variety of diseases, and inhibitors (drugs) have been developed to suppress PDE. Among these, PDE4 breaks down cAMP (cyclic adenosine monophosphate) to produce AMP. PDE4 is present in many inflammatory cells, immune cells, the central nervous system, and other cells, and is involved in the production of substances that cause inflammation.

When there is a lot of PDE4, it can cause immune imbalances and inflammation, so it has been reported that PDE4 inhibitors, by suppressing the activity of PDE4, can restore the body's imbalance and suppress inflammation. For this reason, PDE4 inhibitors are being researched in relation to the treatment of chronic obstructive pulmonary disease (COPD) and rheumatoid arthritis. PDE4 inhibitors are currently used as treatments for psoriasis and atopic dermatitis.

Furthermore, PDE4 inhibitors are known to have cognitive-enhancing and neuroprotective effects, and are being studied for the treatment of central nervous system disorders such as depression, memory impairment, schizophrenia, Parkinson's disease, and Alzheimer's disease.

Compounds with PDE4 inhibitory activity have been reported in, for example, Non-Patent Document 1. Non-Patent Document 1 reports that 3-O-methylquercetin selectively and competitively inhibits PDE4, but that its inhibitory effect is not strong.

Propolis is a glue-like substance made by mixing tree sap, plant buds, and exudates collected by honeybees with beeswax and other materials, and is known to have antibacterial and antioxidant properties. Propolis, which contains natural antibacterial substances, is used in health foods and other products.

The components of propolis are influenced by the type and composition of the plant from which it is made, so it is known that the types and amounts of active ingredients contained in propolis, as well as their physiological activity, can vary greatly depending on the country or region from which it is collected.

In recent years, research has been progressing on the components contained in propolis by region of origin. For example, cinnamic acid derivatives such as p-coumaric acid, artepillin C, drupanin, and baccarin have been detected as the main components of Brazilian propolis, which originates primarily from Baccharis doracunclifolia. On the other hand, flavonoids such as chrysin and galangin have been detected as the main components of propolis from China, Europe, and Australia, which originates from poplar.

Planta Med. 2003 Apr;69(4):310-5.

The present invention aims to provide a PDE4 inhibitor containing propolis.

Since propolis is made from plants harvested by honeybees, its ingredients vary depending on the region of origin, and its effectiveness is therefore expected to vary depending on the region of origin. Therefore, the inventors conducted extensive research to achieve the above-mentioned objective, and discovered that propolis from the State of Bahia in the Federative Republic of Brazil (hereinafter sometimes abbreviated as Bahia propolis) contains three components: 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin, and that combining the two components, 3-O-methylquercetin and 3,6-dimethoxyapigenin, or the three components, 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin, can produce a synergistic effect in PDE4 inhibitory activity.

The present invention was completed based on these findings and through further investigation, and provides the following PDE4 inhibitors.

Item 1. A PDE4 inhibitor containing propolis produced in the state of Bahia, Federative Republic of Brazil.
Item 2. The PDE4 inhibitor according to Item 1, wherein the propolis contains 3-O-methylquercetin or a salt thereof, and at least one selected from the group consisting of 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof.
Item 3. The PDE4 inhibitor according to Item 1 or 2, which is used for improving or preventing at least one condition selected from the group consisting of dementia, Parkinson's disease, inflammatory diseases, psychiatric disorders, and tumor diseases.
Item 4. The PDE4 inhibitor according to Item 3, wherein the inflammatory disease is at least one selected from the group consisting of respiratory inflammatory diseases, skin inflammatory diseases, interstitial pneumonia, and rheumatism.
Item 5. The PDE4 inhibitor according to Item 3, wherein the psychiatric disorder is at least one selected from the group consisting of depression, memory impairment, and schizophrenia.
Item 6. The PDE4 inhibitor according to Item 3, wherein the tumor disease is colon cancer and/or acute lymphocytic leukemia.
Item 7. The PDE4 inhibitor according to Item 4, wherein the respiratory inflammatory disease is asthma and/or chronic obstructive pulmonary disease (COPD).
Item 8. The PDE4 inhibitor according to Item 4, wherein the inflammatory skin disease is psoriasis and/or atopic dermatitis.
Item 9. The PDE4 inhibitor according to any one of Items 1 to 8, which is a cosmetic, a food or drink, a pharmaceutical, or a quasi-drug.

Propolis from Bahia has excellent PDE4 inhibitory properties, making it useful as an active ingredient in PDE4 inhibitors.

In addition, propolis is a naturally derived ingredient, making it highly safe.

1 is a graph showing the PDE4 inhibition rate (%) at the equivalent of 50 μg/mL of Bahia propolis.

The following describes in detail the embodiments of the present invention.

In this specification, the term "comprise" encompasses both the meanings of "essentially consisting of" and "consist of only."

The PDE4 inhibitor of the present invention is characterized by containing propolis from the state of Bahia.

Propolis is a resinous or waxy substance that makes up the walls of beehives, and is made by mixing plant buds and resins collected by bees with their saliva. In the present invention, propolis produced in the state of Bahia is used. In addition to propolis produced in Bahia, propolis from other producing regions such as China, Europe, Oceania, and America may also be used in combination.

Propolis from the State of Bahia is not particularly limited as long as it is collected in the State of Bahia. Among these, propolis from the State of Bahia that contains at least one selected from the group consisting of 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof is preferred, with propolis containing 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof being more preferred. 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin are compounds each represented by the following structural formulas.

Salts of 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin are not particularly limited, and examples include inorganic salts with sodium, potassium, magnesium, calcium, aluminum, etc.; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine, ornithine, and arginine; and ammonium salts. These salts may also be acid addition salts, and specific examples of such salts include acid addition salts with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid. Furthermore, the 3-O-methylquercetin, 3,6-dimethoxyapigenin, and 6-methoxyapigenin and their salts in the present invention also include their hydrates, solvates, crystalline polymorphs, etc.

The propolis of the present invention includes, for example, raw propolis, its pulverized product, supercritical extract, and extract with water and/or hydrophilic organic solvent. Among these, extract with water and/or hydrophilic organic solvent and supercritical extract are preferred, with extract with water and/or hydrophilic organic solvent being more preferred. Extract with water and/or hydrophilic organic solvent is preferred because it extracts propolis components efficiently and in a balanced manner in a short time, and does not require large-scale equipment for scale-up. The propolis extract preferably contains at least one member selected from the group consisting of 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof, and more preferably contains 3-O-methylquercetin or a salt thereof, 3,6-dimethoxyapigenin or a salt thereof, and 6-methoxyapigenin or a salt thereof.

Examples of hydrophilic organic solvents include lower alcohols such as ethanol, methanol, and propanol, ketones (acetone, methyl ethyl ketone, methyl butyl ketone, etc.), esters (methyl acetate, ethyl acetate, etc.), glycols (ethylene glycol, propylene glycol, etc.), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, and dimethyl sulfoxide. Lower alcohols are preferred, with ethanol being particularly preferred.

These extraction solvents can be used alone, or two or more can be used in any combination as a mixture. Water, lower alcohol (preferably ethanol), or a mixture thereof (aqueous alcohol, preferably aqueous ethanol) is preferred. When aqueous alcohol (preferably aqueous ethanol) is used as the extraction solvent, the alcohol concentration is not limited, but is preferably 50 to 100% by volume, and particularly preferably 70 to 100% by volume.

Raw propolis blocks, alcohol-washed raw propolis blocks, etc. can be used for extraction, and extraction efficiency can be improved by using raw propolis blocks that have been cut or crushed to an appropriate size. The volume of extraction solvent used is, for example, 1 to 20 times, and preferably 2 to 10 times, the mass of the raw propolis blocks.

Extraction can be carried out by adding propolis to a solvent and heating it between 0°C and the boiling point of the solvent (usually a temperature below 100°C), preferably between 2 and 40°C. Extraction can be carried out at the above temperature for a specified period of time (for example, one hour or more), with stirring or by leaving the mixture to stand. After extraction, the extract can be obtained by filtering or other processes to remove insoluble matter.

The solvent extract thus obtained can be used as is, or, if necessary, can be further purified to increase the content of active ingredients by ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPLC), dialysis, or a combination of these.

The solvent extract can also be concentrated and dried by freeze-drying, spray-drying, or other methods. Concentration, freeze-drying, and spray-drying can be carried out according to conventional methods. The propolis extract of the present invention also includes powders obtained by powdering such extracts, and granules obtained by granulating the powders.

The PDE4 inhibitor of the present invention may contain other known ingredients as appropriate, provided that the effects of the present invention are not impaired.

The content of propolis in the PDE4 inhibitor of the present invention is not particularly limited as long as the effects of the present invention are obtained, and can be adjusted appropriately depending on the final form, etc. For example, the content of propolis relative to the total solid content of the PDE4 inhibitor is 0.00001% by mass or more, 0.0001% by mass or more, 0.0001% by mass or more, 0.001% by mass or more, 0.01% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, It may be 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, or 90% by mass or more, and 100% by mass or less, 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or more. Below, 55% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, 10% by mass or less, 7% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, or 0.5% by mass or less.

Propolis is preferably applied regularly to inhibit PDE4, with application once a day or in divided doses being particularly preferred. While not particularly limited, for example, propolis can be used at a daily dose of 1 μg to 1,000 mg in solid form for an adult weighing 60 kg, with 9 μg to 300 mg being preferred and 18 μg to 100 mg being more preferred. The dose can be set appropriately within the above range depending on factors such as the recipient's health condition, the method of administration, and the combination with other agents.

The PDE4 inhibitor of the present invention can be used as cosmetics, foods and beverages (particularly foods and beverages intended for health, health maintenance, or promotion (e.g., health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. The PDE4 inhibitor of the present invention also encompasses additives that impart PDE4 inhibitory activity.

In addition to the propolis, the above cosmetics may contain ingredients commonly used in cosmetics, such as disinfectants, preservatives, surfactants, alcohols, aqueous ingredients, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavorings, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), etc., as needed.

Cosmetics include any cosmetic product that is applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, etc. of animals (including humans).

Cosmetics can take a wide range of formulations, including aqueous solutions, solubilized solutions, emulsions, powders, oil solutions, gels, ointments, aerosols, water-oil two-layer systems, and water-oil-powder three-layer systems.

The use of cosmetics is also arbitrary. For example, basic cosmetics include facial cleansers, lotions, emulsions, creams, gels, essences, serums, packs, masks, etc.; makeup cosmetics include foundations, lipsticks, blushers, eye shadows, eyeliners, mascara, etc.; nail cosmetics include nail polish, base coats, top coats, nail polish removers, etc.; and other products include facial cleansers, (paste or liquid) dentifrices, mouthwashes, massage agents, cleansing agents, aftershave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, conditioners, hair treatments, hair styling products, hair tonics, hair growth agents, antiperspirants, bath additives, etc.

The propolis can be used as is in the above-mentioned foods and beverages, or, if necessary, can be blended with minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavorings, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solubilizers, humectants, etc.

Food and beverages include all foods and beverages that can be consumed by animals (including humans). The types of foods and beverages are not particularly limited, and examples include dairy products; fermented foods (yogurt, etc.); beverages (soft drinks such as coffee, juice, and tea drinks, dairy drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and alcoholic beverages such as fruit wine); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western sweets (chocolate, donuts, pies, cream puffs, gum, gummy candies, jelly, candy, cookies, cakes, puddings, etc.); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, popsicles, sorbet, etc.); foods (curry, beef bowls, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

The method for producing foods and beverages is not particularly limited, and any known method can be used as appropriate.

When used as a supplement, the dosage unit form is not particularly limited and can be selected as appropriate. Examples include chewable tablets, troches, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, etc.

The above-mentioned propolis can be used alone in the above medicines, or it can be mixed with other medicinal ingredients listed in the Japanese Pharmacopoeia, such as vitamins and herbal medicines.

When preparing the PDE4 inhibitor of the present invention as a pharmaceutical, the propolis can be prepared together with pharmaceutical acceptable ingredients into a pharmaceutical formulation such as tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, and lozenges), capsules, pills, powders (dispersed medicines), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, and injections (including when mixed with distilled water or infusions such as amino acid infusions or electrolyte infusions to prepare a liquid at the time of use).

Medicinal products may be administered locally or systemically. There are no particular limitations on the method of administration, and they may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intraarterial administration, and transdermal administration.

In addition to propolis, the pharmaceutical product of the present invention may contain, as needed, pharmaceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, antiseptics, stabilizers, surfactants, sweeteners, flavoring agents, and fragrances.

The cosmetics and pharmaceuticals of the present invention also include quasi-drugs.

The PDE4 inhibitors of the present invention described above are applicable to mammals, including humans (preferably humans).

The propolis produced in Bahia, which is contained as an active ingredient in the PDE4 inhibitor of the present invention, has PDE4 inhibitory activity, as demonstrated in the examples described below, and is therefore useful as an active ingredient in PDE4 inhibitors.

The PDE4 inhibitors of the present invention can be used to treat, improve, and prevent various abnormalities, disorders, and diseases caused by the presence of excessive PDE4. Examples of such abnormalities, disorders, and diseases include dementia (Alzheimer's disease, etc.), Parkinson's disease, respiratory inflammatory diseases (asthma, chronic obstructive pulmonary disease (COPD), etc.), inflammatory skin diseases (psoriasis, atopic dermatitis, etc.), interstitial pneumonia, inflammatory diseases such as rheumatism, psychiatric disorders (depression, memory impairment, schizophrenia, etc.), and neoplastic diseases (colon cancer, acute lymphocytic leukemia, etc.).

In addition, the PDE4 inhibitors of the present invention are highly safe because they contain naturally occurring active ingredients.

The present invention will be described in detail below using examples. However, the present invention is not limited to these examples in any way.

Test example : Identifying sample components
Propolis from Bahia, provided by Yamada Bee Farm Co., Ltd., was extracted with aqueous ethanol, and the components were analyzed by HPLC and LC-MS. The aqueous ethanol extract was also used to measure the PDE4 inhibitory activity described below.

As a result, the following compounds were identified as components of propolis from Bahia. Standard specimens of the identified components were purchased and the following activity tests were carried out.
3-O-methylquercetin: C ₁₆ H ₁₂ O ₇
3,6-dimethoxyapigenin: C ₁₇ H ₁₄ O ₇
6-Methoxyapigenin: C ₁₆ H ₁₂ O ₆
The quantitative values of each component in the propolis produced in Bahia were as follows:
3-O-methylquercetin: 1.6% by mass
3,6-dimethoxyapigenin: 3.2% by mass
6-Methoxyapigenin: 5.8% by mass

<Measurement of PDE4 inhibitory activity>
The PDE4B Assay Kit (BPS Bioscience) was used to evaluate the PDE4 inhibitory activity of various materials by fluorescence polarization. The procedure was performed according to the instruction manual included with the kit, but the volume was customized for a 384-well plate. Rolipram (IC ₅₀ 2 μg/mL) was used as a positive control.

The PDE4 inhibitory activity of Bahia propolis (BPP) and the PDE4 inhibitory activity of each component alone or in combination is shown in Table 1 and Figure 1 (comparison at 50 μg/mL BPP equivalent). From the results in Table 1, the main active component was 3-O-methylquercetin.

・Propolis from Bahia: BPP
3-O-methylquercetin: 3O
3,6-dimethoxyapigenin: 3,6DI
6-Methoxyapigenin: 6M

Claims (9)

A PDE4 inhibitor comprising propolis produced in the state of Bahia, Federative Republic of Brazil , which contains 3-O-methylquercetin or a salt thereof and 3,6-dimethoxyapigenin or a salt thereof . The PDE4 inhibitor according to claim 1, wherein the propolis further contains 6 -methoxyapigenin or a salt thereof. The PDE4 inhibitor according to claim 1 or 2, which is used for the improvement or prevention of at least one condition selected from the group consisting of dementia, Parkinson's disease, inflammatory diseases, psychiatric disorders, and tumor diseases. The PDE4 inhibitor according to claim 3, wherein the inflammatory disease is at least one selected from the group consisting of respiratory inflammatory diseases, skin inflammatory diseases, interstitial pneumonia, and rheumatism. The PDE4 inhibitor according to claim 3, wherein the psychiatric disorder is at least one selected from the group consisting of depression, memory impairment, and schizophrenia. The PDE4 inhibitor according to claim 3, wherein the tumor disease is colorectal cancer and/or acute lymphocytic leukemia. The PDE4 inhibitor according to claim 4, wherein the respiratory inflammatory disease is asthma and/or chronic obstructive pulmonary disease (COPD). The PDE4 inhibitor according to claim 4, wherein the inflammatory skin disease is psoriasis and/or atopic dermatitis. The PDE4 inhibitor according to claim 1 or 2, which is a cosmetic, food or beverage, pharmaceutical, or quasi-drug.