JP2023147757A - Anti-influenza virus agents - Google Patents

Abstract

To provide materials allowing for safe administration, prophylaxis of influenza infection and amelioration of influenza symptoms, and to provide anti-influenza virus agents using the materials.SOLUTION: The disclosure provides an anti-influenza virus agent comprising at least one selected from β-nicotinamide mononucleotide, pharmaceutically acceptable salts and solvates thereof as an active constituent and having an activity to inhibit the proliferation of influenza virus in the lung. The invention also provides the anti-influenza virus agent that is administered to prevent infection of influenza virus, or to ameliorate influenza symptoms. Pharmaceutical compositions, health supplementary food and feed are also provided.SELECTED DRAWING: None

Description

The present invention relates to a material that can prevent influenza infection or improve the symptoms of influenza infection, and pharmaceuticals and health supplements using the material.

Influenza is a respiratory tract infection caused by the influenza virus, and causes epidemics every year. In addition to being highly contagious, elderly patients and patients with underlying diseases such as diabetes are more likely to develop severe symptoms, so there is a need for the development of more effective anti-influenza virus agents. In addition, since influenza epidemics occur almost every year, there is a need for an anti-influenza virus agent that can be safely taken over a long period of time.

For example, Patent Document 1 states that lactic acid bacteria produced by Lactobacillus genus lactic acid bacteria and Thermophilus bacteria, which have been widely ingested as yogurt-producing bacteria, suppress the decline in acquired immune function caused by the administration of anti-influenza drugs. It has been disclosed that the lactic acid bacteria product is useful as an anti-influenza virus agent. Further, Patent Document 2 reports that retinoid compounds such as β-carotene, which are contained in vegetables such as carrots and have been ingested by humans since ancient times, have preventive and therapeutic effects against influenza.

On the other hand, nicotinamide mononucleotide (NMN) is a biosynthetic intermediate metabolite of the coenzyme NAD ⁺ . In recent years, it has been reported that NMN has the effect of improving insulin secretion ability in aging mice, and the effect of dramatically improving insulin sensitivity and secretion in mouse models of type 2 diabetes caused by high-fat diet and aging (for example, patent literature (Refer to 3.), it has been reported that it has the effect of significantly enhancing mitochondrial function in aging muscles. Furthermore, it has been reported that by culturing pluripotent stem cells in the presence of NMN, their proliferation ability and differentiation ability can be improved (see, for example, Patent Documents 4 and 5).

Patent No. 6777911 JP2021-91627A US Patent No. 7737158 International Publication No. 2018/143258 International Publication No. 2019/182044

An object of the present invention is to provide a material that can be safely ingested and has the effect of preventing influenza infection and improving symptoms.

As a result of intensive research aimed at solving the above problems, the present inventors found that when mice that were orally ingested with β-NMN were infected with influenza virus, the proliferation of influenza virus in the lungs was significantly suppressed. They discovered that lung lesions can also be suppressed and completed the present invention.

That is, the present invention provides the following drugs and the like.
[1] The active ingredient is one or more selected from β-nicotinamide mononucleotide, its pharmacologically acceptable salts, and their solvates, and has the effect of suppressing the proliferation of influenza viruses in the lungs. An anti-influenza virus agent.
[2] The anti-influenza virus agent according to [1] above, which is administered to prevent influenza infection or to improve symptoms of influenza infection.
[3] The anti-influenza virus agent according to [1] or [2] above, which is orally administered.
[4] A pharmaceutical composition containing the anti-influenza virus agent according to any one of [1] to [3] above, which is administered to prevent influenza infection or to improve symptoms of influenza infection. thing.
[5] A health supplement containing the anti-influenza virus agent according to any one of [1] to [3] above, which is ingested to prevent influenza infection or improve symptoms of influenza infection. .
[6] Feed containing the anti-influenza virus agent according to any one of [1] to [3] above, which is ingested for preventing influenza infection or improving symptoms of influenza infection.
[7] A method for preventing influenza infection, which comprises orally administering the anti-influenza virus agent according to any one of [1] to [3] above.
[8] A method for improving symptoms of influenza infection, which comprises orally administering the anti-influenza virus agent according to any one of [1] to [3] above.

The anti-influenza virus agent according to the present invention contains β-NMN, which originally exists in the body, as an active ingredient, and can significantly suppress the proliferation of influenza viruses in the lungs. Therefore, pharmaceutical compositions, health supplements, and feed containing the anti-influenza virus agent according to the present invention can be safely ingested without causing side effects, and can suppress the incidence of influenza virus infection. , symptoms can be improved.

FIG. 2 is a diagram showing the evaluation results of the general condition of the 500 mg/kg NMN administration group, the 1000 mg/kg NMN administration group, and the control group (group to which NMN was not administered) in Example 1. FIG. 2 is a diagram showing the scores of macroscopic findings of the lungs of the 500 mg/kg NMN administration group, the 1000 mg/kg NMN administration group, and the control group in Example 1. FIG. 2 is a diagram showing the measurement results of the number of viruses in the lungs of the 00 mg/kg NMN administration group, the 1000 mg/kg NMN administration group, and the control group in Example 1.

The anti-influenza virus agent according to the present invention contains NMN (chemical formula: C ₁₁ H ₁₅ N ₂ O ₈ P) as an active ingredient and has the effect of suppressing the proliferation of influenza viruses in the lungs. The greater the amount of influenza virus in the lungs, the more cytokines and the like are secreted excessively due to the large amount of antigens, making it easier to develop influenza, and when symptoms occur, symptoms such as inflammation are more likely to worsen. Anti-influenza virus agents containing NMN as an active ingredient can prevent influenza infections or improve symptoms of influenza infections by suppressing the proliferation of influenza viruses in the lungs. Symptoms of influenza infection that are improved by NMN include, in particular, pulmonary inflammatory symptoms.

There are two types of NMN, α and β, as optical isomers, and the NMN that is the active ingredient of the chromosome stabilizer according to the present invention is β-NMN (CAS number: 1094-61-7). . The structure of β-NMN is shown below.

β-NMN used as an active ingredient may be prepared by any method. For example, artificially synthesized β-NMN purified by chemical synthesis, enzymatic methods, fermentation methods, etc. can be used as the active ingredient. Furthermore, since β-NMN is a component widely present in living organisms, β-NMN obtained by extraction and purification from natural raw materials such as animals, plants, and microorganisms can also be used as an active ingredient. Alternatively, commercially available purified β-NMN may be used.

As a chemical synthesis method for synthesizing β-NMN, for example, β-NMN can be produced by reacting NAM with L-ribose tetraacetate and phosphorylating the obtained nicotinamide mononucleoside. Furthermore, as an enzymatic method, for example, β-NMN can be produced from NAM and 5'-phosphoribosyl-1'-pyrophosphate (PRPP) using nicotinamide phosphoribosyltransferase (NAMPT). As a fermentation method, for example, β-NMN can be produced from NAM using the metabolic system of a microorganism expressing NAMPT.

The active ingredient of the anti-influenza virus agent according to the present invention may be a pharmacologically acceptable salt of β-NMN. The pharmacologically acceptable salt of β-NMN may be an inorganic acid salt or an organic acid salt having a basic moiety such as an amine. Examples of acids constituting such acid salts include acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, and isethion. Acids include lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and the like. Furthermore, the pharmacologically acceptable salt of β-NMN may be an alkali salt or an organic salt having an acidic moiety such as a carboxylic acid. Examples of bases constituting such acid salts include alkali metal salts or alkaline earth metal salts such as sodium hydride, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, and magnesium hydroxide. , zinc hydroxide, ammonia, trimethylammonia, triethylammonia, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine, diethylamine, piperazine, tris( Examples include those derived from bases such as hydroxymethyl)-aminomethane and tetramethylammonium hydroxide.

The active ingredient of the anti-influenza virus agent according to the present invention may be free β-NMN or a solvate of a pharmacologically acceptable salt of β-NMN. Examples of the solvent that forms the solvate include water, ethanol, and the like.

The anti-influenza virus agent according to the present invention may contain other active ingredients in addition to β-NMN. The number of other active ingredients used together with β-NMN may be one type, or a combination of two or more types. Such other active ingredients include, for example, albumin, ascorbic acid, α-tocopherol, insulin, transferrin, sodium selenite, ethanolamine, and Rock inhibitors, which are known to increase the survival efficiency and proliferation efficiency of stem cells. Ingredients known to increase the differentiation efficiency of stem cells, such as valproic acid, dimethyl sulfoxide, dexamethasone, butyric acid, trichostatin A, GSK3 inhibitors, BMP inhibitors, Wnt inhibitors, activin, and noggin. They can be appropriately selected and used.

The anti-influenza virus agent according to the present invention may consist only of active ingredients or may contain other ingredients. For example, the anti-influenza virus agent according to the present invention can be formulated into various dosage forms using conventional pharmaceutical methods by combining the active ingredient with a non-toxic pharmaceutical carrier. Among the dosage forms of the disease prevention agent and symptom improving agent, examples of oral administration include solid preparations such as tablets, granules, powders, capsules, and soft capsules; liquid preparations such as solutions, suspensions, and emulsions. ; Examples include lyophilized preparations. Examples of parenteral agents include injections, suppositories, sprays, transdermal absorption agents, and the like. The dosage form of the anti-influenza virus agent according to the present invention is preferably one suitable for oral administration.

Examples of non-toxic pharmaceutical carriers used in formulation include sugars such as glucose, lactose, sucrose, fructose, and reduced maltose; starch, hydroxyethyl starch, dextrin, β-cyclodextrin, crystalline cellulose, hydroxypropyl cellulose, etc. Carbohydrates; sugar alcohols such as mannitol, erythritol, sorbitol, xylitol; esters such as fatty acid glycerides and polyoxyethylene sorbitan fatty acid ester; polyethylene glycol, ethylene glycol, amino acids, albumin, casein, silicon dioxide, water, physiological saline, etc. Can be mentioned. In addition, in formulating the anti-influenza virus agent according to the present invention, stabilizers, lubricants, wetting agents, emulsifiers, suspending agents, binders, disintegrants, solvents, solubilizing agents, solubilizing agents, etc. Conventional additives such as agents, buffering agents, tonicity agents, preservatives, flavoring agents, and coloring agents can be added as appropriate.

The pharmaceutical composition according to the present invention contains the anti-influenza virus agent according to the present invention in an amount sufficient to exhibit its influenza virus growth suppressing effect. The pharmaceutical composition according to the present invention may contain only the anti-influenza virus agent according to the present invention as an active ingredient, or may contain other active ingredients. Examples of the other active ingredients include various ingredients such as ingredients that have an effect of enhancing immunity and ingredients that suppress inflammation. Specifically, grain-derived alkylresorcinol, yeast-derived β-glucan, agarisque, fucoidan, propolis, arabinoxylan, lactoferrin, mucin, rutin, isoflavone, spiruna, polyphenol, sesamin, allicin, astaxanthin, curcumin, catechin, chitosan, Examples include chitosan oligosaccharide, chitin oligosaccharide, L-ascorbic acid, and coenzyme Q ₁₀ .

The pharmaceutical composition according to the present invention, like the anti-influenza virus agent according to the present invention, can be formulated into various dosage forms by combining the active ingredient with a non-toxic pharmaceutical carrier and using conventional pharmaceutical methods. . The pharmaceutical composition according to the present invention can be formulated in the same manner as described above. The pharmaceutical composition according to the present invention is preferably one that is administered orally.

The anti-influenza virus agent according to the present invention and the pharmaceutical composition containing the same as an active ingredient are preferably administered to humans and non-human animals. Examples of non-human animals include mammals such as cows, pigs, horses, sheep, goats, donkeys, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs. The anti-influenza virus agent and the pharmaceutical composition containing the same as an active ingredient according to the present invention are preferably administered to and ingested by humans, livestock, laboratory animals, and pets; It is more preferable that the

The anti-influenza virus agent according to the present invention and the pharmaceutical composition containing the same as an active ingredient may be administered or ingested in any amount that is effective, and the species, age, weight, symptoms, and disease severity of the animal to be administered. be selected and determined as appropriate depending on the administration schedule, formulation form, etc. The effective amount of the anti-influenza virus agent may be any amount that can reduce the amount of influenza virus in the body of the animal to which it has been administered, compared to when it has not been administered. For example, the daily dose per adult is 0.1 mg to 10 g, preferably 0.5 mg to 7 g, more preferably 10 mg to 5 g, even more preferably 100 mg to 2 g, once or It can be administered in several doses.

Since β-NMN is a biological component and is also contained in foods, it is considered to be highly safe. Therefore, the anti-influenza virus agent according to the present invention can be used as an active ingredient of a health supplement that is taken to prevent influenza infection or improve symptoms of influenza infection. Health supplements are foods and drinks that support nutrition for the purpose of maintaining or improving health conditions, and also include foods for specified health uses, foods with nutritional function functions, and health foods.

Furthermore, the anti-influenza virus agent according to the present invention can be used as an active ingredient in feed ingested by animals in order to prevent influenza infection or improve the symptoms of influenza infection. By giving this feed to livestock, pets, laboratory animals, etc., it is possible to suppress the proliferation of influenza viruses in the lungs of the animals that have ingested it, thereby preventing influenza infection and preventing symptoms after onset. can be improved.

The health supplements and feeds according to the present invention can be prepared into edible forms such as powder, granules, granules, tablets, etc. by adding appropriate auxiliaries to β-NMN etc. using conventional means. It can be manufactured by forming into capsules, soft capsules, pastes, etc. The health supplement according to the present invention may be eaten as is, or mixed with various foods and drinks. For example, powdered health supplements can be ingested in a state in which they are dissolved or dispersed in beverages such as water, alcoholic beverages, fruit juice, milk, and soft drinks. The feed according to the present invention may be ingested by animals as it is, or may be mixed with other solid feed or drinking water.

The health supplement and feed according to the present invention can contain other food materials and various additives. Examples of food materials include vitamins, carbohydrates, proteins, lipids, dietary fibers, fruit juices, and the like. Specifically, for example, vitamin B group vitamins such as vitamin B ₁ derivatives, vitamin B ₂ , vitamin B ₆ , vitamin B ₁₂ , vitamin B ₁₃ , biotin, pantothenic acid, nicotinic acid, and folic acid; vitamin E, vitamin D, or the like; Derivatives, fat-soluble vitamins such as vitamin K ₁ , vitamin K ₂ , β-carotene; minerals such as calcium, potassium, iron, zinc; yeast, L-carnitine, creatine, α-lipoic acid, glutathione, glucuronic acid, taurine, collagen , soy isoflavones, lecithin, peptides, amino acids, γ-aminobutyric acid, diacylglycerol, DHA, EPA, capsaicin, chondroitin sulfate, agaricus mushroom extract, carrot extract, garlic extract, green juice, lecithin, royal jelly, propolis, octacosanol, flavange Examples include nol, pycnogenol, maca, chitosan, garcinia extract, chondroitin, and glucosamine. Examples of additives include sweeteners, acidulants such as organic acids, stabilizers, fragrances, colorants, and the like.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1]
Using a mouse influenza virus infection model, the effectiveness of NMN was evaluated using the general condition score, macroscopic findings in the lungs, and the number of viruses in the lungs as indicators.

<NMN>
A required amount of NMN (manufactured by Oriental Yeast Industry Co., Ltd.) powder was weighed using an electronic balance, and dissolved using water for injection to a concentration of 100 or 50 mg/mL. The prepared NMN solution was stored under refrigerated conditions (2.0 to 8.0°C) and protected from light, and used within 8 days after preparation.

<Influenza virus>
The influenza virus used in the following examples was influenza A virus PR8 (A/PR/8/34, H1N1 subtype) unless otherwise specified. The virus was grown in MDCK cells. In addition, the virus titer was measured by plaque assay using MDCK cells.
The stock solution of the inoculum virus that had been frozen was thawed at the time of use and adjusted to 2×10 ⁴ PFU/mL using DMEM, which was used as the inoculum virus solution.

<Mouse>
The mice used in the following examples are BALB/c strain (BALB/c Cr Slc) SPF mice (purchased from Japan SLC) unless otherwise specified. The mice used were 4-week-old female mice (13.5 to 17.5 g) at the start of breeding.

<Administration of MNM to mice and influenza virus infection>
NMN (500 mg/kg or 1000 mg/kg) was orally administered to mice once a day from 14 days before influenza virus inoculation to 4 days after inoculation (19 times in total). Oral administration of NMN was forcibly performed using a 1 mL disposable syringe (manufactured by Terumo Corporation) equipped with a probe for mice (manufactured by Fuchigami Kikai Co., Ltd.).

On the day of virus inoculation (15th day from the start of NMN administration), the influenza virus was inoculated by dropping 0.05 mL of the inoculated virus solution (1 x 10 ³ PFU/mouse) per mouse into the nasal cavity using a micropipette. Mice were inoculated.

The ability of each mouse to protect against infection was evaluated based on the general condition from the day of virus inoculation to 5 days after inoculation, macroscopic findings in the lungs on the 5th day after inoculation, and the amount of virus in the lungs. As a control, a group to which water for injection was administered in the same manner was set up. The number of animals in each group was 10.

<Evaluation of general condition>
The general condition of the mice was observed once a day and evaluated according to the following general condition score.

<Macroscopic findings of the lungs>
Macroscopic lesion examination of mouse lungs was performed as follows. First, a mouse 5 days after virus inoculation was anesthetized with isoflurane, and about 0.3 mL of blood was collected from the posterior vena cava using a heparin-treated syringe (manufactured by Terumo Corporation). The obtained blood was centrifuged (4°C, 3,000 rpm (2,150xg), 15 minutes) to collect plasma, which was stored frozen at -80°C. After blood collection, the mice were euthanized by exsanguination from the abdominal aorta, and the right and left lungs were removed, and the macroscopic score and virus count of the lungs were examined.

Macroscopic findings of the lungs were evaluated based on the score table below.

<Measurement of the number of viruses in the lungs>
After cutting the extracted right lung into small pieces, 2 mL of HBSS (Hank's Balanced Salt Solution) was added and homogenized using a homogenizer. The obtained homogenate was used as lung tissue fluid and stored frozen.
Lung tissue fluid was thawed and diluted 10, 100, or 1000 times using MEM (Minimum Essential Medium Eagle). The stock solution and the diluted lung tissue fluid were added in an amount of 0.1 mL per well to a 12-well plate seeded with MDCK cells, and the virus was allowed to adsorb to the cells for 1 hour.
Next, medium A (10 mL of 10x MEM, 3 mL of 7.5% sodium bicarbonate, 2 mL of 200 mmol/L L-glutamine, 1 mL of 1% DEAE dextran, 1 mL of 15% glucose, 1 mL of 10% BSA, 40 μL of 2.5% trypsin, Mix equal amounts of antibacterial agent (1 mL, sterile distilled water 31 mL) and medium B (agarose 0.8 g, distilled water 50 mL), layer 1.5 mL each on the cells, and after the agarose solution solidifies, incubate in a carbon dioxide culture device for 2 hours. Cultured for 1 day. Thereafter, a medium prepared by adding neutral red to a mixture of medium A and medium B was overlaid on the cells, and the next day, virus plaques were counted. The number of plaques calculated from the maximum dilution factor at which the number of plaques could be measured was used to calculate the number of viruses in the lungs.

The evaluation results of the general condition of each mouse are shown in FIG. 1, the scores of the macroscopic findings of the lungs are shown in FIG. 2, and the results of measuring the number of viruses in the lungs are shown in FIG. 3, respectively. In both the 500 mg/kg NMN administration group and the 1000 mg/kg NMN administration group, the general condition score and macroscopic lung findings score improved, and the number of viruses in the lungs decreased, compared to the control group (group not administered NMN). was recognized. These results showed that NMN has a growth-inhibiting effect on influenza viruses, thereby improving symptoms caused by influenza infection.

Claims (8)

An anti-inflammatory drug containing one or more selected from β-nicotinamide mononucleotide, its pharmacologically acceptable salts, and their solvates as an active ingredient, and which has the effect of suppressing the proliferation of influenza viruses in the lungs. Influenza virus agent. The anti-influenza virus agent according to claim 1, which is administered to prevent influenza infection or to improve symptoms of influenza infection. The anti-influenza virus agent according to claim 1 or 2, which is orally administered. A pharmaceutical composition containing the anti-influenza virus agent according to any one of claims 1 to 3, which is administered to prevent influenza infection or to improve symptoms of influenza infection. A health supplement containing the anti-influenza virus agent according to any one of claims 1 to 3, which is ingested for preventing influenza infection or improving symptoms of influenza infection. Feed containing the anti-influenza virus agent according to any one of claims 1 to 3, which is ingested for preventing influenza infection or improving symptoms of influenza infection. A method for preventing influenza infection, which comprises orally administering the anti-influenza virus agent according to any one of claims 1 to 3. A method for improving symptoms of influenza infection, which comprises orally administering the anti-influenza virus agent according to any one of claims 1 to 3.

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