WO2017170712A1 - Adjuvant de composé à faible poids moléculaire et vaccin l'utilisant - Google Patents

Adjuvant de composé à faible poids moléculaire et vaccin l'utilisant Download PDF

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WO2017170712A1
WO2017170712A1 PCT/JP2017/012937 JP2017012937W WO2017170712A1 WO 2017170712 A1 WO2017170712 A1 WO 2017170712A1 JP 2017012937 W JP2017012937 W JP 2017012937W WO 2017170712 A1 WO2017170712 A1 WO 2017170712A1
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vaccine
rarγ
agonist
item
antigen
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PCT/JP2017/012937
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English (en)
Japanese (ja)
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典保 平澤
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国立大学法人東北大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a low molecular compound adjuvant and a vaccine using the same.
  • Thymic Stromal Lymphopoietin is a cytokine mainly produced by epithelial cells and activates dendritic cells to enhance humoral immunity. It has been reported that TSLP protein itself enhances antibody production against HIV antigen, and that TSLP protein itself can be used as an adjuvant (Non-patent Documents 1 and 2). However, for the same reason as described above, it is desirable not to administer the TSLP protein itself but to use a low molecular weight compound that induces the TSLP protein.
  • Non-patent Document 3 Non-patent Document 3
  • TSLP Non-patent Document 3
  • other proteins particularly TNF- ⁇ , IL-1 ⁇ , IL-4 etc. involved in allergic reactions
  • TSLP protein
  • certain cytokines cannot be selectively induced not only by TSLP but also by low molecular weight compounds, and such low molecular weight compounds have been desired.
  • An object of the present invention is to provide a chemically synthesized adjuvant that selectively induces TSLP.
  • an agonist of RAR ⁇ selectively induces TSLP and can produce neutralizing antibodies when formulated in a vaccine. Based on this finding, the present inventor has further studied the timing of administration of an RAR ⁇ agonist and the antigen contained in the vaccine, and as a result, completed the present invention.
  • Item 1 A vaccine adjuvant comprising an agonist of RAR ⁇ .
  • Item 2 The vaccine adjuvant according to Item 1, wherein the RAR ⁇ agonist is a retinoid.
  • Item 3. The vaccine adjuvant according to Item 1 or 2, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RAR ⁇ agonist.
  • Item 4. The vaccine adjuvant according to Item 3, wherein the RAR ⁇ agonist is administered as an external preparation.
  • Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Item 5.
  • Vaccine adjuvant is any one of Items 1 to 4, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (types A, B, C) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Vaccine
  • a vaccine comprising a combination of the vaccine adjuvant according to Item 1 or 2 and an antigen.
  • Item 7. The vaccine according to Item 6, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RAR ⁇ agonist.
  • Item 8 The vaccine according to Item 7, wherein the RAR ⁇ agonist is administered as an external preparation.
  • Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Item 9.
  • Item 10 The vaccine according to any one of Items 6 to 8, wherein the RAR ⁇ agonist is a retinoid and the target disease is influenza.
  • Item 11. A combination of an antigen and a vaccine adjuvant according to item 1 or 2 for use as a vaccine.
  • Item 12. The combination according to Item 11, wherein the RAR ⁇ agonist is a retinoid and the target disease is influenza.
  • Item 13 The combination according to Item 11, wherein the RAR ⁇ agonist is a retinoid.
  • Item 14 The combination according to any one of Items 11 to 13, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RAR ⁇ agonist.
  • Item 15. The combination according to Item 14, wherein the RAR ⁇ agonist is administered as an external preparation.
  • Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Any one of Items 11, 13 to 15, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (types A, B, C) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Combination described in.
  • Item 17. Use of an antigen and the vaccine adjuvant according to item 1 or 2 for producing a vaccine.
  • Item 18 The use according to Item 17, wherein the agonist of RAR ⁇ is a retinoid and the target disease is influenza.
  • Item 19 The use according to Item 17, wherein the agonist of RAR ⁇ is a retinoid.
  • Item 20 The use according to any one of Items 17 to 19, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RAR ⁇ agonist.
  • Item 21 The use according to Item 20, wherein the RAR ⁇ agonist is administered as an external preparation.
  • Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Any one of Items 17, 19 to 21, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (A type, B type, C type) and Ebola hemorrhagic fever, dengue fever, Zika fever, etc. Use as described in.
  • Item 23 A method for preventing or treating a disease, comprising a step of administering a vaccine adjuvant according to Item 1 or 2 to a mammal, and a step of administering an antigen to the mammal.
  • Item 24 The method according to Item 23, wherein the agonist of RAR ⁇ is a retinoid and the target disease is influenza.
  • Item 25 The method according to Item 23, wherein the RAR ⁇ agonist is a retinoid.
  • Item 26 The method according to any one of Items 23 to 25, for use in an administration schedule in which the antibody is administered at least 4 hours after administration of the RAR ⁇ agonist.
  • Item 27 The method according to Item 26, wherein the RAR ⁇ agonist is administered as an external preparation.
  • Target diseases are influenza, HIV, chlamydia, malaria, tuberculosis, tumor, measles, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, Item 23, 25 to 27, which is at least one selected from the group consisting of tetanus, Japanese encephalitis, papillomavirus, hepatitis (types A, B, C) and Ebola hemorrhagic fever, dengue fever, zika fever, etc. The method described in 1.
  • a chemically synthesized adjuvant capable of producing neutralizing antibodies when TSLP is selectively induced and blended with a vaccine can be provided.
  • Example 1 The result of Example 1 is shown. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001.
  • the result of Example 2 is shown.
  • the result of Example 3 is shown.
  • the result of Example 4 is shown.
  • the result of Example 5 is shown.
  • the result of Example 6 is shown. ** P ⁇ 0.01, *** P ⁇ 0.001.
  • the result of Example 7 is shown.
  • the result of Example 9 is shown.
  • the result of Example 10 is shown.
  • Vaccine Adjuvant The present invention provides a vaccine adjuvant comprising an agonist of retinoic acid receptor ⁇ (RAR ⁇ ).
  • RAR ⁇ Retinoic acid receptors
  • RXR retinoid X receptors
  • an agonist of RAR ⁇ means a compound having agonist activity for RAR ⁇ belonging to the retinoic acid receptor (RAR) among these receptors.
  • RAR ⁇ agonists include retinoids and RAR ⁇ agonists other than retinoids, and retinoids are preferred.
  • examples of the retinoid include retinoic acid or a salt thereof, retinal, retinol and the like, and retinoic acid or a salt thereof is preferable.
  • the retinoin chain or a salt thereof typically has the following formula:
  • All-trans-retinoic acid (atRA) represented by: 9-cis-retinoic acid, 13-cis-retinoic acid, 9,13-cis-retinoic acid, and salts thereof.
  • RAR ⁇ agonists other than retinoids include 3-fluoro-4-[[2-hydroxy-2- (5,5,8,8-tetramethyl-5,6,7,8, -tetrahydro-2-naphthalenyl) acetyl ] Amino] -benzoic acid (3-fluoro-4-[[2-hydroxy-2- (5,5,8,8-tetramethyl-5,6,7,8, -tetrahydro-2-naphthalenyl) acetyl] amino ] -benzoic acid) (BMS189961, CAS number: 185629-22-5) or a salt thereof; 3-fluoro-4-[[(2R) -hydroxy-2- (5,5,8,8-tetramethyl-5 , 6,7,8, -Tetrahydro-2-naphthalenyl) acetyl] amino] -benzoic acid (3-fluoro-4-[[(2R) -hydroxy (5,5,8,8-tetramethyl-5,6, 7,8-t
  • the salt includes an acid addition salt and a salt with a base.
  • acid addition salts include hydrochloride, hydrobromide, hydroiodide, sulfate, perchlorate, phosphate and other inorganic acid salts, oxalate, malonate, succinic acid Salt, maleate, fumarate, lactate, malate, citrate, tartrate, benzoate, trifluoroacetate, acetate, methanesulfonate, p-toluenesulfonate, trifluoromethane
  • organic acid salts such as sulfonates, and acidic amino acid salts such as glutamates and aspartates.
  • salts with bases include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt or calcium salt, salts with organic bases such as pyridine salt and triethylamine salt, bases such as lysine and arginine. And salts with sexual amino acids.
  • RAR ⁇ agonists are known per se or can be appropriately produced from known compounds.
  • these RAR ⁇ agonists can be used alone or in combination of two or more.
  • the case where the agonist of RAR ⁇ is present in the form of hydrate or solvate is also included.
  • the RAR ⁇ agonist can give stereoisomers, geometric isomers and the like, unless otherwise specified, the stereoisomers and geometric isomers are included in the RAR ⁇ agonists which are the active ingredients of the present invention.
  • the active ingredient RAR ⁇ agonist itself may be used as an adjuvant, but various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH regulators, preservatives). , Antioxidants, solubilizers, thickeners, etc.) may be used as an adjuvant composition. Further, in the present invention, it is preferable to use a RAR ⁇ agonist in combination with a RAR ⁇ agonist because a synergistic action can be brought about.
  • the RAR ⁇ agonist is not particularly limited, and examples thereof include 4-[[(2,3-dihydro-1,1,3,3-tetramethyl-2-oxo-1H-indene-5-nyl) carbonyl] amino].
  • -Benzoic acid (4-[[(2,3-Dihydro-1,1,3,3-tetramethyl-2-oxo-1H-inden-5-yl) carbonyl] amino] -benzoic acid) (BMS753, CAS number : 215307-86-1); 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) amino] carbonyl] benzoic acid salt (4-[[( 5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) amino] carbonyl] benzoic acid) (Tamibarotene, CAS 94497-51-5), 4-[(5,6, 7,8-Tetrahydro
  • the blending ratio of the RAR ⁇ agonist and the RAR ⁇ agonist is not particularly limited.
  • the latter is in the range of 0.1 to 10 mol, preferably 0.5 to 6 mol, preferably 1 to 5 mol with respect to 1 mol of the former. Can be set as appropriate.
  • isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, sodium chloride, potassium chloride, and calcium chloride. Examples include inorganic salts. These tonicity agents can be used alone or in combination of two or more.
  • chelating agents examples include edetate salts such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or salts thereof, hexametalin Examples include acid soda and citric acid. These chelating agents can be used alone or in combination of two or more.
  • Examples of the stabilizer include sodium bisulfite.
  • Examples of the pH regulator include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, and further alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate, or hydrogen carbonate. Salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like. These pH regulators can be used singly or in combination of two or more.
  • preservatives examples include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, chloride Quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, chlorhexidine and the like can be mentioned. These preservatives can be used singly or in combination of two or more.
  • antioxidants examples include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like. These antioxidants can be used alone or in combination of two or more.
  • solubilizer examples include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like.
  • thickening agent examples include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol and the like. These solubilizing agents can be used alone or in combination of two or more. These thickening agents can be used alone or in combination of two or more.
  • the adjuvant composition may further contain a compound known to be used as an adjuvant.
  • the content of the RAR ⁇ agonist in the composition is not particularly limited, and is, for example, 90% by mass or more, 70% by mass or more, 50% by mass or more in terms of the content of the RAR ⁇ agonist. It can be appropriately set from conditions such as 30% by mass or more, 10% by mass or more, 5% by mass or more, 1% by mass or more.
  • the dosage form for administering the agonist of RAR ⁇ is not particularly limited, and for example, oral administration agents such as tablets, pills, capsules, powders, granules, syrups; injections (intravenous injection, intramuscular injection) , Topical injections, etc.), mouthwashes, drops, external preparations (ointments, creams, patches, inhalants), parenteral administration agents such as suppositories, and the like.
  • oral administration agents such as tablets, pills, capsules, powders, granules, syrups
  • injections intravenous injection, intramuscular injection
  • mouthwashes e.g., ointments, creams, patches, inhalants
  • parenteral administration agents such as suppositories, and the like.
  • preferable examples include oral administration agents, injections, and external preparations.
  • the agonist of RAR ⁇ which is an active ingredient of the present invention, can exert TSLP production-inducing action even
  • the administration site is not particularly limited, and can be used at a place where the external preparation is usually applied. Examples thereof include skin, oral cavity, and the like, and skin is preferable.
  • the vaccine adjuvant of the present invention can be used by combining with an antigen to make a vaccine and administering it to a subject such as a mammal. Accordingly, the present invention also provides a method for preventing or treating a disease, comprising the steps of administering the vaccine adjuvant to a mammal and administering an antigen to the mammal. As will be described later, the method of the present invention includes not only a method of administering the vaccine adjuvant and the antigen at intervals, but also a method of administering both at the same time.
  • the step of administering the vaccine adjuvant to a mammal and the step of administering an antigen to the mammal are performed separately (for example, before, simultaneously with and / or after the adjuvant administration step). And a method of performing both steps simultaneously.
  • mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cows, horses, sheep and the like.
  • the vaccine adjuvant of the present invention can be used for any of the conventional diseases for vaccine treatment.
  • target diseases include influenza (types A, B and C), HIV, chlamydia, malaria, tuberculosis, tumor, measles, mumps, chickenpox, yellow fever, rotavirus gastroenteritis, smallpox, pneumococci, rabies, cholera, diphtheria, pertussis, tetanus, Japanese encephalitis, papillomavirus , Hepatitis (A type, B type, C type) and Ebola hemorrhagic fever, dengue fever, Zika fever, and combinations of these diseases.
  • the present invention can be used for the prevention and treatment of the above diseases, particularly for prevention.
  • the content of the RAR ⁇ agonist in the preparation varies depending on the administration route, patient age, body weight, symptoms, etc., and cannot be specified unconditionally, but the RAR ⁇ agonist single dose is usually about 0.001 to 10 mg.
  • the RAR ⁇ agonist which is an active ingredient of the adjuvant, may be administered at the same time as the vaccine, but is preferably administered separately at regular intervals.
  • the present inventor has found that when a RAR ⁇ agonist is administered to a patient, the TSLP production-inducing action is maximized after a certain period of time (typically, about 24 hours after administration in the case of an external preparation). It was. Therefore, in a preferred embodiment of the present invention, the vaccine adjuvant of the present invention comprises at least 4 hours, preferably at least 12 hours, more preferably 23 hours, more preferably 24 hours after administration of the RAR ⁇ agonist. It can be used in an administration schedule in which an antigen is administered after the course.
  • the vaccine adjuvant of the present invention is administered with an antigen before the lapse of 50 hours, preferably before the lapse of 36 hours, more preferably before the lapse of 25 hours after administration of the RAR ⁇ agonist.
  • the active ingredient is continuously absorbed from the skin for a predetermined time after application of the ointment or cream or after application of the patch.
  • “after elapse of t time after administration of RAR ⁇ agonist” means after elapse of t time from the time of applying ointment or cream or the time of applying the patch.
  • a RAR ⁇ agonist is administered to at least one of the plurality of antigen administrations.
  • the antigen is not particularly limited, and examples thereof include carbohydrates, lipids, glycolipids, phospholipids, polypeptides, proteins, glycoproteins, lipoproteins, oligonucleotides, polynucleotides, and chemical or recombinant conjugates thereof. Can be mentioned. Antigens also include viruses, fungi, bacteria, parasitic microorganisms, allergens, self-molecules and the like. Examples of antigens used for influenza vaccines include hemagglutinin (HA), neuraminidase (NA), M1 protein, M2 protein, and nucleoprotein (NP). These antigens can be used alone or in combination of two or more.
  • HA hemagglutinin
  • NA neuraminidase
  • M1 protein M2 protein
  • NP nucleoprotein
  • TSLP can be selectively induced and humoral immunity can be mainly enhanced. Further, in the present invention, it is considered that RAR ⁇ agonists selectively induce TSLP using epithelial cells as an action point, not dendritic cells, lymphocytes and the like.
  • the present invention provides a vaccine comprising a combination of the aforementioned vaccine adjuvant and the aforementioned antigen.
  • the vaccine of the present invention may be in the form of a preparation containing both the vaccine adjuvant and the antigen, or may be in the form of a kit provided with the vaccine adjuvant and the preparation containing the antigen separately.
  • a vaccine comprising a combination of a vaccine adjuvant and an antigen includes both of the above forms.
  • a vaccine preparation containing both a vaccine adjuvant and the antigen, or an antigen-containing preparation provided in a vaccine kit is prepared by using various pharmaceutically acceptable carriers (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.) may be further blended.
  • various pharmaceutically acceptable carriers for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.
  • isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol, sodium chloride, potassium chloride, and calcium chloride. Examples include inorganic salts. These tonicity agents can be used alone or in combination of two or more.
  • chelating agents examples include edetate salts such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or salts thereof, hexametalin Examples include acid soda and citric acid. These chelating agents can be used alone or in combination of two or more.
  • Examples of the stabilizer include sodium bisulfite.
  • Examples of the pH regulator include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, and further alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates such as sodium carbonate, or hydrogen carbonate. Salts, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like. These pH regulators can be used singly or in combination of two or more.
  • preservatives examples include paraoxybenzoic acid esters such as sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate, chlorhexidine gluconate, benzalkonium chloride, chloride Quaternary ammonium salts such as benzethonium and cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, chlorhexidine and the like can be mentioned. These preservatives can be used singly or in combination of two or more.
  • antioxidants examples include sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like. These antioxidants can be used alone or in combination of two or more.
  • solubilizer examples include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol, D-mannitol and the like.
  • thickening agent examples include polyethylene glycol, methyl cellulose, ethyl cellulose, carmellose sodium, xanthan gum, sodium chondroitin sulfate, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol and the like. These solubilizing agents can be used alone or in combination of two or more. These thickening agents can be used alone or in combination of two or more.
  • the upper limit of the content of the antigen in the preparation is not particularly limited, but the antigen amount can be reduced by using the method of the present invention.
  • the antigen amount can be reduced by using the method of the present invention.
  • 1 mass% or less preferably 0.1 mass% or less, more preferably 0.01 mass% or less, more preferably 0.001 mass% or less.
  • the lower limit of the antigen content in the preparation is not particularly limited, but it can be preferably set based on conditions such as 0.1% by mass or more, 0.001% by mass or more, 0.000001% by mass or more.
  • the dosage form of the vaccine preparation is not particularly limited.
  • oral administration agents such as tablets, pills, capsules, powders, granules, syrups; injections (intravenous injection, intramuscular injection, local injection, etc.)
  • various preparation forms such as a mouthwash, an instillation, an external preparation (ointment, cream, patch, inhalant), a parenteral preparation such as a suppository, and the like.
  • preferable examples include oral administration agents, injections, and external preparations.
  • the antigen content in the preparation varies depending on the route of administration, patient age, body weight, symptoms, etc., and cannot be generally specified, but the daily dose of the antigen is usually about 10 to 5000 mg, more preferably about 100 to 1000 mg. It can be an amount. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.
  • the content of the antigen in the preparation is not particularly limited, and can be appropriately set, for example, from an amount equivalent to a normal use amount to an amount of about 1/1000.
  • the upper limit of the content of the antigen in the preparation is, for example, 1% by mass or less, preferably 0.1% by mass or less, more preferably 0.01% by mass or less, more preferably It can be set from conditions such as 0.001% by mass or less.
  • the lower limit of the content of the antigen in the preparation can be set, for example, based on conditions such as preferably 0.1% by mass or more, 0.001% by mass or more, 0.000001% by mass or more.
  • the formulation form of the antigen preparation provided in the vaccine kit in the present invention is not particularly limited, and is an oral administration agent such as a tablet, pill, capsule, powder, granule, syrup, etc .; injection (intravenous injection, intramuscular injection) , Topical injections, etc.), mouthwashes, drops, external preparations (ointments, creams, patches, inhalants), parenteral administration agents such as suppositories, and the like.
  • an oral administration agent such as a tablet, pill, capsule, powder, granule, syrup, etc .
  • injection intravenous injection, intramuscular injection
  • mouthwashes ointments, creams, patches, inhalants
  • parenteral administration agents such as suppositories, and the like.
  • preferable examples include injections.
  • the antigen content in the preparation varies depending on the route of administration, patient age, body weight, symptoms, etc., and cannot be generally specified, but the daily dose of the antigen is usually about 10 to 5000 mg, more preferably about 100 to 1000 mg. It can be an amount. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.
  • the present invention can include other components as necessary.
  • other components include, but are not limited to, a tool for collecting an adjuvant (for example, a syringe) and the like.
  • a tool for collecting an adjuvant for example, a syringe
  • the document etc. which wrote down the above-mentioned administration schedule can also be included.
  • the target disease, administration schedule, etc. of the vaccine of the present invention are the same as those described above for the vaccine adjuvant of the present invention.
  • those in which the RAR ⁇ agonist is a retinoid and the target disease is influenza are preferred.
  • the present invention is not limited to compounds that have TSLP-producing ability, and RAR ⁇ agonists are not only capable of producing TSLP but also other proteins (particularly TNF- ⁇ , IL-1 ⁇ , IL-4 involved in allergic reactions). It has been found that TSLP can be selectively induced over a protein such as a protein, and that neutralizing antibodies are produced when it is actually mixed with a vaccine and administered to a subject.
  • Non-Patent Document 4 (Immunobiology 221 (2016) 161-165) merely describes that retinoic acid induced TSLP, and selectively used TSLP rather than other proteins. There is also no disclosure about guiding to this. In the first place, the non-patent document 4 is not limited to the measurement of blood TSLP in mice sensitized with ovalbumin, and the administration of TSLP together with an antigen associated with some disease is also neutralizing the disease. There is no specific disclosure that the antibody was produced. Therefore, it cannot be said that Non-Patent Document 4 describes that a vaccine containing retinoic acid can be used.
  • Example 1 TSLP expression inducing action of retinoic acid (FIG. 1) 20 ⁇ l of acetone solution containing 0.2 to 20 nmol of All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old). After 24 hours, the mice were exsanguinated and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds.
  • TOMY sample tube
  • Precyllys 24, Bertin tech. Precyllys 24, Bertin tech.
  • the obtained homogenate was centrifuged at 21,600 ⁇ g, 4 ° C. for 20 minutes, and the amount of TSLP in the supernatant was measured with an ELISA kit (R & D Systems) (left).
  • the collected skin sample was homogenized in 500 ⁇ l of RNAiso Plus (Takara Bio) in the same manner using a bead-type cell crusher, and then RNA was extracted according to the protocol attached to RNAiso Plus.
  • the concentration of the obtained RNA solution was measured using NanoDrop (R) spectrophotometer ND-1000 (Thermo Fisher Scientific).
  • the threshold cycle (Ct) value of each sample was calculated by the 2nd derivative maximum method, and after creating a relative calibration curve and confirming that the PCR reaction conditions for each gene were close to 100% amplification efficiency, RNA was obtained by the Ct method. The amount was converted into a relative amount (right side of FIG. 1).
  • FIG. 1 As is clear from FIG. 1, atRA increased TSLP expression in a concentration-dependent manner at the protein level (FIG. 1, left) and the mRNA level (FIG. 1, right).
  • Example 2 Temporal change of TSLP production by retinoic acid (FIG. 2) All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 ⁇ l of acetone. After a certain period of time, the mice were exsanguinated and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds.
  • TOMY sample tube
  • Precyllys 24, Bertin tech. Precyllys 24, Bertin tech.
  • the obtained homogenate was centrifuged at 21,600 ⁇ g, 4 ° C. for 20 minutes, and the amount of TSLP in the supernatant was measured by ELISA kit (R & D Systems). atRA group, black circle, acetone group, white circle.
  • TSLP production by atRA reached a maximum at 24 hours after application and then decreased.
  • Example 3 TSLP selective expression inducing action of retinoic acid (FIG. 3) All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 ⁇ l of acetone. Mice were bled to death after 8 and 24 hours, and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds.
  • TOMY sample tube
  • Precyllys 24, Bertin tech. Precyllys 24, Bertin tech.
  • the obtained homogenate was centrifuged at 21,600 ⁇ g, 4 ° C. for 20 minutes, and the amount of cytokine in the supernatant was measured by ELISA.
  • the ELISA kit used is as follows. TSLP (R & D Systems), IL-1 ⁇ (R & D Systems), TNF- ⁇ (eBioscience), and IL-4 (eBioscience).
  • Example 4 TSLP producing tissue (FIG. 4) All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 ⁇ l of acetone. After 24 hours, the mice were exsanguinated and the auricles were collected. 1 Frozen sections were prepared and fluorescent immunostaining was performed using an anti-TSLP antibody (SantaCruz).
  • TSLP is mainly expressed in epithelial tissues.
  • Example 5 Auricular tissue 24 hours after application of retinoic acid (FIG. 5) All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the auricle of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 2 nmol or 20 ⁇ l of acetone. After 24 hours, the mice were exsanguinated and the auricles were collected. Frozen sections were prepared and Hematoxylin-Eosin staining was performed.
  • retinoic acid atRA, Sigma Aldrich
  • Example 6 Action of RAR ⁇ agonist (FIG. 6) 20 ⁇ l of an acetone solution containing RAR ⁇ agonist BMS753 (100 nnoml) and / or RAR ⁇ agonist BMS189961 (20, 100 nmol) was applied to the ears of ICR mice (Japan SLC, male, SPF, 6 weeks old). After 24 hours, the mice were exsanguinated and the auricles were collected. Weigh it, put it in a sample tube (TOMY), add 10 times the volume (w / v) of homogenizing buffer, and use a bead-type cell crusher (Precyllys 24, Bertin tech.). Homogenization was performed twice for 30 seconds. The obtained homogenate was centrifuged at 21,600 ⁇ g, 4 ° C. for 20 minutes, and the amount of TSLP in the supernatant was measured with an ELISA kit (R & D Systems).
  • RAR ⁇ agonist BMS189961 alone enhanced TSLP production.
  • the RAR ⁇ agonist BMS753 alone did not increase TSLP production, but the effect was enhanced by using it together with the RAR ⁇ agonist BMS189961.
  • Example 7 Retinoic acid enhances production of anti-OVA antibody (IgG1, IgE) (FIG. 7)
  • All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the back of ICR mice (Japan SLC, male, SPF, 6 weeks old) with acetone solution containing 4, 12, 40 nmol or 40 ⁇ l of acetone. 24 hours later, 50 ⁇ l of physiological saline containing 3 ⁇ g of ovalbumin (OVA) was intradermally injected into the same site. Two weeks later, blood was collected, and the serum anti-OVA IgG1 and anti-OVA IgE levels were measured by the respective ELISA kit (Cayman).
  • IgG1 production was slightly observed and IgE production was hardly observed.
  • the production of IgG1 was significantly induced from the pretreatment with atRA-4 nmol.
  • IgE production increased slightly with application of 12 nmol or more.
  • Example 8 Retinoic acid enhances neutralizing antibody production against recombinant influenza hemagglutinin A (H1N1) strain (A / California / 7/2009 (H1N1) or B Victoria strain (B / Brisbane 60/2008) Vaccine activity was evaluated using the influenza hemagglutinin gene recombinant as an antigen All-trans retinoic acid (atRA, Sigma Aldrich) 40 nmol in acetone solution or 40 ⁇ l acetone in BALB / c mice (Japan SLC, female, SPF, 6 weeks old) 24 hours later, 0.1 ml of physiological saline containing 3 ⁇ g of recombinant influenza hemagglutinin was injected subcutaneously, 3 weeks later, all-trans retinoic acid (atRA, Sigma Aldrich) ) Was applied to the back of an acetone solution containing 40 nmol or 40 ⁇ l of acetone, and 24 hours later, 0.1 ml of physiological sa
  • Example 9 Retinoic acid reduces the amount of recombinant influenza hemagglutinin necessary for neutralizing antibody production (FIG. 8) All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the back of BALB / c mice (Japan SLC, female, SPF, 6 weeks old) with acetone solution containing 40 nmol or 40 ⁇ l of acetone.
  • H1N1 hemagglutinin A
  • H1N1 A / California / 7/2009
  • B Victoria strain B / Brisbane 60/2008
  • All-trans retinoic acid atRA, Sigma Aldrich
  • Example 10 Effect of retinoic acid on neutralizing antibody and IgE production in the case of low concentration antigen stimulation (FIG. 9) All-trans retinoic acid (atRA, Sigma Aldrich) was applied to the back of BALB / c mice (Japan SLC, female, SPF, 6 weeks old) with acetone solution containing 4, 12, 40 nmol or 40 ⁇ l of acetone. 24 hours later, 0.1 ml of physiological saline containing recombinant influenza hemagglutinin A (H1N1) strain (A / California / 7/2009 (H1N1) [0.003 ⁇ g] was injected subcutaneously at the same site.
  • H1N1 physiological saline containing recombinant influenza hemagglutinin A
  • the present invention is the first invention that a low-molecular-weight organic compound having high purity and extremely low flammability can be used as a vaccine.
  • the present invention can be expected to reduce the side effects caused by the unpurified antigen itself that has been conventionally used as a vaccine, and to reduce the amount of antigen to be used. Expected to be.
  • the vaccine adjuvant of the present invention can be applied as an external preparation, it is useful in terms of low invasiveness, suppression of nonspecific adjuvant effect expression, convenience, and the like. Therefore, the present invention is extremely useful in industry.

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Abstract

La présente invention traite le problème de la fourniture d'un adjuvant chimiosynthétique pour induire sélectivement TSLP. La présente invention concerne un adjuvant de vaccin comprenant un agoniste de gamma RAR, et un vaccin obtenu par combinaison de l'adjuvant de vaccin et d'un antigène.
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Citations (4)

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JP2011500733A (ja) * 2007-10-26 2011-01-06 ケマファー インコーポレーテッド 免疫応答を増強するための組成物および方法
WO2011051657A1 (fr) * 2009-10-26 2011-05-05 St George's Hospital Medical School Protéine a comme adjuvant de vaccin
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WO2001078700A2 (fr) * 2000-04-13 2001-10-25 Frederic Geissmann Compositions et procedes utilises pour moduler la fonction du systeme immunitaire
JP2011500733A (ja) * 2007-10-26 2011-01-06 ケマファー インコーポレーテッド 免疫応答を増強するための組成物および方法
WO2011051657A1 (fr) * 2009-10-26 2011-05-05 St George's Hospital Medical School Protéine a comme adjuvant de vaccin
WO2016021601A1 (fr) * 2014-08-04 2016-02-11 日東電工株式会社 Composition favorisant l'induction immunitaire et comprenant un ligand des récepteurs nucléaires, et composition pharmaceutique vaccinale

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MARTIN MARIA DEL P. ET AL.: "Adjuvanted Influenza Vaccine Administered Intradermally Elicits Robust Long-Term Immune Responses that Confer Protection from Lethal Challenge", PLOS ONE, vol. 5, no. 5, 2010, pages e10897, XP055429093 *
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