JP7233043B2 - anti-influenza agent - Google Patents

Description

IPOD FERM BP-22254

The present invention relates to anti-influenza agents. Specifically, it relates to an anti-influenza virus agent using a substance derived from microalgae and its use.

Influenza is a respiratory infectious disease that repeats epidemics every year, and causes symptoms such as sudden fever, headache, and general malaise. It is likely to become severe and may be complicated by influenza encephalopathy and pneumonia, and the mortality rate is particularly high among the elderly and infants. Vaccination is used to prevent influenza, but its preventive effect is limited because antigens change easily and it is difficult to produce sufficient antibodies when the immune system is weakened. On the other hand, anti-influenza agents such as oseltamivir phosphate (trade name Tamiflu) and zanamivir hydrate (trade name Relenza) have been developed (see, for example, Non-Patent Document 1) and have been clinically applied, but resistant strains have emerged. and side effects, and there is an urgent need to develop new anti-influenza agents.

Von Itzstein M., et al., Nature. 1993 Jun 3;363(6428):418-23.

Under the above background, an object of the present invention is to provide a new effective means for treating or preventing influenza.

As a result of intensive studies aimed at solving the above problems, dry powder (alga bodies) of the genus Coccomyxa microalgae showed excellent anti-influenza activity in animal experiments (mouse models). In addition, high anti-influenza activity was also exhibited when thymidine was used in combination. On the other hand, in individuals with decreased viral load, a significant increase in neutralizing antibody titers in serum and airway lavage fluid was observed, revealing part of the mechanism of action of the dry powder of Coccomyxa microalgae, and its efficacy. was substantiated.
The following inventions are provided mainly based on the above results and considerations.
[1] Containing as an active ingredient an algal body of microalgae belonging to the genus Coccomyxa or a dry powder thereof,
An anti-influenza agent in which thymidine is used in combination.
[2] The anti-influenza agent of [1], wherein the microalgae is Coccomyxa sp. KJ strain or a mutant strain thereof.
[3] The anti-influenza agent of [1] or [2], which is a combination drug containing the active ingredient and thymidine.
[4] The anti-influenza agent according to any one of [1] to [3], wherein the target influenza virus is influenza A virus .

Results of influenza virus infection experiments. Changes in body weight were compared and evaluated. Test plot #1: control (distilled water), test plot #2: Tamiflu (0.2 mg/0.4 ml/day), test plot #3: algal body (5.5 mg/0.4 ml/day), test plot #4: algae body (4.6 mg/0.4 ml/day), test group #5: alga body and thymidine mixed at 90:10 (weight ratio) (5.5 mg/0.4 ml/day), test group #6: alga body and thymidine Mixed at 80:20 (weight ratio) (5.5 mg/0.4 ml/day), test group #7: algae and thymidine mixed at 70:30 (weight ratio) (5.6 mg/0.4 ml/day), test group #8: Mix algae and lactose at 95:5 (weight ratio) (5.5 mg/0.4 ml/day), test group #9: Mix algae and lactose at 90:10 (weight ratio) (5.5 mg/ 0.4 ml/day), test group #10: algae and lactose mixed at 80:20 (weight ratio) (5.5 mg/0.4 ml/day), test group #11: algae, thymidine and lactose at 70:10 : 20 (weight ratio) (5.5 mg/0.4 ml/day), test group # 12: algal cells, thymidine, and lactose mixed at 60:20:20 (weight ratio) (5.5 mg/0.4 ml/day) . In addition, test plots (#8 to #12) were also provided in which lactose, which is frequently used as an excipient during formulation, was added. Results of influenza virus infection experiments. We compared and evaluated the amount of virus in the lungs 3 days after infection. *p< 0.05, ***p< 0.001 vs. control Results of influenza virus infection experiments. Viral load in airway lavage fluid (BALF) 3 days after infection was compared and evaluated. *p< 0.05, **p< 0.01, ***p< 0.001 vs. control Results of influenza virus infection experiments. Neutralizing antibody titers in sera 14 days after infection were compared and evaluated. *p< 0.05, **p< 0.01, ***p< 0.001 vs. control Results of influenza virus infection experiments. Neutralizing antibody titers in airway lavage fluid (BALF) 14 days after infection were compared and evaluated. *p< 0.05, **p< 0.01 vs. control

1. Terminology and actions Anti-influenza agents are antiviral agents that target influenza viruses. As shown in Examples below, the anti-influenza virus agent of the present invention can be expected to have a therapeutic or prophylactic effect against influenza. Although not bound by theory, it can be said that the anti-influenza agent of the present invention exerts its effect through suppression of influenza virus proliferation in view of the experimental results shown in the examples below. In addition, the anti-influenza agent of the present invention can be expected to have the effect of promoting the production of neutralizing antibodies against influenza virus.

Influenza viruses are single-stranded RNA viruses, and are broadly divided into three genera, namely, influenza A virus, influenza B virus, and influenza C virus, which differ in the protein, antigenicity, morphology, etc. that make up the virus particles. be. Influenza A viruses have hosts such as humans, birds, horses, and pigs, and the symptoms (pathology) caused by their infection are generally more severe than those of other types. Influenza A causes epidemics every year. In addition, there is concern about a global pandemic (pandemic), and countermeasures are urgently needed. Influenza A viruses are divided into two groups according to the type of HA molecule: Group 1 consisting of H2, H5, H1, H6 (H1 cluster; H1a), H13, H16 and H11 (H1 cluster; H1b). , H8, H12, H9 (all above H9 cluster), H4, H14, H3 (all above H3 cluster), H15, H7 and H10 (all above H7 cluster). Examples of types of influenza A viruses include H1N1, H1N2, H2N2, H3N2, H5N1, H5N2, H6N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H9N1. can be done.

2. Active Ingredient of Anti-Influenza Agent The anti-influenza agent of the present invention contains algal bodies of microalgae belonging to the genus Coccomyxa or a dry powder thereof as an active ingredient. The Coccomyxa genus microalgae is not particularly limited, but preferred examples include Coccomyxa sp. KJ strains or mutant strains thereof. Coccomyxa sp. KJ strain (KJ Denso) was established on June 4, 2013 by National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Biotechnology Center, Patent Organism Depository (NITE-IPOD) (2-5-Kazusa Kamatari, Kisarazu City, Chiba Prefecture). 8 120) under Accession No. FERM P-22254 and transferred to the International Deposit on June 2, 2015 under the provisions of the Putabest Treaty under Accession No. FERM BP-22254.

Mutants of Coccomyxa sp. KJ strain are obtained by irradiation with ultraviolet rays, X-rays, gamma rays, mutagen treatment, heavy beam irradiation, genetic manipulation (introduction of foreign genes, gene disruption, genetic modification by genome editing, etc.), etc. be able to. As long as a mutant strain exhibiting anti-influenza activity can be obtained, the method for obtaining the mutant strain, its characteristics, etc. are not particularly limited.

The method for culturing the Coccomyxa microalgae is not particularly limited. The medium for culturing the genus Coccomyxa microalgae may be one commonly used for culturing microalgae. For example, a medium for known freshwater microalgae containing various nutrients, trace metal salts, vitamins, etc. , any medium for marine microalgae can be used. Examples of the medium include AF6 medium. The composition of AF6 medium (per 100 ml) is as follows.
_NaNO3 14mg
_{NH4NO3 2.2mg
3} mg of _{MgSO4.7H2O
KH2PO4 1mg
K2HPO4 0.5mg
CaCl2.2H2O 1mg
CaCO3} 1mg
Fe-citrate 0.2mg
Citric acid 0.2mg
Biotin 0.2 μg
Thiamine HCl 1 μg
Vitamin _B6 0.1μg
Vitamin _B12 0.1μg
Trace metals 0.5mL
Distilled water 99.5mL

Nutrients include, for example, _NaNO3 , _KNO3 , _NH4Cl , nitrogen sources such as urea, and phosphorus sources such as _K2HPO4 , _{KH2PO4 ,} sodium glycerophosphate _. Trace metals include iron, magnesium, manganese, calcium, zinc and the like, and vitamins include vitamin B1, vitamin B12 and the like.

In the culture method, agitation may be performed while supplying carbon dioxide under an aeration condition. At that time, culture is performed by irradiating light with a fluorescent lamp for 12 hours, irradiating light with a light-dark cycle such as dark conditions for 12 hours, or irradiating with continuous light. The culture conditions are not particularly limited as long as they do not adversely affect the growth of the genus Coccomyxa microalgae.

In addition, regarding the method of culturing the Coccomyxa sp. KJ strain, see JP 2015-15918, WO 2015/190116 A1, Satoh, A. et al., Characterization of the Lipid Accumulation in a New Microalgal Species, Pseudochoricystis ellipsoidea (Trebouxiophyceae) J. Jpn.Inst.Energy (2010) 89:909-913.

The dry powder of algal bodies can be prepared by subjecting the collected algal bodies to drying treatment and crushing (crushing) treatment. As the drying treatment, for example, drum drying, spray drying, freeze drying, or the like can be employed. A bead crusher, homogenizer, French press, mixer/blender, pulverizer, etc. can be used for the crushing treatment. The order of drying treatment and crushing treatment does not matter. Alternatively, a device having drying and crushing functions may be used to perform the drying treatment and the crushing treatment at the same time.

The particle size of the dry powder is not particularly limited. For example, dry powder having an average particle size of 0.2 μm to 2 mm, preferably 0.4 μm to 400 μm.

3. Combination with Thymidine In one aspect of the present invention, thymidine is used in combination with the above active ingredient (alga bodies of microalgae belonging to the genus Coccomyxa or dry powder thereof). In other words, the anti-influenza agent of this aspect contains algal bodies of microalgae belonging to the genus Coccomyxa or dry powder of algal bodies and thymidine as active ingredients. The concomitant use of thymidine can be expected to enhance the anti-influenza activity (that is, increase the therapeutic effect/prophylactic effect against influenza). Thymidine (CAS No. 50-89-5) is one of DNA nucleosides, and has a structure in which deoxyribose is connected to thymine, which is a pyrimidine base.

The amount of thymidine to be used in combination is not particularly limited, but the weight ratio of the above active ingredient and thymidine (provided as dry weight) is, for example, 100:1 to 1:1, preferably 10:1 to 10:3. be.

Typically, the anti-influenza agent of this aspect is provided as a combination drug in which the above active ingredient and thymidine are mixed. However, the anti-influenza agent of this aspect can also be provided in the form of a kit comprising a first component containing the above active ingredient and a second component containing thymidine. In this case, the first component and the second component are used simultaneously. Simultaneous here does not require strict simultaneity, and the active ingredient of the first component (algal body of microalgae belonging to the genus Coccomyxa or its dry powder) and the active ingredient of the second component in the application target As long as a state in which (thymidine) coexists is formed and the effect of the combined use of thymidine is exhibited, the condition of "simultaneous" is satisfied. For example, when a medicament is constructed using the anti-influenza agent of the present invention, the subject (typically a human) to be treated or prevented is administered, for example, after mixing both elements, or by administering one of them. After that, the other should be promptly administered. In addition, as long as the effect of combined use with thymidine can be obtained, administration of one may be followed by administration of the other with a predetermined time lag. In this case, it is preferable to set the time difference as short as possible, for example, one is administered within 1 hour (preferably within 30 minutes) after administration of the other.

4. Applications and methods of use of anti-influenza agent The anti-influenza agent of the present invention can be used for various purposes as a composition containing it. Examples of compositions herein are medicaments (therapeutic or prophylactic), disinfectants, disinfectant cleaners, foodstuffs, baits. Considering its use, the composition of the present invention contains acyclovir, ganciclovir, valacyclovir, vitarabine, foscarnet, trifluridine, oseltamivir phosphate (trade name Tamiflu), zanamivir hydrate (trade name Relenza), peramivir Antiviral agents such as hydrate (trade name Rapiacta), laninamivir octanoate hydrate (trade name Inavir), baloxavir marboxil (trade name Xofluza), benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethyl Antibacterial or sterilizing ingredients such as phenol and chlorhexidine gluconate may be contained.

(1) Medicine The medicine of the present invention is used for treatment or prevention of influenza. The medicament of the present invention can exert a therapeutic effect or a preventive effect (these two effects are collectively referred to as "medicinal effect") against influenza. The medicinal effect here includes (1) prevention of influenza virus infection, (2) prevention, suppression or delay of onset of influenza, (3) mitigation (reduction) of symptoms characteristic of influenza or associated symptoms, (4) Prevention, suppression or delay of exacerbation of symptoms characteristic of influenza or accompanying symptoms, etc. are included. Since the therapeutic effect and the prophylactic effect are partially overlapping concepts, it is sometimes difficult to clearly distinguish between them, and there is little practical benefit from doing so.

Formulation of pharmaceuticals can be carried out according to conventional methods. When formulating, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline solution, etc.). Lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used as excipients. Starch, carboxymethyl cellulose, calcium carbonate and the like can be used as the disintegrant. Phosphate, citrate, acetate and the like can be used as buffers. Gum arabic, sodium alginate, tragacanth and the like can be used as emulsifiers. As suspending agents, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used. Benzyl alcohol, chlorobutanol, sorbitol and the like can be used as analgesics. Propylene glycol, ascorbic acid and the like can be used as stabilizers. Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. As antiseptics, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used.

The dosage form for formulation is also not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations (ointments, creams, lotions, liquids, gels, poultices, plasters, tapes , aerosols, etc.), and suppositories. Pharmaceuticals are administered orally or parenterally according to the dosage form (local injection to the affected area, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) applied to the subject by Systemic administration and local administration are also indicated by the subject. These administration routes are not mutually exclusive, and two or more arbitrarily selected routes can be used in combination.

The medicament of the present invention contains an amount of active ingredient necessary to obtain the expected effect (that is, a therapeutically or prophylactically effective amount). Although the amount of active ingredient in the pharmaceutical of the present invention generally varies depending on the dosage form, the amount of active ingredient is set, for example, within the range of about 0.1% to about 99% by weight so as to achieve the desired dosage.

The dosage of the medicament of the present invention is set so as to obtain the expected effects. Symptoms, patient's age, sex, weight and the like are generally taken into consideration in setting a therapeutically or prophylactically effective dose. A person skilled in the art can take these matters into consideration and set an appropriate dosage. To give an example of dosage, the dosage can be set so that the amount of the active ingredient per day is 1 mg to 20 mg, preferably 2 mg to 10 mg, for adults (body weight of about 60 kg). As an administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the dosing schedule, the patient's condition and duration of effect of the active ingredient can be taken into consideration.

Other drugs (e.g., oseltamivir phosphate (trade name Tamiflu), zanamivir hydrate (trade name Relenza), peramivir hydrate (trade name Rapiacta), laninami in parallel with treatment or prevention with the medicament of the present invention) Anti-influenza agents such as viroctanoate hydrate (trade name Inavir) and baloxavir marboxil (trade name Xofluza) may be administered. Combining the active ingredient of the present invention with a drug having a different mechanism of action provides multiple actions/effects, and when applied to the treatment of influenza, it is possible to increase the therapeutic effect.

As is clear from the above description, the present application is characterized by administering a therapeutically or prophylactically effective amount of a medicament containing the anti-influenza agent of the present invention to a subject who has or is likely to have influenza. Also provided are methods of treating or preventing influenza. Subjects for treatment or prevention are typically humans, but non-human mammals (e.g., monkeys, cows, pigs, horses, goats, sheep, dogs, cats, rabbits, etc.), birds (chickens, quail, turkeys, etc.) , geese, ducks, ostriches, ducks, parakeets, Java sparrows, etc.).

(2) Disinfectant, sterilizing detergent The disinfectant or sterilizing detergent of the present invention is used, for example, for disinfection or sterilization cleaning of living rooms (including hospital rooms), kitchens, toilets, washrooms, bathrooms, tableware, cutlery (knives , forks, spoons, etc.), cooking utensils (kitchen knives, knives, pots, mixers, microwave ovens, ovens, etc.), medical instruments and devices, etc., and disinfecting or sterilizing hands and fingertips. The disinfectant or sterilizing cleaning agent of the present invention can be, for example, liquid (eg, spray, lotion), gel, or solid (eg, powder), and applied by coating, spraying, spraying, or the like. The disinfectant or disinfectant detergent of the present invention may be supported or adhered to a carrier (for example, sheet-shaped) made of natural fibers, synthetic fibers, etc., and used for wiping or the like.

Antibacterial or disinfecting ingredients such as benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethylphenol, chlorhexidine gluconate, etc., pH adjusters, surfactants, adsorbents, carriers, etc. are added to the disinfectant or disinfectant detergent of the present invention. You may decide to add.

The disinfectant or sterilizing agent of the present invention contains an amount of active ingredient that can be expected to have a disinfecting or sterilizing effect. The amount to be added can be determined in consideration of its use, form, and the like.

(3) Food and feed Examples of the food of the present invention include general foods (cereals, vegetables, meat, various processed foods, confectionery and desserts, milk, soft drinks, fruit juice drinks, coffee drinks, vegetable juice drinks, alcoholic beverages, etc. ), nutritional supplements (supplements, nutritional drinks, etc.), food additives, pet food, and pet nutritional supplements. In the case of nutritional supplements or food additives, they can be provided in the form of powder, granule powder, tablet, paste, liquid, or the like. By providing it in the form of a food composition, it becomes easier to take the active ingredient of the present invention on a daily basis or continuously.

Examples of feeds of the present invention are feeds (for example feeds for livestock, poultry, etc.), pet foods.

The food or feed of the present invention preferably contains an amount of active ingredient that can be expected to have a therapeutic or preventive effect. The amount to be added can be determined in consideration of the condition, age, sex, weight, etc. of the person to whom it is used.

1. Preparation of dry powder (algal body) of Coccomyxa microalgae Coccomyxa sp. strain KJ was cultured according to the previously reported method. Specifically, after planting Coccomyxa sp. KJ strain in AF6 medium, 2% CO ₂ (v/v) was aerated, and the medium was heated to room temperature (25°C) while irradiating with light (300 μmol/m ² /s). was cultured for 48 hours. Alga bodies were recovered from the culture solution by centrifugation. The collected alga bodies were dried with a drum dryer and pulverized with a pulverizer to form a powder (dry powder of alga bodies).

2. Influenza Virus Infection Test Algal bodies (dry powder) of Coccomyxa sp. KJ strain were evaluated for effectiveness against influenza. Changes in efficacy when combined with thymidine were also examined.

<Method>
A type A influenza virus (A/NWS/33, H1N1 subtype) was used as the virus strain.
(1) Set up the following test groups, and give BALB/c mice (6 weeks old, female) (10 mice per group) twice a day (9:00 and 18:00) from 7 days before to 7 days after virus infection. ) Administer the sample orally.
Test plot #1: control (distilled water)
Trial #2: Tamiflu (0.2 mg/0.4 ml/day)
Test plot #3: algal body (5.5 mg/0.4 ml/day)
Test plot #4: algal body (4.6 mg/0.4 ml/day)
Test plot #5: algae and thymidine mixed at 90:10 (weight ratio) (5.5 mg/0.4 ml/day)
Test plot #6: Alga bodies and thymidine mixed at 80:20 (weight ratio) (5.5 mg/0.4 ml/day)
Test plot #7: algal cells and thymidine mixed at 70:30 (weight ratio) (5.6 mg/0.4 ml/day)
Test Group #8: Algae and lactose mixed at 95:5 (weight ratio) (5.5 mg/0.4 ml/day)
Test Group #9: Algae and lactose mixed at 90:10 (weight ratio) (5.5 mg/0.4 ml/day)
Test group #10: Algae and lactose mixed at 80:20 (weight ratio) (5.5 mg/0.4 ml/day)
Test plot #11: algal body, thymidine, and lactose mixed at 70:10:20 (weight ratio) (5.5 mg/0.4 ml/day)
Test plot #12: algal cells, thymidine, and lactose mixed at 60:20:20 (weight ratio) (5.5 mg/0.4 ml/day)
(2) On day 0, mice are nasally inoculated with virus (2 x ¹⁰⁴ PFU/50 µl/mouse) under anesthesia.
(3) Record body weight and mortality for 14 days after infection.
(4) Three days after infection, airway lavage fluid (BALF) and lungs are collected from 5 mice in each group and subjected to virus quantification.
(5) 14 days after infection, BALF and serum are collected from the remaining mice (5 or 4 mice) of each group and subjected to neutralizing antibody titer measurement.

<Results/Discussion>
(1) #3 to #12 showed the same degree of weight loss as #1 (control group) in the acute phase up to 7 days after infection, but after 8 days, a rapid weight recovery process was observed. traced (Fig. 1).
(2) One animal in #1 died 8 days after infection, but all animals in the other administration groups survived.
(3) In all of #3 to #12, the viral load in the lung (Fig. 2) and airway lavage fluid (Fig. 3) decreased, but there was no significant difference between the administration groups.
(4) Neutralizing antibody titers in serum (Fig. 4) and airway lavage fluid (Fig. 5) of #3 to #12 were both significantly higher than #1 (control group).

As described above, good anti-influenza activity was observed in algal bodies (dry powder) of Coccomyxa sp. KJ strain. In addition, the algal body (dry powder) of Coccomyxa sp. KJ strain showed high anti-influenza activity even when thymidine was used in combination.

The anti-influenza agent of the present invention contains a substance derived from microalgae as an active ingredient, and can exhibit anti-influenza activity through a mechanism of action different from that of existing anti-influenza agents. Therefore, it is suitable for combined use with existing anti-influenza drugs and provides a new therapeutic strategy. On the other hand, the anti-influenza agent of the present invention can be expected to be highly safe because the active ingredient is algal bodies of microalgae.

The present invention is by no means limited to the description of the above embodiments and examples of the invention. Various modifications are also included in the present invention within the scope of those skilled in the art without departing from the description of the claims. The contents of articles, published patent publications, patent publications, etc., identified herein are incorporated by reference in their entirety.

Claims (4)

Containing algal bodies of microalgae belonging to the genus Coccomyxa or a dry powder thereof as an active ingredient,
An anti-influenza agent characterized by being used in combination with thymidine. 2. The anti-influenza agent according to claim 1, wherein the microalgae is Coccomyxa sp. KJ strain or a mutant strain thereof. The anti-influenza agent according to claim 1 or 2, which is a combination drug containing the active ingredient and thymidine. The anti-influenza agent according to any one of claims 1 to 3, wherein the target influenza virus is influenza A virus .

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