WO1997029773A1

WO1997029773A1 - Vaccine-containing emulsion and vaccine-containing powder for oral administration and process for producing the same

Info

Publication number: WO1997029773A1
Application number: PCT/JP1997/000351
Authority: WO
Inventors: Katsuhiko Ooyama; Masashi Umemura; Miho Sakurada; Kumiko Kurokawa
Original assignee: The Nisshin Oil Mills, Ltd.
Priority date: 1996-02-13
Filing date: 1997-02-10
Publication date: 1997-08-21
Also published as: AU1671197A

Abstract

An oil-in-water type vaccine-containing emulsion for oral administration prepared by emulsifying an immunogen with which human or animals can be immunized and a fat in an aqueous medium containing a polyhydric alcohol with the use of at least one hydrophilic nonionic surfactant and at least one lipophilic nonionic surfactant; and a vaccine-containing powder for oral administration obtained by drying this emulsion.

Description

Specification

Emulsion containing vaccine for oral administration,

TECHNICAL FIELD The present invention relates to a vaccine-containing emulsion for oral administration, a vaccine-containing powder for oral administration, and methods for producing the same. More particularly, the present invention relates to a vaccine-containing emulsion for oral administration which is excellent in formulation stability, a vaccine-containing powder for oral administration which can be easily redispersed, and a method for producing them. An immunogen for immunizing a human body or an animal is usually used in a state of being suspended in a buffered physiological saline. Such suspensions are referred to as vaccines, which include inactivated vaccines (eg, typhoid vaccine, 100 days Cough vaccine, rabies vaccine, etc.), attenuated vaccine (eg, pathogen, raw polio vaccine, etc.), detoxified vaccine (eg, diphthene vaccine) Rear toxoid, tetanus toxoid, etc.) are known.

Current vaccines are usually administered in the form of injections, especially in the form of subcutaneous injections, which makes them inconvenient to administer, as well as redness, fever, muscular atrophy or shock at the site of administration. There are drawbacks that easily cause side effects such as.

As a method for solving this drawback, an administration agent not by injection has been proposed in, for example, Japanese Patent Application Laid-Open No. 5-294845, but furthermore it is safe and convenient. Development of Oral Drugs That Have Both Properties It has been.

However, development of oral administration has been difficult because of the adverse effects such as inactivation of immunogens in vaccines by gastric acid and plowing of foreign substances in the small intestine. Yes.

Therefore, for oral administration, a study has been made on preparations using ribosome, microsphere, etc., and those using adjuvants so far. However, it has not yet reached the stage of practical use.

On the other hand, micro-fluidizers and ultrasonic emulsifiers are used to emulsify micelles for the purpose of enhancing the immune response and stabilizing the emulsion. Methods have been used for injections and the like [WO90Z148337 (published publication No. 5-5083885) and the like].

However, there is no particular problem when the vaccine-containing emulsion obtained in this way is used as an injection, but it does not have long-term formulation stability or oral administration. This was not enough. In other words, emulsions are unstable because of a heterogeneous dispersion system, which causes poor stability of the formulation itself and easily separates water and oil. On the other hand, removing water from emulsions has been considered technically.However, once the water has been removed and the powdered emulsion has been dispersed again in the water system. However, it was difficult to obtain an emulsion in which oil droplets were uniformly dispersed so that the emulsion could be applied to a vaccine for the U-line. In addition, such a vaccine-containing emulsion has not been obtained not only in terms of pharmaceutical properties but also in terms of effect. won.

In addition, liquid emulsions are difficult to enterolate, making it difficult to deal with gastric acid and the like in oral administration. According to the present invention, an immunogen for immunizing a human or an animal and an oil or fat are mixed with at least one hydrophilic non-ionic surfactant in an aqueous medium containing a polyhydric alcohol. And at least one lipophilic nonionic surfactant (provided that the mixing weight ratio is 20: 1 to 1: 1, preferably 9: 1 to 1: 1). There is provided an oil-in-water orally-administered emulsion for oral administration, which is obtained by emulsification.

In addition, an actin-containing powder for oral administration which can be obtained by adding two or more kinds of oil droplet interface adsorbents to the above-mentioned vaccine-containing emulsion and drying the same is provided. It is.

Further, according to the present invention, (i) a hydrophilic and lipophilic non-ionic surfactant (provided that the mixing weight ratio is 20: 1 to 1: 1 and preferably 9: 1 to 1: a step of melt-mixing 1) to obtain a mixture,

(ii) adding a polyhydric alcohol containing a small amount of water to the mixture, or separately adding a small amount of water and the polyhydric alcohol,

(iii) a step of gradually adding fats and oils to the obtained mixture while gently stirring;

(i V) and a step of adding a predetermined amount of water.

Providing an immunogen for immunizing a human or animal after step (i), The above-mentioned vaccine-containing emulsion for oral administration which can be added before step (ii), during any of steps (ii) to (iv), or after step (iv). Is provided.

In addition, the method comprises the steps (i) to (iv) described above, wherein an immunogen and an oil droplet surface adsorbent for immunizing a human or animal are simultaneously or separately provided after the step (i). , Which is added before step (ii), during any of steps (ii) to (iv), or after step (iv), and is subjected to a drying treatment. A method for producing a tin-containing powder is provided. In the vaccine-containing emulsion and the vaccine-containing powder of the present invention, the immunogen is also referred to as an antigen, and produces an antibody or a sensitized lymphocyte against a human or animal. A substance that induces humoral immunity or cellular immunity. Specifically, it is not particularly limited as long as it is used for the prevention of infectious diseases, and a vaccine is used. Examples of vaccines include inactive vaccines (eg, influenza vaccine, Japanese encephalitis vaccine, whooping cough vaccine, and triple vaccine vaccine). , Cholera vaccine, pneumococcal vaccine, rosin vaccine, etc.), attenuated vaccines (for example, polio vaccine, measles vaccine, Vaccine vaccine, BCG vaccine, typhus vaccin, pox vaccine, etc., detoxified vaccine (for example, diphtheria vaccine) , Tetanus vaccine, butoxoid, typhoid vaccine, botulinum vaccine, etc.) and component vaccines (Influenza HA vaccine) , Hepatitis B vaccine, non-A non-B hepatitis vaccine, herpes vaccine, AIDS vaccine, cancer vaccine, etc.) Can be listed. These immunogens can be lyophilized vaccine solution or vaccine solution, or when this lyophilized product is used, phosphate buffer, physiological saline, It can be used by dissolving it in purified water. At this time, although the immunogen varies depending on the content or type, etc., the immunogen is contained in an amount of about 0.001 to about 0.1% by weight based on the total amount of the vaccine-containing emulsion. Preferably, about 0.027 to about 0.011% by weight is particularly preferred.

The fats and oils are not particularly limited as long as they are pharmaceutically low-toxic and suitable for oral administration. Among them, vegetable oils such as olive oil, soybean oil, sesame oil and the like are preferred. The amount of fats and oils added is about 2 to about 30% by weight, preferably about 2 to about 20% by weight, and more preferably about 5% by weight, based on the total amount of the emulsion containing the vaccine. About 15% by weight. If the oil content of the vaccine-containing emulsion is more than 30% by weight, it may cause digestive problems such as diarrhea when administered orally. If the content is less than 2% by weight, a sufficient effect cannot be obtained, which is not preferable.

Furthermore, in the vaccine-containing emulsion for oral administration and the vaccine-containing powder of the present invention, both the hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant are simultaneously used. It is necessary to use them. The hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant can each be used alone or in combination of two or more. The hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant are preferably in a weight ratio of 20: 1 to 1: 1 and more preferably 9: 1 to 1: 1. , 7: 3 to 1: 1 are more preferred. This These surfactants are preferably contained in a total amount of about 0.5 to about 4.5% by weight, and preferably about 2.5% by weight, based on the total amount of the emulsion containing the vaccine. ~ 3.5% by weight is more preferred. The surfactant is preferably a combination having an HLB value in the range of 7 to 18 when a hydrophilic and lipophilic nonionic surfactant is mixed, and 11 to 14 A range of 5 is more preferred.

Examples of the hydrophilic non-ionic surfactant include polyoxyethylene sorbitan fatty acid ester (the number of moles of oxyethylene added to the polyoxyethylene is from 1 to 1). And the fatty acid esters are cabronic acid, carboxylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, It is derived from a saturated or unsaturated fatty acid having 6 to 18 carbon atoms such as palmitooleic acid, oleic acid, linoleic acid, linolenic acid, etc.), Polyoxyethylene sorbitol fatty acid esters (the number of moles of oxyethylene added is the same as described above, and the fatty acid esters are the same or mixed fatty acid esters of the same fatty acids as described above). Sucrose fatty acid ester (HLB value is 10 or more, flg fatty acid ester is above The same applies), polyoxyethylene fatty acid ester (the number of moles of added oxyethylene, fatty acid ester is the same as above), and polyoxyethylene fatty acid ester (oxyethylene). The number of moles of added len and fatty acid ester are the same as described above), polyglycerin fatty acid ester (H and B values are 10 or more, and the amount of glycerin in polyglycerin is The number of moles to be added is from 1 to 100, and the fatty acid ester is the same or a mixed fatty acid ester of the same fatty acids as described above .: Polyoxyethylene alkyl Ether (Alkyl ether is an alkyl ether having 10 to 18 carbon atoms in the same manner as above for the number of moles of added oxyethylene): Polyoxyethylene hardened castor oil (Oxyethylene The number of moles of added len is the same as described above). Among them, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene hydrogenated castor oil are preferred, and specifically, polyoxyethylene. Preference is given to styrene ethylene bitan monooleate esters, lauromacrogol, and polyoxyethylene hydrogenated castor oil. These hydrophilic non-ionic surfactants can be used alone or in combination of two or more. For example, if two or more types are used in combination, polyoxyethylene hardened castor oil and sucrose fatty acid ester, polyoxyethylene alkyl ether and polyoxy Silicone bitumen fatty acid ester, polyoxyethylene bitumen fatty acid ester and sucrose fatty acid ester, polyoxyethylene alkyl ether and polyoxyethylene hardened castor oil A combination of a polyoxyethylene alkyl ether and a sucrose fatty acid ester is preferred.

Examples of lipophilic nonionic surfactants include sorbitan fatty acid esters (fatty acid esters are as described above), glycerin fatty acid esters (fatty acid esters are as described above), Glycerin fatty acid ester (HLB value less than 10; fatty acid ester is the same or mixed fatty acid ester of the same fatty acids as above); sucrose fatty acid ester (HLB value less than 10; fatty acid ester Same as above), glycerin / lower mono or dicarboxylic acid / fatty acid ester (one of them) The ester is a fatty acid ester similar to the above, and the other ester is a lower mono- or di-carboxylic acid such as succinic acid, acetic acid, acetic acid, citric acid, tartaric acid, etc. And dicarboxylic acid is an ester of which may be substituted with a hydroxyl group, an acetyl group, etc.). Among them, Solbitan

Fatty acid esters and glycerin fatty acid esters are preferred, specifically, solvitan sesquioleate and glycerine monostearate. These lipophilic nonionic surfactants can be used alone or in combination of two or more. For example, when two or more kinds are used in combination, a combination of sorbitan fatty acid ester and glycerin fatty acid ester is preferable.

Preferred examples of the combination of a hydrophilic non-ionic surfactant and a lipophilic non-ionic surfactant include polyoxyethylene sorbitan fatty acid ester and sorbitan. Fatty acid esters, polyoxyethylene alkyl ethers and sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters and glycerin fatty acid esters, polyoxyethylene alkyl ethers And glycerin fatty acid esters. These are preferably combined in such a way that the HLB is in the range of 〜 to 18 and, among them, the Lauromac mouth goal and the solvent, which are polyoxyethylene alkyl ethers. Preference is given to a combination with bitumen fatty acid ester, solvitan sesquioleate.

Further, the vaccine-containing emulsion of the present invention is formed into an emulsion in an aqueous medium containing a polyhydric alcohol. Multivalent Al Due to its water retention function, it is considered that the addition of the surfactant improves the association property of the surfactant and makes it easier for oil droplets to be retained in the surfactant phase. It is also thought that it has helped to lower the interfacial tension, which is essential for the formation of the emulsion. It is thought that this will keep the immunogen in good condition in the emulsion and maintain its effect in oral administration. Examples of the polyhydric alcohol include glycerin, polyethylene glycol, propylene glycol, and the like. Glycerin is particularly preferred. The content of the polyhydric alcohol is from about 0.2 to about 1.4% by weight, preferably from about 0.6 to about 1.0% by weight, based on the total amount of the vaccine-containing emulsion. %.

Further, the vaccine-containing emulsion of the present invention contains water as a basic medium together with each of the above-mentioned components. Therefore, the vaccine-containing emulsion of the present invention is of the oil-in-water type (o / w). In this case, distilled water or deionized water is preferable as the water. The added amount of water is used to constitute the balance of the content of each component as described above. However, it is preferable to use PBS in consideration of administering the obtained emulsion.

Further, the vaccine-containing emulsion of the present invention further contains two or more types of oil droplet surface adsorbents and is subjected to a drying step such as freeze-drying, whereby the vaccine is obtained. It may be in the form of a contained powder. This oil droplet interfacial adsorbent can reproduce fine and uniform emulsions when the vaccine-containing emulsion of the present invention is powdered into a vaccine-containing powder. It is included for the purpose of realizing oil droplets and A compound that is adsorbed on the boundary, has high water dispersibility, and is solid at ordinary temperature. Specific examples include natural polymer compounds such as polysaccharides and proteins, and synthetic polymer compounds such as polyvinyl alcohol. Among them, arabia rubber, cyclodextrin, and dextrin are preferred, and a combination of two or three of these is preferred. In particular, a combination of Arabic rubber and /? — Cyclodextrin is preferred. The oil droplet surface adsorbent is preferably used in an amount of about 0.8 to about 3 parts by weight, more preferably about 1 part by weight (equal amount), based on 1 part by weight of the fat or oil in the emulsion containing the vaccine. I prefer it.

The vaccine-containing emulsion for oral administration of the present invention can be produced, for example, by the following method.

(i) First, hydrophilic and lipophilic non-ionic surfactants at a temperature above their melting point in a ratio of 20: 1 to 1: 1 and more preferably 9: 1 to 1: 1. Melt and mix in proportions to obtain a mixture. The temperature at this time varies depending on the nonionic surfactant used, but is generally preferably room temperature to 70 * C.

(ii) Next, a polyhydric alcohol containing a small amount of water is added to the obtained mixture. At this time, the ratio of polyhydric alcohol to water is preferably about 1: 9 to 1: 1 by weight, more preferably 1: 4 to 2: 3, and about 3: 7. Is even more preferred. The addition is preferably carried out at room temperature with stirring. At this time, a small amount of water and a polyhydric alcohol may be separately added in any order so as to have the above ratio.

(iii) Subsequently, the fats and oils are not gently stirred into the obtained mixture. Add it. Slow stirring means that natural emulsification is promoted without applying the mechanical pressure of a conventional device such as a microfilter or an ultrasonic emulsifier. Means this. At this time, since the temperature may rise due to the heat generated during stirring, if an immunogen that is not stable to heat is used, the fats and oils should not be cooled so that the temperature does not rise too quickly. It is preferable to add it by force. In this case, the addition is preferably performed gradually. Here, the term “gradually” means a force that can be appropriately adjusted because it differs depending on the manufacturing scale. It is sufficient that the force is not more than about lOOgZ, and 0.1g to 100g. / Min, and more preferably about 1 to 10 g Z min.

(iv) Further, a predetermined amount of water is added to the obtained mixture. Also in this case, it is preferable that the stirring is performed gradually while stirring gently. Here, the predetermined amount is an amount sufficient to adjust the content of each component determined in advance. Also, gradually means the same as in the above case. The immunogen in the present invention may be any of the immunogens after the heating and melting and mixing of the nonionic surfactant (i) in the above-mentioned step. Specifically, in any one of the step of adding a polyhydric alcohol containing a small amount of water (ii), the step of adding fats and oils (iii), the step of adding water (iv), or It may be added before step (ii) or after step (iV). However, it is preferred to add it as much as possible in the subsequent step, and most preferably to add it after step (iV) or step (iV).

In addition, another alternative is to use hydrophilic and lipophilic non-ionic surfactants. , A polyhydric alcohol and a small amount of water are melted and mixed at a temperature higher than the melting point of the nonionic surfactant, generally at room temperature to 70 to obtain a mixture, and the fats and oils are moderated By gradually adding the mixture while stirring, and adding and mixing a predetermined amount of water, a fine and stable oil-in-water emulsion can be obtained. In this case, the immunogen may be added at any step after the melting and mixing, but is preferably added after the step (iV) or after the step (iV), if possible. Is the most preferred.

In addition, the above-mentioned method uses natural emulsifying power, so that it has a vaccine effect の the use of oils and fats or surfactants for extracting it, as compared with the conventional emulsion method using mechanical pressure. The addition amount can be reduced, and it is excellent in safety, and also excellent in cost of manufacturing equipment and the like. Furthermore, it is excellent in preventing the deactivation of vaccines.

The resulting vaccine-containing emulsion can be used as it is as an orally administered drug. However, it is preferable to apply powder for enteric formulation to prevent inactivation of vaccine due to stomach acid, or to improve storage stability.

Hereinafter, a method for producing a vaccine-containing powder for oral administration in the present invention will be described.

The oil-drop surface adsorbent is added to the vaccine-containing emulsion obtained as described above in its original form, or in the form of a suspension or the like, and mixed. Alternatively, during the process of producing a vaccine-containing emulsion, after melting and mixing a non-ionic surfactant, the polyvalent alpha is added. After adding or simultaneously with the alcohol, melting and mixing the nonionic surfactant and the polyhydric alcohol, after adding or simultaneously adding the fat or oil, or after adding the water or At the same time, an oil droplet interfacial adsorbent may be added. In these cases, the immunogen may be added together with the oil droplet surface adsorbent or separately after the oil droplet surface adsorbent is added.

Next, after the oil droplet interfacial adsorbent is added in this way, it can be made into a powder by subjecting it to, for example, a freeze drying method, a spray drying method, a dehydration drying method, or the like. Of these, the freeze-drying method is preferred. However, in order to avoid denaturation of the protein in the immunogen, it is desirable to carry out the method of dehydrating and drying the mixture at 50 under the following conditions. Before pulverization by a freeze-drying method or the like, water or PBS is added to the obtained emulsion vaccine to obtain an emulsion. It is preferable to adjust the amount of fats and oils to 1 to 5% by weight, preferably about 2% by weight, based on the total amount of cutin.

The vaccine-containing powder obtained in this way can be easily redispersed in an aqueous medium. It can be used in the form of granules, tablets, pills, microcapsules, hard or soft capsules, and the like.

On the other hand, vaccine-containing emulsions can be used in the form of hard or soft capsules.

These preparations are preferably enteric-coated preparations in order to expect a sufficient effect of the vaccine.

For preparing granules, tablets, and pills, usually a pharmaceutically acceptable excipient or carrier (eg, monosaccharides such as glucose, lactose, etc.) Disaccharides, polysaccharides such as pullulan and dextrin, starches such as corn starch and potato starch), binders (eg, methylcellulose, histamine) Doxypropyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, hydroxypropylmethylcellulose, etc., disintegrants (for example, carboxymethylcellulose, Various additives such as ruboxime methylcellulose sodium, crystalline cellulose, partially pregelatinized starch, etc., and lubricants (eg, magnesium stearate, talc, etc.) are appropriately used. . In addition, pharmaceutically acceptable excipients, film-forming agents, solvents, and the like as described above are appropriately selected and used in order to obtain microcapsules. Hard or soft capsules are generally formed of gelatin. As the film-forming agent used for enteric solubilization of each preparation, for example, those known in the art can be appropriately selected and used. For example, methacrylic acid / methacrylic acid alkyl ester copolymers, hydroxypropyl propyl cellulose phthalate, hydroxypropyl propyl cellulose acetyl cellulose succinate, Carboxymethyl ethylcellulose, cellulose acetate phthalate, refined shellac, white shellac, ethylcellulose, aminoalkyl methyl acrylate polymer, etc.げEnteric solubilization is generally performed after preparation of each preparation in advance. Each of the above-mentioned preparations can be produced using a method known in the art.

The oral dosage of the vaccine-containing emulsion and the vaccine-containing powder of the present invention varies depending on the type of the immunogen used, but usually, the immunogen is administered once per adult. Is about 12.5 to 10000 (g) It is preferable to use it in the amount. In addition, the dose varies depending on other components, the number of doses, the sex, age, etc. of the recipient. For example, when influenza is used as an immunogen, it usually contains 12.5-100 000 g of influenza once per adult. If the dose is given orally and no increase in antibody titer is observed, additional dose can be given 2 to 4 weeks later. Hereinafter, the vaccine-containing emulsion and the vaccine-containing powder for oral administration of the present invention and the production method thereof will be described with reference to Examples, but the present invention is not limited thereto. is not.

Example 1

9 g of Lauromacrogol (trade name: BL-21; manufactured by Nikko Chemicals; the same applies hereinafter) and sorbitol sesquioleate (trade name: SO-15); Mix 9 g of the mixture and heat and melt at 60 ° C. Add 3 and 6 g of glycerin (manufactured by Nacalai Tesque, hereinafter the same) and a small amount of water. Mix at room temperature. While stirring, 60 g of soybean oil (manufactured by Nisshin Oil Co., Ltd .; the same applies hereinafter) was gradually added.

In addition, an aqueous solution of influenza enzyme (A / Guizhou / 54 / 89H3N2, the same applies hereinafter) is gradually added and mixed, and the oil content becomes 10% by weight. As described above, dilution was performed with a phosphate buffer (PBS) to obtain an emulsion containing influen- vacactin with an antigen amount of 27 µg / l. Example 2

Mix 9 g of lauromacrogol and 9 g of sorbitan sesquioleate, heat and melt at 60, add 3.6 g of glycerin and a small amount Of water were mixed at room temperature. To this, 6 Og of olive oil (manufactured by Gokyo Sangyo, the same applies hereinafter) was gradually added while stirring.

Furthermore, an aqueous solution of influenzae protein is gradually added and mixed, and diluted with PBS so that the oil amount becomes 10% by weight, and the antigen amount is adjusted to 27%. An emulsion containing g / m 1 of influenza enquinone was obtained.

Example 3

12.6 g of lauromacrogol and 5.4 g of sesquioleic acid sorbitan were mixed, heated and melted at 60 X: and added with 3.6 g of glycerin. g and a small amount of water were mixed at room temperature. Here, 60 g of soybean oil was gradually added with stirring.

Further, an aqueous solution of influenzae tactic vaccine is gradually added and mixed, and diluted with PBS so that the oil amount becomes 10% by weight, and the antigen amount is 27 / xg. An emulsion containing 1 / ml of the influenza enzyme was obtained.

Example 4

Mix 16.2 g of lauromacrogol and 1.8 g of sorbitan sesquioleate, heat and melt with, add 3.6 g of glycerin and a small amount Of water were mixed at room temperature. Here, 60 g of soybean oil was gradually added with stirring. Further, an influenzae evening aqueous vaccine solution was gradually added and mixed, and diluted with PBS so that the oil amount became 10% by weight, and the antigen amount was adjusted to 27%. An emulsion containing ng / m 1 of influenzawatin was obtained.

Example 5

Mix 9 g of lauromacrogol and 9 g of sorbitan sesquioleate, heat and melt at 60 ° C, and add 3.6 g of glycerin and a small amount of water to room temperature. And mixed. Here, 18 Og of soybean oil was gradually added with stirring.

Further, an influenza enzyme vaccine aqueous solution was gradually added and mixed, and diluted with PBS so that the oil amount became 30% by weight. The antigen amount was 27 g / An emulsion containing m1 influenzawacchin was obtained.

Example 6

A mixture of 13.5 g of lauromacrogol and 13.5 g of sorbisoen sesquioleate was heated and melted at 60 ° C, and then added with 3.6 g of glycerin. g and a small amount of water were mixed at room temperature. Here, 180 g of soybean oil was gradually added with stirring.

Furthermore, the aqueous solution of influenzae protein is gradually added and mixed, and diluted with PBS so that the oil amount becomes 30% by weight, and the antigen capacity is 27 g. An influenza enzymatic emulsion containing Zml was obtained.

Example 7

Lauromacrogol 12.6 g, Sesquioleic acid Sorbi Evening 5.4 g of glycerin, 3.6 g of glycerin and a small amount of water were mixed, and the mixture was heated and melted to 60. Here, 180 g of soybean oil was gradually added with stirring.

Furthermore, an influenza enzyme vaccine aqueous solution is gradually added and mixed, and diluted with PBS so that the oil amount becomes 30% by weight, and the antigen amount is adjusted to 27 g / An emulsion containing m1 influenza vaccine was obtained.

Example 8

Mix 18.9 g of lauromacrogol and 8.lg of solvitan sesquioleate, heat and melt at 60, and add 3.6 g of daricelin And a small amount of water were mixed at room temperature. Here, 60 g of sesame oil (manufactured by Kuki Sangyo, the same applies hereinafter) was gradually added with stirring.

Further, the influenzaein evening vaccine solution was gradually added and mixed, and diluted with PBS so that the oil amount became 10% by weight, and the antigen amount was adjusted to 27 g / An emulsion containing m1 influenza vaccine was obtained.

Example 9

9 g of lauromacrogol and 9 g of sorbitan sesquioleate were mixed with 3.6 g of glycerin and a small amount of water at room temperature, and heated and melted at 60 * C. I let you. Here, 60 g of sesame oil was gradually added with stirring.

Furthermore, the aqueous solution of influenza intact vaccine is gradually added and mixed, and diluted with PBS so that the oil amount becomes 10% by weight. Thus, an emulsion containing influenza enzyme with an antigen amount of 27 g / m 1 was obtained.

Example 10

9 g of lauromacrogol and 9 g of sorbitan sesquioleate are mixed, heated and melted at 60, and 3.6 g of glycerin and a small amount of water are added thereto. Mix at room temperature. Here, 180 g of sesame oil was gradually added with stirring.

Further, an influenza enzyme vaccine aqueous solution is gradually added and mixed, and diluted with PBS so that the oil amount becomes 30% by weight, and the antigen amount is adjusted to 27 X g An emulsion containing / m 1 of influenza actin was obtained.

Example 1 1

12.6 g of lauromacrogol, 5.4 g of solyretan sesquioleate, 3.6 g of glycerin and a small amount of water were mixed and heated and melted to 60. I let you. Here, 6 Og of sesame oil was gradually added with stirring.

Furthermore, an aqueous solution of influenza enzyme vaccine is gradually added and mixed, and diluted with PBS so that the oil amount becomes 10% by weight, and the antigen amount is 27 g. Influenza enquinone-containing emulsion was obtained.

Example 1 2

Mix 9 g of lauromacrogol and 9 g of sorbitan sesquioleate, heat and melt at 60, add 3.6 g of glycerin and a small amount Water was mixed at room temperature. Here, stirring Then, 180 g of olive oil was gradually added.

Further, an aqueous solution of influenzae protein is gradually added and mixed, and diluted with PBS so that the oil amount becomes 30% by weight, and the antigen amount is adjusted to 27% by weight. An emulsion containing u-S / m1 of influenza enquintin was obtained.

Example 13

13.5 g of lauromacrogol, 13.5 g of sorbitan sesquioleate, 6 g of glycerin and a small amount of water are mixed and heated and melted at 60. I let it. Here, 180 g of olive oil was gradually added with stirring.

Further, an influenza enzymatic vaccine solution was gradually added and mixed, and diluted with PBS so that the oil amount became 30% by weight, and the antigen amount was adjusted to 27 gm 1. Thus, an influenza enzyme-containing emulsion was obtained.

Example 14

Polyoxyethylene sorbitan monooleate (trade name: TO-10M; manufactured by Nikko Chemicals, the same applies hereinafter) 24.3 g and sesquioleate sorbitan The mixture was heated and melted at 60 ° C., and 1.2 g of glycerin and a small amount of water were mixed at room temperature. Here, 60 g of soybean oil was gradually added with stirring.

Further, an aqueous solution of influenzae protein is gradually added and mixed, and diluted with PBS so that the oil amount becomes 10% by weight, and the antigen amount is adjusted to 27%. An emulsion containing ug / m 1 of influenza enzyme was obtained. Example 15

Polyoxyethylene sorbitan monooleate (12.6 g) and glycerine monostearate (trade name: MGS-B; manufactured by Nikko Chemicals, Inc.) 5.4 g were mixed and heated and melted at 60, and 3.6 g of glycerin and a small amount of water were mixed at room temperature. Here, 60 g of soybean oil was gradually added with stirring.

Further, an influenza enzyme vaccine aqueous solution was gradually added and mixed, and diluted with PBS so that the oil amount became 10% by weight, and the antigen amount was adjusted to 27 g / Thus, an emulsion containing m 1 influenza wax was obtained.

Example 16

Mix 9 g of lauromacrogol and 9 g of glycerin monoostearate, heat and melt at 60 "C, and add 3.6 g of glycerin. Then, a small amount of water was mixed at room temperature, and while stirring, 60 g of soybean oil was gradually added.

Further, an influenza intact vaccine aqueous solution is gradually added and mixed, and diluted with PBS so that the oil amount becomes 10% by weight, and the antigen amount is adjusted to the ml amount of the antigen. An emulsion containing fluen- zactin was obtained.

Example 17

9 g of lauromacrogol and 9 g of sorbitan sesquioleate are mixed and heated and melted at 60, and 3.6 g of glycerin is mixed at room temperature. Was. After cooling, the amount of influenza enzyme is finally reduced to 27 ug / m1. A small amount of water in which the vaccine was dissolved was added and mixed.

While cooling, 6 Og of soybean oil was gradually stirred and mixed. Further, the oil was diluted with PBS so as to have an oil amount of 10% by weight to obtain an emulsion containing influenza enquintin having an antigen amount of 27 g / ml.

Example 18

A rubber (dispersed in Sanei Pharmaceutical Trading Co., Ltd., manufactured by Sanei Pharmaceutical Co., Ltd.) dispersed in water so that the oil power becomes 2% by weight was added to the emulsion containing 30 ml of the influenzawacchin-containing emulsion prepared in Example 1. 1.35 g and β-cyclodextrin (manufactured by Nippon Shokuhin Kako Co., Ltd .; the same applies hereinafter) (1.65 g) were added thereto, followed by stirring and mixing. This was freeze-dried to obtain an emulsion containing powdered influenza vaccin.

Example 19

9 g of lauromacrogol, 9 g of sorbitan sesquioleate, 3.6 g of glycerin and a small amount of water were mixed, and the mixture was heated and melted at 60 ° C. To this, 27 g of arabia rubber dispersed in water and 33 g of 0-cyclodextrin were added, followed by stirring and mixing. 60 g of soybean oil was gradually added to this mixture with stirring.

Further, an aqueous solution of influenza enzyme vaccine is gradually added and mixed, and diluted with PBS so that the oil amount becomes 2% by weight, and the antigen amount is adjusted to 5.4 ng / m 2. Thus, an emulsion containing 1 influenza vaccine was obtained. This was freeze-dried to obtain an emulsion containing powdered influenza vaccine. Example 20

9 g of lauromacrogol, 9 g of sorbitan sesquioleate, 3.6 g of glycerin and a small amount of water were mixed, and the mixture was heated and melted to 60. To this, 30 g of arabia rubber dispersed in water, 30.6 g of dextrin (manufactured by Matsutani Chemical), and 25.4 g of 3-cyclodextrin were added. Stir and mix. 60 g of soybean oil was gradually added to this mixture with stirring.

Further, an aqueous solution of influenzae protein is gradually added and mixed, and diluted with PBS so that the oil amount becomes 2% by weight, and the antigen amount is adjusted to 5.4 g / antigen. Thus, ml of emulzine containing influenza enquin was obtained. This was freeze-dried to obtain an emulsion containing powdered influenza vaccine.

Example 2 1

Polyoxyethylene sorbitan monooleate 24.3 g, sorbitan sesquioleate 2.7 g, glycerin 3.6 g and a small amount of water Were mixed and heated and melted at 60 ° C. To this, 79 g of arabia rubber and 12.7 g of 3-cyclodextrin dispersed in water were added and mixed with stirring. 60 g of soybean oil was gradually added to this mixture with stirring.

Further, an influenza enzyme vaccine aqueous solution is gradually added and mixed, and the mixture is diluted with PBS so that the oil amount becomes 2% by weight, and the antigen amount is 5.4 ug / m 1. An emulsion containing INFLUENWA ACTIN was obtained.

This is freeze-dried to give a powder A modified vaccine was obtained.

Example 22

9 g of lauromacrogol, 9 g of sorbitan sesquioleate, 3.6 g of glycerin and a small amount of water were mixed, and the mixture was heated and melted to 60. While stirring, 60 g of soybean oil was gradually added thereto, followed by stirring and mixing. To this mixture was added 57.6 g of arabia gum dispersed in water and 57.9 g of j3-cyclodextrin.

Furthermore, an influenza enzyme vaccine aqueous solution is gradually added and mixed, and diluted with PBS so that the oil amount becomes 2% by weight, and the antigen amount is adjusted to 5.4 / xg. Z ml of the influenza enantione containing emulsion was obtained. This was freeze-dried to obtain an emulsion containing powdered influenza vaccin.

Example 23

Polyoxyethylene hardened castor oil (trade name: HCO — 60: manufactured by Nikko Chemicals) 8. lg, 0.9 g of solvitan sesquioleate, and sugar-powered prill Mix 9.0 g of acid ester (trade name: SM-880: manufactured by Mitsubishi Chemical Foods Co., Ltd.), heat and melt at 6, mix 3.6 g of glycerin and a small amount Of water were mixed at room temperature. Here, 60 g of soybean oil (manufactured by Nakarai Tesk) was gradually added with stirring.

Furthermore, an aqueous solution of influenza enzyme vaccine was gradually added and mixed, and diluted with PBS so that the oil amount became 10% by weight, and the antigen amount was adjusted to 2%. An emulsion containing 7 g / m 1 of influenza enzyme was obtained. Example 2 4

When the powdered influenza vaccine-containing emulsion lg prepared in Example 18 was dissolved in water or 900 g of PBS, it was promptly dissolved within 30 minutes. It re-dispersed, and no oil was found 1 hour after re-dispersion.

Furthermore, changes in the particle size of the emulsion before pulverization and the particle size of the emulsion after re-dispersion were observed with a sub-micron particle analyzer (COULTERN 4 SD type). It was confirmed that a uniform emulsion state was maintained.

Table 1 below shows the results. Unit: (nm)

【table 1 】

Example 2 5

A centrifugal flow-type coating granulator (SPIR-A-FLOW: manufactured by Freund Sangyo Co., Ltd.) was added to the emulsion containing powdered influenza enquin obtained in Example 18. ) And enteric coating was applied. Table 2 shows the formulation of the enteric coating solution when applying the enteric coating.

[Table 2]

The enteric coating amount was adjusted so that the content of HPMC-AS was about 15% by weight based on the powdered influenza emulsion-iodinated vaccine. The obtained emulsion preparation containing enteric powder influenza vaccin was suitable for the disintegration test according to the disintegration test method of the Japanese Pharmacopoeia method in accordance with the section on enteric preparations.

Example 26

The emulsion containing powdered influenza en- quatin obtained in Example 18 was filled in a hard capsule, and the PAN coating apparatus (HI—CORTER.48N type: Freon) was used. Enteric coating was applied to the product. Enteric coating formulation was performed in the same manner as above.

Test example 1

The influenzawacutin-containing emulsion prepared in Example 1 was applied to a mouse (ddY female, 6 to 7 weeks old: 10 mice per group, the same applies hereinafter) at a dose of 27 g / ml via lml. (Primary immunization). Blood was collected 3 weeks after administration, a secondary immunization was performed under the same conditions as the primary immunization, and blood was collected 2 weeks later (5 weeks after the primary immunization). Blood bleeding obtained 3 and 5 weeks after these immunizations was evaluated. Table 3 shows the results of evaluation of the amount of these specific antibodies in the blood by measuring the HI titer (hemarginitin-in-hip-pass assay). Generally, this HI titer is considered to be effective (antibody amount capable of protecting infection) above 128 HI.

[Table 3]

Test example 2

The influenzazawacin-containing emulsion prepared in Example 2 was orally administered (primary immunization) with 27 g of Zml to the mouse. Blood was collected 3 weeks after the administration, a secondary immunization was performed under the same conditions as the primary immunization, and blood was collected 2 weeks later (5 weeks after the primary immunization). Serum obtained 3 and 5 weeks after these immunizations was used for evaluation. The amounts of specific antibodies in the blood were evaluated by measuring the HI titer. The results are shown in Table 4.

[Table 4]

Test example 3

The emulsion containing influenza vaccine prepared in Example 3 The mouse was orally administered with 27 ml of izg Zml (primary immunization). Blood was collected three weeks after administration, and the obtained serum was used for evaluation. The amount of specific antibodies in these blood was evaluated by measuring the HI titer. Table 5 shows the results.

[Table 5]

Test example 4

The influenza warpactin-containing emulsion prepared in Example 4 was orally administered (primary immunization) to the mouse with 27 ml of Zml in an amount of lml. Blood was collected 3 weeks after administration, and the obtained serum was used for evaluation. The amount of specific antibodies in these blood was evaluated by measuring the HI titer. Table 6 shows the results.

[Table 6]

Test example 5

The emulsion containing the influenza vaccine prepared in Example 5 was orally administered (primary immunization) to the mouse with 27 ml of Z ml in an amount of 1 ml. Blood was collected 3 weeks after administration, and the obtained serum was used for evaluation.The amount of specific antibodies in the blood was evaluated by measuring the HI titer. Was performed. Table 7 shows the results.

[Table 7]

Test example 6

The influenza enzym- cin-containing emulsion prepared in Example 6 was orally administered (primary immunization) with 27 ig Zml to the mouse. Three weeks after administration, blood was collected, and the obtained serum was used for evaluation. The amount of the specific antibody in the blood was evaluated by measuring the HI titer. Table 8 shows the results.

[Table 8]

Test example 7

The emulsion containing the influenza warpactin prepared in Example 7 was orally administered (primary immunization) to the mouse with 1 ml / 27 / xg Z ml. Three weeks after administration, blood was collected, and the obtained serum was used for evaluation. The amount of specific antibodies in these blood was evaluated by measuring the HI titer. Table 9 shows the results. [Table 9]

In Test Examples 3 to 5 above, the effective amount of the antibody in the blood was not reached by one immunization, but the results of Examples 1 and 2 indicate that after the second immunization, Since two to four times the amount of antibody is produced, it is considered that effective secondary antibody immunization will surely produce an effective amount of antibody.

Test Example 8. The mice were administered orally (primary immunization) with 27 wg Zml of the emulsion containing influenzacactin prepared in Example 23 to the mouse at 27 wg Zml. Blood was collected 3 weeks after administration, a secondary immunization was performed under the same conditions as the primary immunization, and blood was collected 2 weeks later (5 weeks after the primary immunization). The sera obtained 3 and 5 weeks after these immunizations were used for evaluation.

The specific antibody levels in these blood were evaluated by measuring the HI titer. Table 10 shows the results.

[Table 10]

H I value

3 weeks later Serum <32 HI

5 weeks after serum 2 5 6 HI Test Example 9 (Invitation test for powdered chemicals)

Using the sample of Example 18 to confirm the antigenicity of the emulsion before pulverization and the redispersion emulsion after pulverization in vitro, it was confirmed that the influenza enzyme was used. The amount of antigen was almost the same as that of the control solution. Thus, it is considered that there is no decrease in antigenicity due to powderization. • experimental method

Each sample was prepared with PBS so as to be 5 CCA nom 1, a calibration curve was prepared using an influenza vaccine solution, and the working curve was determined from the absorbance measured by the ELISA method. The antigen amount of the sample was calculated using the antigen amount of the tin solution as a theoretical value.

(EUISA method)

1) 96 エル The primary antibody is anti-HA protein (mouse IgG clone G7G12) 10 g / m1 (X200, in PBS (1)). ) Was added in 50 ^ 1 wells, coated, and left for 1 で with 4.

2) Block Ace (X4, distilled water) was added to 200 1 1 ゥ Z well and left at 4 for 1 晚.

3) The wells were washed using TPBS (PBS + 0.005% Tween 20) (three times).

4) Samples were added at 100 / X1 / ゥ.

5) Left at 37 for 1 hour.

6) The well was washed with TPBS (3 times).

7) Piotinylated anti-HA protein (X200, TP

(In BS), add 1001 noel and leave at 37 for 1 hour Was.

8) The cell was washed with PBS (3 times).

9) Add 0.11 Z ゥ of Streptavidin- Horseradish Peroxidase Conjugate (x4,000 in PBS), which is a color-forming enzyme, for 1.5 hours at 37 t I left it.

10) Wash the wells with TPBS (6 times).

1 1) Add 1 µl of a color-forming substrate [TMB (tetramethyl benzidine): mixed with Perox i dase Solut ion B = 1: 1] and let stand at room temperature for 20 minutes. 12) 1 M / l of a 1 M phosphoric acid solution was added as a reaction stop solution.

13) The absorbance was measured using a microplate reader (450 nm / 595 nm).

14 4) A calibration curve was prepared, and the antigen amount of the sample was expressed in% using the control sample as the theoretical value.

• The results are shown in Table 11. [Table 11] Antigen content% vaccine solution 94% Target emulsion (before powdering) 101% Redispersion emulsion (after powdering) 93%

Claims

The scope of the claims

1. An immunogen that immunizes humans or animals and fats and oils are mixed with at least one hydrophilic non-ionic surfactant in an aqueous medium containing a polyhydric alcohol. Both are oil-in-water type, orally-administered, emulsions containing actinide for oral administration, which are emulsified with one kind of lipophilic nonionic surfactant.

2. The vaccine according to claim 1, wherein the hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant are contained in a mixed weight ratio of 20: 1 to 1: 1. Emulsion containing.

3. The vaccine according to claim 2, wherein the hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant are contained in a mixed weight ratio of 9: 1 to 1: 1. Contained emulsion.

4. Claims 1 to 3 wherein the total amount of the hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant is contained in the emulsion at 0.5 to 4.5% by weight. A vaccine-containing emulsion according to any of the preceding claims.

5. The work according to any one of claims 1 to 4, wherein the hydrophilic non-ionic surfactant and the lipophilic non-ionic surfactant are combined so as to have an HLB of 7 to 18. Tin-containing emulsion.

6. Hydrophilic non-ionic surfactant and lipophilic non-ionic surfactant are combined with polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, and polyoxyethylene alkyl. Ether and sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester and glycerin fatty acid ester or polyoxyethylene The vaccine-containing emulsion according to any one of claims 1 to 5, wherein the emulsion is a combination of an alkyl ether and a glycerin fatty acid ester.

7. The emulsion containing a peptide according to any one of claims 1 to 6, wherein the immunogen is contained in the emulsion in an amount δ of 0.001 to 0.1% by weight. .

8. The emulsion containing a vaccine according to any one of claims 1 to 7, wherein the oil or fat is contained in an amount of 2 to 30% by weight in the emulsion. 09. The oil or fat is contained in the emulsion. The vaccine-containing emulsion according to claim 8, which is contained in an amount of 5 to 15% by weight.

10. The vaccine-containing emulsion according to any one of claims 1 to 9, wherein the oil or fat is one or a combination of two or more of oil, soybean oil, and sesame oil.

51. The zinc-containing alcohol according to any one of claims 1 to 10, wherein the polyhydric alcohol is contained in the emulsion in an amount of 0.2 to 1.4% by weight. Emulsion.

12. The method according to any one of claims 1 to 11, wherein the polyvalent alcohol is glycerin, polyethylene glycol, or propylene glycol. Emulsion containing vaccine.

13. A process which can be obtained by adding two or more kinds of oil droplet interfacial adsorbents to the vaccine-containing emulsion according to any one of claims 1 to 12, followed by drying. Vaccine-containing powder for oral administration.

1 4. The oil droplet interfacial adsorbent is combined with Arabic rubber and / 3-cyclodextrin 14. The vaccine-containing powder for oral administration according to claim 13, which is a combination with phosphorus.

15. (i) a step of melt-mixing a hydrophilic and lipophilic nonionic surfactant (provided that the mixing weight ratio is 20: 1 to 1: 1) to obtain a mixture, (ii) the mixture Then adding a polyhydric alcohol containing a small amount of water, or separately adding a small amount of water and a polyhydric alcohol,

(iv) further comprising a step of adding a predetermined amount of water,

An immunogen for immunizing a human or animal is provided after step (i), before step (ii), during any of steps (ii) to (iv), or in step (iv). A method for producing a vaccine-containing emulsion for oral administration, which can be added after the step.

16. The peptide-containing emulsion for oral administration according to claim 15, wherein the mixed weight ratio of the hydrophilic and lipophilic nonionic surfactants in the step (i) is from 9: 1 to 1: 1. Production method.

17. Steps (i) and (ii) are performed simultaneously, and then steps (iii) and (iV) are performed sequentially.

From adding the immunogen after step (ii) but before step (iii), during any of the steps (iii) or (iv), or after step (i V). A method for producing a vaccine-containing emulsion for oral administration according to any one of claims 15 and 16.

18- (i) Hydrophilic and lipophilic non-ionic surfactants (however, mixed Melt blending the weight ratio of 20: 1 to 1: 1) to obtain a mixture, (ii) adding a polyhydric alcohol containing a small amount of water to the mixture, or adding a small amount of water and The process of adding the polyhydric alcohol separately,

(iiii) a step of gradually adding fats and oils to the obtained mixture while gently stirring;

(iv) a step of further adding a predetermined amount of water,

An immunogen and an oil droplet surface adsorbent for immunizing a human or animal may be used simultaneously or separately, after step (i), before step (ii), in steps (ii) to Πν). A method for producing a powder containing a vaccine for oral administration which can be added during any step or after step (iv) and dried.

19. The pentank-containing tablet for oral administration according to claim 18, wherein the mixed weight ratio of the hydrophilic and lipophilic nonionic surfactants in the step (i) is from 9: 1 to 1: 1. Emulsion manufacturing method.

20. Steps (i) and (ii) are performed simultaneously, and then steps (iii) and (iv) are sequentially performed.

The immunogen and the oil droplet surface adsorbent may be added simultaneously or separately, after step (ii) but before step (iii), during any of step (iii) or (iv), or 10. The method for producing a vaccine-containing powder for oral administration according to claim 18 or 19, which is added after (iv).