JP2014152117A - Immunostimulator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunostimulator useful for the prevention/treatment of a variety of diseases, on which immunopotentiation through the expression enhancing action for a type I interferon gene or interleukin-3 gene has effect.SOLUTION: A compound is represented by the general formula (I) or general formula (II).

Description

  The present invention relates to an immunostimulant useful as a prophylactic / therapeutic agent for various diseases, in which the immunostimulatory effect by the type I interferon gene expression enhancing action or interleukin 3 gene expression enhancing action is effective.

  In recent years, it has been clarified that endogenous interferon plays a central role in living body defense mechanisms against viral and microbial infections and also plays an important role in immune regulation in vivo. Since the technology for mass production of interferon has been established and natural interferon can be easily obtained from cultured cells, and mass production of interferon using microorganisms has become possible, many studies using these interferons have been conducted. Results have been accumulated. Specifically, interferon has been confirmed to have various biological effects such as antiviral action, cell growth inhibitory action, and immunomodulatory action, and clinically, therapeutic agents for viral diseases such as hepatitis B and C, Alternatively, it has already been put into practical use as a therapeutic agent for cancer and immune diseases. It has also been suggested that interferon has an effect of suppressing carcinogenesis in hepatitis B and C. Interferon is especially used for many of the above diseases because there is no other effective treatment.

  Known substances that induce biosynthesis of interferon include various animal viruses, microorganisms such as bacteria and protozoa, and extracts thereof, mitogens, specific antigens, and immunostimulants. For example, various natural double-stranded RNAs, synthetic double-stranded RNAs such as poly I: C, and anionic polymer compounds such as polyacrylic acid and chlorite oxyamylose have interferon-inducing activity. It is known. On the other hand, among low molecular compounds, fluorenones, pyrimidine derivatives, anthraquinones, acridine compounds and the like have been found to have an interferon-inducing action. However, when these compounds were used in clinical trials, they were successfully developed as interferon inducers due to unexpectedly low interferon-inducing ability and strong side effects or reduced interferon-inducing ability due to repeated administration. Not. In addition, imidazoquinolines are also known as low-molecular interferon inducers, but these compounds are known to have low interferon-selective ability and induce other cytokines simultaneously.

  On the other hand, interleukin 3 (IL-3) is a hematopoietic factor having a molecular weight of 20-32 kDa known as multi-CSF. IL-3 is produced by activated T cells, mast cells, and eosinophils. The most significant biological activity of IL-3 is to stimulate the differentiation of hematopoietic cells, and to promote the differentiation of erythrocytes, megakaryocytes, granulocytes, macrophages, basophils, neutrophils, eosinophils, mast cells, and monocytic cells. It is known to lead to colony formation. From these effects, enhancement of IL-3 production ability leads to enhancement of basic immune activity, and is considered to be effective for various infectious diseases. However, a component that specifically increases IL-3 has not been found, and a safe and effective component has been demanded.

  Incidentally, diterpenes are compounds containing four isoprene units, and diterpenes having various structures are naturally distributed. Diterpenes having a labdan-type basic skeleton are known to be contained in plants of the family Ginger, and Patent Document 1 discloses Gyogatrial, which is a labdan-type diterpene aldehyde contained in the ginger of the family Ginger. It has been disclosed to have antibacterial action, platelet aggregation inhibitory action, and 5-lipoxygenase inhibitory action. Patent Document 2 discloses the possibility that galanal contained in Albinia galanga, a ginger family plant, becomes an antitumor agent. Patent Document 3 discloses that an extract from an inflorescence or cocoon of myoga exhibits an antihypertensive action, a central nerve inhibitory action, a cholinergic nerve excitatory action, a cardiac inhibitory action, and an anti-inflammatory action. However, labdan-type diterpene aldehyde or Ginger plant extract enhances the expression of type I interferon gene or interleukin 3 gene, increases the production ability of type I interferon or interleukin 3, and has an immunostimulatory effect. That has never been known.

JP 2006-241068 A JP 63-162644 A JP-A-55-009021

  An object of the present invention relates to providing an immunostimulant useful for prevention / treatment of various diseases, in which an immunostimulatory action by an expression enhancing action of a type I interferon gene or an interleukin 3 gene is effective.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that a labdan-type diterpene aldehyde having a specific structure isolated from a ginger of a ginger family plant is a type I interferon gene and an interleukin 3 gene. Various diseases (virus infections, microbial infections, viruses) that have an expression enhancing action, specifically increase the ability to produce type I interferon and interleukin 3 in vivo, and these gene expression enhancing actions are effective And the present invention has been completed.

That is, this invention is an immunostimulant which contains the compound or its salt represented by the following general formula (I), or the compound or its salt represented by the following general formula (II) as an active ingredient.

  The present invention is also an anti-infective, viral hepatitis preventive / therapeutic agent, food or drink or animal feed containing the immunostimulant.

  According to the immunostimulant of the present invention, various diseases in which type I interferon or interleukin 3 is effective while maintaining the functions and homeostasis of animals including humans (particularly mammals) such as virus infections, microorganisms Infectious diseases, viral hepatitis, viral encephalitis and the like can be prevented or treated. In addition, the active ingredient of the immunostimulant of the present invention is an ingredient contained in myoga that has been edible since ancient times, and since it has no harmful side effects and is highly safe even when continuously taken, the immunity of the present invention The activator can be used not only as a medicament such as an anti-infective agent and a viral hepatitis preventive / therapeutic agent, but also as a food and drink and animal feed.

FIG. 1 is a graph showing changes in the expression level of type I interferon genes in human-derived cells. FIG. 2 is a graph showing changes in the expression level of interleukin 3 gene in mouse-derived cells.

  The aging inhibitor of the present invention is a compound represented by the above general formula (I) (hereinafter also referred to as compound I) or a salt thereof, or a compound represented by the above general formula (II) (hereinafter also referred to as compound II). Or a salt thereof as an active ingredient. The aging inhibitor of the present invention may contain only one of “Compound I or a salt thereof” and “Compound II or a salt thereof”, or may contain both. In addition, the aging inhibitor of the present invention may be in the form of only an active ingredient (Compound I or II or a salt thereof), or may be in the form of containing other ingredients in addition to the active ingredient.

Compounds I and II, which are active ingredients of the immunostimulant of the present invention, are both labdan-type diterpene aldehydes. Compound I is (4aS, 6aS, 7S, 11aR, 11bS) -4,4,11b-trimethyl-7-hydroxy-1,2,3,4,4a, 5,6,7,8,11,11a, 11b-dodecahydro-6aH-cyclohepta [a] naphthalene-6a, 9-dicarbaldehyde [CAS registration number: 104086-74-0, chemical formula: C ₂₀ H ₃₀ O ₃ , common name: Galalanal A], Or (4aS, 6aS, 7R, 11aR, 11bS) -4,4,11b-trimethyl-7-hydroxy-2,3,4,4a, 5,6,6a, 7,8,11,11a, 11b-dodecahydro -1H- cyclohepta [a] naphthalen -6A, 9-di-carbaldehyde [CAS Registry number: 104113-52-2, chemical formula: C ₂₀ H ₃₀ O _3, trivial name: Garanaru B ( Galanal B)], or a mixture (including a racemate) thereof, and has a chemical structure represented by the above general formula (I). Compound II is (8S) -14,20-cyclolabdan-12,14 (20) -diene-15,16-dial [chemical formula: C ₂₀ H ₂₈ O ₂ , common name: Mioganal], It has the chemical structure shown in the general formula (II).

  The type of the salt of Compound I and the salt of Compound II is not particularly limited, but specific examples include, for example, addition salts of alkali metals such as sodium salts and potassium salts; alkaline earth metals such as magnesium salts and calcium salts And addition salts thereof. Preferred is a pharmaceutically acceptable salt.

  Compounds I and II can be obtained by isolation (extraction / purification) from ginger family plants such as ginger by a known method. That is, the active ingredient of the immunostimulant of the present invention may be a ginger plant extract. However, the compounds I and II and salts thereof used in the present invention are not limited to such plant-derived natural substances, and are chemically synthesized substances produced using known chemical synthesis techniques or gene recombination techniques. Alternatively, it may be a genetically modified substance. When compounds I and II are isolated from plants, compound I can be isolated from plants according to, for example, the method described in Patent Document 2, and compound II can be isolated from, for example, Biosci. Biotechnol. Biochem (72) 10, 2181-2186, 2008. It can be isolated from a plant according to the method described in 2008.

  Examples of the plant containing the compounds I and II include plants belonging to the family Ginger, and more specifically, ginger of the genus Ginger, ginger genus (scientific name: Alpinia galanga), hanamyoga, ghetto etc. Is mentioned. Among these ginger plants, in particular, ginger (especially the edible flower buds) and Nankyo (especially the rhizomes that are edible parts) contain relatively large amounts of compounds I and II. Therefore, it is preferably used in the present invention as these sources.

  Compounds I and II have an action of enhancing the expression of type I interferon gene, as is clear from the test results described in the following [Examples] section, and specifically increase the ability to produce type I interferon in vivo. In addition, it has an interleukin 3 gene expression enhancing action and can specifically increase the ability to produce interleukin 3 in vivo. Therefore, the immunostimulant of the present invention containing compound I or II or a salt thereof exhibits an excellent immunostimulatory effect due to the above-described gene expression enhancing effect, and type I interferon or interleukin 3 is effective for prevention / treatment. It is possible to prevent various diseases and conditions that act, such as viral infections, microbial infections, viral hepatitis, viral encephalitis, etc., and to treat and improve these various diseases that have already occurred. Can also be used.

  In addition, since compounds I and II are components contained in ginger family plants such as myoga and nankyo with abundant dietary experience, there is a concern that even if ingested continuously for a long period of time, they may cause harmful effects on the living body. It is a low and safe compound. Therefore, the immunostimulant of the present invention containing Compound I or II or a salt thereof can be safely and continuously ingested not only by healthy persons and adults but also by elderly persons and sick persons, In addition to pharmaceuticals for humans or animals, they are useful as human foods and drinks or animal feeds, or as raw materials for producing them. The present invention includes pharmaceuticals, foods and drinks, and animal feeds containing the immunostimulant of the present invention described above. As used herein, “animal” includes mammals other than humans.

  Examples of the medicament of the present invention include an anti-infective agent and a viral hepatitis preventive / therapeutic agent in addition to the above-described immunostimulant itself. The dosage form of the pharmaceutical immunostimulant and the anti-infective and viral hepatitis preventive / treating agent containing the same is not particularly limited, and includes all dosage forms that can contain Compound I or II or a salt thereof. Oral preparations such as tablets, capsules, granules, powders, syrups, dry syrups, solutions, suspensions, etc .; parenterals such as inhalants, transdermal preparations, suppositories, drops, injections, etc. Can be mentioned.

  The medicament of the present invention contains, as necessary, various pharmaceutically acceptable carriers such as excipients, stabilizers, other additives, etc., in addition to the active ingredient Compound I or II or a salt thereof. Or may contain other medicinal ingredients such as various vitamins, minerals, herbal medicines and the like. The medicament of the present invention can be produced by blending the above-mentioned carrier and other medicinal ingredients with Compound I or II or a salt thereof, or a plant extract containing these, according to a conventional method.

  In addition, as the food and drink and animal feed of the present invention, for example, health foods, functional foods and drinks, and specified health indications that are intended to prevent or improve infection with viruses and microorganisms, and improve viral hepatitis. Food and drink for children, food and drink for the sick, feed for domestic animals, racehorses, appreciation animals, and pet food. The form of the food and drink and animal feed of the present invention is not particularly limited, and includes all forms that can contain Compound I or II or a salt thereof, and can be, for example, solid, semi-solid, or liquid, or , Tablets, chewable tablets, powders, capsules, granules, drinks, gels, syrups, liquid foods for enteral nutrition.

  Specific examples of the form of the food and drink according to the present invention include tea drinks such as green tea, oolong tea, and tea, coffee drinks, soft drinks, jelly drinks, sports drinks, milk drinks, carbonated drinks, fruit juice drinks, lactic acid bacteria drinks, and fermented milk drinks. , Powdered beverages, cocoa beverages, alcoholic beverages, beverages such as purified water, butter, jam, sprinkles, margarine spreads, mayonnaise, shortening, cream, dressings, breads, cooked rice, noodles, pasta, miso soup, tofu , Milk, yogurt, soups or sauces, confectionery (for example, biscuits and cookies, chocolate, candy, cake, ice cream, chewing gum, tablets). In addition, since animal feed can be utilized with a composition and form substantially the same as food and drink, the description regarding the food and drink in this specification is similarly applied to animal feed unless otherwise specified.

  The food and drink and the animal feed of the present invention are compounds I or II or salts thereof, or other foods or other animal feeds that are usually eaten by plant extracts containing these compounds. Food / beverage materials, various nutrients, various vitamins, minerals, amino acids, various fats and oils, various additives (eg, flavoring ingredients, sweeteners, acidulants such as organic acids, surfactants, pH adjusters, stabilizers, antioxidants) Agent, pigment, flavor) and the like, and can be produced according to a conventional method. Alternatively, the food / drink or animal feed of the present invention can be obtained by adding Compound I or II or a salt thereof, or a plant extract containing these to a general food / drink or animal feed that is usually eaten. Can be manufactured.

  The content of compound I or II, or a salt thereof, which is an active ingredient in the pharmaceutical, food or drink or animal feed of the present invention (when both compounds are contained, the total content of each compound) has a desired effect. Any amount can be used as long as it can be expected, and can be appropriately changed according to the pharmaceutical dosage form, the form of food or drink or animal feed, the species, symptoms, age, sex, etc. of the individual to be administered or ingested. When targeting humans, the dose of compound I or II or a salt thereof (when both compounds are contained, the total content of each compound) is usually from 10 mg to 1 adult (in terms of 60 kg) per day. It can be 10 g. The daily dose may be administered once or divided into several times. It is desirable that the pharmaceutical dosage form or administration regimen of the present invention, or the form of the food or drink or animal feed of the present invention be in a form that allows the dose to be appropriately managed.

  Examples are given to specifically describe the present invention, but the present invention is not limited by the following examples.

[Examples 1 and 2]
In accordance with the following (isolation method of active ingredients), the compound represented by the above general formula (I) (galana racemate) and the compound represented by the above general formula (II) from the ginger of the ginger family plant ( (Myoganal) was isolated, and the former was designated as Example 1 and the latter as Example 2.

(Method for isolating active ingredients)
1. Extraction treatment Fresh florets were prepared, washed thoroughly with water, drained, and sliced to obtain a sample for extraction. After adding 3 times the amount of hexane to 600 g of this extraction sample and homogenizing, extraction processing (first extraction processing) was performed for 30 minutes while stirring on a magnetic stirrer at normal temperature and normal pressure. The solid content obtained in the first extraction process and the extract are separated, and the solid content is further extracted with hexane under the same conditions (second extraction process), and obtained in the first extraction process. The extract obtained by the second extraction process was mixed.
2. Purification process The extract obtained by the above extraction process was filtered and dehydrated, and concentrated and dried with a rotary evaporator. Dissolved in 2 ml of 15% acetone-hexane and equilibrated with hexane, silica gel 7729 (Merck) flash chromatography. Applied to 50 g column. Thereafter, elution was performed with 300 ml of 15% acetone-hexane. The eluted fraction was concentrated to 1/10 volume by a rotary evaporator and purified by preparative HPLC (manufactured by Shimadzu Corporation). Regarding preparative HPLC, silica-60 (TOSOH) 7.8 × 300 mm was used as a column, and 3.5% acetone-hexane was used as a mobile phase at a flow rate of 4.0 ml / min. The detection wavelength was 232 nm. The compound represented by the above general formula (I) (galanar racemate) was obtained by referring to the HPLC elution pattern of a standard product separately isolated by the method described in Patent Document 2, and the above general formula (II) The fraction represented by the formula (myoganal) was prepared by referring to the HPLC elution pattern of a standard product separately isolated by the method described in Biosci. Biotechnol. Biochem (72) 10, 2181-2186, 2008. Sorted. The collected fraction was concentrated to dryness under a nitrogen stream to obtain an oily substance having a purity of 95 to 98%.

[Test Example 1: Type I interferon (IFNβ-1) gene expression confirmation test]
As a cell, human-derived monocyte-like cell THP-1 was used. Cells were dispensed in 0.5 ml aliquots on a culture plate at 1 × 10 ⁶ cells / ml per well and cultured in RPMI 1640 medium containing 10% FBS. Example 1 (galanar racemate) or Example 2 (myoganal) was added as a test substance so that the final concentrations would be 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and no test substance was added. The cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas for 6 hours. Total RNA is extracted from the cultured cells using TaKaRa FastPure (registered trademark) RNA Kit (manufactured by Takara Bio Inc.), and reverse transcription reaction is performed using PrimeScript RT reagent Kit (manufactured by Takara Bio Inc.) using this total RNA as a template. To prepare cDNA. The amount of mRNA was quantified by quantitative PCR using the prepared cDNA as a template. For quantitative PCR, SYBR (registered trademark) Premix Ex Taq ^™ II (manufactured by Takara Bio Inc.) was used, and 2-step PCR at 95 ° C. for 5 seconds and 60 ° C. for 30 seconds was used and measurement was performed at N = 2. Primers having the following sequences were used at a final concentration of 0.4 μM. The quantitative result was corrected with the quantitative result of β-actin. The results are shown in FIG.
Human IFNβ-1: 5'-CATCTAGCACTGGCTGGAATGAG-3 '/ 5'-TCCAGGACTGTCTTCAGATGGTTTA-3'
β-Actin: 5'-TGCACCACACCTTCTACAATGA-3 '/ 5'-CAGCCTGGATAGCAACGTACAT-3'

  As is apparent from FIG. 1, the galanal racemate (Example 1) added group and the myoganal (Example 2) added group were compared with the control without addition of these test substances (negative control). IFNβ-1) gene expression was significantly increased. From this, it is clear that the compound represented by the above general formula (I) (galanar) and the compound represented by the above general formula (II) (myoganal) have an action of enhancing expression of type I interferon gene. It was confirmed that type I interferon is promising as a prophylactic / therapeutic agent for various mammalian diseases.

[Test Example 2: Interleukin 3 (IL-3) gene expression confirmation test]
Mouse-derived WEHI-3 cells were used as the cells. Cells were dispensed in 0.5 ml aliquots on a culture plate at 1 × 10 ⁶ cells / ml per well and cultured in RPMI 1640 medium containing 10% FBS. Example 1 (galanar racemate) or Example 2 (myoganal) was added as a test substance to a final concentration of 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and no test substance added cells The cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas for 6 hours. Total RNA is extracted from the cultured cells using TaKaRa FastPure (registered trademark) RNA Kit (manufactured by Takara Bio Inc.), and reverse transcription reaction is performed using PrimeScript RT reagent Kit (manufactured by Takara Bio Inc.) using this total RNA as a template. To prepare cDNA. The amount of mRNA was quantified by quantitative PCR using the prepared cDNA as a template. For quantitative PCR, SYBR (registered trademark) Premix Ex Taq ^™ II (manufactured by Takara Bio Inc.) was used, and 2-step PCR at 95 ° C. for 5 seconds and 60 ° C. for 30 seconds was used and measurement was performed at N = 2. Primers having the following sequences were used at a final concentration of 0.4 μM. The quantitative result was corrected with the quantitative result of β-actin. The results are shown in FIG.
Mouse IL-3: 5'-ACCGTTTAACCAGAACGTTGAATTG-3 '/ 5'-TCCACGAATTTGGACAGGTTTACTC-3'
β-Actin: 5'-TGCACCACACCTTCTACAATGA-3 '/ 5'-CAGCCTGGATAGCAACGTACAT-3'

  As apparent from FIG. 2, the galanal racemate (Example 1) added group and the myoganal (Example 2) added group had interleukin 3 (as compared to the control without addition of the test substance (negative control)). IL-3) gene expression was significantly increased. From this, it is clear that the compound represented by the general formula (I) (galanal) and the compound represented by the general formula (II) (myoganal) have an interleukin 3 gene expression enhancing action. Thus, it was confirmed that interleukin 3 is promising as a prophylactic / therapeutic agent for various mammalian diseases.

Claims (7)

An immunostimulant comprising a compound represented by the following general formula (I) or a salt thereof or a compound represented by the following general formula (II) or a salt thereof as an active ingredient.

  The immunostimulant according to claim 1, which has an action of enhancing expression of type I interferon gene.   The immunostimulant according to claim 1 or 2, which has an interleukin 3 gene expression enhancing action.   The immunostimulant according to any one of claims 1 to 3, wherein the active ingredient is an extract of a ginger family plant.   The anti-infective agent containing the immunostimulant of any one of Claims 1-4.   Viral hepatitis preventive / therapeutic agent containing the immunostimulant according to any one of claims 1 to 4.   The food-drinks or animal feed containing the immunostimulant of any one of Claims 1-4.

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