JP2014040396A - Adjuvant composition containing dyslipidemia therapeutic agent - Google Patents

Adjuvant composition containing dyslipidemia therapeutic agent Download PDF

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JP2014040396A
JP2014040396A JP2012183875A JP2012183875A JP2014040396A JP 2014040396 A JP2014040396 A JP 2014040396A JP 2012183875 A JP2012183875 A JP 2012183875A JP 2012183875 A JP2012183875 A JP 2012183875A JP 2014040396 A JP2014040396 A JP 2014040396A
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antigen
adjuvant
composition according
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peptide
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Junichi Matsuda
純一 松田
Kazuyoshi Kaminaka
一義 上仲
Chikahide Nozaki
周英 野崎
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Chemo-Sero-Therapeutic Research Institute
一般財団法人化学及血清療法研究所
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Abstract

PROBLEM TO BE SOLVED: To provide a new adjuvant in place of a conventionally used alum adjuvant, that is, an adjuvant which has a high effect of enhancing the immunogenicity of an antigen and is rich in safety to the human body.
An adjuvant containing a dyslipidemic agent exhibits an adjuvant effect equivalent to or higher than that of alum adjuvant. In addition, since dyslipidemic drugs have been used by a great number of people as pharmaceuticals, the safety to the human body is also excellent. Therefore, the adjuvant containing the therapeutic agent for dyslipidemia of the present invention is excellent in antibody production ability and safety.
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Description

  The present invention relates to an adjuvant composition containing a therapeutic agent for dyslipidemia.

  Substances added for the purpose of enhancing the immune response in an antigen-antibody reaction are generally called immunostimulants and adjuvants. An adjuvant refers to a substance that enhances an immune response to an administered antigen when mixed with the antigen and administered to a living body.

  Adjuvants such as adsorbing antigens to enhance the uptake of antigen-presenting cells, maintaining antigen stimulation by gradually releasing the antigen locally for a long period of time, or directly activating immunocompetent cells It depends on the type. Therefore, an adjuvant is very useful in terms of reducing the dose and frequency of administration of the vaccine and the amount of antigen in the vaccine. As a typical adjuvant, aluminum hydroxide (hereinafter referred to as alum adjuvant) has been used for many vaccines for a long time, but it is difficult to say that it is an ideal adjuvant from the viewpoint of immunity induction effect and convenience. Other than alum adjuvants, there are those using squalene, MPL (monophosphoryl lipid), etc., but they have a weak point that the adjuvant activity is strong but the side reaction is also strong. Therefore, in the medical field, development of an adjuvant that elicits a high immune response, has few side effects, and has improved convenience is eagerly desired.

  On the other hand, dyslipidemia is an excess or deficiency of lipids in the blood, and this symptom itself is not particularly painful, and it does not show any particular subjective symptoms, Arteriosclerosis (and cerebral infarction, angina pectoris, myocardial infarction) and complications are likely to occur. At present, dyslipidemia is classified into hypercholesterolemia, high LDL (low density lipoprotein) cholesterolemia, low HDL (high density lipoprotein) cholesterolemia, and high TG (triglyceride) emia. Hypercholesterolemia is a type of dyslipidemia that has a high total cholesterol level in the blood. Many dyslipidemias due to lifestyle are of this type. Hyper-LDL cholesterolemia is a type of dyslipidemia in which a large amount of LDL, a carrier of cholesterol, is present in the blood. It is the only absolute risk factor for cardiovascular disease among the test values for cholesterol, and is clearly more important than other test values for HDL and TG. Low HDL cholesterolemia is a type of dyslipidemia with low HDL in the blood. HyperTG is a type of dyslipidemia in which a lot of TG is present in the blood. Treatment includes diet, exercise, and medication.

  Currently used therapeutic agents for dyslipidemia include HMG-CoA (hydroxymethylglutaryl CoA) reductase inhibitor (statin), fibrate, anion exchange resin (resin), and probucol. In addition, there are preparations that have indications for health insurance in Japan, such as fish oil (EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid)), plant sterols, and vitamins.

  It is not known that such a dyslipidemic agent has an adjuvant activity. Rather, statins have been reported to suppress immunity (Non-patent Document 1).

International Publication 2008/133208 Pamphlet International Publication 2010/044464 Pamphlet

Okazaki H., Nagashima T. and Minota S., Jpn.J.Clin.Immunol., 27 (6) 357-360 (2004)

  The present inventor provides a new adjuvant that replaces the conventionally used alum adjuvant, that is, an adjuvant that is highly effective in enhancing the immunogenicity of the antigen, is highly safe to the human body, and has excellent solubility in a solvent. It is a problem to do.

  In view of such circumstances, the present inventors have intensively studied, and as a result, extremely new findings that have not been reported in the prior art that a therapeutic drug for dyslipidemia exerts an adjuvant effect equivalent to or better than that of alum adjuvant. I found. In addition, since dyslipidemic drugs have been used by a great number of people as pharmaceuticals, the safety to the human body is also excellent. In addition, dyslipidemic agents are easy to dispense because of their excellent solubility in solvents. Therefore, in the present invention, it is possible to provide an adjuvant excellent in adjuvant effect, safety to the human body, and solubility in a solvent by using the therapeutic agent for dyslipidemia as an adjuvant.

That is, the present invention
[1] An adjuvant composition containing a therapeutic agent for dyslipidemia.
[2] The adjuvant composition according to [1], wherein the therapeutic agent for dyslipidemia is one or more compounds selected from the group consisting of statins, fibrates, fish oil, and metformin.
[3] The adjuvant according to [2], wherein the statin is one or more compounds selected from the group consisting of atorvastatin, simvastatin, pitavastatin, fluvastatin, pravastatin, mevastatin, lovastatin, and the salts and hydroxides described above. Composition.
[4] The adjuvant composition according to [2], wherein the fibrate is one or more compounds selected from the group consisting of fenofibrate, bezafibrate, and the above salts and hydroxides.
[5] The adjuvant composition according to [2], wherein the fish oil is one or more compounds selected from the group consisting of eicosapentaenoic acid (EPA), ethyl icosapentate, and the above salts and hydroxides.
[6] The adjuvant composition according to any one of [1] to [5], wherein the adjuvant composition is excellent in solubility in a solvent.
[7] A vaccine composition comprising the adjuvant composition according to any one of [1] to [6] and an antigen.
[8] The vaccine composition according to [7], wherein the antigen is an influenza virus antigen.
[9] The vaccine composition according to [8], wherein the influenza virus antigen is influenza virus hemagglutinin (HA).
[10] The vaccine composition according to [7], wherein the antigen is a peptide antigen.
[11] The vaccine composition according to [10], wherein the peptide antigen is an Aβ peptide.
[12] The vaccine composition according to [11], wherein the Aβ peptide is a peptide consisting of a partial amino acid sequence of the Aβ peptide, or a peptide obtained by adding or inserting one or several cysteines to the peptide.
[13] The vaccine composition according to [11] or [12], wherein the Aβ peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
[14] The Aβ peptide has an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. The fusion protein described.

  According to the present invention, it is possible to improve immunogenicity by administering a therapeutic drug for dyslipidemia and an antigen together as compared with the case of administering the antigen alone. In addition, depending on the type of dyslipidemic agent selected, the adjuvant effect is much higher than that of the existing adjuvant (Alum adjuvant).

  A dyslipidemic drug is a drug already administered to the human body, and since its drug efficacy and side effects are known, it is considered that the risk of occurrence of side effects is reduced. In addition, dyslipidemic drugs have substances with various properties such as water solubility and lipid solubility, and it is thought that it is possible to select those that are easy to dispense with antigens compared to conventional alum adjuvants and oil-based adjuvants. . Therefore, the adjuvant containing the therapeutic agent for dyslipidemia of the present invention and the vaccine containing the same have improved convenience and superior safety to the human body compared to the conventional adjuvant and the vaccine containing the same. it is conceivable that.

  The present invention includes an adjuvant composition containing a dyslipidemic agent.

  The concentration of the dyslipidemic agent may be appropriately changed according to the type of antigen, the drug dosage form, the administration method, the storage conditions, and the like. Preferably, A to B μg / mL (A or B is 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500 , 4000, 4500, 5000, 10000, 20000, 25000, 50000, and A is a numerical value smaller than B).

  The dose of the therapeutic agent for dyslipidemia may be appropriately changed according to the type of antigen, the dosage form of the drug, the administration method, storage conditions, and the like. For example, the dose may be set by immunizing a test animal after mixing with an antigen at various concentrations and evaluating an antibody titer to the antigen or an infection protection test. Preferably, A to B μg / individual (A or B is selected from 1, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, and A is a numerical value smaller than B).

  The antibody titer is measured by applying the antiserum collected from the test animal to the antigen-immobilized plate, a secondary antibody labeled with an enzyme or fluorescent dye, and if necessary, a chromogenic substrate, and measuring the absorbance. The method of doing is illustrated.

  Nunc Immunoplate Maxi Soap (Thermo scientific), ELISA Plate (Sumitomo Bakelite Co., Ltd.), ELISPOT (MERCK), Immunoplate (Cosmo Bio Co., Ltd.), Elisa Plate (IWAKI), ELISA Plate (ExtraGene) The antigen may be bound to the plate by a method commonly practiced by those skilled in the art.

  Secondary antibodies include Calbiochem (EMD Millipore), Chemicon (Millipore), anti-mouse IgG-POD labeled antibody (Thermo), GOAT Anti-MOUSE IgG (Beckman Coulter, Inc.), Whole IgG anti-immunoglobulin (Iwai Chemical) Co.) is exemplified.

  HMG-CoA reductase inhibitor (statin), fibrate, anion exchange resin (resin), probucol, fish oil, plant sterol, vitamin, ezetimibe, unapproved or under development in Japan Drugs (TAK-475, ISIS 301012). In addition, among drugs classified as antidiabetic drugs, for example, those that are applied to the improvement of dyslipidemia, such as the biguanide drug metformin, are known. Preferred dyslipidemic agents for use in adjuvants are statins, fibrates, fish oils, and metformin.

  Statins have the effect of inhibiting hepatocyte HMG-CoA reductase, reducing intracellular cholesterol content, up-regulating LDL receptors (increasing receptors), and taking cholesterol from the blood. Since the discovery of mevastatin, the first statin by Akira Endo in Japan in 1973, various types of statins have been developed and used around the world. In addition to the pharmacological action as a dyslipidemic agent, statins are said to have various actions such as anti-inflammatory action, NO (oxygen monoxide) synthesis promotion action (coronary artery dilation), and immunosuppressive action. Statins include atorvastatin, simvastatin, cerivastatin, pitavastatin, fluvastatin, pravastatin, mevastatin, rosuvastatin, lovastatin, the above salt (sodium salt, calcium salt), or the above hydroxide. Atorvastatin, atorvastatin sodium, atorvastatin calcium, simvastatin, pitavastatin, fluvastatin, pravastatin, mevastatin, lovastatin, and lovastatin hydroxy acid are preferable.

  Fibrates act on α-type peroxisome proliferator-activated receptors (PPAR-α), which are nuclear receptors, and control the synthesis of proteins involved in lipid synthesis. It also increases LPL (lipoprotein lipase) expression and promotes VLDL (very low density lipoprotein) and chylomicron catabolism in the vascular endothelium. Fibrates include clinofibrate, clofibrate, clofibrate aluminum, fenofibrate, and besafibrate. Preferable are fenofibrate and bezafibrate.

  Resins have the longest history as drugs for treating dyslipidemia. Since it is safe in that it is not absorbed by the body, it can be used with peace of mind even in patients with familial hyperlipidemia who are planning puberty or pregnancy. It is the drug used in the clinical trial LRC-CTTP (Lipid Research Clinics Coronary Primary Prevention Trial), where lipid lowering therapy was first proven to lead to prevention of ischemic heart disease. Cholesterol is excreted in bile but is reabsorbed while flowing to the terminal ileum (enterohepatic circulation). If the reabsorption is inhibited and excreted in the stool, the cholesterol level decreases. Furthermore, it upregulates the LDL receptor and increases cholesterol uptake from the blood. As the resin, cholestyramine and colestimide are used.

  The mechanism of action of probucol is still unclear, but it increases the expression of SR-BI (scavenger receptor class B type I) receptor, which is a type of scavenger receptor and also involved in HDL uptake, and catabolizes HDL It is said to activate the reverse cholesterol transfer circuit. The regression of the arteriosclerotic lesion is seen more than the degree of cholesterol lowering. It is also said to have an antioxidant effect.

  Fish oil is a fatty oil collected from fish, and the fatty acid component is mainly palmitic acid, but includes stearic acid, myristic acid, arachidic acid, oleic acid, hexadecenoic acid and the like. In addition, the content of highly unsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), such as highly unsaturated fatty acids and unsaponifiable compounds, is also high. Fish oil suppresses fatty acid synthesis via the transcription factor SREBP-1 (sterol regulatory element binding protein-1). It is also metabolized as PUFA (polyunsaturated fatty acid) to become PG13 (prostaglandin 13), which exhibits antiplatelet action. It also has the benefits of preventing blood clots associated with rupture of vaginal species and improving blood circulation in chronic obstructive arteriosclerosis. There is EPA which refined sardine fish oil as a preparation, and the relapse prevention effect of ischemic heart disease was confirmed in the JELIS test conducted in Japan. In addition, DHA and EPA have the effect of improving learning ability / memory ability, suppressing visual loss, suppressing thrombus, improving exercise ability, improving / preventing senile dementia, antiallergy, anti-inflammatory action, and improving atopy It has been known. Fish oil may be used as the adjuvant composition of the present invention, but it is preferable to use fatty acids contained in fish oil. As the adjuvant composition, eicosapentaenoic acid (EPA) or ethyl icosapentate which is an ethyl ester compound thereof is preferable.

  Plant sterols, such as rice oil γ oryzanol, dissolve in bile micelles circulating in the intestinal liver and competitively inhibit the uptake of cholesterol, which is an animal lipid, in the intestinal tract, thus lowering cholesterol levels.

  Ezetimibe inhibits the function of NPC1L1 (Niemann-Pick C1 Like 1), a small intestine cholesterol transporter that expresses cholesterol absorption, which is expressed on the epithelial cell lumen side of the small intestine, and absorbs cholesterol and plant sterols more than placebo. % Suppress. It is a small tablet, unlike a bulky resin that is difficult to squeeze, and a combination with statin has also been developed.

  TAK-475 (generic name: lapaquistat acetate) suppresses “squalene synthase” downstream of HMG-CoA reductase, reduces intracellular cholesterol content, upregulates LDL receptor, and reduces cholesterol from blood Incorporate. Development was discontinued on March 28, 2008.

  ISIS 301012 is an antisense RNA that inhibits the synthesis of Apo-B required to carry cholesterol.

  The adjuvant composition of the present invention exhibits an adjuvant effect equivalent to or higher than that of alum adjuvant. The adjuvant effect refers to an effect of improving antibody production against an antigen when an antigen and an adjuvant composition are administered in combination as compared to when administered with an antigen alone.

  The present invention includes a vaccine composition comprising a therapeutic agent for dyslipidemia and an antigen. Vaccine compositions of the present invention can include therapeutic vaccines, prophylactic vaccines, and vaccines with both therapeutic and prophylactic uses.

  The method for preparing the vaccine composition of the present invention comprises a method of mixing an antigen solution and a liquid adjuvant composition containing a therapeutic agent for dyslipidemia, a method of dissolving a solid (powder, etc.) adjuvant composition in the antigen solution, a solid And a method of dissolving a solid antigen (powder or the like) in a liquid adjuvant composition, a method of mixing a solid antigen and a solid adjuvant composition, and then dissolving them in a solvent. Preferably, the antigen solution and the liquid adjuvant composition are mixed.

  The type of antigen may be inactivated antigen, attenuated antigen, subunit antigen, protein antigen, peptide antigen, virus-like particle, or virosome. Chemically, it may be a carbohydrate, glycolipid, glycoprotein, lipid, lipoprotein, phospholipid, polypeptide, protein, peptide, polynucleotide, oligonucleotide, or a chemical or recombinant conjugate thereof. . The method for obtaining the antigen may be obtained from genetic recombination means, chemical synthesis, or natural products.

  Antigens may be viruses, bacteria, fungi, parasitic microorganisms, cancer cells, tumor cells, allergens, self molecules, pathogens that infect cells (eg, tumor cells or normal cells).

  The types of bacteria include, for example, Actinobacillus pleuropneumoniae, Aroiocox otiditis, Haemophilus influenzae (both typeable and nontypeable), Yersinia, Parrot disease Chlamydia, Campylobacter, Chlamydia pneumonia Pathogens, Clostridia species, Vibrio cholerae, Salmonella choleresius, Giardia, Diphtheria, Pseudomonas species, Streptococcus gordonii, Streptococcus thermophilus, Streptococcus bovis, Streptococcus agalactiae, Trachoma chlamydia, T. tuberculosis・ Hemolytica, Pasteurella multocida, Mycobacterium tuberculosis, Streptococcus porcine, Proteus bulgaris, Proteus mirabilis, Hemophilus somnus, Helicobacter Helicobacter pylori, Borrelia burgdorferi, Mycoplasma galicepticum, Moraxella catararis, Leptospira interrogans, Staphylococcus aureus, Streptococcus pyogenes, Neisseria meningitidis, Shigella, Streptococcus, Escherichia coli, Bacillus anthracis, Salmonella typhi, Tetanus , Streptococcus pneumoniae, Bordetella pertussis, Staphylococcus epidermidis, Streptococcus faeces, Green streptococci, and Neisseria gonorrhoeae.

  The types of viruses include, for example, hepatitis virus, RS virus, adenovirus, abra virus, isa virus, canine distemper virus, influenza virus AC, equine arteritis virus, Ebola virus, enterovirus, calicivirus, coronavirus, simian immunodeficiency virus , Sogotovirus, Dengue virus, Toga virus, Avian infectious bursa disease virus, Avian pneumonia virus (formerly turkey rhinotracheitis virus), Nipper virus, Newcastle disease virus, Pneumovirus feline infectious peritonitis virus, Feline leukemia virus, No Walk virus, papilloma virus, papova virus, parainfluenza virus type 1-3, parvovirus, picornavirus human cytomegalovirus, human post-pneumonia virus , Human immunodeficiency virus, porcine respiratory injury / reproductive syndrome virus, flavivirus, henipavirus, hepadnavirus, herpesvirus, Hendra virus, poliovirus, Marek's disease virus, metapneumovirus, morbillivirus, rhinovirus , Rubra virus, respirovirus, retrovirus, rotavirus, vaccinia, yellow fever virus, infectious rhinotracheitis virus, rinderpest virus, rabies virus, varicella virus, encephalitis virus, rubella virus, measles virus, epidemic parotid gland It may be a flame virus. An influenza virus is preferred.

  The types of parasites are, for example, Shigella amoeba, Plasmodium, Forest type tropical Leishmania, Ascaris, Trichinella, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma, Pneumocystis carini There may be.

  Influenza virus is an RNA envelope virus belonging to the Orthomyxoviridae family and having a particle size of about 100 nm in diameter, and is classified into A, B, and C types based on the antigenicity of the internal protein. Among them, A and A are highly diverse infecting both humans and animals. The type A has two types of envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Due to the difference in antigenicity, there are 16 types in HA and 9 types in NA. There is an influenza A virus. When a combination of influenza viruses that has not occurred in the past appears, it is a pandemic because we are not immune to it. It is a so-called influenza pandemic. In the present invention, the type and subtype of influenza virus are not particularly limited, and the antigen may be not only HA and NA but also proteins constituting viruses such as M1, M2, and NP. Preferably, it is HA used in the current influenza vaccine.

  The method for preparing the influenza virus antigen is not particularly limited, and any known method can be used without limitation. For example, a virus strain isolated from an influenza-infected animal or an influenza patient may be infected with chicken eggs or cells and cultured by a conventional method, and an antigen may be prepared from a purified virus stock solution (Okuno Y., Japan Thoracic Clinic) , 59 (9) 645-652 (2000)). In addition, an influenza virus recombined by a genetic engineering technique such as reverse genetics may be used as a starting material.

  A peptide peptide is exemplified by Aβ peptide. Examples of the Aβ peptide include an Aβ peptide (for example, SEQ ID NO: 1), a peptide consisting of a part of the amino acid sequence of the Aβ peptide (for example, a peptide at positions 1 to 28 counted from the N terminus of the Aβ peptide, SEQ ID NO: 2), A peptide in which cysteine is added or inserted into a part of the amino acid sequence of the above-mentioned Aβ peptide is disclosed in International Publication No. 2008/133208 (Patent Document 1) and International Publication No. 2010/044464 (Patent Document 2). Aβ peptide. Preferable is peptide 28AACys (SEQ ID NO: 3) in which one cysteine residue is added to the C-terminal side of a peptide consisting of 28 amino acids counted from the N-terminus of Aβ peptide. The above Aβ peptides can be obtained by chemical synthesis.

  In the vaccine composition of the present invention, the antigen concentration and the antigen dosage may be appropriately changed according to the type of antigen, the dosage form and dosage form of the vaccine, etc., and are set based on the antibody titer and the infection protective effect as an index. It doesn't matter. When the antigen is influenza virus HA, the antigen concentration is A to B μg / mL (A or B is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, (5, 6, 7, 8, 9, 10, 50, 100, A is a number smaller than B). In addition, the antigen dose is A to B μg / individual (A or B is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10 and A is a numerical value smaller than B). When the antigen is 28AACys, the antigen concentration is A to B μg / mL (A or B is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, (Choose from 600, 700, 800, 900, 1000, A is smaller than B). The antigen dose is A to B μg / mL (A or B is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800. , 900, 100, and A is a numerical value smaller than B).

  The vaccine composition of the present invention may contain a single antigen or a plurality of types of antigens. When multiple types of antigens are included, there are cases where multiple types of antigens such as different types of viruses and bacteria are included (multivalent vaccine), and cases where multiple types of antigens such as different types of viruses and bacteria are included (mixed vaccines). May be. Viruses and bacteria may be selected from those described above. In the case of an influenza virus vaccine, it is preferable that antigens of different types, type A and type B, are included.

  The dosage form of the vaccine composition of the present invention may be in a state where the adjuvant composition containing the therapeutic agent for dyslipidemia and the antigen are formulated in a single container, and the adjuvant composition and the antigen are formulated in separate containers. It may be in a state of being converted.

  The dosage form of the adjuvant composition or vaccine composition of the present invention may be, for example, liquid, powder (freeze-dried powder, dry powder), capsule, tablet, frozen state or the like. When the adjuvant composition of the present invention is in a powder form, it may be used by dissolving in an appropriate solvent. Solvents for therapeutic agents for dyslipidemia that are active ingredients of the adjuvant composition are known to those skilled in the art, and examples thereof include ethanol, distilled water for injection, and DMSO.

  The adjuvant composition and vaccine composition of the present invention may contain a pharmaceutically acceptable carrier. As the pharmaceutically acceptable carrier, carriers usually used for vaccine production can be used without limitation, and specifically, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer and Combinations thereof may be mentioned, and additives such as adjuvants, emulsifiers, preservatives (eg, thimerosal), isotonic agents, pH adjusters, and inactivating agents (eg, formalin) may be appropriately blended. . Adjuvants include Emulsigen (MVP Laboratories), tocopherol acetate, alum, saponin (QS21, ISCOM), CpG oligo and the like.

  The administration subject of the adjuvant composition or vaccine composition of the present invention can be any immunizable organism. In particular, it may be humans and other mammals (eg livestock, pets and wild animals), etc.

  The administration route of the adjuvant composition or vaccine composition of the present invention is, for example, transdermal administration, sublingual administration, eye drop administration, intradermal administration, intramuscular administration, oral administration, enteral administration, nasal administration, intravenous administration. , Subcutaneous administration, intraperitoneal administration, inhalation administration from the mouth to the lung, and the like.

  Examples of the administration method of the adjuvant composition and vaccine composition of the present invention include stents, catheters, transdermal patches, microneedles, implantable sustained-release devices, syringes, syringes with microneedles, needle-free devices, A method of administration by spraying may be used. When the antigen and the adjuvant are formulated in separate containers, the antigen and the adjuvant may be administered simultaneously, or the other may be administered after a certain period of time after the antigen or adjuvant is administered.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples at all.

≪Adjuvant effect study with influenza HA antigen 1≫
(1) Preparation of dyslipidemic drug As dyslipidemic drugs, statins simvastatin (Wako: 193-12051) and atorvastatin (Wako: A791725) were used. Simvastatin was dissolved in ethanol (Nacalai: 147-13) and atorvastatin was dissolved in distilled water for injection (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a concentration of 5 mg / mL. It was stored frozen at −80 ° C. until use and mixed with an antigen solution prepared with physiological saline. ALHYDROGEL '85' 2% (BRENNTAG) was used as a control adjuvant (Alum adjuvant) (hereinafter abbreviated as Alum). Alum was mixed at a volume ratio of 1: 1 with the antigen solution.

(2) Preparation of antigen Influenza virus HA antigen was prepared as an antigen (strain: A / Solomon strain, composition: 1260 μg / mL HA) (hereinafter abbreviated as HA).

(3) Adjustment of immunity
A 5 mg / mL atorvastatin solution and an HA solution were mixed (Composition 1). 5 mg / mL simvastatin and HA solution were mixed (Composition 2). 0.02 g / mL Alum and HA solution were mixed (Composition 3). The HA solution was diluted with PBS (gibco: 09037) (Composition 4). The adjustment results are shown in Table 1.

(4) Administration mouse
BALB / c mice 6-week-old females were used as 3 mice per group.

(5) Immunization group composition First group: HA + atorvastatin (10 μg / individual) administration group, second group: HA + simvastatin (10 μg / individual) administration group, third group: HA + Alum administration group, fourth group: HA single administration group It was.

(6) Immunization method and schedule Each immunized product was administered into the abdomen subcutaneously (or intradermally) using a 1 mL tuberculin syringe (Terumo, SS-01T2613S), 200 μL per mouse (Table 2). The mice were immunized twice at 2-week intervals.

(7) Blood collection
On the 14th day after the second administration, all mice were bled from the abdominal vena cava under anesthesia with pentobarbital sodium (Kyoritsu Seiyaku, Somnopentyl) and killed. The collected blood was transferred to Microtina (BECTON DICKINSON), sufficiently coagulated at room temperature, and then centrifuged at 5,000 rpm for 10 minutes. Separated sera were dispensed into two 0.5 mL tubes and stored at −80 ° C. until measurement.

(8) Measurement of anti-HA antibody 50 μL / well of the same immunized antigen as that of the immunized antigen was added to an ELISA plate at a concentration of 1 μg / mL, and allowed to stand overnight at 4 ° C. by natural adsorption to be immobilized. After washing 3 times with PBS (prepared on the day), 200 μL / well of Blocker Casein in PBS (Thermo) was added and allowed to react at room temperature for 1 hour. After washing with PBS three times, the specimen was diluted with Blocker Casein in PBS, added at 50 μL / well, and allowed to react at room temperature for 2 hours. An anti-mouse IgG-POD labeled antibody (Thermo) diluted 2,000 times with Blocker Casein in PBS was added at 50 μL / well and allowed to react at room temperature for 1 hour. After washing 4 times with PBS, 50 μL / well of substrate TMB (BioFX) was added, and after 15 minutes of reaction at room temperature, 50 μL / well of 1N H 2 SO 4 was added to stop the reaction. Absorbance at 450 nm was measured. When the measurement was carried out after diluting the specimen 100 times or more, the antibody titer (OD) was corrected to the value at the time of 100-fold dilution.

  The results are shown in Table 3. The group immunized with atorvastatin, simvastatin and the antigen showed a clear adjuvant effect compared with the antigen alone group, and an adjuvant effect almost equal to or higher than that of the Alum group.

≪Adjuvant effect study with influenza HA antigen 2≫
(1) Preparation of therapeutic agent for dyslipidemia Lovastatin (Wako: 125-04581) and ethyl icosaventate (Evadale S300: Mochida Pharmaceutical) were used as therapeutic agents for dyslipidemia. Lovastatin was dissolved in ethanol at a concentration of 5 mg / mL, and ethyl icosabentoate was dispersed in distilled water for injection at a concentration of 5 mg / mL. It was stored frozen at −80 ° C. until use and mixed with an antigen solution prepared with physiological saline. Alum was used as a control adjuvant.

(2) Preparation of antigen The antigen was prepared according to Example 1.

(3) Adjustment of immunity
A 5 mg / mL lovastatin solution and an HA solution were mixed (Composition 5). 5 mg / mL ethyl icosaventate and HA solution were mixed (Composition 6). The adjustment results are shown in Table 4.

(4) BALB / c mice (7 weeks old, SPF), male mice, were purchased from Nippon Charles River and reared in an SPF environment. Divide 32 mice into 4 groups, 1 group: HA + lovastatin (100 μg / individual) administration group, 2 group: HA + ethyl icosaventate (100 μg / individual) group 3: HA + Alum administration group, 4 Group: HA administration group.

(5) Immunization method and schedule It implemented according to Example 1 (Table 5).

(6) Blood collection was performed according to Example 1.

(7) Measurement of anti-HA antibody The measurement was performed according to Example 1. The results are shown in Table 6. The group immunized with lovastatin, ethyl icosaventate and the antigen showed a clear adjuvant effect compared to the group with the antigen alone.

≪Adjuvant effect study with Aβ peptide antigen 1≫
(1) Preparation of dyslipidemic drug As the dyslipidemic drug, statins lovastatin, simvastatin and atorvastatin were used. Robustine and simvastatin were dissolved in ethanol and atorvastatin was dissolved in distilled water for injection at a concentration of 5 mg / mL. It was stored frozen at −80 ° C. until use and mixed with an antigen solution prepared with physiological saline. Alum was used as a control adjuvant.

(2) Preparation of antigen A peptide in which one molecule of cysteine was added to the C-terminal of the 28th sequence (SEQ ID NO: 2) from the N-terminal of Aβ peptide was used as an antigen (28AACys: SEQ ID NO: 3, synthesized at PH Japan) ).

(3) Adjustment of immunity
A 5 mg / mL lovastatin solution and 28AACys were mixed (Composition 7). 5 mg / mL simvastatin and 28AACys were mixed (Composition 8). 5 mg / mL atorvastatin and 28AACys were mixed (Composition 9). 0.02 g / mL Alum and 28AACys were mixed (Composition 10). 28AACys was diluted (Composition 11). The adjustment results are shown in Table 7.

(4) C57BL / 6 (7-week-old, SPF), a male mouse administered, was purchased from Japan Charles River Co., Ltd. and reared in an SPF environment.

(5) Immune group composition
20 mice were divided into 4 groups of 4 mice, 1st group: 28AACys + lovastatin (100μg / individual) administration group, 2nd group: 28AACys + simvastatin (100μg / individual) administration group, 2nd group: 28AACys + atorvastatin (100μg / individual) Individual) Administration group, Group 4: 28AACys + Alum administration group, Group 5: 28AACys administration group.

(6) Immunization method and schedule 200 μL of each immunized product was administered subcutaneously (or intradermally) in the abdomen using a 1 mL tuberculin syringe (Terumo, SS-01T2613S) (Table 8, per individual) Administration antigen amount: 10 μg). The mice were immunized twice at 2-week intervals.

(7) Blood collection
On the 14th day after the second administration, all mice were bled from the abdominal vena cava under anesthesia with pentobarbital sodium (Kyoritsu Seiyaku, Somnopentyl) and killed. The collected blood was transferred to Microtina (BECTON DICKINSON), sufficiently coagulated at room temperature, and then centrifuged at 5,000 rpm for 10 minutes. Separated sera were dispensed into two 0.5 mL tubes and stored at −80 ° C. until measurement.

(8) Measurement of anti-Aβ IgG antibody Aβ peptide (amino acid sequence 1-40 from the N-terminus (SEQ ID NO: 4), invitrogen: 03-136) was diluted to 10 μg / mL with 0.1 M Carbonate buffer, pH 9.6, and 8 well strips (Nalge-Nunc, Immobilizer Amino) was added at 100 μL / well and allowed to stand overnight at 4 ° C. to be solid-phased. The next day, each well was washed 3 times with 300 μL of 0.05% Tween20-containing PBS (PBST), 10 mM ethanolamin was added by 300 μL / well and allowed to stand at room temperature for 1 hour.

  After 1 hour, 10 mM ethanolamin was sufficiently removed, and the sample was diluted with PBST and added at 100 μL / well (each sample was duplicated). After reaction at room temperature for 1 hour, each diluted serum added was discarded and washed 3 times with 300 μL / well PBST. After washing, thoroughly remove the wash solution in the well, add HRP-labeled anti-mouse IgG goat antibody (American Qualex, A131PS) diluted 2000-fold with the sample diluent at 100 μL / well, and react at room temperature for 1 hour did. After the reaction, the labeled antibody diluent is discarded, washed twice with 300 μL / well of PBST and twice with the same amount of distilled water, added with 100 μL / well of chromogenic substrate solution TMB + (Dako), and protected from light at room temperature for 30 minutes. Reacted for 1 minute. Thereafter, 1N sulfuric acid was added at 100 μL / well to stop color development, and the absorbance at 450 nm (OD450 value) was measured.

  A commercially available monoclonal antibody against Aβ (CHEMICON MAB1560) was used as standard serum. Standard serum was diluted with PBST to 0.156, 0.3125, 0.625, 1.25, 2.5, 5, 10 ng / mL to prepare a standard for antibody titer measurement. Simultaneously with the measurement of the anti-Aβ IgG antibody of each test mouse serum, the OD450 value of each diluted specimen was measured with duplicate. The anti-Aβ IgG antibody titer of each mouse serum was calculated from the standard units obtained and the standard line of OD450 values.

  Table 9 shows the anti-Aβ antibody titer in the mouse serum in each immunization group calculated. The group immunized with lovastatin, simvastatin or atorvastatin together with the antigen showed a clear adjuvant effect compared with the antigen alone group, and an adjuvant effect almost equal to or higher than that of the Alum administration group.

≪Adjuvant effect study with Aβ peptide antigen 2≫
(1) Preparation of dyslipidemic drugs The dyslipidemic drugs are the statins atorvastatin, fluvastatin sodium (Nacalai: 24185-15), mevastatin (convertin, Wako: 033-17301) and pitavastatin calcium (Wako: 163- 24861) was used. Mevastatin was dissolved in ethanol, atorvastatin and fluvastatin sodium were dissolved in distilled water for injection, and pitavastatin calcium was dissolved in dimethyl sulfoxide ((DMSO) Nacalai: 13407-45) at a concentration of 5 mg / mL. It was stored frozen at −80 ° C. until use and mixed with an antigen solution prepared with physiological saline.

(2) Preparation of antigen 28AACys was used as an antigen in the same manner as in Example 3.

(3) Adjustment of immunity
A 5 mg / mL atorvastatin solution and 28AACys were mixed (Composition 12). 5 mg / mL fluvastatin sodium and 28AACys were mixed (Composition 13). 5 mg / mL mevastatin and 28AACys were mixed (Composition 14). 5 mg / mL pitavastatin calcium and 28AACys were mixed (Composition 15). The adjustment results are shown in Table 10.

(4) C57BL / 6 (7-week-old, SPF), a male mouse administered, was purchased from Japan Charles River Co., Ltd. and reared in an SPF environment.

(5) Immune group composition
20 mice were divided into 5 groups of 4 mice, group 1: 28AACys + atorvastatin (100μg / individual) administration group, group 2: 28AACys + pitavastatin (100μg / individual) administration group, group 3: 28AACys + fluvastatin (100μg / Individual) administration group, group 4: 28AACys + mevastatin (100 μg / individual) administration group, group 5: 28AACys administration group.

(6) Immunization method and schedule It implemented according to Example 3 (Table 11).

(7) Blood collection It was carried out according to Example 3.

(8) Measurement of anti-Aβ IgG antibody The measurement was performed according to Example 3. Table 12 shows the calculated anti-Aβ antibody titers in the mouse serum in each immunization group. The group immunized with atorvastatin, pitavastatin, fluvastatin or mevastatin together with the antigen showed a clear adjuvant effect as compared with the antigen alone group.

≪Adjuvant effect study with Aβ peptide antigen 3≫
(1) Preparation of dyslipidemic drug The dyslipidemic drug is ethyl icosaventate, bezafibrate of fibrates (SIGMA: B7273-1G), fenofibrate (SIGMA: F6020-5G), rosuvastatin of statins (Crestor 5 mg tablets) : AstraZeneca), atorvastatin sodium (the aforementioned atorvastatin), atorvastatin calcium (Wako: A791725), and pravastatin (Nacalai: 59718-84) were used. Ethic icosaventate and rosuvastatin were dispersed in distilled water for injection. Atorvastatin sodium and pravastatin were dissolved in distilled water for injection, and bezafibrate, fenofibrate and atorvastatin calcium were dissolved in DMSO at a concentration of 5 mg / mL. It was stored frozen at −80 ° C. until use and mixed with an antigen solution prepared with physiological saline. Alum was used as a control adjuvant.

(2) Preparation of antigen 28AACys was used as an antigen in the same manner as in Example 3.

(3) Adjustment of immunity
5 mg / mL ethyl icosaventate solution and 28AACys were mixed (composition 16). 5 mg / mL bezafibrate and 28AACys were mixed (composition 17). 5 mg / mL fenofibrate and 28AACys were mixed (Composition 18). 5 mg / mL rosuvastatin and 28AACys were mixed (Composition 19). 5 mg / mL atorvastatin sodium and 28AACys were mixed (Composition 20). 5 mg / mL atorvastatin calcium and 28AACys were mixed (Composition 21). 5 mg / mL pravastatin and 28AACys were mixed (Composition 22). The adjustment results are shown in Table 13.

(4) C57BL / 6 (7-week-old, SPF), a male mouse administered, was purchased from Japan Charles River Co., Ltd. and reared in an SPF environment.

(5) Immune group composition
40 mice were divided into 10 groups of 4 mice, 1st group: 28AACys + ethyl icosaventate (100μg / individual), 2nd group: 28AACys + bezafibrate (100μg / individual), 3rd group: 28AACys + fenofibrate (100μg / individual) administration group, Group 4: 28AACys + rosuvastatin (100μg / individual) administration group, Group 5: 28AACys + atorvastatin sodium (100μg / individual) administration group, Group 6: 28AACys + atorvastatin calcium (100μg / individual) administration Group, Group 7: 28AACys + pravastatin (100 μg / individual) administration group, Group 8: 28AACys + Alum administration group, Group 9: 28AACys administration group.

(6) Immunization method and schedule It implemented according to Example 3 (Table 14).

(7) Blood collection It was carried out according to Example 3.

(8) Measurement of anti-Aβ IgG antibody The measurement was performed according to Example 3. Table 15 shows the anti-Aβ antibody titer in the mouse serum in each immunization group calculated. In all of the dyslipidemia drugs performed, the group immunized together with the antigen showed a clear adjuvant effect as compared with the antigen alone group. Except for fenofibrate and pravastatin, all adjuvant effects were higher than those of the Alum group.

≪Adjuvant effect study with Aβ peptide antigen 4≫
(1) Preparation of dyslipidemic agent Lovastatin (described above), lovastatin hydroxy acid (Wako: L472250), and metformin (Wako: 138-15581) were used as dyslipidemic agents. Lovastatin hydroxy acid, metformin was dissolved in distilled water for injection, and lovastatin was dissolved in ethanol at a concentration of 5 mg / mL. It was stored frozen at −80 ° C. until use and mixed with an antigen solution prepared with physiological saline. Alum was used as a control adjuvant.

(2) Preparation of antigen 28AACys was used as an antigen in the same manner as in Example 3.

(3) Adjustment of immunity
25 mg / mL or 5 mg / mL lovastatin and 28AACys were mixed (compositions 23 to 26). A 5 mg / mL lovastatin hydroxy acid solution and 28AACys were mixed (Composition 27). 5 mg / mL metformin and 28AACys were mixed (Composition 28). The adjustment results are shown in Table 16.

(3) C57BL / 6 (7-week-old, SPF), a male mouse administered, was purchased from Japan Charles River Co., Ltd. and bred in an SPF environment.

(4) Immune group composition
32 mice were divided into 8 groups of 4 mice, group 1: 28AACys + lovastatin (500μg / individual) administration group, group 2: 28AACys + lovastatin (100μg / individual) administration group, group 3: 28AACys + lovastatin (10μg / individual) Individual) administration group, Group 4: 28AACys + lovastatin (1μg / individual) administration group, Group 5: 28AACys + lovastatin hydroxy acid (100μg / individual) administration group, Group 6: 28AACys + metformin (100μg / individual) administration group, Group Group 7: 28AACys + Alum administration group, Group 8: 28AACys administration group.

(5) Immunization method and schedule It implemented according to Example 3 (Table 17).

(6) Blood collection was performed according to Example 1.

(7) Measurement of anti-Aβ IgG antibody The measurement was performed according to Example 5. Table 18 shows the anti-Aβ antibody titers in the mouse sera calculated for each immunization group. In all the dyslipidemic drugs performed, the group immunized together with the antigen showed a clear adjuvant effect, except for the group administered with 1 μg of lovastatin, as compared to the group of the antigen alone. Moreover, the adjuvant effect higher than that of the Alum group was observed except for the group administered with 1 μg of lovastatin, 100 μg of lovastatin hydroxy acid and 100 μg of metformin.

  From the results of Examples 1 to 6, a clear adjuvant effect was observed for the therapeutic agents for dyslipidemia. The effect is considered to have an effect equal to or higher than that of the existing alum adjuvant depending on the type and concentration. In addition, no abnormalities are observed in mice administered with a dyslipidemic agent, and the safety is excellent.

  In the present invention, an adjuvant composition containing a therapeutic agent for dyslipidemia and a vaccine containing the adjuvant composition are provided. Drugs for treating dyslipidemia are useful and safe for the living body, and can be selected from fat-soluble and water-soluble, and are safer than conventional alum adjuvants and oil-based adjuvants. It is possible to provide a vaccine and an adjuvant that are excellent in terms of convenience.

Claims (14)

  1. An adjuvant composition containing a therapeutic agent for dyslipidemia.
  2. The adjuvant composition according to claim 1, wherein the therapeutic agent for dyslipidemia is one or more compounds selected from the group consisting of statins, fibrates, fish oil, and metformin.
  3. The adjuvant composition according to claim 2, wherein the statin is one or more compounds selected from the group consisting of atorvastatin, simvastatin, pitavastatin, fluvastatin, pravastatin, mevastatin, lovastatin, and the above salts and hydroxides.
  4. The adjuvant composition according to claim 2, wherein the fibrate is one or more compounds selected from the group consisting of fenofibrate, besafibrate, and the above salts and hydroxides.
  5. The adjuvant composition according to claim 2, wherein the fish oil is one or more compounds selected from the group consisting of eicosapentaenoic acid (EPA), ethyl icosapentate, and the above salts and hydroxides.
  6. The adjuvant composition according to any one of claims 1 to 5, wherein the adjuvant composition is excellent in solubility in a solvent.
  7. A vaccine composition comprising the adjuvant composition according to any one of claims 1 to 6 and an antigen.
  8. The vaccine composition according to claim 7, wherein the antigen is an influenza virus antigen.
  9. 9. The vaccine composition according to claim 8, wherein the influenza virus antigen is influenza virus hemagglutinin (HA).
  10. The vaccine composition according to claim 7, wherein the antigen is a peptide antigen.
  11. The vaccine composition according to claim 10, wherein the peptide antigen is an Aβ peptide.
  12. The vaccine composition according to claim 11, wherein the Aβ peptide is a peptide consisting of a partial amino acid sequence of the Aβ peptide, or a peptide in which one or several cysteines are added or inserted into the peptide.
  13. The vaccine composition according to claim 11 or 12, wherein the Aβ peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  14. The fusion according to claim 13, wherein the Aβ peptide has an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. protein.
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