JP2020158494A - Oral mucosa-adhering vaccine preparation - Google Patents

Abstract

To provide an oral mucosa-adhering vaccine preparation, which is an oral mucosa-adhering type preparation, and stably exposes vaccine antigen to a mucosal surface by attaching high-concentration drug solution to the oral mucosa for a fixed time, and can be completely dissolved within a predetermined time period.SOLUTION: There is provided an oral mucosa-adhering vaccine preparation having a drug layer containing vaccine antigen and a drug cover layer. The drug cover layer has a thickness of 15 μm or more. The disintegration property (A) of the drug layer and the disintegration property (B) of the drug cover layer against distilled water at 37°C have a relationship of (A)>(B). When a test piece of the drug cover layer having an area of 20 cm2 and a thickness of 50 μm is immersed in 500 mL of distilled water at 37°C, the residual rate (C) thereof 7 minutes after the start of immersion is 50% or more and 90% or less.SELECTED DRAWING: None

Description

The present invention relates to an oral mucosa-adhering vaccine preparation.

Conventionally, a method of using a liquid agent, an ointment agent, a jelly agent, a spray agent, a troche agent, a buccal tablet, a sublingual tablet, or the like has been known as a method for administering a drug into the oral cavity. In recent years, many oral mucosa-adhering preparations based on water-soluble or water-swelling polymers have been developed or proposed as preparations that show good adhesion to oral mucosa wet with water such as saliva. Has been done.

Oral mucosal adhesion type preparations are often used for the purpose of protecting and treating oral mucosal diseases such as so-called stomatitis. Therefore, it is desired that the application site is physically protected even when water or saliva is contained after application, and that the application site remains for a long time. Therefore, in the recent development of oral mucosal adhesion type preparations, oral mucosal adhesion type preparations that have sustained release of drugs and physically remain in the oral cavity for a long time have been developed or proposed.

Further, since the oral mucosa site has a relatively high drug absorbency except when the oral mucosa site is a disease site, for example, an attempt has been made to use it as an administration route of a drug that is difficult to be absorbed orally. As such an oral mucosa-adhering preparation, for example, by having Patent Document 1 a lower alkyl or lower hydroxyalkyl ether of cellulose and a drug layer composed of an acrylic acid polymer and a drug, the drug is gradually added. It is taught to satisfy release and fast-acting properties.
However, it has not been disclosed or suggested that a high-concentration drug solution is adhered to the oral mucosa for a certain period of time by controlling the disintegration property of the drug layer and the cover layer in the oral mucosal adhesion type preparation. Furthermore, no disclosure or suggestion of an oral mucosa-adherent vaccine formulation containing a vaccine antigen in such a drug layer is also disclosed.

On the other hand, drug administration by oral mucosal adhesion type preparation has almost no physical and psychological burden on the patient such as pain, fear, injection scar and subsequent scarring as compared with drug administration by injection, and needle stick of a medical worker. Since there is no risk of infection accidents and medical waste such as injection needles is not generated, it is attracting attention as an administration method instead of injection.
In particular, injections such as subcutaneous or intradermal injection and intramuscular injection are generally used for inducing immunity by vaccination. Vaccines need to be invasively administered into the body, as microorganisms and viruses are blocked from entering through the skin due to their size.
However, intradermal injection, which has a high immune effect, is difficult to administer, and intramuscular injection causes severe pain to patients. Therefore, a vaccine administration method instead of injection has been desired.

Here, the oral mucosa has evolved to block the induction of specific immune responses against harmless antigens such as food and inhaled particles. As a result, many antigens introduced into the oral mucosal surface induce "tolerance" rather than productive T and B cell responses. Therefore, it is necessary to overcome these natural processes in order to create an effective vaccine. Therefore, in the formulation of the oral mucosal vaccine, it is essential to effectively deliver the vaccine antigen to immune cells, that is, to stably expose the vaccine antigen to the oral mucosal surface. As a technique for formulating such a vaccine, for example, Patent Document 2 discloses a low-viscosity liquid dosage form that can be orally administered, and teaches that the absorption of an active substance is promoted with low viscosity. In addition, Patent Document 3 discloses an oral vaccine for inducing an immunogenic response, utilizing starch as an immune response-enhancing matrix-forming agent for delivery of the vaccine, and a fast-soluble dosage form. Is taught to be.
However, there is no disclosure or suggestion of an oral mucosal-attached vaccine preparation that stably exposes the vaccine antigen to the mucosal surface for a certain period of time.

The biggest obstacle to administering a drug using an oral mucosa-adhering preparation is that the placed drug is washed away by the time it is absorbed due to the expansion and contraction of the mucosal part due to secretions such as saliva or conversation. In addition, a sufficient pharmacological effect cannot be obtained as compared with the amount of the drug. In addition, even if the oral mucosal adhesion type preparation is maintained in the oral cavity, the drug in the preparation may dissolve and be swallowed due to exposure of the preparation to a large amount of saliva. In some cases it was unclear if it was being administered. Therefore, there is also a drawback that it is difficult to adjust the type and amount of the drug to be administered depending on the type or symptom of the disease.

Further, in medical institutions, as a response to a drug-induced anaphylactic reaction, it is usually required to be observed by a doctor for 30 minutes after administration of the drug. Therefore, medical institutions also manage time and physical condition for patients after drug administration. Therefore, a method of reducing the burden of time management after drug administration has also been desired.

JP-A-58-043915 Japanese Unexamined Patent Publication No. 2011-251989 Japanese Unexamined Patent Publication No. 2013-5397666

In view of the above situation, it is an object of the present invention to provide an oral mucosa-adhering vaccine preparation capable of stably exposing a vaccine antigen to the oral mucosa for a certain period of time.

As a result of diligent studies, the present inventors have made a specific relationship between the drug layer and the drug cover layer constituting the oral mucosa-adhering vaccine preparation so that the disintegration property of each layer with respect to water has a specific relationship, thereby producing a high-concentration drug solution in the oral cavity. It was found that the drug can be retained on the mucosa and exposed to the mucosal surface. Furthermore, by setting the residual rate (C) under specific conditions showing the disintegration property of the drug cover layer to a specific range, the drug cover layer does not disintegrate for a certain period of time, and the high-concentration drug solution is stabilized for a certain period of time. It was found that the drug cover layer collapses after a certain period of time and does not remain in the oral cavity, facilitating time management after drug administration.

The present invention is an oral mucosal adhesion type vaccine preparation having a drug layer containing a vaccine antigen and a drug cover layer, wherein the thickness of the drug cover layer is 15 μm or more and the temperature is 37 ° C. disintegration of the drug layer (a) and disintegration of the drug cover layer (B) has a relationship of (a)> (B), a test piece of the drug cover layer area 20 cm ² thickness 50 [mu] m, It is an oral mucosal adhesion type vaccine preparation characterized in that the residual rate (C) 7 minutes after the start of immersion in 500 mL of distilled water at 37 ° C. is 50% or more and 90% or less.

Further, it is preferable that the drug layer has a solution viscosity (D) of 20 mPa · s or less when a single dose is dissolved in 1.0 mL of distilled water at 37 ° C.
Further, the drug layer preferably further contains a polymer component.
The polymer component is preferably at least one selected from the group consisting of dextran, pectin and sodium alginate.
The polymer component is preferably dextran.
The drug layer preferably further contains at least one immunostimulatory agent.
The vaccine antigen is preferably at least one selected from the group consisting of peptide antigens, protein antigens and nucleic acids.
The present invention will be described in detail below.

The oral mucosal adhesion type vaccine preparation of the present invention has a drug layer containing a vaccine antigen and a drug cover layer, and the thickness of the drug cover layer is 15 μm or more, and the above-mentioned is applied to distilled water at 37 ° C. The disintegration property (A) of the drug layer and the disintegration property (B) of the drug cover layer have a relationship of (A)> (B). Furthermore, the drug cover layer, a test piece of the drug cover layer area 20 cm ² Thickness 50 [mu] m, were immersed in distilled water 500mL of 37 ° C., the residual rate after 7 minutes from start of immersion (C) is 50% or more It is 90% or less.
With the above configuration, in the oral mucosal adhesion type vaccine preparation of the present invention, the drug layer is disintegrated by saliva on the mucosa immediately after being placed in the oral cavity. On the other hand, since the drug cover layer does not disintegrate for a certain period of time, a high-concentration drug solution can be stably retained on the mucous membrane for a certain period of time.

That is, in the oral mucosal adhesion type vaccine preparation of the present invention, the disintegration (B) of the drug cover layer is smaller than the disintegration (A) of the drug layer, so that the mucous membrane to which the vaccine preparation of the present invention is applied. When the drug layer is disintegrated by the above saliva and a high-concentration drug solution is formed, the drug cover layer surely prevents the oral saliva from being mixed with the drug solution in an amount larger than necessary. Therefore, it is possible to prevent the drug solution from being diluted by saliva for a certain period of time, and to retain a high concentration of the drug solution on the mucous membrane. Therefore, even in the oral route, the drug can be reliably administered without being affected by saliva. Furthermore, since it is not affected by saliva, it is possible to obtain a pharmacological effect according to the drug dose even when it is administered orally.
If the disintegrating (A) and (B) relationships between the drug layer and the drug cover layer deviate from the specific relationship, the drug layer is disrupted by saliva on the mucous membrane to which the preparation is attached. The drug cover layer is rapidly destroyed by saliva in the oral cavity, and a high-concentration drug solution cannot be adhered to the mucous membrane.

Further, when the residual rate (C) of the drug cover layer in a specific size is 50% or more and the thickness of the drug cover layer in the oral mucosal adhesion type vaccine preparation is 15 μm or more, for example, the oral cavity. The shape of the drug cover layer is retained for 2 minutes or longer in the internal environment.
Here, in order to efficiently obtain an immunoinducing effect as a vaccine preparation, a drug solution (also referred to as a vaccine antigen solution) formed by disintegrating the drug layer is prepared at a high concentration for a certain period of time (2 minutes or more). It is necessary to be exposed on the mucous membrane. Therefore, if saliva of a certain amount (saliva existing on the mucosa serving as the administration site) or more is mixed in the drug layer of the oral mucosa-adhering vaccine preparation administered into the oral cavity within a certain period of time (2 minutes), It causes a decrease in the concentration of the drug solution, and there is a possibility that immunity induction may not be performed efficiently. In addition, there is a possibility that the pharmacological effect according to the amount of the administered drug cannot be surely obtained.
That is, when the residual rate (C) of the drug cover layer is lower than 50%, the time until the drug cover layer is disintegrated by saliva in the oral cavity becomes shorter than a certain time (2 minutes), and the concentration is high. The drug solution cannot be retained on the mucous membrane for a certain period of time, and there is a possibility that the immunoinducing effect cannot be sufficiently obtained.
Note the residual rate (C) is to prepare a test piece of area 20 cm ² Thickness 50μm using a composition that constitutes the drug cover layer, and weighed (mg) of the test piece, 37 ° C. distilled water It is immersed in 500 mL, and the residual weight (mg) 7 minutes after the start of immersion is expressed in 100% with respect to the weight of the test piece before immersion.

Further, in medical institutions, as a response to a drug-induced anaphylactic reaction, an observation time that requires observation by a doctor after administration of the drug is set. The observation time may vary depending on the type of vaccine and the vaccine recipient, but is usually 30 minutes.
In intraoral adhering vaccine formulations of the present invention, the drug cover layer, a test piece of the drug cover layer area 20 cm ² Thickness 50 [mu] m, it was immersed in distilled water 500mL of 37 ° C., the residual from the start of immersion after 7 minutes When the rate (C) is 90% or less and the thickness of the drug cover layer in the oral mucosal adhesion type vaccine preparation is 15 μm or more, the drug cover layer is disintegrated in, for example, about 30 minutes in the oral environment. Can be configured to As described above, since an observation time is required after the administration of the vaccine, the oral mucosa-adhering vaccine preparation that disintegrates at a desired time contributes to reducing the burden of time management of medical institution personnel. In addition, it can also contribute to reducing the time management burden of the administration subject himself / herself. The residual rate (C) may be 50% or more and 90% or less.
The residual rate (C) is preferably 50% or more and 90% or less, and is preferably determined according to the observation time required after administration of the preparation.

The residual ratio of the drug cover layer to 500 mL of distilled water at 37 ° C. and the residual ratio to saliva in the oral cavity are strictly different because their components are different, but 99.5% of saliva is water. Therefore, the residual rate with respect to distilled water at 37 ° C. is considered to exhibit the same characteristics as the residual rate with respect to saliva in the human oral cavity, for example.

In addition, the thickness of the drug cover layer in the oral mucosa-adhering vaccine preparation of the present invention may be 15 μm or more, preferably 20 μm or more.
The thickness of the drug cover layer in the oral mucosa-adhering vaccine preparation is preferably 30 μm or less, and preferably 25 μm or less.
When the thickness of the drug cover layer in oral mucoadhesive vaccine formulation is 15μm or more, the test piece of the drug cover layer area 20 cm ² Thickness 50 [mu] m, it was immersed in distilled water 500mL of 37 ° C., from start of immersion 7 This is because the shape of the drug cover layer having a residual rate (C) of 50% or more and 90% or less after minutes is retained for 2 minutes or more in the oral environment.

The method for measuring the residual rate (C) of the drug cover layer is as follows.
(Measuring method of residual rate (C) of drug cover layer)
1. 1. 500 mL of distilled water was placed in the vessel of the dissolution tester and warmed to 37 ° C.
2. 2. After measuring the weight of the watch glass (6 cmφ),
3. 3. A drug cover layer having an area of 20 cm ² and a thickness of 50 μm was fixed to a watch glass with double-sided tape.
The drug cover layer fixed to the watch glass was put into the paddle while rotating at a speed of 4.50 rpm, and was taken out after 7 minutes.
5. After the taken-out drug cover layer was dried at 100 ° C. for 1 hour, the weight was measured, the ratio to the weight of the drug cover layer before the test was calculated, and the value was taken as the residual ratio (C).

The drug layer in the oral mucosal adhesion type vaccine preparation of the present invention preferably has a rapid disintegration property with respect to saliva on the mucosa to which the preparation is attached. In the present specification, the concentration of the drug eluted in the distilled water after 30 seconds after adding the drug layer for one dose to 1.0 mL of distilled water at 37 ° C and stirring with a stirrer is distillation at 37 ° C. When the concentration of the drug dissolved in distilled water is 80% or more when the entire amount of the drug for one dose is dissolved in 1 mL of water, the drug layer has a rapid disintegration property with respect to saliva on the mucous membrane. Suppose you have. The specific measurement method will be described in detail in Examples.
The drug layer in the present invention preferably has a mass of 5 to 500 mg per dose from the viewpoint of ease of administration, handleability and immune induction.

The drug layer in the oral mucosa-adhering vaccine preparation of the present invention is preferably at least one selected from the group containing peptide antigens, protein antigens and nucleic acids as vaccine antigens. The oral mucosa is generally considered to be difficult to immunize, and even if it can induce immune tolerance against these antigens, it is difficult to activate immunity. However, the oral mucosa of the present invention is considered to be difficult to activate. Since the adherent vaccine preparation can hold the drug solution on the oral mucosa at a high concentration for a certain period of time, the mucosa is stably maintained without being diluted by saliva even when administered to the oral mucosa. A high concentration drug solution can be exposed on the surface, and a systemic immune response and a mucosal immune response can be effectively induced against the vaccine antigen. Among them, a vaccine antigen that can induce anaphylactic shock and can be suitably used for a vaccine antigen that requires time management after vaccine administration.

The vaccine antigen is not particularly limited as long as it is used as a vaccine antigen in the technical field to which the present invention belongs, but it is preferably at least one selected from the group consisting of peptide antigens, protein antigens and nucleic acids. .. In addition, for the purpose of preventing diseases, it is necessary to form an antibody in the patient's body in advance by administering a preparation, so it is preferable to use an infectious disease pathogen-derived antigen as the vaccine antigen, for example, infectious. Examples include pathogen and infectious pathogen-derived proteins, human endogenous-derived proteins, and the like.

The disease caused by the infectious agent of the above-mentioned infectious agent-derived antigen is not particularly limited, and is, for example, adenovirus, herpesvirus (for example, HSV-I, HSV-II, CMV, or VZV), poxvirus (for example, for example). Orthopox virus such as psoriasis or vaccinia, or infectious ligamentoma), picornavirus (eg, rhinovirus or enterovirus), paramixovirus (eg, parainfluenza virus, mumps virus, goblin virus, respiratory organs) Synthetic virus (RSV)), coronavirus (eg, SARS), papovavirus (eg, human papilloma (papilloma) virus such as genital tract, vulgaris vulgaris, or those that cause sole swelling), hepadna Viral diseases such as diseases caused by viral infections such as viruses (eg, hepatitis B virus), flaviviruses (eg, hepatitis C virus or dengue virus), or retroviruses (eg, lentiviruses such as HIV), Escherichia, entero Bacter, salmonella, staphylococcus, diarrhea, listeria, aerobacter, helicobacter, clebusiera, proteus, pseudomonas, streptococcus, chlamydia, mycoplasma, pneumococcus, nyseria, crostrium, bacillus, corynebacterium, mycobacterium, campylobacter, vibrio , Seratia, Providenceia, Chromobacterium, Brucella, Elsina, Hemophilus, or Bacterial diseases such as those caused by bacterial infections such as Bordetella, Chlamydia, Candidosis, Aspergillosis, Histoplasmosis, Cryptocox meningitis, etc. However, the present invention includes, but is not limited to, fungal diseases, malaria, pneumocystiscarini pneumonia, Leishmaniasis, cryptospolidium disease, toxoplasmosis, and tripanosoma infection.

In addition, the infectious disease pathogen-derived antigen may be an influenza virus-derived antigen.
Here, the influenza virus is an RNA enveloped virus belonging to the Orthomyxoviridae family and having a particle size of about 100 nm in diameter, and is classified into A, B and C types based on the antigenicity of an internal protein.
The influenza virus comprises a core of ribonucleic acid (RNA) associated with an internal nucleocapsid or nucleoprotein surrounded by a viral envelope having a lipid bilayer structure, and an external glycoprotein. The inner layer of the viral envelope is mainly composed of matrix proteins, and the outer layer is mostly composed of host-derived lipid substances.
In addition, the RNA of the influenza virus has a segmental structure. Influenza A that is prevalent all over the world is mainly caused by influenza A virus, which has two types of enveloped glycoproteins, hemagglutinin (HA) and neuraminidase (NA). It is classified into 16 subtypes in HA and 9 subtypes in NA according to the difference in antigenicity.
In the present invention, influenza A and influenza B virus-derived antigens are preferably used as the infectious disease pathogen-derived antigens. The subtypes of the above-mentioned influenza A and B viruses are not particularly limited, and may be a subtype isolated so far or a subtype isolated in the future. The influenza virus used for the influenza vaccine antigen preferably contains two or more types of influenza vaccine antigens, one or more types A and one or more types B. Among them, H1N1 type and H3N2 type are preferable as the influenza A vaccine antigen, and Yamagata strain and Victoria strain are preferable as the influenza B vaccine antigen.

In the present invention, the influenza virus-derived antigen is not particularly limited as long as it is at least a part of the various components constituting the influenza virus. For example, a live virus or purified virus particles are an organic solvent / surface activity. Examples thereof include whole virus particles inactivated by an agent or other reagent, or virus subunits prepared by removing impurities from the whole virus particles and purifying HA and / or NA. From the viewpoint of immunogenicity, HA subunit or whole virus particles are preferable. It is more preferable that the whole virus particles are inactivated by formalin or the like. It is also particularly effective for HA subunits (splits), which have few impurities and require adjuvants such as immunostimulators.
The method for preparing the influenza virus-derived antigen is not particularly limited, and known methods can be used without limitation. For example, a method in which an influenza virus strain isolated from an influenza-infected animal or an influenza patient is infected with chicken eggs or the like, cultured by a conventional method, and an antigen is prepared from a purified virus stock solution. Further, a virus-derived antigen prepared in cultured cells by genetic engineering may be used.

In the oral mucosa-adhering vaccine preparation of the present invention, the drug layer may contain an effective amount of the vaccine antigen. For example, the total amount of the vaccine antigen is 0.001 μg or more in the drug layer. It is preferably contained in the range of 1.0 mg. If it is less than 0.001 μg, its function as a preventive or therapeutic agent for infectious diseases may be insufficient, and if it exceeds 1.0 mg, it may pose a safety problem. The more preferable lower limit of the antigen content is 0.01 μg, the further preferable lower limit is 0.1 μg, the more preferable upper limit is 500 μg, and the further preferable upper limit is 100 μg.
When the vaccine antigen is an antigen other than the influenza virus-derived antigen, it is relatively difficult to induce immunity by oral administration as compared with the influenza virus-derived antigen depending on the type, and the amount of the vaccine antigen is large. Needs consideration.
The "mass of vaccine antigen" referred to in the present specification is the total mass of all vaccine antigen proteins contained in the drug layer, unless otherwise specified. Therefore, when the antigen is a biological substance such as a virus, it means the mass of all proteins contained in the antigen. When it contains a plurality of types of antigens, it means the total amount thereof.

The effect of inducing the systemic immune response and the mucosal immune response can be measured by an immune induction experiment using a model animal for immune evaluation and the ELISA method (antigen-specific IgG and IgA antibody).
Samples for measuring the immune-inducing effect are blood and nasal lavage fluid of model animals for immunological evaluation.

The drug layer in the oral mucosa-adhering vaccine preparation of the present invention preferably has a solution viscosity (D) of 20 mPa · s or less when dissolved in 1.0 mL of distilled water at 37 ° C. for a single dose. .. When the oral mucosa-adhering vaccine preparation of the present invention is applied into the oral cavity, the drug layer is dissolved by saliva on the mucosa to form a drug solution. However, since the solution viscosity is as low as 20 mPa · s or less, the drug solution. The diffusivity of the vaccine antigen in the vaccine is improved, and the immune response and the mucosal immune response can be induced more effectively. It is considered that the exposure of the vaccine antigen to the mucosa is promoted by retaining the drug solution as a low-viscosity solution in the oral mucosa.
The drug layer in the present invention preferably has a mass of 5 to 500 mg per dose from the viewpoint of ease of administration, handleability and immune induction.

Since human saliva secretes about 0.8 to 1.2 mL per minute, in the present invention, the solution obtained by dissolving in 1.0 mL of purified water is used as an intraoral viscosity measurement model. did.
The solution viscosity for distilled water at 37 ° C and the solution viscosity for saliva in the oral cavity are strictly different because their components are different, but since 99.5% of saliva is water, it is relative to distilled water at 37 ° C. The solution viscosity is considered to exhibit the same characteristics as the solution viscosity for saliva in the human oral cavity, for example.
The solution viscosity can be measured by a commonly used method and apparatus. Specifically, for example, the solution viscosity at 37 ° C. can be measured using a viscosity measuring device (RheoStress 600, manufactured by THEROMO HAAKE).

The drug layer preferably contains a polymer component, and the polymer component is preferably a water-soluble edible polymer. By making the drug layer water-soluble, when the oral mucosa-adhering vaccine preparation is placed in the oral cavity, the drug layer is dissolved by saliva on the mucosa, and a high-concentration drug solution can be adhered to the mucosa. Because it can be done.
The water-soluble edible polymer is not particularly limited, but specifically, for example, polyvinyl alcohol, carboxyvinyl polymer, hydroxypropylmethyl cellulose (hereinafter referred to as “HPMC”), hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, low starch. Degree of Substitution Synthetic polymers such as hydroxypropyl cellulose, crystalline cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl starch, sodium alginate, dextran, casein, gelatin, purulan, pectin, guar gum, xanthan gum, tragancant Examples thereof include natural polymers such as gum, acacia gum, arabic gum and starch, polysaccharides and derivatives thereof.

Further, it is more preferable that the drug layer contains at least one selected from the group consisting of dextran, pectin and sodium alginate as a polymer component. This is because the inclusion of such a component can improve the disintegration property of the drug layer with respect to distilled water at 37 ° C. The content of the specific polymer component may be appropriately blended so that the drug layer in the oral mucosa-adhering vaccine preparation of the present invention has a desired disintegration property, which is within the knowledge of those skilled in the art. ..
Furthermore, by containing the above-mentioned specific polymer component, the viscosity of the solution when the drug layer is dissolved in saliva (distilled water at 37 ° C.) is lowered, which is suitable for administration of a vaccine preparation through the oral mucosal route.

The dextran is generally a partial decomposition of a polysaccharide produced by fermentation of sucrose by Leuconostoc mesenteroides Van Thieghem (Lactobacillaceae), and is mainly composed of D-glucose. The above dextran can be used without any problem if the mass average molecular weight is 10,000 or more, more specifically 10,000 to 100,000, but it is listed in the pharmaceutical additive standard from the viewpoint that the present invention is a preparation. Dextran 40 (average molecular weight 40,000) or dextran 70 (mass average molecular weight 70,000) is preferably used.

When a lyophilized preparation is used as the drug layer, the content of the dextran is preferably a total amount based on the total mass of the vaccine-containing preparation solution immediately before drying in the process of producing the lyophilized preparation. Is 1.0 to 35.0 parts by mass, more preferably 2.5 to 22.5 parts by mass. If it is less than 1.0 part by mass, a sufficient dosage form as a pharmaceutical product may not be formed after drying, while if it exceeds 35.0 parts by mass, it is not uniformly dispersed or dissolved in the production solution, and the product is produced. It may be a problem.

The pectin is a high molecular weight polysaccharide generally obtained by extracting from citrus fruits or apples with water, and is composed of galacturonic acid and its methyl ester.
The mass average molecular weight of the pectin is about 30,000 to 100,000. It is divided into LM pectin and HM pectin according to the rate of methyl esterification. Of the total galacturonic acid, those in which methylated galacturonic acid accounts for more than 50% are called HM pectin, and those in which 50% or less are called LM pectin. Either pectin may be used in the present invention, but since LM pectin forms a heat irreversible gel in the presence of calcium ions, it is necessary to consider the combined use with an organic acid salt containing calcium ions.

When a lyophilized preparation is used as the drug layer, the content of the LM pectin is a total amount based on the total mass of the vaccine-containing preparation solution immediately before drying in the process of producing the lyophilized preparation. It is preferably 0.5 to 15.0 parts by mass, and more preferably 1.0 to 10.0 parts by mass. If it is less than 0.5 parts by mass, a sufficient dosage form as a pharmaceutical product may not be formed after drying, while if it exceeds 15.0 parts by mass, immunity may not be sufficiently induced.

When a lyophilized preparation is used as the drug layer, the content of the HM pectin is a total amount based on the total mass of the vaccine-containing preparation solution immediately before drying in the process of producing the lyophilized preparation. It is preferably 0.25 to 7.50 parts by mass. If it is less than 0.25 parts by mass, a sufficient dosage form as a pharmaceutical product may not be formed after drying, while if it exceeds 7.50 parts by mass, immunity may not be sufficiently induced.

When a lyophilized preparation is used as the drug layer, the content of the sodium alginate is a total amount based on the total mass of the vaccine-containing preparation solution immediately before drying in the process of producing the lyophilized preparation. It is preferably 0.1 to 15.0 parts by mass, and more preferably 0.2 to 10.0 parts by mass. If it is less than 0.1 parts by mass, a sufficient dosage form as a pharmaceutical product may not be formed after drying, while if it exceeds 15.0 parts by mass, immunity may not be sufficiently induced.

The polymer component in the drug layer of the oral mucosa-adhering vaccine preparation of the present invention preferably contains dextran. This is because it is highly disintegrating with respect to a small amount of saliva (for example, 1 mL) adhering to the mucous membrane, and the solution viscosity of the drug solution formed by dissolving the drug layer is low.

The drug layer in the oral mucosal adhesion type vaccine preparation of the present invention has the effect of the present invention in addition to the above-mentioned specific polymer component (at least one selected from the group consisting of dextran, pectin and sodium alginate). As long as it does not inhibit, an appropriate amount of edible polymer soluble in water and / or an organic solvent can be used in combination.
The blending amount of the other edible polymer is preferably 0.1 to 10.0 parts by mass based on the total mass of the drug layer in the oral mucosa-adhering vaccine preparation of the present invention.

The drug layer preferably contains at least one immunostimulatory agent (adjuvant) in addition to the vaccine antigen. The above-mentioned immunostimulatory agent is administered together with an antigen and has an effect of enhancing the effect of a vaccine by activating the entire immunity, but in the present invention, it is not particularly limited as long as it has an immunostimulatory effect. As an example, it contains a TOLL-like receptor (TLR) agonist (eg, a TLR2 / 6 agonist, a TLR4 agonist) and at least one selected from the group consisting of cyclic dinucleotides or salts or derivatives thereof.

The TLR2 / 6 agonist may be an extract from a mycoplasma cell membrane or a variant thereof, or a synthetic product.
Examples of the mycoplasma, for example, Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma gallisepticum, Mycoplasma hyopneumoniae, Mycoplasma laboratorium, Mycoplasma mycoides, Mycoplasma ovipneumoniae and the like.

Moreover, as the TLR4 agonist, lipopolysaccharide or a salt thereof is preferable. The lipopolysaccharide referred to in the present specification is a concept including lipopolysaccharide itself and its derivatives or variants as long as it has its properties. The salt referred to herein may be any organic or inorganic acid salt, but is preferably a pharmaceutically acceptable salt.
In addition, the lipopolysaccharide may be an extract from the cell wall of Gram-negative bacteria or a variant thereof, or may be a synthetic product.
Examples of the gram-negative bacteria include the genus Acetobacter, the genus Achromobacter, the genus Acidicaldus, the genus Acidiphilium, the genus Acidisphaera, the genus Acidocola, the genus Acidomonas, the genus Aglobacterium, the genus Asia, the genus Bacillus, and the genus Bacillus. , Clostridium spp., Craurococcus spp., Chlamydia spp., Enterobacter spp., Escherichia spp., Flavobacterium sp., Francisella sp., Gluconacetobacter genus, Gluconobacter spp., Haemophilus spp., Kozakia spp., Klebsiella spp., Leahibacter genus, Leclercia spp., Legionella spp., Methanoculleus genus, Methanosarcina spp., Micrococcus sp., Muricoccus spp., Neisseria spp., Neoasaia genus, Oleomonas spp., Pantoea spp., Plesiomonas spp., Paracraurococcus spp., Pseudomonas spp., Prophyromonas spp, Proteus spp., Rahnella genus, Rhodopila genus, Roseococcus genus, Rubritepida spp., Salmonella spp., The genus Shigella, the genus Stenortophomonas, the genus Saccharibacter, the genus Serratia, the genus Stella, the genus Swamithania, the genus Vibrio, the genus Vparahaemolyticus, the genus Teichococcus, the genus Teichococcus, the genus Xanasomas. The gram-negative bacteria are preferably of the genus Escherichia, the genus Shigella, the genus Salmonella, the genus Klebsiella, the genus Proteus, the genus Yersinia, the genus Vibrio, the genus Vparahaemoryticus, the genus Haemophilius, the genus Pseudomella, the genus Pseudomella , Bacteroides, Neisseria, Chlamydia, Plesiomonas, Prophyromonas, Pantoea, Agrobacterium, Stenortophomonas, Enterobacter, Acetomona, etc.
Among them, those derived from the genus Escherichia, the genus Salmonella, the genus Pantoea, the genus Acetobacter, the genus Zymomonas, the genus Xanthomonas, or the genus Enterobacter are preferable. These have been contained in many foods and Chinese herbs since ancient times, and their safety to the living body is guaranteed. In particular, those derived from the genus Pantoea are currently used as health foods and can be said to be more effective. It is also possible to use the extracts derived from these bacteria or their variants as they are.

When the extract from the cell wall of Gram-negative bacteria or the purified lipopolysaccharide is used as the lipopolysaccharide, it is generally necessary to consider the safety to the living body, and a detoxified variant thereof. Can also be used as. On the other hand, Acetobacter genus (Acetobacter aceti, Acetobacter xylinum, Acetobacter orientalis, etc.), Zymomonas genus (Zymomonas mobilis, etc.), Xanthomonas genus (Xanthomonas campestris, etc.), Enterobacter spp (Enterobacter cloacae, etc.), Pantoea sp (Pantoea agglomerans, etc.), ancient It is contained in more foods and Chinese herbs, and its safety to the living body is guaranteed. Extracts derived from these bacteria or purified lipopolysaccharide can be used as they are.

Examples of the lipopolysaccharide derivative include a derivative from which the polysaccharide portion has been removed, specifically, lipid A, monophosphoryl lipid A, 3-de-acylated monophosphoryl lipid A (3D-MPL), and glucopyra. Glucopyranosyl lipid (GLA), alkyl glucosaminide phosphate (AGP) and the like can be mentioned.
As Lipid A from which the polysaccharide portion of the lipopolysaccharide has been removed, an isolate derived from the Gram-negative bacteria or a product synthesized so as to have the same structure as the isolate derived from these Gram-negative bacteria may be used. ..
Further, as the variant of Lipid A, dephosphorylated monophosphoryl lipid or a salt thereof is also preferably used. The derivative of monophosphoryl lipid referred to in the present specification can be used in the present invention as long as it has the properties of monophosphoryl lipid. In particular, 3-de-acylated monophosphoryl lipid A (3D-MPL), which has already been proven as an immunostimulator in medical applications, or deacylation proposed in US Patent Application Publication No. 2010/0310602. Glucopyranosyl lipid, which has not been used, is preferable from the viewpoint of safety to the living body.
Further, as the monophosphoryl lipid, those derived from Salmonella, which have safety and precedent for use, are also preferably used.
The alkyl glucosaminide phosphate (AGP) is disclosed in International Publication No. 9850399 or US Pat. No. 6,303,347 (which also discloses a method for preparing AGP), and AGP. Pharmaceutically acceptable salts, such as those disclosed in US Pat. No. 6,764,840, are also preferably used.
As the cyclic dinucleotide, a cyclic dipurine nucleotide is preferable, and a salt or derivative thereof may be used as long as it has the characteristics thereof. As the cyclic di-purine nucleotide, for example, from the viewpoint of safety, c-di-GMP which is a cyclic diguanosine monophosphate and c-di-AMP which is a cyclic di-adenosine monophosphate are preferably used.

The amount of the immunostimulatory agent in the drug layer is preferably in the range of 0.01 μg to 100 mg with respect to the total amount of the drug layer, for example, for once administration to one individual. If it is less than 0.01 μg, a sufficient function as a preventive or therapeutic agent for infectious diseases may not be obtained, and if it exceeds 100 mg, a safety problem may occur. The more preferable lower limit of the content of the immunostimulatory agent is 0.03 μg, and the more preferable upper limit is 50 mg.

The drug cover layer preferably covers the sides and the upper part of the drug layer and is laminated on the drug layer. Further, the drug cover layer preferably has an outer peripheral edge outside the outer peripheral portion of the drug layer. With such a configuration, when the oral mucosa-adhering vaccine preparation of the present invention is applied into the oral cavity, the adhesion to the oral mucosa is enhanced and the vaccine preparation is more reliably retained in the oral cavity. is there. In addition, with such a configuration, it is possible to more reliably prevent saliva in the oral cavity from coming into contact with the drug layer in excess of the required amount, and to maintain a high concentration of the drug solution when the drug layer is dissolved. Because.
The width of the outer peripheral edge of the drug cover layer is preferably 1 mm or more, and more preferably 2 mm or more. Further, the width of the outer peripheral edge of the drug cover layer is preferably 5 mm or less, and more preferably 4 mm or less.

The drug cover layer does not contain the vaccine antigen contained in the drug layer. Therefore, the oral mucosa-adhering vaccine preparation of the present invention is different from the mucosal-attached preparation having two or more drug layers such as a fast-dissolving drug layer and a long-acting drug layer.

The drug cover layer contains at least one selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxypropylmethylcellulose phthalate (HPMCP), carboxymethylethylcellulose (CMEC) and ethylcellulose (EC) as the water-soluble polymer. Is preferable.
Such water-soluble drug cover layer containing a polymer is easy to adjust the solubility 37 ° C. distilled water, the specimen of the drug cover layer area 20 cm ² Thickness 50μm is immersed in distilled water 500mL of 37 ° C., This is because the residual rate (C) 7 minutes after the start of immersion can be easily adjusted to 50% or more and 90% or less.
In particular, the drug cover layer preferably contains a combination of HPMCP and HPMC, a combination of HPMC and CMEC, a combination of EC and HPMC, or CMEC alone, and among them, a combination of HPMCP and HPMC or HPMC. And CMEC are more preferably included.

The blending amount of hydroxypropylmethylcellulose phthalate (HPMCP) and hydroxypropylmethylcellulose (HPMC) in the cover layer is preferably 60:40 to 87:13.
The blending amount of hydroxypropylmethyl cellulose (HPMC) and carboxymethyl ethyl cellulose (CMEC) in the drug cover layer is preferably 50:50 to 0: 100.
The blending amount of ethyl cellulose (EC) and HPMC in the drug cover layer is preferably 40:60 to 50:50.

The drug cover layer is preferably laminated with the drug layer via a mucosal adhesion layer.
FIG. 1 is a cross-sectional view showing one form of the oral mucosal adhesion type vaccine preparation of the present invention, which has a drug layer 1, a mucosal adhesion layer 2, and a drug cover layer 3.
The mucosal adhesion layer is preferably a water-soluble polymer. This is because the vaccine preparation can be completely disintegrated within the observation time after administration of the vaccine preparation of the present invention.
As the water-soluble polymer used for the mucosal adhesion layer, celluloses such as hydroxypropyl cellulose (HPC), hypromellose (HPMC), hydroxyethyl cellulose (HEC), carmellose, carmellose sodium, carboxyvinyl polymer, polyvinylpyrrolidone, polyvinyl alcohol and the like. Contains at least one selected from the group consisting of polymers such as polymers, sodium alginate, pullulan, guar gum, glucono-δ-lactone, pectin, dextrin, tamarind gum, xanthan gum and other polysaccharides, gum arabic powder, and tragant powder. Is preferable. This is because it has adhesion to the mucous membrane and water solubility.
The mucosal adhesion layer preferably has an outer peripheral edge on the outer side of the outer peripheral portion of the drug layer, similarly to the drug cover layer. With such a configuration, when the oral mucosa-adhering vaccine preparation of the present invention is applied into the oral cavity, the adhesion to the oral mucosa is further enhanced, and the retention in the oral cavity is more ensured. Can be done. The width of the outer peripheral edge of the mucosal adhesion layer is preferably 1 mm or more, and more preferably 2 mm or more. Further, the width of the outer peripheral edge of the mucosal adhesion layer is preferably 5 mm or less, and more preferably 4 mm or less.

The drug layer in the oral mucosa-adhering vaccine preparation of the present invention is preferably in the form of a dry preparation.
In the present specification, the dry preparation means that the water content is 15 parts by mass or less. Among the above-mentioned dry preparations, those having a water content of 10 parts by mass or less are particularly called low water content dry preparations.
The "moisture content" referred to here is determined in accordance with the 16th revised Japanese Pharmacopoeia, general test method, and dry weight loss test method (hereinafter, also simply referred to as "dry weight loss method"). That is, the water content is determined by the rate of decrease in mass when the test piece of the drug layer in the present invention in the form of a dry preparation is heated at 105 ° C. for 3 hours.

In the oral mucosa-adhering vaccine preparation of the present invention, the container used is not particularly limited, but a sealable container is preferable, and examples thereof include aluminum packaging materials, blister containers, and freeze-drying vials.

The oral mucosa-adhering vaccine preparation of the present invention is administered to the oral mucosa of humans or animals (mammalia, birds, etc.), and is supragmatic, sublingual, buccal, pharyngeal mucosa, and gingival mucosa. Administration to. The administration site can be selected according to the type of drug contained in the oral mucosa-adhering vaccine preparation of the present invention. For example, when the purpose is to induce strong immunity, sublingual administration is preferable.
It should be noted that the oral mucosa is generally considered to be difficult to immunize, and even if it can induce these immune tolerances, it is difficult to activate immunity. However, the oral mucosa-attached type of the present invention has been considered. Vaccine preparations can retain a high concentration of a drug solution containing at least one antigen on the oral mucosa, so that even when administered to the oral mucosa, the systemic immune response and mucosa are effectively maintained. It can induce an immune response.

The oral mucosa-adhering vaccine preparation of the present invention may have an appropriate shape depending on the administration site of the oral mucosa. For example, as shown in FIG. 2, the diameter φ or one side x is 10. It can be approximately circular or approximately square with a diameter of ~ 15 mm.

The oral mucosal adhesion type vaccine preparation of the present invention has excellent compliance, for example, non-invasive administration, painlessness, relief from fear of injection, and easy administration, so that the patient can administer it by himself / herself, and the needle stick of a medical worker. The risk of infection accidents can be avoided, the frequency of hospital visits for repeated administration can be reduced, which can contribute to improving the quality of life of patients, and has the advantage of not generating medical waste that requires special disposal such as injection needles. Has.
Further, in the oral mucosal adhesion type vaccine preparation of the present invention, the drug layer is dissolved by saliva on the mucosa and a high concentration drug solution is retained in the oral cavity for a certain period of time, so that the vaccine is effectively vaccined by the oral mucosal route. The antigen can be administered, the pharmacological effect on the amount of the vaccine antigen can be easily predicted, and the type and amount of the vaccine antigen can be appropriately adjusted according to the purpose.
Furthermore, the oral mucosa-adhering vaccine preparation containing the vaccine antigen in the drug layer has an advantage that strong immunity can be induced as compared with injection administration.
Furthermore, since the oral mucosa-adhering vaccine preparation of the present invention can be controlled so as to be completely dissolved within a predetermined time, it is easy to control the time after administration, and in particular, administration of a drug that can induce anaphylactic shock. Can be suitably used for.

It is sectional drawing which shows one aspect of the oral mucosal adhesion type vaccine preparation of this invention. It is a bottom schematic which shows one aspect of the oral mucosa-adhesive vaccine preparation of this invention. It is a graph which shows the effect of the antigen-specific IgG antibody induction of the oral adhesion type vaccine preparation which concerns on Example 1-2 and Comparative Example 1-3. It is a graph which shows the effect of the antigen-specific IgA antibody induction of the drug layer which concerns on Experimental Examples 12 to 15 and Comparative Experimental Examples 14 and 15. It is a graph which shows the effect of the antigen-specific IgG antibody induction of the drug layer which concerns on Experimental Examples 12 to 15 and Comparative Experimental Examples 14 and 15. It is a graph which shows the effect of the antigen-specific IgA antibody induction of the drug layer which concerns on Experimental Examples 16-18 and Comparative Experimental Examples 16 and 17. It is a graph which shows the effect of the antigen-specific IgG antibody induction of the drug layer which concerns on Experimental Examples 16-18 and Comparative Experimental Examples 16 and 17. It is a graph which shows the effect of the antigen-specific IgA antibody induction of the drug layer which concerns on Experimental Examples 19-21 and Comparative Experimental Examples 18 and 19. It is a graph which shows the effect of the antigen-specific IgG antibody induction of the drug layer which concerns on Experimental Examples 19-21 and Comparative Experimental Examples 18 and 19. It is a graph which shows the effect of the antigen-specific IgA antibody induction of the drug layer which concerns on Experimental Examples 22-24 and Comparative Experimental Examples 20 and 21. It is a graph which shows the effect of the antigen-specific IgG antibody induction of the drug layer which concerns on Experimental Examples 22-24 and Comparative Experimental Examples 20 and 21. It is a graph which shows the effect of the antigen-specific IgA antibody induction of the drug layer which concerns on Experimental Examples 25-32. It is a graph which shows the effect of the antigen-specific IgG antibody induction of the drug layer which concerns on Experimental Examples 25-32.

The present invention will be specifically described with reference to the following Examples and Experimental Examples, but the present invention is not limited to these Examples and Experimental Examples.

(Experimental Examples 1 to 4, Comparative Experimental Examples 1 to 3)
A drug layer having the composition shown in Table 1 below was prepared. Specifically, the polymer components and distilled water in the amounts shown in Table 1 below were added to the container, and the mixture was appropriately stirred and dissolved. Next, 50 parts by weight of a 1.0 mg / mL_OVA solution (OVA: egg white albumin protein, manufactured by Sigma-Aldrich) was added to the obtained solution as a vaccine antigen, and the mixture was thoroughly mixed with stirring. Further, 5 parts by weight of 1.0 mg / mL adjuvant solution (ND002, lipopolysaccharide derived from Pantoea agglomeras, manufactured by Nitto Denko KK) was added to the obtained solution and mixed thoroughly to obtain a vaccine-containing preparation solution.
1.0 g of the obtained vaccine-containing preparation solution was dispensed into an aluminum packaging material and freeze-dried to obtain a drug layer. The water content of the obtained drug layer was 5 parts by mass or less.

The polymers listed in Table 1 are as follows.
Hydroxypropyl Cellulose / HPC-L: Hydroxypropyl Methyl Cellulose manufactured by Nippon Soda Co., Ltd./HPMC (5R): Dextran manufactured by Shin-Etsu Chemical Co., Ltd./(Mass average molecular weight 70000): LM Pectin manufactured by Meishu Sangyo Co., Ltd./(LM-102AS-J) : CP Kelco Sodium Alginate / (IL-2): Kimika

(Measurement of drug layer disintegration)
Put 1 mL of distilled water in a vial and heat it in an incubator at 37 ° C. The obtained drug layer is placed in a place where the distilled water in this vial is stirred with a stirrer (300 rpm), and after 30 seconds, a filter (0.22 μm) is applied to remove undissolved components, and the solution is prepared. Recovered.
The drug concentration (mg / mL) in the solution was quantified by HPLC.
When the drug concentration was 80% or more of the concentration when the amount of the drug contained in the drug layer was completely dissolved in 1 mL of distilled water, it was determined that the drug layer had a rapid disintegration property.
The evaluation results are shown in Table 1 below.

(Experimental Examples 5-11, Comparative Experimental Examples 4-13)
A drug cover layer having the composition shown in Table 2 below was prepared. Specifically, polyethylene glycol 400 (PEG400, manufactured by Sanyo Chemical Industries, Ltd.), distilled water, and ethanol in the amounts shown in Table 2 below were added to a glass bottle and mixed thoroughly. Next, the bases in the blending amounts shown in Table 2 below were added to the obtained solution and mixed thoroughly to obtain a composition for a drug cover layer. The obtained composition for a drug cover layer was applied to one side of polyethylene terephthalate (PET) having a thickness of 75 μm so as to have a thickness of 50 μm after drying, and dried for 30 minutes to prepare a drug cover layer.

(Base used)
Hydroxypropyl methylcellulose / HPMC (5R), (5E): Shin-Etsu Chemical Co., Ltd. Carboxymethylethylcellulose / CMEC: Freund Sangyo Co., Ltd. Ethylcellulose EC (7P), (10P): Nissin Kasei Co., Ltd. Hydroxypropylmethylcellulose phthalate / HPMCP ( HP-55): Made by Shin-Etsu Chemical Co., Ltd.

(Measurement of disintegration of drug cover layer)
500 mL of distilled water was placed in a vessel of an dissolution tester (manufactured by Agilent Technologies) and warmed to 37 ° C. Next, after measuring the weight of the watch glass (6 cmφ), the obtained drug cover layer was cut into an area of 20 cm ² and a thickness of 50 um, and fixed to the watch glass with double-sided tape.
The drug cover layer fixed to the watch glass was put in while rotating the paddle at a speed of 50 rpm, and was taken out after 7 minutes.
After the taken-out drug cover layer was dried at 100 ° C. for 1 hour, the weight was measured, the ratio to the weight of the drug cover layer before the test was calculated, and the value was taken as the residual ratio of the drug cover layer. The survival rate is shown in Table 2 below.

(Preparation method of mucosal adhesion layer)
1 part by weight of polyethylene glycol 400 (PEG400, manufactured by Sanyo Chemical Industries, Ltd.) and 90 parts by weight of ethanol were added to a glass bottle and mixed sufficiently. Next, 4.5 parts by weight of hydroxypropyl cellulose (HPC-M, manufactured by Shin-Etsu Chemical Co., Ltd.) and 4.5 parts by weight of polyvinylpyrrolidone (PVP_K-90F, manufactured by Shin-Etsu Chemical Co., Ltd.) were added to the obtained solution. It was added and mixed thoroughly to obtain a composition A for a mucosal adhesion layer.
To another glass bottle, 6 parts by weight of polyethylene glycol 400 (PEG400, manufactured by Sanyo Chemical Industries, Ltd.) and 80 parts by weight of ethanol were added and mixed sufficiently. Next, 7 parts by weight of hydroxypropyl cellulose (HPC-L, manufactured by Nippon Soda Co., Ltd.) and 7 parts by weight of polyvinylpyrrolidone (PVP_K-90F, manufactured by BASF Japan Ltd.) were added to the obtained solution and mixed sufficiently. Then, the composition B for the mucosal adhesion layer was obtained.
The mucosal adhesion layer composition A was applied to one side of a polyethylene terephthalate (PET) film having a thickness of 75 μm so as to have a thickness of 20 μm after drying, and dried for 30 minutes to obtain a plurality of mucosal adhesion layer A.
Next, the composition B for the mucosal adhesion layer was applied to one side of a polyethylene terephthalate (PET) film having a thickness of 75 μm so as to have a thickness of 50 μm after drying, and dried for 30 minutes to form a plurality of mucosal adhesion layers B. I got one.
The mucosal adhesive layer A, the mucosal adhesive layer B, the mucosal adhesive layer A, the mucosal adhesive layer B, and the mucosal adhesive layer A were laminated in this order to obtain a mucosal adhesive layer.

(Method of preparing mucosal adherent drug cover layer)
The obtained drug cover layer and the mucosal adhesion layer were laminated to obtain a drug cover layer having the mucosal adhesion layer.
Next, the obtained drug cover layer having the mucosal adhesion layer was punched to 1 cm ² to obtain a mucosal adhesion drug cover layer.
Furthermore, a disintegration test of the obtained mucosal-attached drug cover layer was carried out on the panelists by the method shown below.

(Collapse test of mucosal adherent drug cover layer on humans)
The obtained mucosal-attached drug cover layer was administered under the sublingual of the panelist, and the disintegration property of the mucosal-adhered drug cover layer was visually confirmed 2 minutes and 30 minutes after the administration.
If the remaining area is less than 50%, it is judged to be collapsed. The results are shown in Table 2 below.

From the above, the drug layers according to Experimental Examples 1 to 4 in Table 1 showed rapid disintegration.
On the other hand, in the Table 2 Experimental Examples 5-11 and Comparative Experiment Examples 4-13, both area 20 cm ² drug cover layer having a thickness of 50μm was immersed in distilled water 500mL of 37 ° C. from the start of immersion after 7 minutes residual rate Since it was not 0%, it was confirmed that it had no rapid disintegration property. Therefore, the orally adherent vaccine preparation using the drug layer according to Experimental Examples 1 to 4 and the drug cover layer according to Experimental Examples 5 to 11 and Comparative Experimental Examples 4 to 13 is applied to distilled water at 37 ° C. , The disintegration of the drug layer (A)> the disintegration of the drug cover layer (B) is satisfied.
The mucosal-adhered drug cover layer having the drug cover layer of Experimental Examples 5 to 11 retains its shape 2 minutes after being administered under the sublingual of the panelist, but the mucosal-adhered drug cover layer is maintained 30 minutes later. Was collapsing. Therefore, it was confirmed that the drug cover layer of Experimental Examples 5 to 11 is a drug cover layer that does not disintegrate for a certain period of time but disintegrates within a specific time (30 minutes) after a certain period of time has elapsed.

(Examples 1 and 2, Comparative Examples 1 to 3)
An oral mucosal adhesion type vaccine preparation having the composition shown in Table 3 below was prepared. Specifically, a drug layer, a mucosal adhesion layer and a drug cover layer were prepared as follows to prepare an oral mucosal adhesion type vaccine preparation. For the oral mucosa-adhering vaccine preparations according to Examples and Comparative Examples, a size that is easy to administer to a test animal (rabbit) is selected. Therefore, the size may differ from the size described in the above test example.

(Method of preparing drug layer)
The polymer components and distilled water in the amounts shown in Table 3 below were added to the container, and the mixture was appropriately stirred and dissolved. Next, 50 parts by weight of 1.0 mg / mL_OVA solution and 5 parts by weight of 1.0 mg / mL adjuvant solution (ND002, manufactured by Nitto Denko Co., Ltd.) were added to the obtained solution as a vaccine antigen, and the mixture was thoroughly mixed to obtain a vaccine. A containing lumber solution was obtained.
About 2 μL (2 mg) of the obtained vaccine-containing preparation solution was dispensed into a container (2 mmφ) and freeze-dried to obtain a drug layer. The diameter of the obtained drug layer was 1.5 mmφ, and the water content was 10% or less of the total mass of the drug layer. In addition, the obtained drug layer contained 1.0 μg of vaccine antigen and 0.1 μg of adjuvant.

(Preparation method of mucosal adhesion layer)
1 part by weight of polyethylene glycol 400 (PEG400, manufactured by Sanyo Chemical Industries, Ltd.) and 90 parts by weight of ethanol were added to a glass bottle and mixed sufficiently. Next, 4.5 parts by weight of hydroxypropyl cellulose (HPC-M, manufactured by Shin-Etsu Chemical Co., Ltd.) and 4.5 parts by weight of polyvinylpyrrolidone (PVP_K-90F, manufactured by Shin-Etsu Chemical Co., Ltd.) were added to the obtained solution. It was added and mixed thoroughly to obtain a composition A for a mucosal adhesion layer.
To another glass bottle, 6 parts by weight of polyethylene glycol 400 (PEG400, manufactured by Sanyo Chemical Industries, Ltd.) and 80 parts by weight of ethanol were added and mixed sufficiently. Next, 7 parts by weight of hydroxypropyl cellulose (HPC-L, manufactured by Nippon Soda Co., Ltd.) and 7 parts by weight of polyvinylpyrrolidone (PVP_K-90F, manufactured by BASF Japan Ltd.) were added to the obtained solution and mixed sufficiently. Then, the composition B for the mucosal adhesion layer was obtained.
The mucosal adhesion layer composition A was applied to one side of a polyethylene terephthalate (PET) film having a thickness of 75 μm so as to have a thickness of 20 μm after drying, and dried for 30 minutes to obtain a plurality of mucosal adhesion layer A.
Next, the composition B for the mucosal adhesion layer was applied to one side of a polyethylene terephthalate (PET) film having a thickness of 75 μm so as to have a thickness of 50 μm after drying, and dried for 30 minutes to form a plurality of mucosal adhesion layers B. I got one.
The mucosal adhesive layer A, the mucosal adhesive layer B, the mucosal adhesive layer A, the mucosal adhesive layer B, and the mucosal adhesive layer A were laminated in this order to obtain a mucosal adhesive layer.

(Preparation method of drug cover layer)
Polyethylene glycol 400 (PEG400, manufactured by Sanyo Chemical Industries, Ltd.), distilled water, and ethanol in the amounts shown in Table 3 below were added to the glass bottle and mixed thoroughly. Next, the bases in the blending amounts shown in Table 3 below were added to the obtained solution and mixed thoroughly to obtain a composition for a drug cover layer. The obtained composition for a drug cover layer was applied to one side of polyethylene terephthalate (PET) having a thickness of 75 μm so as to have a thickness of 20 μm after drying, and dried for 30 minutes to prepare a drug cover layer.

(Preparation method of vaccine preparation)
The obtained drug cover layer and the mucosal adhesion layer were laminated to obtain a drug cover layer having the mucosal adhesion layer.
Next, the drug cover layer having the obtained mucosal adhesion layer was punched to a diameter of 3 mmφ, and the drug cover layer and the obtained drug layer were adhered via the mucosal adhesion layer to adhere the oral mucosa according to Examples and Comparative Examples. An adherent vaccine formulation was obtained.
Furthermore, a disintegration test and an immunoinduction test of the oral mucosa-adhering vaccine preparation according to the obtained Examples and Comparative Examples were carried out on the model animals for immune evaluation by the method shown below.
The results are shown in Table 4.

(Collapse test of oral mucosa-adhering vaccine preparation for rabbits)
The obtained orally adherent vaccine preparation was administered under the sublingual of rabbits, and the disintegration property of the drug layer and the drug cover layer was visually confirmed 2 minutes and 30 minutes after the administration.
If the remaining area is less than 50%, it is judged to be collapsed.
The disintegration results are shown in Table 4 below.

(Evaluation of mucosal immunity of oral mucosal adherent vaccine preparation for mice)
After 2 μL of distilled water was placed under the sublingual of a mouse (C57 / BL6 NCr female 7 weeks old), the obtained oral adhesion type vaccine preparation (drug layer: diameter 1.5 mmφ, drug cover layer 3 mmφ) was attached. From above, 20 μL of mouse saliva was added. One week after administration, an orally adherent vaccine preparation was similarly administered under the tongue of the mice.
One week after the second administration, mouse sera were collected and the antigen-specific IgG antibody titer in the sera was measured by the ELISA method.
The measurement results are shown in Table 4 and FIG. 3 below.

(Method for measuring antigen-specific IgG titer in mouse serum (ELISA method))
To a 96-well plate for ELISA, 100 μL of an OVA-containing solution (100 μg / mL) diluted with a carbonate buffer is added to separate plates, and the mixture is allowed to stand overnight.
The wells were washed 3 times with a pre-prepared washing solution (PBS containing Room 20), and 200 μL of a blocking solution (Block Ace, manufactured by Sumitomo Dainippon Pharma) diluted with purified water to 4 g / 100 mL was added for 2 hours. It was left at room temperature. Then, the wells were washed 3 times with a washing solution.
Serum collected from mice in advance was centrifuged at 3000 g for 10 minutes at 4 ° C., and the supernatant was collected. Using a solution obtained by diluting the blocking agent with a phosphate buffer solution (manufactured by Nacalai Tesque, Inc.) to 0.4 g / 100 mL, the above-mentioned supernatant or nasal lavage fluid was serially diluted 2-fold, and 50 μL of each solution was added. It was left at room temperature for 2 hours.
Then, the wells were washed 3 times with a washing solution, and the blocking agent was diluted to 0.4 g / 100 mL with a phosphate buffer solution (manufactured by Nacalai Tesque) to HRP-labeled anti-mouse IgG antibody (Goat-anti mouse IgG Fc HRP). (BETHYL) was diluted 10000 times, 100 μL was added, and the mixture was left at room temperature for 1 hour.
Then, the wells were washed 3 times with a washing solution, 100 μL of a TMB solution (ELISA POD TMB kit, manufactured by Nacalai Tesque) was added, and the mixture was left in a dark place for 30 minutes.
Then, 100 μL of a 1 M sulfuric acid solution was added, and the 96-well plate was measured for absorbance at 450 nm with a microplate reader (SpectraMax M2 ^e , manufactured by Molecular Devices). The IgG antibody titer in mouse serum was determined by Log2 based on the absorbance at the time of serial dilution.

From the results in Table 4 above, the drug layer prepared in Experimental Example 1 described above was used as the drug layer, and the drug cover layer prepared in Experimental Example 6 described above was used as the drug cover layer (residual rate (C) was 51%). In the orally adherent vaccine preparation according to Example 1 using the above, the drug layer had collapsed when visually observed 2 minutes after the preparation was administered under the tongue of the rabbit, but the shape of the drug cover layer was Was held. Moreover, when the shape of the preparation was confirmed after 30 minutes, the drug cover layer collapsed and the preparation disappeared from the oral cavity of the rabbit. Further, according to Example 1, since the high-concentration drug solution was retained for a certain period of time, a sufficient immunoinducing effect on mice was confirmed.
In addition, an example in which the drug layer prepared in Experimental Example 1 described above was used as the drug layer, and the drug cover layer prepared in Experimental Example 10 described above (residual rate (C) was 64%) was used as the drug cover layer. In the orally adherent vaccine preparation according to No. 2, the drug layer had collapsed when visually observed 2 minutes after the preparation was administered under the tongue of the rabbit, but the shape of the drug cover layer was maintained. Moreover, when the shape of the preparation was confirmed after 30 minutes, the drug cover layer collapsed and the preparation disappeared from the oral cavity of the rabbit. Further, according to Example 2, since the high-concentration drug solution was retained for a certain period of time, a sufficient immunoinducing effect on mice was confirmed.

From the results in Table 4 above, the drug layer prepared in Experimental Example 1 described above was used as the drug layer, and the drug cover layer prepared in Comparative Experimental Example 7 described above (residual rate (C) was 38%) as the drug cover layer. ) Was used for the orally adherent vaccine preparation according to Comparative Example 1, and the drug layer was collapsed when visually observed 2 minutes after the preparation was administered under the tongue of the rabbit, but the drug cover layer was also collapsed. Was. According to Comparative Example 1, since the high-concentration drug solution was not retained for a certain period of time, a sufficient immunostimulatory effect on mice could not be obtained.
Further, the orally adherent vaccine preparation according to Comparative Example 3 which uses the drug layer prepared in Experimental Example 1 as the drug layer and does not have a drug cover layer is prepared after the preparation is administered under the sublingual of a rabbit. After 2 minutes, the drug layer had collapsed. According to Comparative Example 3, since the high-concentration drug solution was not retained for a certain period of time, a sufficient immunostimulatory effect on mice could not be obtained.
On the other hand, a comparison using the drug layer prepared in Experimental Example 1 described above as the drug layer and the drug cover layer prepared in Comparative Experimental Example 5 described above (residual rate (C) 95%) as the drug cover layer. In the orally adherent vaccine preparation according to Example 2, the drug layer had collapsed when visually observed 2 minutes after the preparation was administered under the tongue of the rabbit, but the shape of the drug cover layer was maintained. .. Further, when the shape of the preparation was confirmed after 30 minutes, the drug cover layer kept its shape, and the preparation remained in the oral cavity of the rabbit. According to Comparative Example 2, since the high-concentration drug solution was retained for a certain period of time, a sufficient immunostimulatory effect was confirmed on the mice, but the shape of the drug layer was retained 30 minutes after the administration of the vaccine preparation. , A vaccine formulation that disintegrates at the desired time could not be obtained.

From the above, the thickness of the drug cover layer in the vaccine preparation is 15 μm or more, and the disintegration property (A) of the drug layer and the disintegration property (B) of the drug cover layer have a relationship of (A)> (B). The oral mucosal adhesion type vaccine preparation according to Examples 1 and 2 in which the residual rate (C) of the drug cover layer is 50% or more and 90% or less is high without the drug cover layer in the preparation being disintegrated for a certain period of time. The drug solution having a high concentration could be stably exposed to the mucosal surface for a certain period of time. As a result, a sufficient immunostimulatory effect could be obtained according to the amount of the administered drug without diluting the high-concentration drug solution with saliva in the oral cavity. In addition, the drug cover layer collapsed at the desired time, reducing the burden of time management.
On the other hand, in the orally adherent vaccine preparation according to Comparative Example 1 in which the residual rate (C) of the drug cover layer is less than 50%, the drug cover layer collapses and is high 2 minutes after being administered orally. The concentration of drug solution was not retained in the oral cavity for a period of time. Therefore, a sufficient immunostimulatory effect on mice could not be obtained. Similarly, the mucosal-adhering vaccine preparation according to Comparative Example 3 having no drug cover layer did not have a sufficient immunostimulatory effect on mice.
In addition, the orally adherent vaccine preparation according to Comparative Example 2 in which the residual rate (C) of the drug cover layer exceeded 90% remained in the oral cavity even after a desired time had passed after the oral administration. .. That is, the preparation did not reduce the burden of time management.

(Experimental Example 12)
A pharmaceutical composition was prepared with the component concentrations and formulations shown in Table 5. That is, 5 parts by mass of Dextran 70 (manufactured by Meito Sangyo Co., Ltd.) was added as an excipient to 40 parts by mass of purified water, and the mixture was appropriately stirred and dissolved.
To the obtained solution, 50 parts by mass of a 1 mg / mL OVA solution and 5 parts by mass of a 1 mg / mL adjuvant (ND002) solution were added, and the mixture was thoroughly mixed.
1.0 g of the obtained antigen-containing preparation solution was dispensed into an aluminum packaging material and freeze-dried to obtain a pharmaceutical composition. The water content of the obtained pharmaceutical composition was 10% by mass or less.
After measuring the mass of the obtained pharmaceutical composition, it was dissolved in 1.0 mL of purified water, and the solution viscosity at 37 ° C. was measured using a viscosity measuring device (RheoStress 600, manufactured by THEROMO HAAKE).
Furthermore, an immune induction test was carried out using a model animal for immunological evaluation by the method shown below.
The results are shown in FIGS. 4 and 5.

(Mice immunological test by sublingual administration)
Using the pharmaceutical composition produced as described above, an immunoinduction test was conducted using a mouse as a model animal.
First, the pharmaceutical composition was dissolved in 1 mL of purified water to obtain a vaccine preparation solution.
Next, after anesthetizing the mice prepared in advance (BALB / c mice, female 7 weeks old), 20 μL of the obtained vaccine preparation solution was administered under the sublingual of the mice. In addition, as a positive comparative example (PC), a solution having the same concentration of OVA and adjuvant ND002 as the vaccine preparation solution was prepared and administered in the same manner.
One week after the administration, the mice were anesthetized again and administered in the same manner. One week after the second administration, mouse serum and nasal lavage fluid were collected. Blood was centrifuged at 3000 G at 4 ° C. for 10 minutes, and 300 μL of phosphate buffer (manufactured by Nacalai Tesque) was added to 20 μL of the supernatant to prepare a serum sample.
As the nasal lavage fluid, a notch was made in the lower part of the respiratory tract of the mouse, 200 μL of phosphate buffer (manufactured by Nacalai Tesque, Inc.) was poured, and the sample that appeared in the nasal cavity was collected and used as a nasal lavage fluid sample.
The systemic immune response was evaluated by measuring OVA-specific IgG antibodies in mouse sera. The mucosal immune response was evaluated by measuring the immunogen-specific IgA titer in the mouse nasal cavity lavage fluid.

(Method for measuring antigen-specific IgG titer in mouse serum (ELISA method))
The same procedure as described above was carried out to measure the antigen-specific IgG titer.

(Antigen-specific IgA titer measurement method in mouse nasal cavity lavage fluid (ELISA method))
It is basically the same as the antigen-specific IgG titer measurement method, but the measurement sample is a nasal lavage fluid, and the HRP-labeled anti-mouse IgA antibody (Goat-anti-mouse IgA α) is used instead of the HRP-labeled anti-mouse IgG antibody. HRP, manufactured by BETHYL) was used.
All other operations were the same as the antigen-specific IgG titer measurement method.

(Experimental Examples 13 to 24)
A solution was prepared at the component concentrations and blending amounts shown in Tables 5 to 8 below in the same procedure as in Experimental Example 12, and freeze-dried to obtain a pharmaceutical composition. As excipients, Dextran 70 (manufactured by Meishu Sangyo Co., Ltd.) (mass average molecular weight 70000) in Experimental Examples 13 to 15, and LM pectin (GENU Pectin Type LM-102AS-J, CP Kelco) in Experimental Examples 16 to 18 In Experimental Examples 19 to 21, HM pectin (GENU Pectin Type USP-H, manufactured by CP Kelco) was used, and in Experimental Examples 22 to 24, sodium alginate (Kimika algin IL-2, manufactured by Kimika) was used. After measuring the mass of the pharmaceutical compositions obtained in Experimental Examples 13 to 24, they were dissolved in 1.0 mL of purified water, and the solution viscosity at 37 ° C. was measured by the method described above. The water content of the obtained pharmaceutical compositions was 10% by mass or less.
Furthermore, an immune induction test was carried out using a model animal for immunological evaluation by the method described above.
The results are shown in FIGS. 4-11.

(Comparative Experimental Examples 14 to 21)
A solution was prepared in the same procedure as in Experimental Example 12 with the blending amounts shown in Tables 5 to 8, and freeze-dried to obtain a pharmaceutical composition. After measuring the mass of the obtained pharmaceutical composition, it was dissolved in 1.0 mL of purified water, and the solution viscosity at 37 ° C. was measured by the method described above. The water content of the obtained pharmaceutical compositions was 10% by mass or less.
Furthermore, an immune induction test was carried out using a model animal for immunological evaluation by the method described above.
The results are shown in FIGS. 4-11.

(Experimental Examples 25 to 32)
A solution was prepared at the component concentrations and blending amounts shown in Table 9 in the same procedure as in Experimental Example 12, 1.0 g each was dispensed into an aluminum packaging material, and freeze-dried to obtain a pharmaceutical composition. The water content of the obtained pharmaceutical compositions was 10% by mass or less.
After measuring the mass of the obtained pharmaceutical composition, it was dissolved in 1.0 mL of purified water, and the solution viscosity at 37 ° C. was measured using the above-mentioned viscosity measuring device.
Furthermore, an immune induction test was carried out using a model animal for immunological evaluation by the method described above.
The results are shown in FIGS. 12 and 13.

As shown in Tables 5 to 8, it was found that the solution viscosity of the pharmaceutical composition was increased by increasing the blending amount of the excipient. As a result, it was found that sufficient immunity induction can be obtained with a solution having a solution viscosity of 20 mPa · s or less.
As shown in Table 9, even if the solution viscosity exceeds 20 mPa · s in Tables 5 to 8, the solution viscosity of a single dose can be reduced to 20 mPa · s or less by reducing the mass of the preparation. It was found that it decreased and sufficient immune induction was obtained.
That is, it was shown that the size of the preparation and the viscosity of the solution obtained by dissolution are important parameters, not the ratio of the excipient in the compounding solution or the pharmaceutical composition.

According to the above Experimental Examples 12 to 32 and Comparative Experimental Examples 14 to 21, the solution viscosity (D) of the drug layer when a single dose was dissolved in 1.0 mL of distilled water at 37 ° C. was 20 mPa · s or less. It was confirmed that this is suitable for administering a vaccine preparation and inducing immunity through the oral mucosal pathway.

1 Drug layer 2 Mucosal adhesion layer 3 Drug cover layer

Claims (7)

An oral mucosa-adhering vaccine preparation having a drug layer containing a vaccine antigen and a drug cover layer.
The thickness of the drug cover layer is 15 μm or more,
The disintegration property (A) of the drug layer and the disintegration property (B) of the drug cover layer with respect to distilled water at 37 ° C. have a relationship of (A)> (B).
Test pieces of the drug cover layer area 20 cm ² Thickness 50 [mu] m, wherein the immersed in distilled water 500mL of 37 ° C., the residual rate after 7 minutes from start of immersion (C) is less than 90% 50% Oral mucosal adhesion type vaccine preparation. The oral mucosa-adhering vaccine preparation according to claim 1, wherein the drug layer has a solution viscosity (D) of 20 mPa · s or less when a single dose is dissolved in 1.0 mL of distilled water at 37 ° C. The oral mucosa-adhering vaccine preparation according to claim 1 or 2, wherein the drug layer further contains a polymer component. The oral mucosa-adhering vaccine preparation according to claim 3, wherein the polymer component is at least one selected from the group consisting of dextran, pectin and sodium alginate. The oral mucosa-adhering vaccine preparation according to claim 4, wherein the polymer component is dextran. The oral mucosa-adhering vaccine preparation according to claim 1, 2, 3, 4 or 5, wherein the drug layer further comprises at least one immunostimulatory agent. The oral mucosa-adhering vaccine preparation according to claim 1, 2, 3, 4, 5 or 6, wherein the vaccine antigen is at least one selected from the group consisting of peptide antigens, protein antigens and nucleic acids.

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