WO2020111248A1

WO2020111248A1 - Vaccine preparation

Info

Publication number: WO2020111248A1
Application number: PCT/JP2019/046846
Authority: WO
Inventors: 孝広中田; 後藤　雅宏; 秀斗小坂
Original assignee: 小林製薬株式会社; 国立大学法人九州大学
Priority date: 2018-11-29
Filing date: 2019-11-29
Publication date: 2020-06-04
Also published as: JPWO2020111248A1

Abstract

The purpose of the present invention is to provide a vaccine preparation that can induce excellent immunity through transdermal administration. This vaccine preparation includes: an aqueous phase having an antigen dissolve therein; glycerin fatty acid ester and/or poly-glycerin fatty acid ester; and an oil phase, wherein the aqueous phase is dispersed in the oil phase. The vaccine preparation can, when being administered transdermally, induce excellent immunity in a manner better than administration through injection.

Description

Vaccine formulation

The present invention relates to a vaccine preparation capable of excellent immunity induction by transdermal administration.

The three major therapies, surgery, radiation therapy, and chemotherapy, have been established as cancer treatment methods, but in recent years, cancer immunotherapy has attracted attention as an effective method with few side effects. Among cancer immunotherapy, cancer vaccine therapy that induces antibody production and cell-mediated immunity against cancer cells has few side effects and can be used in combination with the above three major therapies. There is.

In addition, conventionally, for the prevention of infectious diseases, a vaccine immunization method that induces antibody production against specific pathogens and cell-mediated immunity by artificial artificial infection has been widely used.

In this way, for the treatment of cancer and prevention of infectious diseases, the method of inducing adaptive immunity by administering a vaccine is effective, and conventionally, vaccine preparations have been vigorously studied. However, most of vaccine formulations that have been conventionally developed are injectable formulations, and such injectable formulations have the drawback that they require medical personnel for administration and are accompanied by pain due to injection.

On the other hand, the skin has a strong immune function to resist the invasion of foreign substances from the outside, and Langerhans cells present in the epidermis are more strongly cytotoxic than other dendritic cells. It has been reported that a response can be induced (see Non-Patent Documents 1 and 2). Therefore, if the vaccine preparation can be transdermally administered and the antigen can be permeated from the skin surface and delivered to the Langerhans cells, effective induction of an immune response can be expected. Further, the transdermal administration of the vaccine preparation can be performed by the patient himself and is non-invasive, so that there is an advantage that the administration is simple and the burden on the patient is small. As described above, the vaccine preparation for transdermal administration can be expected to overcome the drawbacks of the injection preparation and achieve both excellent vaccine effect and convenience.

However, the stratum corneum located in the outermost layer of the epidermis prevents foreign substances from entering from the outside as a strong biological barrier, and can only penetrate oil-soluble substances or low molecular weight substances having a molecular weight of 500 Da or less (non-patent reference) Reference 3). Since protein antigens and peptide antigens generally used as vaccines are hydrophilic and have a high molecular weight, it is difficult to deliver them even to Langerhans cells existing under the stratum corneum just by applying them to the skin. In fact, it has been reported that the hydrophilic gel patch containing an antigen has an overwhelmingly low utilization rate of the antigen as compared with an injection (see Non-Patent Document 4). In addition, conventionally, attempts have been made to improve the immunity-inducing ability by transdermal administration by a transdermal vaccine preparation using microneedles, but such a vaccine preparation complicates the manufacturing process and increases the manufacturing cost. Has the drawback of inviting. Against the background of such conventional techniques, development of a vaccine for transdermal administration capable of excellent immunity induction without using microneedles has been earnestly desired.

An object of the present invention is to provide a vaccine preparation capable of excellent immunity induction by transdermal administration.

The inventors of the present invention have conducted extensive studies to solve the above problems, and include an aqueous phase in which an antigen is dissolved, a glycerin fatty acid ester and/or a polyglycerin fatty acid ester, and an oil phase, and the aqueous phase is the oil phase. It has been found that the vaccine preparation dispersed in A. can induce immunity superior to that by injection when administered transdermally. The present invention has been completed by further studies based on such findings.

That is, the present invention provides the inventions of the following modes.
Item 1. A vaccine preparation for transdermal administration, comprising an aqueous phase in which an antigen is dissolved, a glycerin fatty acid ester and/or a polyglycerin fatty acid ester, and an oil phase, wherein the aqueous phase is dispersed in the oil phase.
Item 2. Item 2. The vaccine preparation for transdermal administration according to Item 1, wherein the origin of the antigen is a cancer cell, a pathogenic virus, a pathogenic bacterium, a pathogenic organism, or pollen.
Item 3. Item 3. The vaccine preparation for transdermal administration according to

Item

1 or 2, wherein the antigen is an antigen peptide or an antigen protein.
Item 4. Item 4. The vaccine preparation for transdermal administration according to any one of Items 1 to 3, wherein the glycerin fatty acid ester is glyceryl monooleate.
Item 5. Item 5. The vaccine preparation for transdermal administration according to any one of Items 1 to 4, wherein the content of the glycerin fatty acid ester and/or the polyglycerin fatty acid ester is 0.1 to 30% by weight.
Item 6. For the production of a vaccine preparation for transdermal administration of a composition comprising an aqueous phase in which an antigen is dissolved, glycerin fatty acid ester and/or polyglycerin fatty acid ester, and an oil phase, wherein the aqueous phase is dispersed in the oil phase Use of.
Item 7. An immune-inducing effective amount of a composition containing an aqueous phase in which an antigen is dissolved, glycerin fatty acid ester and/or polyglycerin fatty acid ester, and an oil phase, the aqueous phase being dispersed in the oil phase, An immunity-inducing method, comprising the step of transdermal administration to an animal in need.

According to the vaccine preparation of the present invention, transdermal administration enables superior immunity induction over injection administration, and thus is excellent in allergen immunotherapy for allergy such as cancer immunotherapy, infectious disease prevention, and pollinosis. It is possible to exert a therapeutic or preventive effect. Further, since the vaccine preparation of the present invention can effectively induce immunity by transdermal administration, it can be used in a non-invasive, safe and simple manner.

FIG. 3 is a diagram schematically showing a schedule of an immunity induction experiment in Test Example 1. FIG. 5 is a diagram showing the results of measuring the tumor volume over time in the immunity induction experiment in Test Example 1. In the immunity induction experiment in Test Example 1, for the transdermal administration group of Example 1 and the injection administration group of Reference Example 1, tumors taken out 14 days after transplantation were stained with APC-labeled anti-CD8 antibody and DAPI, and observed with a fluorescence microscope. FIG. FIG. 6 is a diagram schematically showing a schedule of an immunity induction experiment in Test Example 2. In the immunity induction experiment in Test Example 2, for the transdermal administration group of Example 2, the injection administration group of Reference Example 2, and the control group, the serum concentration of Crij1-specific IgE antibody and IgG1 antibody and the spleen cell culture medium It is a figure which shows the result of having measured the cytokine (IL-4 and IL-5) concentration. FIG. 8 is a diagram schematically showing a schedule of an immunity induction experiment in Test Example 3. FIG. 8 is a diagram showing the results of measuring the serum concentration of anti-influenza A(H3N2) antibody in the transdermal administration group of Example 3 and the transdermal administration group of Comparative Example 3 in the immunity induction experiment in Test Example 3.

The vaccine preparation of the present invention is a vaccine preparation for transdermal administration, comprising an aqueous phase in which an antigen is dissolved, a glycerin fatty acid ester and/or a polyglycerin fatty acid ester, and an oil phase, the aqueous phase being the oil phase. It is characterized by being dispersed. Hereinafter, the vaccine preparation of the present invention will be described in detail.

[Water phase]
In the vaccine preparation of the present invention, the aqueous phase is composed of a solution in which the antigen is dissolved. In the vaccine preparation of the present invention, the hydrophilic portion of the glycerin fatty acid ester and/or the polyglycerin fatty acid ester is associated with the water phase, and the water phase is in a WO emulsion form in which the water phase is dispersed in the oil phase. In the vaccine preparation of the present invention, a reverse micelle coated with glycerin fatty acid ester and/or polyglycerin fatty acid ester around the aqueous phase is formed and emulsified, or around the aqueous phase, glycerin fatty acid ester and/or polyglycerin fatty acid ester is formed. It is presumed that the aqueous phase is in an emulsified state in which it is emulsified particles even if the glycerin fatty acid ester is not completely covered. Hereinafter, each component contained in the aqueous phase will be described.

-Antigen The antigen used in the present invention is not particularly limited as long as it is capable of inducing an immune response in vivo as an immunogen of a vaccine and is water-soluble. Can be mentioned. In the present invention, the antigen protein refers to a protein that is specifically expressed or present in a pathogen or an allergen and induces an immune response. In addition, the antigen peptide refers to a peptide derived from an antigen protein (for example, an antigen protein having a reduced molecular weight). In the present invention, the antigen protein and the antigen peptide may be those capable of inducing antigen-specific T cells by directly forming a complex with the MHC molecule (HLA molecule) on the cell surface of the antigen-presenting cell. In addition, a peptide fragment that is taken up into cells and then decomposed into cells binds to MHC molecules to form a complex, and the complex is presented on the cell surface to generate antigen-specific T cells. It may be inducible.

From the viewpoint of enhancing the efficiency of percutaneous absorption and inducing even better transdermal immunity, an antigen peptide is a suitable example of the antigen. The number of amino acid residues of the antigen peptide is not particularly limited, but examples thereof include 2 to 50, preferably 7 to 31, and more preferably about 8 to 20.

The origin of the antigen is not particularly limited, but examples thereof include cancer cells, pathogenic viruses, pathogenic bacteria, pathogenic organisms, pollen, and the like. As the origin of the antigen, specifically, melanoma, pancreatic cancer, lung cancer, osteosarcoma, colon cancer, colon cancer, gastric cancer, rectal cancer, liver cancer, breast cancer, bladder cancer, prostate cancer, cervical cancer, head and neck cancer, bile duct cancer. , Cancer cells such as solid cancers such as gallbladder cancer, oral cancer, brain tumors, and blood cancers such as leukemia and malignant lymphoma; influenza virus, avian influenza virus, parainfluenza virus, adenovirus, SARS virus, AIDS virus, cytomegalo Viral, hepatitis virus, Japanese encephalitis virus, measles virus, rubella virus, varicella-zoster virus, polio virus, papilloma virus, herpes virus, mumps virus, rotavirus, cholera virus, rabies virus, HIV and other pathogenic viruses; diphtheria , Tetanus, tubercle bacillus, pneumococcus, meningococcus, staphylococcus, aeruginosa, pertussis, anthrax, salmonella and other pathogenic bacteria; malaria parasites, mites and other pathogenic organisms; cedar pollen, Examples include pollen such as cypress pollen and birch pollen. Among these antigens, cancer cells, influenza virus, and pollen are preferable.

In the vaccine preparation of the present invention, the content of the antigen in the aqueous phase (dissolution) is, for example, 0.001 to 60% by weight, preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight. %, more preferably 0.1 to 25% by weight, particularly preferably 0.1 to 5% by weight.

The content of the antigen in the vaccine preparation of the present invention may be appropriately set according to the type of the antigen, the condition of the patient, the dose, etc., but is, for example, 0.0001 to 5% by weight, preferably 0.0001 to 3. 5% by weight, more preferably 0.0001 to 2.5% by weight, particularly preferably 0.01 to 2% by weight.

-Aqueous phase base The aqueous phase contains an aqueous phase base for dissolving the antigen. The type of aqueous phase base is not particularly limited as long as it can dissolve the antigen, and examples thereof include water, a monohydric lower alcohol, and a mixed solution thereof.

The water used as the aqueous phase base may be purified water, ultrapure water, or the like, as well as a buffer solution, physiological saline, or the like.

Examples of monohydric lower alcohols used as the aqueous phase base include monohydric alcohols having 2 to 5 carbon atoms. As the monohydric lower alcohol, more specifically,
Examples thereof include ethanol, n-propanol, isopropanol, n-butanol, sec-butanol, tert-butanol, n-amyl alcohol, sec-amyl alcohol, isoamyl alcohol, tert-amyl alcohol and neopentyl alcohol. These monohydric lower alcohols may be contained alone or in combination of two or more.

A preferable embodiment of the aqueous phase base is a mixed solution of water and a monohydric lower alcohol, more preferably a mixed solution of water and isopropanol. In addition, another preferable embodiment of the aqueous phase base is water or an aqueous solution (buffer solution, physiological saline, etc.) containing no monohydric lower alcohol.

When a mixed liquid of water and a monohydric lower alcohol is used as the aqueous phase base, the mixing ratio of these is not particularly limited, but for example, 1 to 50 parts by weight of the monohydric lower alcohol per 1 part by weight of water. The ratio is preferably 1 to 45 parts by weight, more preferably 1 to 40 parts by weight, and particularly preferably 1 to 20 parts by weight.

The content of the aqueous phase base in the aqueous phase is, for example, 40 to 99.999% by weight. From the viewpoint of inducing even more excellent transdermal immunity, the lower limit of the content of the aqueous phase base in the aqueous phase is preferably 50% by weight or more, more preferably 60% by weight or more, and further preferably 95% by weight. The above is mentioned. From the viewpoint of inducing even more excellent transdermal immunity, the upper limit of the content of the aqueous phase base in the aqueous phase is preferably 99.9% by weight or less, more preferably 99% by weight or less. More specifically, the content of the aqueous phase base in the aqueous phase is preferably 50 to 99.999% by weight, more preferably 60 to 99.999% by weight, and further preferably 75 to 99.9% by weight. , Particularly preferably 95 to 99.9% by weight.

The content of the aqueous phase base in the vaccine preparation of the present invention is, for example, 0.1 to 50% by weight. From the viewpoint of inducing even more excellent transdermal immunity, the content of the aqueous phase base in the vaccine preparation of the present invention is preferably 0.1 to 40% by weight, more preferably 0.1 to 30% by weight, and further preferably Is 0.5 to 30% by weight, particularly preferably 5 to 20% by weight.

[Glycerin fatty acid ester and/or polyglycerin fatty acid ester]
In the vaccine preparation of the present invention, the glycerin fatty acid ester and/or the polyglycerin fatty acid ester plays a part or all in association with the aqueous phase, plays a role of dispersing the aqueous phase in the oil phase and emulsifying, and transdermal administration. Plays a role in enabling superior immunity induction.

Glycerin fatty acid ester is a monoester, diester, or triester of fatty acid and glycerin. The number of carbon atoms of the fatty acid constituting the glycerin fatty acid ester is, for example, 6 to 24, preferably 8 to 22 and more preferably 12 to 18. Specific examples of the glycerin fatty acid ester include glyceryl monomyristate, glyceryl monostearate, glyceryl monoisostearate, glyceryl monooleate, glyceryl dioleate, glyceryl trioleate, and glyceryl distearate. Among these glycerin fatty acid esters, monoesters of fatty acids and glycerin are preferable, and glyceryl monooleate is more preferable.

“Polyglycerin fatty acid ester” is an ester of fatty acid and polyglycerin. In the polyglycerin fatty acid ester, the number of ester bonds (the number of fatty acids bound to one molecule of polyglycerin) is, for example, 1 to 10, preferably 1 to 6, and more preferably 1 to 3. The carbon number of the fatty acid constituting the polyglycerin fatty acid ester is, for example, 6 to 24, preferably 8 to 22, and more preferably 12 to 18. The degree of polymerization of polyglycerin that constitutes the polyglycerin fatty acid ester is, for example, 2 to 30, and preferably 2 to 10. Specifically, as the polyglycerin fatty acid ester, polyglyceryl-2 stearate (diglyceryl monostearate), polyglyceryl-2 oleate (diglyceryl monooleate), polyglyceryl-4 oleate (tetraglyceryl monooleate), Polyglyceryl oleate-10 (decaglyceryl monooleate), polyglyceryl-5 trioleate (pentaglyceryl trioleate), polyglyceryl-10 trioleate (decaglyceryl trioleate), polyglyceryl-10 palmitate (monopalmitic acid) Decaglyceryl), polyglyceryl-2 isostearate, polyglyceryl-2 triisostearate, polyglyceryl-4 stearate, polyglyceryl tristearate-6, polyglyceryl pentastearate-10, polyglyceryl pentahydroxystearate-10, polyglyceryl pentaisostearate- 10, polyglyceryl-10 pentaoleate, polyglyceryl-6 polyricinoleate, polyglyceryl-10 polyricinoleate, and the like. Among these polyglycerin fatty acid esters, polyglyceryl trioleate and polyglyceryl isostearate-2 are preferable.

In the present invention, one of the glycerin fatty acid ester and the polyglycerin fatty acid ester may be used alone, or two or more thereof may be used in combination.

Among glycerin fatty acid ester and polyglycerin fatty acid ester, from the viewpoint of inducing even more excellent transdermal immunity, preferably glycerin fatty acid ester, more preferably monoester of fatty acid and glycerin, more preferably glyceryl monooleate. Can be mentioned.

In the vaccine preparation of the present invention, the ratio of the aqueous phase (dissolved liquid) to the glycerin fatty acid ester and/or polyglycerin fatty acid ester is, for example, the total amount of glycerin fatty acid ester and/or polyglycerin fatty acid ester per 1 part by weight of the aqueous phase. At 0.5 to 200 parts by weight, preferably 0.5 to 75 parts by weight, more preferably 0.5 to 50 parts by weight, further preferably 0.5 to 40 parts by weight, particularly preferably 0.5 to 3 parts by weight. Parts.

The content of glycerin fatty acid ester and/or polyglycerin fatty acid ester in the vaccine preparation of the present invention is, for example, 0.1 to 30% by weight, preferably 0.25 to 27.5% by weight, more preferably 0.5. Up to 25% by weight.

[Oil phase]
In the vaccine preparation of the present invention, the oil phase serves as a dispersion medium for the aqueous phase, and is formed of an oil phase base such as liquid oil, solid oil and higher alcohol.

-Liquid oil is an oil that maintains a liquid form at 25°C. The liquid oil used in the present invention may be one normally used in cosmetics and external medicines, for example, avocado oil, camellia oil, macadamia atsutu oil, olive oil, almond oil, soybean oil, jojoba oil, Vegetable oils such as cottonseed oil, rapeseed oil, sesame oil, perilla oil, cinnamon oil, corn oil, peanut oil, sunflower oil, cacao oil, mint oil, bergamot oil, fennel oil, etc.; fatty acids such as oleic acid, isostearic acid; ethylhexanoic acid Cetyl, ethylhexyl palmitate, octyldodecyl myristate, neopentyl glycol diethylhexanoate, glyceryl tri-2-ethylhexanoate, octyldodecyl oleate, isopropyl myristate, glyceryl triisostearate, diparamethoxycinnamate-monoethylhexane Ester oils such as glyceryl acid; silicone oils such as dimethylpolysiloxane, methylhydrogenpolysiloxane, methylphenylpolysiloxane and octamethylcyclotetrasiloxane; liquid hydrocarbon oils such as liquid paraffin, squalene and squalane. These liquid oils may be used alone or in combination of two or more. Among these liquid oils, ester oil is preferable, and isopropyl myristate is more preferable.

Solid oil is an oil that maintains a solid form at 25°C. The solid oil used in the present invention may be one normally used for cosmetics and external medicines, for example, candelilla wax, rice bran wax, beeswax, cotton wax, carnauba wax, lanolin, shellac wax, ozokerite, ceresin, Polyethylene wax, microcrystalline wax, paraffin, petrolatum, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, 12-hydroxystearic acid, undecylenic acid, myristyl myristate, cetyl myristate, stearyl stearate, cetyl stearate. , Solid oils such as cetyl palmitate, cholesteryl stearate, cholesteryl oleate, dextrin palmitate, inulin stearate, hydrogenated jojoba oil, ceresin wax, solid paraffin wax, polyethylene wax and silicone wax. These solid oils may be used alone or in combination of two or more.

Higher alcohol is a monohydric alcohol having 6 or more carbon atoms in one molecule. The number of carbon atoms in one molecule in the higher alcohol used in the present invention may be 6 or more, preferably 6 to 34, more preferably 14 to 22.

The higher alcohol used in the present invention may be one normally used in cosmetics and external medicines, for example, lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, cetostearyl alcohol, cetanol, oleyl. Examples thereof include linear higher alcohols such as alcohols; branched higher alcohols such as nostearyl glycerin ether (batyl alcohol). These higher alcohols may be used alone or in combination of two or more.

Among these oil phase bases, liquid oil is preferable.

In the vaccine preparation of the present invention, the ratio of the water phase and the oil phase is, for example, 1 to 1000 parts by weight, preferably 1.5 to 1000 parts by weight, and more preferably 2 to 1 part by weight of the oil phase. The amount is 1000 parts by weight, more preferably 2.5 to 200 parts by weight, and particularly preferably 2.5 to 10 parts by weight.

The content of the oil phase in the vaccine preparation of the present invention is not particularly limited and may be appropriately set according to the type of the oil phase base used, for example, 40 to 99.8% by weight, preferably 50 to 99.7% by weight, more preferably 55 to 99.4% by weight.

[Other ingredients]
The vaccine preparation of the present invention may contain an adjuvant in order to further improve the induction of transdermal immunity. Examples of the adjuvant include CpG oligodeoxynucleotide, Freund, aluminum hydroxide (Alum), alum and the like.

Further, the vaccine preparation of the present invention may contain other bases and additives required for formulation and the like, in addition to the above-mentioned components, if necessary. Such additives are not particularly limited as long as they are pharmaceutically or cosmetically acceptable, for example, preservatives (methylparaben, propylparaben, benzoic acid, sodium benzoate, sorbic acid, etc.), Flavoring agents (citral, 1,8-cionele, citronellal, farnesol, etc.), colorants (tar pigments (brown 201, blue 201, yellow 4, yellow 403, etc.), cacao pigments, chlorophyll, aluminum oxide, etc. ), thickeners (carboxyvinyl polymer, hypromellose, polyvinylpyrrolidone, sodium alginate, ethyl cellulose, sodium carboxymethyl cellulose, xanthan gum, carrageenan, etc.), pH adjusters (phosphoric acid, hydrochloric acid, citric acid, sodium citrate, succinic acid, tartaric acid) , Sodium hydroxide, potassium hydroxide, triethanolamine, triisopropanolamine, etc.), wetting agent (sodium dl-pyrrolidonecarboxylate solution, D-sorbitol solution, macrogol, etc.), stabilizer (dibutylhydroxytoluene, butylhydroxy) Anisole, sodium edetate, sodium metaphosphate, L-arginine, L-aspartic acid, DL-alanine, glycine, sodium erythorbate, propyl gallate, sodium sulfite, sulfur dioxide, chlorogenic acid, catechin, rosemary extract, etc.) , Polyhydric alcohols (glycerin, propylene glycol, dipropylene glycol, butylene glycol, polyethylene glycol, etc.), antioxidants, UV absorbers, chelating agents, adhesives, buffers, solubilizers, preservatives and other additives Can be mentioned.

When the vaccine preparation of the present invention contains an adjuvant or an additive, the water-soluble component may be added to the water-soluble fraction, and the lipophilic component may be added to the oil phase. It may be set appropriately according to

[Formulation form]
The vaccine preparation of the present invention is a water-in-oil type (W/O type) emulsified form in which an aqueous phase is dispersed in an oil phase. The vaccine preparation of the present invention may be further dispersed in an aqueous phase according to a conventional method to give a W/O/W type preparation.

The dosage form of the vaccine preparation of the present invention is not particularly limited as long as it can be applied transdermally, and examples thereof include creams, ointments, gels, poultices, lotions, liniments, and aerosols. Can be mentioned. Of these, creams, ointments, emulsions and lotions are preferable.

[Production method]
The vaccine preparation of the present invention can be produced according to a known formulation method for emulsified preparations. For example, as a method for producing the vaccine preparation of the present invention, the components to be contained are divided into a water-soluble component and an oil component, a solution containing the water-soluble component (aqueous phase component), an oil component and a glycerin fatty acid ester and/or Examples include a method of preparing a mixed solution containing a polyglycerin fatty acid ester (oil phase component and glycerin fatty acid ester and/or polyglycerin fatty acid ester), and emulsifying them according to a known method. Specifically, a solution in which an antigen is dissolved in an aqueous phase base, and a mixed solution containing glycerin fatty acid ester and/or polyglycerin fatty acid ester and an oil phase base are prepared, and the solution and the mixed solution are mixed. The vaccine formulation of the present invention can be produced by mixing and emulsifying.

[Dose/Usage]
The vaccine formulation of the present invention is transdermally administered and used to induce immunity. The conventional vaccine preparation for general transdermal administration has a drawback that the antigen utilization rate is low and immunity cannot be sufficiently induced, but the vaccine preparation of the present invention induces immunity superior to injection administration by transdermal administration. It is possible to produce an effect.

The vaccine preparation of the present invention is used for cancer immunotherapy, infectious disease prevention, allergen immunotherapy for allergies such as hay fever, etc., depending on the type of antigen contained, but it can be suitably used for cancer immunotherapy. ..

The subject of administration of the vaccine preparation of the present invention is not particularly limited as long as it is an animal for which the immunity induction is required, and examples thereof include humans, monkeys, mice, rats, dogs, rabbits, cats, cows, horses, goats and the like. Mammals: Birds such as chickens and ostriches are mentioned.

The dose of the vaccine preparation of the present invention is appropriately set according to the type of antigen used, symptoms of the subject to be administered, age, body weight, etc., but when the subject animal is a human, it is usually administered once a day. The antigen may be transdermally administered in an amount equivalent to 3 to 12 mg/kg once to several times, preferably once a day (primary immunization). In addition, the vaccine preparation of the present invention may be re-administered usually 1 to 2 weeks after the initial immunization under the same conditions as the initial immunization (boost).

The skin site to which the vaccine preparation of the present invention is administered is not particularly limited, but, for example, in the case of humans, the upper arm, chest, back, etc. may be mentioned, and in the case of non-human animals, the back, ear. An intermediary part and the like can be mentioned.

The present invention will be described below with reference to examples. However, the present invention is not construed as being limited to the following examples.

Test example 1
1. Preparation of formulation Each formulation having the composition shown in Table 1 was prepared by the following method.

[Example 1 and Comparative Example 1]
A predetermined amount of melanoma antigen peptide (K-TRP-2, amino acid sequence: KKKGSVYDFFVWL, tyrosinase-related protein 2 at the N-terminal side of the peptide consisting of amino acids 180 to 188) was added to an aqueous phase base (purified water and isopropanol). A lysate was prepared by dissolving a glycine residue and a peptide to which three lysine residues were added. Separately, a predetermined amount of a surfactant (glyceryl monooleate or polyoxyethylene sorbitan monooleate (Tween 80)) was added to liquid oil (isopropyl myristate) and heated at 70° C. for 10 minutes to completely dissolve it. . While stirring a predetermined amount of the solution, the solution was dropped little by little. By stirring this until it reached room temperature, a vaccine formulation in a water-in-oil emulsified form containing melanoma antigen peptide (a form in which the water phase was dispersed in the oil phase and emulsified by the reverse micelle) was obtained.

[Comparative Example 2 and Reference Example 1]
An aqueous vaccine preparation was obtained by adding a predetermined amount of a predetermined amount of melanoma antigen peptide (K-TRP-2) to a phosphate buffer (PBS) and dissolving it.

2. Immune induction experiment 6-week-old mice (C57BL/6N, female) were divided into four test groups shown in Table 2 (6 mice per group), and the first day (Day-14) under the conditions shown in Table 2 Immunization, one week later (Day -7), booster immunization was performed. The amount of each component administered in each of the primary immunization and the booster immunization is as shown in Table 1.

Seven days after the booster immunization, 100 μl of a cell suspension of mouse melanoma cells (B16F10) suspended in HBSS(−) medium at 1.0×10 ⁶ cells/ml was transplanted subcutaneously to each mouse. (Day 0). The major axis and minor axis of the tumor were measured every 2 days from 5 days after the transplantation of mouse melanoma cells, and the tumor volume was calculated based on the following formula.

For the transdermal administration group of Example 1 and the injection administration group of Reference Example 14, 14 days after the transplantation of mouse melanoma cells, the tumor was taken out from the skin, the tumor was cut to a thickness of 20 μm, and placed on a slide glass. It was stained with a phycocyanin (APC)-labeled anti-CD8 antibody and DAPI (4′,6-diamidino-2-phenylindole) (Day 14).

Fig. 1 shows the experimental schedule, Fig. 2 shows the results of measuring the tumor volume over time, and Fig. 3 shows the results of staining the tumors taken out 14 days after transplantation with the APC-labeled anti-CD8 antibody and DAPI. As can be seen from FIG. 2, in the subcutaneous administration group of Comparative Example 1 and the subcutaneous administration group of Comparative Example 2, the tumor volume was large and the induction of cancer immunity was insufficient as compared with the injection administration group of Reference Example 1. On the other hand, in the transdermal administration group of Example 1, the tumor volume was smaller than that in the injection administration group of Reference Example 1, and cancer immunity could be effectively induced. Furthermore, as can be seen from FIG. 3, in the injection administration group of Reference Example 1, fluorescence of APC was not observed in the tumor, and cytotoxic T cells were not migrated into the tumor. In the group, fluorescence of APC was observed in the tumor, and it was confirmed that cytotoxic T cells migrated in the tumor.

Further, in the vaccine preparation for transdermal administration of Example 1, in place of glyceryl monooleate, polyglyceryl-10 trioleate or polyglyceryl isostearate-2 was used to prepare a vaccine preparation for transdermal administration, When the same immunity induction experiment as described above was carried out, excellent induction of cancer immunity was confirmed even with a vaccine preparation for transdermal administration using polyglyceryl-10 trioleate or polyglyceryl isostearate-2. Furthermore, in the vaccine preparation for transdermal administration of Example 1, a vaccine preparation for transdermal administration is prepared by replacing the aqueous phase base from a mixed solution of a phosphate buffer and isopropanol with a phosphate buffer alone. When an immunity induction experiment similar to that was conducted, excellent induction of cancer immunity was confirmed.

From the above results, a vaccine preparation in which a water-soluble fraction containing a solution in which an antigen is dissolved in water and a glycerin fatty acid ester and/or a polyglycerin fatty acid ester are dispersed in an oil phase is effective for immunity by transdermal administration. It was confirmed that it can be induced.

Test example 2
1. Preparation of formulation Each formulation having the composition shown in Table 3 was prepared by the following method.

[Example 2]
A solution was prepared by dissolving a predetermined amount of pollen antigen peptide (pepA, amino acid sequence: SMKVTVAFNQFGP) in an aqueous phase base (purified water and isopropanol). Separately, a predetermined amount of a surfactant (glyceryl monooleate) was added to a liquid oil (isopropyl myristate) and heated at 70° C. for 10 minutes to completely dissolve it. While stirring a predetermined amount of the solution, the solution was dropped little by little. By stirring this to room temperature, a vaccine formulation in a water-in-oil emulsified form (form in which the water phase was dispersed in the oil phase by the reverse micelle and emulsified) containing the pollen antigen peptide was obtained.

[Comparative Example 3]
An aqueous vaccine preparation was obtained by adding a predetermined amount of a predetermined amount of pollen antigen peptide (pepA) to a phosphate buffer (PBS) and dissolving it.

2. Immunity induction experiment 40 6-week-old mice (B10.S, female) were bred for 1 week in an environment where they could freely ingest water and food, and then acclimatized, and then 10 μg of pollen antigen extract and Inject? 200 μl of a phosphate buffer solution (PBS) containing 4 mg of Alum Adjuvant (Thermo Fisher Scientific) was subcutaneously administered to the roots of both legs once a week for a total of 3 times to prepare a pollinosis model mouse. Histamine (20 ng/head) was intranasally administered 6 days after the third subcutaneous administration, and from the next day, pollen exposure was performed once a day for 5 days. For pollen exposure, the pollen solution was prepared by dissolving a cedar pollen extract (trade name "Cedar pollen extract-Cj", manufactured by LSS Co., Ltd.) in a PBS solution to a concentration of 0.1 mg/mL in both nostrils. By nasal exposure of 10 μL each. One week after the end of the pollen exposure, blood was collected from the tail of the mouse, and the IgE antibody titer in the blood was measured by ELISA. Twelve mice were selected from the 5th to 34th pollinosis model mice in descending order of IgE antibody titers, and the two groups (1 group) shown in Table 4 were selected so that the average IgE antibody titers in each group were the same. 6 animals) and were bred for 8 days in a normal environment, and then subjected to the initial immunization and booster immunization under the conditions shown in Table 4.

Histamine (20 ng/head) was intranasally administered 6 days after the completion of the second booster immunization (completion of a total of 3 immunizations), and from the next day, pollen exposure was performed once a day for 5 days. For pollen exposure, a pollen solution prepared by dissolving a cedar pollen extract (trade name “Sugi pollen extract-Cj”, manufactured by ELS Co., Ltd.) in a PBS solution to a concentration of 0.1 mg/mL was applied to both nostrils. By nasal exposure of 10 μL each. One week after the end of pollen exposure, blood was collected from the tail of the mouse, and the spleen was extracted.

Serum was prepared from the obtained blood, and serum concentrations of Crij1-specific IgE antibody and IgG1 antibody were measured by ELISA.

Also, the obtained spleen was ground and subjected to hemolytic treatment, and then cells were collected by removing impurities with a 70 μm cell strainer. After culturing the obtained cells in a PRMI1640 medium containing 10% inactivated FBS (fetal bovine serum), 50 μM 2-mercaptoethanol, and 1% antibiotic/antimycotic mixed solution at 37° C. for 3 days, The culture medium (spleen cell culture medium) was collected. The cytokine (IL-4 and IL-5) concentration in the obtained culture solution was measured by ELISA.

FIG. 4 shows the experimental schedule, and FIG. 5 shows the results of measuring the serum concentrations of Crij1-specific IgE antibody and IgG1 antibody and the cytokine (IL-4 and IL-5) concentrations in the spleen cell culture medium. As a result, in the transdermal administration group of Example 2, the antibody concentration in the serum was lower than that in the transdermal administration group of Comparative Example 3, and it was confirmed that the production amount of cytokines involved in allergic reaction can be suppressed. It was

Test example 3
1. Preparation of formulation Each formulation having the composition shown in Table 5 was prepared by the following method.

[Example 3]
A solution was prepared by dissolving a predetermined amount of influenza virus antigen protein (Influenza A H3N2 (A/Aichi/1968) Hemagglutinin/HA Protein (His tag), Sino Biological) in an aqueous phase base (purified water and isopropanol). did. Separately, a predetermined amount of a surfactant (glyceryl monooleate) was added to a liquid oil (isopropyl myristate) and heated at 70° C. for 10 minutes to completely dissolve it. While stirring a predetermined amount of the solution, the solution was dropped little by little. By stirring this to room temperature, a vaccine formulation in a water-in-oil emulsified form (form in which the water phase was dispersed in the oil phase by the reverse micelle and emulsified) containing the pollen antigen peptide was obtained.

[Control]
A vaccine preparation in a water-in-oil emulsified form (form in which an aqueous phase is dispersed in an oil phase by reverse micelles and emulsified) is prepared in the same manner as in Example 3 except that the influenza virus antigen protein is not included. Obtained.

2. Immunity induction experiment Six-week-old mice (BALB/c, female) were bred for 1 week in an environment where they could freely ingest water and food and acclimated, and then divided into two test groups shown in Table 6 (per group). , 6), and the initial immunization and booster immunization were performed under the conditions shown in Table 6.

After the second booster immunization, the animals were raised in a normal environment for 3 weeks. Blood was collected from the tail of the mouse once a week from the day of the first immunization. Serum was prepared from the obtained blood and the serum concentration of anti-influenza A(H3N2) antibody was measured by ELISA.

FIG. 6 shows the experimental schedule, and FIG. 7 shows the measurement results of the serum concentration of anti-influenza A(H3N2) antibody. As a result, in the transdermal administration group of Example 3, hyou
Three weeks after the initial immunization, production of anti-influenza A(H3N2) antibody was observed, and it was confirmed that the antibody concentration increased with time.

Claims

A vaccine preparation for transdermal administration, which comprises an aqueous phase in which an antigen is dissolved, a glycerin fatty acid ester and/or a polyglycerin fatty acid ester, and an oil phase, and the aqueous phase is dispersed in the oil phase.
The vaccine preparation for transdermal administration according to claim 1, wherein the origin of the antigen is a cancer cell, a pathogenic virus, a pathogenic bacterium, a pathogenic organism, or pollen.
The vaccine preparation for transdermal administration according to claim 1 or 2, wherein the antigen is an antigen peptide.
The vaccine preparation for transdermal administration according to any one of claims 1 to 3, wherein the glycerin fatty acid ester is glyceryl monooleate.
The vaccine preparation for transdermal administration according to any one of claims 1 to 4, wherein the content of the glycerin fatty acid ester and/or the polyglycerin fatty acid ester is 0.1 to 30% by weight.
For the production of a vaccine preparation for transdermal administration of a composition comprising an aqueous phase in which an antigen is dissolved, glycerin fatty acid ester and/or polyglycerin fatty acid ester, and an oil phase, wherein the aqueous phase is dispersed in the oil phase Use of.
An immune-inducing effective amount of a composition containing an aqueous phase in which an antigen is dissolved, glycerin fatty acid ester and/or polyglycerin fatty acid ester, and an oil phase, the aqueous phase being dispersed in the oil phase, A method for inducing immunity, comprising the step of transdermal administration to an animal in need.