JP2020019730A - Anti-influenza agent - Google Patents

Abstract

To provide new means effective to treatment or prevention of influenza.SOLUTION: There is provided an anti-influenza agent containing, as an active ingredient, an alga body or its dry powder of microalgae belonging to genus Coccomyxa.SELECTED DRAWING: None

Description

  The present invention relates to anti-influenza agents. More specifically, the present invention relates to an anti-influenza virus agent using a substance derived from microalgae, its use, and the like.

  Influenza is a respiratory infection that recurs every year and causes symptoms such as sudden fever, headache, and general malaise. The disease tends to be severe and may be accompanied by influenza encephalopathy or pneumonia. In particular, the mortality of the elderly and infants is high. Vaccination is performed to prevent influenza, but its preventive effect is limited because antigens are easily changed and sufficient antibodies cannot be produced when immunity is reduced. On the other hand, oseltamivir phosphate (trade name: Tamiflu), zanamivir hydrate (trade name: Relenza) and the like have been developed as anti-influenza agents (for example, see Non-Patent Document 1) and clinically applied, but emergence of resistant strains The development of new anti-influenza drugs is urgently needed.

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 means effective for treating or preventing influenza.

As a result of intensive studies to solve the above-mentioned problems, in an animal experiment (mouse model), a dry powder (algae) of the microalgae of the genus Kompani showed excellent anti-influenza activity. 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 the neutralizing antibody titer in serum and in the airway washings was observed, revealing a part of the mechanism of action of the dry powder of the microalgae of the genus Kokkokusami, and its efficacy. Was supported.
The following inventions are provided mainly based on the above results and considerations.
[1] An anti-influenza agent comprising, as an active ingredient, alga bodies of microalgae belonging to the genus Kokkomikusa or a dry powder thereof.
[2] The anti-influenza agent according to [1], wherein the microalgae is a strain of Kokko-mixa sp. KJ or a mutant thereof.
[3] The anti-influenza agent according to [1] or [2], wherein thymidine is used in combination.
[4] The anti-influenza agent according to [3], which is a combination drug containing the active ingredient and thymidine.
[5] The anti-influenza agent according to any one of [1] to [4], wherein the target influenza virus is an influenza A virus.
[6] A composition comprising the anti-influenza agent according to any one of [1] to [3].
[7] The composition according to [6], which is a medicament against influenza.
[8] The composition according to [6], which is a disinfectant or a sterilizing detergent.
[9] The composition according to [6], which is a food or a feed.

Influenza virus infection experiment results. 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: Algae (5.5 mg / 0.4 ml / day), Test plot # 4: Algae (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: Algae and thymidine Mixed at 80:20 (weight ratio) (5.5 mg / 0.4 ml / day), Test plot # 7: Algae and thymidine mixed at 70:30 (weight ratio) (5.6 mg / 0.4 ml / day), Test plot # 8: Algae and lactose were mixed at a ratio of 95: 5 (weight ratio) (5.5 mg / 0.4 ml / day), and Test Group # 9: Algae and lactose were mixed at a ratio of 90:10 (weight ratio) (5.5 mg / weight). 0.4 ml / day), Test plot # 10: Algae and lactose mixed at 80:20 (weight ratio) (5.5 mg / 0.4 ml / day), Test plot # 11: Algae, thymidine, lactose 70:10 : 20 (weight ratio), mixed (5.5 mg / 0.4 ml / day), Test group # 12: Algae, thymidine, and lactose mixed at 60:20:20 (weight ratio) (5.5 mg / 0.4 ml / day) ). In addition, test groups (# 8 to # 12) to which lactose which is frequently used as an excipient at the time of formulation was added were also provided. Influenza virus infection experiment results. Three days after infection, the amount of virus in the lung was compared and evaluated. * p <0.05, *** p <0.001 vs. control Influenza virus infection experiment results. Three days after infection, the amount of virus in the airway lavage fluid (BALF) was compared and evaluated. * p <0.05, ** p <0.01, *** p <0.001 vs. control Influenza virus infection experiment results. Neutralizing antibody titers in serum 14 days after infection were compared and evaluated. * p <0.05, ** p <0.01, *** p <0.001 vs. control Influenza virus infection experiment results. Neutralizing antibody titers in the airway lavage fluid (BALF) 14 days after infection were compared and evaluated. * p <0.05, ** p <0.01 vs. control

1. Terms, effects Anti-influenza agents are anti-viral agents that target the influenza virus. As shown in the Examples below, the anti-influenza virus agent of the present invention can be expected to have a therapeutic or preventive effect on influenza. Although not wishing to be bound by theory, it can be said that the anti-influenza agent of the present invention exerts its effects through suppression of influenza virus growth in view of the results of the experiments described in the examples below. Further, the anti-influenza agent of the present invention can be expected to have an effect of promoting the production of neutralizing antibodies against influenza virus.

  Influenza virus is a single-stranded RNA virus, and is roughly classified into three genera having different proteins, antigenicity, morphology, etc. constituting virus particles, namely, influenza A virus, influenza B virus and influenza C virus. You. Influenza A virus uses humans, birds, horses, pigs, and the like as hosts, and the symptoms (pathology) of the infection are generally more serious than those of other types. Influenza A causes an epidemic every year. In addition, there is a concern about a pandemic, and measures are urgently needed. Influenza A virus is divided into two groups according to the type of HA molecule: H2, H5, H1, H6 (or more, H1 cluster; H1a), H13, H16, and H11 (or more, H1 cluster; H1b). , H8, H12, H9 (H9 cluster), H4, H14, H3 (H3 cluster), H15, H7 and H10 (H7 cluster). Examples of influenza A viruses include H1N1, H1N2, H2N2, H3N2, H5N1, H5N2, H6N1, H7N2, H7N3, H7N7, H7N9, H9N2 and H9N1. Can be.

2. Active ingredient of anti-influenza agent The anti-influenza agent of the present invention comprises an alga body of a microalga belonging to the genus Coccomyxa or a dry powder thereof as an active ingredient. Although there is no particular limitation on the microalgae of the genus Kokkomikusa, preferred examples include Kokkomikusa sp. KJ strain or a mutant thereof. Kokkomikusa sp. KJ shares (KJ Denso) was established on June 4, 2013 by the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation Biotechnology Center Patent Organism Depositary Center (NITE-IPOD) (2-5 Kazusa Kamasa, Kisarazu-shi, Chiba) No. 8120) and deposited under accession number FERM BP-22254 under the provisions of the Putavest Treaty on June 2, 2015 under the accession number FERM BP-22254.

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

The method for cultivating Microalgae of the genus Kokkomushi is not particularly limited. The medium for cultivating the microalgae of the genus Kokkomikusa may be any of those commonly used for culturing microalgae.For example, various nutrients, trace metal salts, known media for freshwater microalgae containing 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.
NaNO ₃ 14mg
NH ₄ NO ₃ 2.2mg
MgSO ₄ · 7H ₂ O 3mg
KH ₂ PO ₄ 1mg
K ₂ HPO ₄ 0.5mg
CaC _l2 · 2H ₂ O 1mg
CaCO ₃ 1mg
Fe-citrate 0.2mg
Citric acid 0.2mg
Biotin 0.2μg
Thiamine HCl 1μg
Vitamin B ₆ 0.1μg
Vitamin B ₁₂ 0.1μg
Trace metals 0.5mL
Distilled water 99.5mL

Examples of the nutrients include nitrogen sources such as NaNO ₃ , KNO ₃ , NH ₄ Cl and urea, and phosphorus sources such as K ₂ HPO ₄ , KH ₂ PO ₄ and sodium glycerophosphate. The trace metals include iron, magnesium, manganese, calcium, zinc and the like, and the vitamins include vitamin B1, vitamin B12 and the like.

  As the culture method, stirring may be performed while supplying carbon dioxide under aeration conditions. At this time, the cells are cultured by irradiating with a fluorescent lamp for 12 hours, irradiating with light and dark cycles such as 12 hours of dark conditions, or irradiating with continuous light. The culture conditions are not particularly limited as long as they do not adversely affect the growth of the microalgae of the genus Kokkosuma. For example, the pH of the culture solution is 3 to 9, and the culture temperature is 10 to 35 ° C.

  In addition, regarding the culture method of the Kkkomikusa sp. KJ strain, JP-A-2015-15918, WO 2015/190116 A1, Satoh, A. et al. Jpn. Inst. Energy (2010) 89: 909-913.

  The dry powder of alga bodies can be prepared by subjecting the collected alga bodies to a drying treatment and a crushing (crushing) treatment. As the drying treatment, for example, drum drying, spray drying, freeze drying and the like can be employed. For the crushing treatment, a bead crusher, a homogenizer, a French press, a mixer / blender, a fine crusher, or the like can be used. The order of the drying treatment and the crushing treatment does not matter. Alternatively, a drying and crushing process may be performed simultaneously using an apparatus having drying and crushing functions.

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

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

  Although the amount of thymidine used in combination is not particularly limited, the amount of the active ingredient and the thymidine is in a weight ratio (however, dry weight), for example, 100: 1 to 1: 1, preferably 10: 1 to 10: 3. To be.

  Typically, the anti-influenza agent of this embodiment will be provided as a combination preparation obtained by mixing the active ingredient and thymidine. However, the anti-influenza agent of this embodiment can be provided in the form of a kit comprising the first component containing the active ingredient and the second component containing thymidine. In this case, the first component and the second component are used simultaneously. Simultaneous here does not require strict synchronicity, and the active ingredient of the first component (algae of microalga belonging to the genus Kokkomikusa or its dry powder) and the active ingredient of the second component are not required for application. 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 drug is constituted by using the anti-influenza agent of the present invention, for example, administration may be performed after mixing both components, or administration may be performed to a target to be treated or prevented (typically, a human). Thereafter, the other may be administered immediately. In addition, as long as the effect of the combined use of thymidine can be obtained, the administration of one may be followed by administration of the other with a predetermined time difference. In this case, it is preferable to set the time difference as short as possible. For example, the other is administered within one hour (preferably within 30 minutes) after administration of one.

4. Use and usage of anti-influenza agent The anti-influenza agent of the present invention can be used for various applications as a composition containing it. Examples of compositions herein are pharmaceuticals (therapeutic or prophylactic), disinfectants, germicidal cleaners, foods, baits. Considering its use, the composition of the present invention may be added to acyclovir, ganciclovir, valacyclovir, vitarabine, foscarnet, trifluridine, oseltamivir phosphate (trade name Tamiflu), zanamivir hydrate (trade name Relenza), peramivir Antivirals such as hydrate (trade name Rapiacta), laninamivir octanoate hydrate (trade name Inavir), valoxavir marboxil (trade name Zofluza), benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethyl An antibacterial or antibacterial component such as phenol and chlorhexidine gluconate may be contained.

(1) Medicine The medicine of the present invention is used for treating or preventing influenza. The medicament of the present invention can exert a therapeutic effect or a prophylactic effect on influenza (these two effects are collectively referred to as “pharmaceutical effects”). The medicinal effects here include (1) prevention of influenza virus infection, (2) prevention, suppression or delay of influenza onset, (3) relief of symptoms characteristic of influenza or associated symptoms (mildness), (4) Including, for example, preventing, suppressing or delaying the deterioration of symptoms or accompanying symptoms characteristic of influenza. It should be noted that since the therapeutic effect and the preventive effect are partially overlapping concepts, it may be difficult to distinguish them clearly and the benefits of doing so are small.

  Pharmaceutical formulations can be made according to conventional methods. When formulated, other pharmaceutically acceptable components (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological Saline, etc.). As an excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As a disintegrant, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. Phosphates, citrates, acetates and the like can be used as buffers. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As a suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate, and the like can be used. Benzyl alcohol, chlorobutanol, sorbitol and the like can be used as a soothing agent. As a stabilizer, propylene glycol, ascorbic acid, or the like can be used. As a preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl paraben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  The dosage form in the case of formulation is not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, and external preparations (ointments, creams, lotions, solutions, gels, cataplasms, plasters, tapes) , Aerosols, etc.) and suppositories. Depending on the dosage form, the drug is administered orally or parenterally (local injection into the affected area, intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, transnasal, transmucosal, etc.) Applied to the subject. Systemic administration and local administration are also adapted to the subject. These administration routes are not mutually exclusive, and arbitrarily selected two or more can be used in combination.

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

  The dose of the medicament of the present invention is set so as to obtain an expected effect. In setting a therapeutically or prophylactically effective dose, symptoms, the age, sex, and weight of the patient are generally considered. Those skilled in the art can determine an appropriate dose in consideration of these matters. As an example of the dose, the dose can be set so that the daily amount of the active ingredient is 1 mg to 20 mg, preferably 2 mg to 10 mg for an adult (body weight: about 60 kg). The administration schedule may be, for example, once to several times a day, once every two days, or once every three days. In preparing the administration schedule, the condition of the patient, the duration of the effect of the active ingredient, and the like can be considered.

  In parallel with the treatment or prevention by the medicament of the present invention, other medicines (for example, oseltamivir phosphate (trade name Tamiflu), zanamivir hydrate (trade name Relenza), peramivir hydrate (trade name Rapiacta), laninami Treatment with an anti-influenza agent such as biruoctanoic acid ester hydrate (trade name Inavir) or baloxavir marboxil (trade name Zofluza) may be performed. When the active ingredient of the present invention is used in combination with a drug having a different mechanism of action, a combined action / effect can be obtained, and when applied to the treatment of influenza, the therapeutic effect can be increased.

  As is evident from the above description, the present application is characterized in that a subject containing an anti-influenza agent of the present invention is administered a therapeutically or prophylactically effective amount to a subject suffering from or likely to suffer from influenza. And methods for treating or preventing influenza. The target for treatment or prevention is typically a human, but a mammal other than a human (eg, monkey, cow, pig, horse, goat, sheep, dog, cat, rabbit, etc.), bird (chicken, quail, turkey) , Geese, ducks, ostriches, ducks, parakeets, birds, etc.).

(2) Disinfectant or disinfectant cleaning agent The disinfectant or disinfectant cleaning agent of the present invention includes, for example, disinfecting or disinfecting cleaning of living rooms (including sick rooms), cooking rooms, toilets, washrooms, bathrooms, etc., tableware, cutlery (knife) , Forks, spoons, etc.), cooking utensils (knives, knives, pans, mixers, microwave ovens, ovens, etc.), disinfecting or disinfecting and cleaning of medical instruments and devices, and disinfecting or disinfecting and cleaning of hands and fingertips. The disinfectant or the disinfectant cleaning agent of the present invention is formed, for example, in a liquid (for example, a spray or a lotion), a gel, or a solid (for example, a powder), and is applied by coating, spraying, or spraying. The disinfectant or the germicidal detergent of the present invention may be carried or adhered to a carrier (for example, a sheet) made of a natural fiber, a synthetic fiber, or the like to obtain a product used for wiping or the like.

  Antibacterial or antibacterial components such as benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethylphenol, and chlorhexidine gluconate, pH adjusters, surfactants, adsorbents, carriers and the like are used as the disinfectant or disinfectant cleaning agent of the present invention. It may be added.

  The disinfectant or germicidal detergent of the present invention contains an effective ingredient in an amount that can be expected to have a disinfecting or germicidal washing effect. The amount of addition can be determined in consideration of its use, form, and the like.

(3) Foods and baits Examples of the foods 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 drinks, etc. ), Dietary supplements (supplements, nutritional drinks, etc.), food additives, pet food for pets, and dietary supplements for pets. In the case of dietary supplements or food additives, they can be provided in the form of powders, granules, tablets, pastes, liquids and the like. By providing in the form of a food composition, it becomes easy to take the active ingredient of the present invention on a daily basis or continuously.

  Examples of feeds of the present invention are feed (eg feed for livestock, poultry, etc.), pet food.

  The food or feed of the present invention preferably contains an active ingredient in an amount 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, and the like of the person to whom it is used.

1. Preparation of dried powder (algae) of Microalgae of the genus Kokkomikusa sp. KJ strain was cultured according to the method previously reported. Specifically, after inoculating the A. spp. KJ strain in the AF6 medium, 2% CO ₂ (v / v) was aerated and room temperature (25 ° C.) while irradiating with light (300 μmol / m ² / s). For 48 hours. Algae were collected from the culture solution by centrifugation. The collected algal cells were dried with a drum dryer and pulverized with a fine pulverizer to obtain a powder (dry powder of algal cells).

2. Influenza virus infection experiment The alga body (dry powder) of Kokkomixa sp. KJ strain was evaluated for its effectiveness against influenza. Changes in efficacy when used in combination with thymidine were also investigated.

<Method>
The virus strain used was influenza A virus (A / NWS / 33, H1N1 subtype).
(1) Establish the following test plots, and give BALB / c mice (6 weeks old, female) (10 mice per group) twice a day (9 o'clock and 18 o'clock) from 7 days before to 7 days after virus infection. ) Administer the sample orally.
Test plot # 1: control (distilled water)
Test plot # 2: Tamiflu (0.2 mg / 0.4 ml / day)
Test plot # 3: Algae (5.5 mg / 0.4 ml / day)
Test plot # 4: Algae (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: Algae and thymidine mixed at 80:20 (weight ratio) (5.5 mg / 0.4 ml / day)
Test plot # 7: Algae and thymidine mixed at 70:30 (weight ratio) (5.6 mg / 0.4 ml / day)
Test plot # 8: Algae and lactose mixed at 95: 5 (weight ratio) (5.5 mg / 0.4 ml / day)
Test plot # 9: Algae and lactose mixed at 90:10 (weight ratio) (5.5 mg / 0.4 ml / day)
Test plot # 10: Algae and lactose mixed at a weight ratio of 80:20 (5.5 mg / 0.4 ml / day)
Test plot # 11: Algae, thymidine, and lactose mixed at 70:10:20 (weight ratio) (5.5 mg / 0.4 ml / day)
Test plot # 12: Algae, thymidine, and lactose mixed at a weight ratio of 60:20:20 (5.5 mg / 0.4 ml / day)
(2) On day 0, mice are intranasally inoculated with virus (2 × 10 ⁴ PFU / 50 μl / mouse) under anesthesia.
(3) Record body weight and deaths 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 used for virus quantification.
(5) 14 days after infection, BALF and serum are collected from the remaining mice (5 or 4) in each group, and used for measurement of neutralizing antibody titers.

<Results and Discussion>
(1) In the acute period up to 7 days after infection, # 3 to # 12 showed the same weight loss progress as in # 1 (control group), but from day 8 onward, a rapid weight recovery process was observed. (Figure 1).
(2) One animal died 8 days after infection in # 1, but all survived in the other administration groups.
(3) In all of # 3 to # 12, the viral loads in the lung (FIG. 2) and the airway lavage fluid (FIG. 3) decreased, but there was no great difference between the administration groups.
(4) The neutralizing antibody titers in the sera # 3 to # 12 (FIG. 4) and in the airway lavage fluid (FIG. 5) were all significantly higher than in # 1 (control group).

  As described above, good anti-influenza activity was observed in the alga bodies (dry powder) of Kokkomixa sp. KJ strain. In addition, the alga body (dry powder) of the strain Kokkomikusa sp. KJ 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 by a different mechanism of action from existing anti-influenza agents. Therefore, it is suitable for 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 have high safety because the active ingredient is a microalgae alga body.

  The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. The contents of articles, published patent gazettes, and patent gazettes specified in this specification are all incorporated by reference.

Claims (9)

  An anti-influenza agent comprising, as an active ingredient, an alga body of a microalga belonging to the genus Kokkomikusa or a dry powder thereof.   2. The anti-influenza agent according to claim 1, wherein the microalgae is a strain of Kokko-mixa sp. KJ or a mutant thereof.   The anti-influenza agent according to claim 1 or 2, wherein thymidine is used in combination.   The anti-influenza agent according to claim 3, which is a combination preparation containing the active ingredient and thymidine.   The anti-influenza agent according to any one of claims 1 to 4, wherein the target influenza virus is an influenza A virus.   A composition comprising the anti-influenza agent according to claim 1.   The composition according to claim 6, which is a medicament against influenza.   The composition according to claim 6, which is a disinfectant or a germicidal detergent.   The composition according to claim 6, which is a food or a feed.

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