JP4092367B1 - Liposomes and compositions for immunostimulatory activity comprising the same. - Google Patents

Abstract

【選択図】 なしPROBLEM TO BE SOLVED: To further enhance the immunostimulatory activity of glycosphingolipid.
A liposome containing at least one glycosphingolipid represented by the following formula (1) inside and outside a lipid membrane.

[Selection figure] None

Description

 The present invention relates to a liposome containing a glycosphingolipid having a specific structure inside and outside a lipid membrane, and an immunostimulatory active composition containing the liposome.

 The present applicants have revealed that glycosphingolipids isolated from specific sphingomonas genus have immunostimulatory activity and the like (Patent Documents 1 to 5). However, these immunostimulatory activities and the like are not always sufficient, and further activation has been demanded.

International Publication No. WO92 / 12986 Japanese Patent Laid-Open No. 11-43437 JP 2000-51676 A Japanese Patent Laid-Open No. 2002-010797 Japanese Patent Laying-Open No. 2005-267397

  The object of the present invention is to solve the above-mentioned problems, and to increase the immunostimulatory activity of the glycosphingolipid.

（式（１）中、Ｒ ²は、水素原子または、α−ガラクトース基、α−グルコース基、α−マンノース基、α−グルコサミン基若しくはβ−グルコサミン基またはこれらの１以上の組み合わせからなる基である。）

（２）前記式（１）において、Ｒ²は、水素原子または、α−ガラクトース基、α−グルコース基、α−マンノース基、α−グルコサミン基若しくはβ−グルコサミン基またはこれらの２つまたは３つの組み合わせからなる基である、（１）に記載のリポソーム。
（３）前記式（１）において、Ｒ²は、水素原子、下記Ｒ⁶²、Ｒ⁶³、Ｒ⁶⁴またはＲ⁶⁵である、（１）に記載のリポソーム。

（４）前記式（１）において、Ｒ²は、水素原子である、（１）に記載のリポソーム。
（５）前記脂質膜を構成する脂質は、１，２−ジオレイル−３−（トリメチルアンモニウム）プロパンおよび／または３β−［Ｎ−（ジメチルアミノエタン）カルバモイル］コレステロールがモル比で０．４以上を占める（１）〜（４）のいずれか１項に記載のリポソーム。
（６）前記脂質膜を構成する脂質の少なくとも１種が、１，２−ジオレイル−３−（トリメチルアンモニウム）プロパンである、（１）〜（５）のいずれか１項に記載のリポソーム。
（７）前記脂質膜を構成する脂質は、１，２−ジオレイル−３−（トリメチルアンモニウム）プロパン、３β−［Ｎ−（ジメチルアミノエタン）カルバモイル］コレステロールおよび１，２−ジオレオイル−ｓｎ−グリセロ−３−ホスファチジルエタノールアミンがモル比で０．７５以上を占める、（１）〜（６）のいずれか１項に記載のリポソーム。
（８）さらに、前記脂質膜内外に、抗原を含む、（１）〜（７）のいずれか１項のリポソーム。
（９）（１）〜（８）のいずれか１項に記載のリポソームを含む免疫賦活活性用組成物。
（１０）（１）〜（８）のいずれか１項に記載のリポソームを含むＩＦＮ−γ産生促進用組成物。
（１１）（１）〜（８）のいずれか１項に記載のリポソームを含むＩＬ−４産生促進用組成物。
（１２）（１）〜（８）のいずれか１項に記載のリポソームを含むＩＬ−１２産生促進用組成物。
（１３）（１）〜（８）のいずれか１項に記載のリポソームを含む抗体産生促進作用組成物。
（１４）（１）〜（８）のいずれか１項に記載のリポソームを含む抗腫瘍作用組成物。
（１５）（１）〜（８）のいずれか１項に記載のリポソームを含むＮＫＴ細胞活性化用組成物。
（１６）（１）〜（８）のいずれか１項に記載のリポソームを含む樹状細胞活性化用組成物。
（１７）（１）〜（８）のいずれか１項に記載のリポソームを含む抗ウイルス作用組成物。
（１８）（１）〜（８）のいずれか１項に記載のリポソームを含む抗アレルギー作用組成物。
Based on the above problems, the present inventors have found that the above problems can be solved by the following means.
(1) 1,2-dioleoyl-3-lipid transmembrane comprising at least one (trimethylammonium) propane and 3.beta .- [N-(dimethylamino ethane) carbamoyl] cholesterol, it is represented by the following formula (1), R ¹ is R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁷⁰ , R ⁷¹ , R ⁷² , R ⁷³ , R ⁷⁴ , R ^75, R ^76, at least one of glycosphingolipids is Ru represented by R ⁷⁷ or R ^78, liposomes containing.
Formula (1)

(In formula (1) , R ² is a hydrogen atom or a group consisting of an α-galactose group, an α-glucose group, an α-mannose group, an α-glucosamine group, a β-glucosamine group, or one or more combinations thereof. is there.)

(2) In the formula (1), R ² is a hydrogen atom, an α-galactose group, an α-glucose group, an α-mannose group, an α-glucosamine group, a β-glucosamine group, or two or three of them. The liposome according to (1), which is a group consisting of a combination.
(3) The liposome according to (1), wherein, in the formula (1), R ² is a hydrogen atom, the following R ⁶² , R ⁶³ , R ⁶⁴ or R ⁶⁵ .

(4) The liposome according to (1), wherein in the formula (1), R ² is a hydrogen atom.
( 5 ) The lipid constituting the lipid membrane has a molar ratio of 1,2-dioleyl-3- (trimethylammonium) propane and / or 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol of not less than 0.4. The liposome according to any one of (1) to ( 4) .
( 6 ) The liposome according to any one of ( 1) to (5), wherein at least one lipid constituting the lipid membrane is 1,2-dioleyl-3- (trimethylammonium) propane.
( 7 ) The lipids constituting the lipid membrane are 1,2-dioleyl-3- (trimethylammonium) propane, 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol and 1,2-dioleoyl-sn-glycero- The liposome according to any one of (1) to ( 6) , wherein 3-phosphatidylethanolamine accounts for 0.75 or more in molar ratio.
( 8 ) The liposome according to any one of (1) to ( 7) , further comprising an antigen inside and outside the lipid membrane.
( 9 ) A composition for immunostimulatory activity comprising the liposome according to any one of (1) to ( 8) .
( 10 ) A composition for promoting IFN-γ production comprising the liposome according to any one of (1) to ( 8) .
( 11 ) A composition for promoting IL-4 production, comprising the liposome according to any one of (1) to ( 8) .
( 12 ) A composition for promoting IL-12 production, comprising the liposome according to any one of (1) to ( 8) .
( 13 ) An antibody production promoting composition comprising the liposome according to any one of (1) to ( 8) .
( 14 ) An antitumor composition comprising the liposome according to any one of (1) to ( 8) .
( 15 ) A composition for activating NKT cells, comprising the liposome according to any one of (1) to ( 8) .
( 16 ) A composition for activating dendritic cells, comprising the liposome according to any one of (1) to ( 8) .
( 17 ) An antiviral composition comprising the liposome according to any one of (1) to ( 8) .
( 18 ) An antiallergic composition comprising the liposome according to any one of (1) to ( 8) .

  In the present invention, GSL immunostimulatory activity is achieved by using liposomes containing at least one glycosphingolipid represented by formula (1) (hereinafter sometimes referred to as “GSL”) inside and outside the lipid membrane. It became possible to increase it further.

  Hereinafter, the contents of the present invention will be described in detail. In the present specification, “to” is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.

In the present invention, “liposome” is a substance having a lipid (sometimes referred to as “lipid membrane” in the present specification) in which hydrophobic portions are gathered inward toward each other, and is composed of an internal aqueous layer. Means.
Further, in this specification, “containing at least one glycosphingolipid represented by the following formula (1) inside and outside the lipid membrane” means that GSL is the surface and / or inside of the lipid membrane, and the liposome. It is said that it is contained in any of the insides, and usually part or all of it is contained in the lipid membrane of the liposome.
The lipid membrane in the present invention may have a single layer structure or a multilayer structure.

  In the present invention, as described above, it has been surprisingly found that the immunostimulatory activity of GSL is enhanced by making GSL into a liposome. Glycolipids are sometimes used as the lipid component of lipid membranes when liposomes are formed, but it is rare that the glycolipids themselves are the target substances for liposome formation as in the present invention. Furthermore, it is very surprising that the GSL immunostimulatory activity is remarkably enhanced by GSL liposome formation.

（式（１）中、Ｒ ²は、水素原子または、α−ガラクトース基、α−グルコース基、α−マンノース基、α−グルコサミン基若しくはβ−グルコサミン基またはこれらの１以上の組み合わせからなる基である。）
First, the GSL employed in the present invention will be described in detail. GSL is I glycosphingolipid der represented by formula (1), R ^{1 is, R 51, R 52, R} 53, R 54, R 55, R 56, R 57 employed in the present invention, It is a glycosphingolipid represented by R ⁵⁸ , R ⁵⁹ , R ⁷⁰ , R ⁷¹ , R ⁷² , R ⁷³ , R ⁷⁴ , R ⁷⁵ , R ⁷⁶ , R ⁷⁷ or R ⁷⁸ .
Formula (1)

(In formula (1) , R ² is a hydrogen atom or a group consisting of an α-galactose group, an α-glucose group, an α-mannose group, an α-glucosamine group, a β-glucosamine group, or one or more combinations thereof. is there.)

R ^{51 to} R ⁷⁸ are as follows.

On the other hand, in the formula (1), R ² is a hydrogen atom, an α-galactose group, an α-glucose group, an α-mannose group, an α-glucosamine group, a β-glucosamine group, or a combination thereof. The group is preferably a combination, more preferably a hydrogen atom, the following R ⁶² , R ⁶³ , R ⁶⁴ or R ⁶⁵ , and even more preferably a hydrogen atom. By adopting a hydrogen atom with R ^2, the effects of various immunostimulatory activities are particularly preferred for improving.

 Furthermore, only one type of glycosphingolipid of the present invention may be employed, or two or more types may be employed. The ratio of each component in the case of containing 2 or more types in combination is not particularly limited.

 GSL can be obtained by extracting from bacterial cells having glycosphingolipids. For example, the method described in International Publication WO92 / 12986 pamphlet, Japanese Patent Application Laid-Open No. 2002-10797, or Japanese Patent Application Laid-Open No. 2005-267397 can be employed. Glycosphingolipids are contained in bacterial cells belonging to the family Sphingomonas, so that if extracted using any of the bacteria belonging to the family Sphingomonas, a glycosphingolipid represented by the formula (1) is obtained. Can do. Here, the bacteria belonging to the family Sphingomonas are classified as those belonging to the same family as the bacteria other than those generally belonging to the genus "Sphingomonas". The purpose is to include bacteria. As a strain that can be employed in the present invention, for example, any strain shown in Microbiol. Immunol., 2000, 44, 563-575 can be employed.

 Since GSL is insoluble in acetone, it is preferable to wash the cells with acetone before the extraction operation. The solvent used for GSL extraction is preferably an alcohol solvent such as methanol or a mixed solvent of an alcohol solvent and a polar solvent such as chloroform from the viewpoint of yield. However, other solvents may be used as long as they are glycosphingolipid-soluble solvents.

In the present invention, GSL described in the table below can be preferably used. In the table, the part of “ceramide” is one or more of R ^{51 to} R ⁷⁸ described above. Moreover, it cannot be overemphasized that GSL employ | adopted by this invention is not limited to what was obtained from the strain described below.

  In addition, when a mixture of glycosphingolipids is obtained, each component can be separated according to a method well known in the art. For example, it can be performed according to the description in JP-A-2005-267397. Specifically, glycosphingolipids can be completely separated by chromatography. When a chloroform / methanol mixed solution is used as the eluent, each glycosphingolipid is eluted in the order of (1) GSL-1, 6-13, (2) GSL-3, (3) GSL-2, 4, 5 To do. Moreover, since GSL-2, 4, and 5 produce a different strain, they can be separated very easily. The chromatographic separation conditions such as the filler, eluent, elution rate, pressure, and temperature can be appropriately adjusted. In addition, a reagent that selectively reacts only with a specific substance contained in a mixture of glycosphingolipids is allowed to act to prepare a derivative of the substance, and separation is performed using the chemical or physical properties of the derivative. You can also.

 GSL can also be synthesized by combining known synthesis methods. For example, GSL can be prepared by synthesizing a sugar and a sphingosine moiety in advance or extracting from a microbial cell and forming an amide bond.

Next, the liposome of the present invention and the method of forming a liposome will be described.
The diameter (“volume average particle diameter”) of the liposome of the present invention is usually 20 to 2000 nm, preferably 40 to 800 nm. By setting it as such a range, when using as a pharmaceutical compound (pharmaceutical formulation), the effect is easy to be exhibited.
The "volume average particle diameter" of liposomes can be determined based on the principle such as dynamic light scattering method (see DDLasic, "liposomes: from basic to applications", Elsevier Science Publishers, pp. 1-171 (1993)) .

 In the liposome of the present invention, the volume average particle size and the proportion of particles having a particle size of ± 5% preferably occupy 50% or more of the total particles.

 The content of GSL in the liposome of the present invention is not particularly limited, but is preferably 0.01 to 30% with respect to the total amount of lipid (not including the content of GSL) constituting the lipid membrane.

The lipid constituting the liposome lipid membrane in the present invention includes at least one of 1,2-dioleyl-3- (trimethylammonium) propane and 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol. In addition, other lipids that can be made into liposomes may be included, and synthetic and natural lipids can be widely adopted. For example, the following lipids can be used.

1,2-dioleoyl-3- (trimethylammonium) propane (1,2-dioleoyl-3- (trimethylammonium) propane, DOTAP), phosphatidylcholine (soybean phosphatidylcholine, egg yolk phosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine ( DPPC) or distearoylphosphatidylcholine), phosphatidylethanolamine (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, DOPE), dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanol Amine (DPPE) or distearoylphosphatidylethanolamine), Phosphatidylserine (dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine or distearoylphosphatidylserine), phosphatidic acid, phosphatidylglycerol (dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol (DPPG) Stearoylphosphatidylglycerol, etc.), phosphatidylinositol (dilauroylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol or distearoylphosphatidylinositol), lysophosphatidylcholine, sphingomyelin , Soy lecithin, digalactosyl diglycerides (such as digalactosyl dilauroyl glyceride, digalactosyl dimyristoyl glyceride, digalactosyl dipalmitoyl glyceride or digalactosyl distearoyl glyceride), or galactosyl diglycerides (galactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride) Galactosyl dipalmitoyl glyceride or galactosyl distearoyl glyceride), galactosyl cerebroside, lactosyl cerebroside or ganglioside, cholesterol, cholesterol hemisuccinate, 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol (DC cholesterol), ergosterol or Lanosterol, dialkyl Dimethylammonium (dialkyl dimethylammnonium amphiphiles), polyglycerol alkyl ether, polyoxyethylene alkyl ether, alkyl glycoside, alkyl methyl glucamide, alkyl sucrose ester, dialkyl polyoxyethylene ether, dialkyl polyglycerol ether, polyoxyethylene-polylactic acid, Long chain alkylamines (tetradecylamine, hexadecylamine, stearylamine, etc.), long chain fatty acid hydrazides (myristic acid hydrazide, palmitic acid hydrazide, stearic acid hydrazide, etc.), etc. are mentioned.
These lipids can be used alone or in combination of two or more.

Among these, the lipid membrane in the present invention preferably contains a cationic lipid in order to improve immunostimulatory activity, and more preferably the cationic lipid occupies 0.4 or more in molar ratio.
The cationic lipid preferably contains at least DOTAP.
In particular, in the present invention, it is more preferable that DOTAP, DC cholesterol, and DOPE occupy 0.75 or more by molar ratio, and it is further preferable that DOTAP, DC cholesterol, and DOPE occupy 0.90 or more by molar ratio.
By setting it as such a lipid composition, there exists a tendency for immunostimulatory activity to become higher, and it is preferable.

 Furthermore, in the present invention, as a component of the lipid membrane, other substances can be added as necessary in addition to the lipid. Specific examples include lipid membrane stabilizers, various functional substances, charge control agents and the like. These other substances are preferably contained in a range of about 0.0001 to 30% with respect to the total amount of lipid constituting the liposome.

The liposome of the present invention may encapsulate various substances without departing from the spirit of the present invention. The substance to be encapsulated is not particularly limited, and examples thereof include substances used for pharmaceuticals. Specifically, contrast compounds, antitumor compounds, antioxidant compounds, antibacterial compounds, anti-inflammatory compounds, blood circulation promoting compounds, whitening compounds, rough skin prevention compounds, anti-aging compounds, hair growth promoting compounds, moisturizing compounds, hormone agents, Vitamins, pigments, proteins and the like. The substance to be encapsulated is desirably a water-soluble compound, or a water-soluble salt form or a water-soluble form by the addition of a solubilizing agent. It may be a hydrophobic or fat-soluble compound.
In particular, in the present invention, it is preferable to encapsulate an antigen. Encapsulating the antigen is preferable because it has the advantage that an antigen-specific immune response enhancing action can be induced. The antigen to be encapsulated is not particularly limited, and examples thereof include antigens used for vaccination, tumor antigens, autoimmune disease autoantigens, and allergens.

 The liposome of the present invention can be produced according to a known production method. For example, the method described in Bangham, AD, Standish, MM and Watkins, JC “Diffusion of univalent ions across the lamellae of swollen phospholipids.” (1965) J. Mol. Biol. 13,238-252 can be employed. . More specifically, although it can manufacture according to the following procedures, the description does not necessarily limit the manufacturing method of the liposome of the present invention.

1) Dissolve GSL in a solvent. The solvent at this time can dissolve lipid and preferably has a low boiling point. Specific examples of the organic solvent include chloroform, methanol and the like. Moreover, it is preferable to ultrasonically process after heating at 30-70 degreeC.
2) Dissolve or suspend components (excluding GSL) constituting the lipid membrane in an organic solvent. Here, it is preferable that the organic solvent can dissolve lipid and has a low boiling point. Specific examples of the organic solvent include chloroform, methanol, ethanol and the like.
3) A lipid membrane is formed by mixing the solutions obtained in 1) and 2) above and removing the solvent.
4) A liposome suspended in an aqueous solvent can be obtained by adding an aqueous solvent (preferably a buffer) to the lipid membrane obtained in 3) above and stirring.

 By passing the aqueous solvent (suspension) containing the liposomes obtained above through a filter, the particle size of the liposomes can be made uniform. Moreover, the liposome can be isolate | separated as a deposit by carrying out the ultracentrifugation process of the water-soluble solvent containing a liposome. You may wash | clean the isolate | separated liposome as needed.

  Confirmation of the formation of liposomes can be performed by a known method. For example, for a substance prepared by a known method for forming liposomes, the particle diameter is measured by light scattering, and when the average particle diameter is 2000 nm or less, the liposome in the present invention is formed. Further, the particles here do not necessarily have to be completely spherical.

  The liposome of the present invention has an effect that the immunostimulatory activity is further enhanced. Among immunostimulatory activities, in particular, IFN-γ production promotion, IL-4 production promotion, IL-12 production promotion, antibody production promotion action, antitumor action, NKT cell activity, dendritic cell activity, for enhancing infection resistance, Antiviral action, IL-10 production promotion, NK cell activity, antiallergic action, IL-6 production promotion, NO production promotion can be enhanced, IFN-γ production promotion, IL-4 production promotion, IL-12 production promotion , Antibody production promoting action, antitumor action, NKT cell activity, dendritic cell activity, infection resistance enhancement, antiviral action can be further enhanced, IFN-γ production promotion, IL-4 production promotion, IL-12 Production promotion, antibody production promotion action, antitumor action, NKT cell activity, dendritic cell activity, infection resistance enhancement can be further enhanced, IFN-γ production promotion, IL-4 production promotion, IL-12 production Susumu, antibody production promoting effect, can be increased in particular an anti-tumor effect.

Examples of those promoted by the promotion of IFN-γ production include Th1 induction and macrophage activation action promoted by IFN-γ.
Examples of those promoted by the promotion of IL-4 production include induction of Th2 and induction of antibody class switch.
In the present specification, the promotion of antibody production refers to an enhancement effect of an antigen-specific immune response and an adjuvant effect.
In this specification, the antitumor action is intended to include, for example, helper T cell activation and killer T cell activation.
NKT cells include, for example, human Vα24 ⁺ NKT cells and mouse Vα14 ⁺ NKT cells. NKT cell activation is intended to include enhancement of cytotoxic activity, enhancement of cytokine production, and promotion of NKT cell proliferation.
In the present specification, dendritic cell activation is intended to include, for example, enhancement of antigen presentation ability.
In the present specification, the effect of enhancing resistance to infection is intended to include, for example, activation of helper T cells and killer T cells. The composition for enhancing infection resistance of the present invention is particularly useful for use in Salmonella infection and tuberculosis.
In this specification, the antiviral action is intended to include, for example, enhancement of type I interferon production, activation of helper T cells and killer T cells. The antiviral composition of the present invention is particularly useful for use in herpesviridae infections such as cytomegalovirus infection and herpesvirus infection.
In this specification, NK cell activation is intended to include, for example, infected cell injury.
In this specification, the antiallergic effect is intended to include, for example, eosinophil infiltration suppression, mast cell suppression effect, IgE production suppression, and chemical transmitter release suppression. Further, the antiallergic composition of the present invention tends to have weak side effects and is particularly useful for use in hay fever, bronchial asthma and the like.

  In addition, when the liposome of the present invention and the composition containing the liposome are used as a pharmaceutical or a quasi-drug or an active ingredient thereof, it can be preferably administered as a pharmaceutical composition that can be produced by methods well known to those skilled in the art . Examples of the pharmaceutical composition include tablets, capsules, powders, fine granules, granules, liquids, and syrups. Said pharmaceutical composition can be manufactured by adding a pharmacologically and pharmaceutically acceptable additive. Examples of pharmacologically and pharmaceutically acceptable additives include, for example, excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, and dissolution. Examples thereof include agents or solubilizers, tonicity agents, pH adjusters, stabilizers, propellants, and pressure-sensitive adhesives. You may mix | blend 1 type (s) or 2 or more types of components which have another effect in the said pharmaceutical composition within the range which does not deviate from the meaning of this invention. The dosage of the medicament of the present invention is not particularly limited, and can be appropriately selected according to the type of active ingredient, and various other factors that should normally be taken into consideration, such as patient weight and age, type and symptom of disease, and administration route Depending on the factor, it can be appropriately increased or decreased. Generally, it can be used in the range of 0.001 to 1000 mg, preferably 0.01 to 100 mg per adult day. Also, the administration method is not particularly limited, and it may be administered by intravenous injection or orally as an injection, an infusion, or the like.

  The present invention will be described more specifically with reference to the following examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the specific examples shown below.

In the present Example, it carried out using GSL-7 of Table 1. These GSLs were separated according to the methods described in International Publication WO92 / 12986, Japanese Patent Application Laid-Open No. 2002-010797, and Japanese Patent Application Laid-Open No. 2005-267397.
Moreover, in each table | surface of an Example of this application, "control" does not add any of liposome and non-liposomal GSL-7 (P20), others have shown the comparative example performed on the same conditions, “GLS-7 (P-20)” is a comparative example in which only GSL-7 (P20) that was not made into liposomes was added, and the others were performed under the same conditions.

Example 1 Induction of IFN-γ production from splenocytes (preparation of liposomes)
GSL-7 was dissolved in a physiological saline for injection (manufactured by Otuka Pharmaceutical) to which Polysorbate-20 (manufactured by Wako) was added (0.5% solution), and was heated in a thermostatic bath at 60 ° C. for 10 minutes at the time of use. The sample was sonicated with a sonicator for 20 minutes and used for the experiment. Hereinafter, GSL-7 dissolved by the above method is abbreviated as GSL-7 (P20).
Next, with respect to 0.1 mol of GSL-7, the lipid having the composition shown in the following table was dissolved in a solvent (chloroform: methanol (volume ratio 2: 1)), placed in an eggplant type flask, and at room temperature, a rotary evaporator. The solvent was removed with to form a lipid film.
Saline was added, mixed by vortexing, and sonicated for 1 minute to prepare liposomes.

(Lipid composition)
Lipids used for liposome preparation were cholesterol (Chol, manufactured by Wako), 1,2-dioleyl-3- (trimethylammonium) propane (DOTAP, manufactured by Avanti polar lipids), 3β- [N- (dimethylaminoethane) carbamoyl]. Cholesterol (DC cholesterol, Sigma), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE, Avanti polar lipids). Moreover, the following table | surface has shown as the quantity (mol) with respect to 0.1 mol GSL-7.

(Measurement of IFN-γ production promoting ability)
Spleen cells of C57BL / 10ScSn mice (8-10 weeks old, male) (autologous breeding) (2 × 10 ⁶ ) In addition, liposomes A to E prepared by the above method and non-liposomal GSL-7 (P20) were added, respectively, and IFN-γ in the culture supernatant after 48 hours was measured. The concentration at this time was such that the concentration of GSL-7 was 100 ng / ml. IFN-γ was measured by using purified anti-IFN-γ antibody (R4-6A2) (manufactured by Nippon Becton Dickinson Co., Ltd.) as a capture antibody and biotin-conjugated anti-IFN-γ antibody (AN-18) (Nippon Becton) as a detection antibody. -The sandwich ELISA method using Dickinson Co., Ltd. was used. In the reaction following the biotin-conjugated antibody, alkaline phosphatase-labeled streptavidin (manufactured by SIGMA) was bound, and color was developed using paranitrophenyl phosphate as a substrate. Absorbance at a wavelength of 405 nm was measured with MICROPLATE READER Model 550 (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.). The IFN-γ concentration of the test sample was calculated from the standard curve of IFN-γ from the recombinant mouse.
As comparative experiments, (1) none of liposomes A to E and non-liposomal GSL-7 (P20) were added, and the other was performed in the same manner (control), and (2) non-liposomal GSL- 7 (P20) alone was added, and the others performed in the same manner (GSL-7 (P-20)) are also shown below.

(result)
The results are shown in the table below. Although these results were performed under the same conditions, they were performed in three steps, so that the results performed at the same time are summarized in a table.
As shown in the table below, even when the spleen cells were stimulated with GSL-7 (P20), at the concentration of 100 ng / ml, only the same amount as the control induced IFN-γ production.
On the other hand, liposomes A and B significantly induced INF-γ from splenocytes. In particular, liposome B was remarkable.
In addition, regarding the production amount of IFN-γ, in the following table, when GSL-7 (P-20) was added to the control, no significant difference was observed, but GSL-7 (P-20) A significant difference was observed when the addition amount was 1 μg / ml or more. That is, it was confirmed that the liposome of the present invention promotes the production of IFN-γ in a significantly smaller amount than when using non-liposomal GSL-7.

Example 2 Induction of IFN-γ Production by Changing Liposome Concentration In Example 1, Liposome B or GSL-7 (P20) was changed so that the equivalent amount of GSL-7 was the amount described in the table below, and the like. Were performed in the same manner, and the IFN-γ concentration of each sample was calculated. Moreover, only the lipid composition of the liposome B was added (GSL-7 was not included), and others were performed similarly.

上記表から明らかなとおり、リポソームＢの添加量を増やすことにより、ＩＦＮ−γの誘導が促進されることが確認された。
尚、上記表では、ＩＦＮ−γの産生量に関し、ＧＳＬ−７（Ｐ−２０）を添加した場合、コントロールに対して有意差が認められていないが、ＧＳＬ−７（Ｐ−２０）の添加量を１μｇ／ｍｌ以上とすることにより、コントロールに対して有意差が認められた。 (result)
These results are shown in the table below.

As apparent from the above table, it was confirmed that the induction of IFN-γ was promoted by increasing the amount of liposome B added.
In the above table, when GSL-7 (P-20) was added with respect to the production amount of IFN-γ, no significant difference was observed with respect to the control, but the addition of GSL-7 (P-20) When the amount was 1 μg / ml or more, a significant difference was recognized with respect to the control.

Example 3 Influence on IFN-γ production promotion effect by anti-CD1dmAb and isotype mAb When Liposome B of Example 2 was added so that the equivalent amount of GSL-7 was 100 ng / ml, along with the addition of Liposome B The anti-CD1dmAb (manufactured by BD Pharmingen) or isotypemAb (manufactured by BD Pharmingen) was added so that the concentration was 40 μg / ml, and the others were performed in the same manner. Here, the addition of isotype mAb was performed in order to confirm whether the activity of liposome B was inhibited by another antibody that was not related at all.

これらの結果より、抗ＣＤ１ｄ ｍＡｂを添加することにより、リポソームＢの活性が有意に阻害されることが確認された。また、ｉｓｏｔｙｐｅ ｍＡｂを添加しても、リポソームＢのＩＦＮ−γの産生が阻害されることはなかった。 (result)
These results are shown in the following table together with comparative examples.

From these results, it was confirmed that the activity of liposome B was significantly inhibited by adding anti-CD1d mAb. Further, addition of isotype mAb did not inhibit IFN-γ production of liposome B.

Example 4 IFN-γ induction by various mice In Example 1, C57BL / 10ScSn mice were replaced with C57BL / 10ScSn (WT) mice or C57BL / 10ScN (TLR4 ^{− / −} mice), and liposome B (GSL-7 equivalent concentration) : 500 ng / ml), IFN-γ induction from splenocytes was measured in the same manner.
At the same time as the addition of liposome B, 20 μg / ml of anti-IL-12 p40 / p70 mAb (BD Pharmingen) was added, and the others were performed in the same manner.

(result)
As shown in the table below, liposome B promotes the production of IFN-γ by the addition of liposome B and significantly increases the addition of anti-IL-12 p40 / p70 mAb in any mouse. The amount added was reduced.

Example 5 Induction of IL-12 production from splenocytes (measurement of IL-12 production promoting ability)
C57BL / 10ScSn (WT) mouse (self-propagating) splenocytes (2 × 10 ⁶ ) Were added with liposome B prepared by the method described in Example 1 and non-liposomal GSL-7 (P20), and IL-12p40 in the culture supernatant after 24 hours was measured. The concentration at this time was adjusted so that the concentration of GSL-7 would be the amount described in the following table. IL-12p40 was measured using an OptEIA mouse cytokine detection set (BD Bioscience). Others were performed according to the measurement of IFN-γ in Example 1.

リポソームＢの添加によって、ＩＬ−１２ｐ４０の産生が有意に促進されていることが認められた。また、リポソームＢの添加量を増やすことにより、ＩＬ−１２ｐ４０の産生量が増加することが認められた。 (result)
The results are shown in the table below.

It was observed that the addition of liposome B significantly promoted the production of IL-12p40. Moreover, it was recognized that the production amount of IL-12p40 increases by increasing the addition amount of liposome B.

Example 6 Influence on IL-12 Production Promotion Effect by Anti-CD1dmAb or IsotypeAb When Liposome B of Example 5 was added so that the amount of GSL-7 was 500 ng / ml, simultaneously with the addition of Liposome B Anti-CD1 dmAb (manufactured by BD Pharmingen) or isotypemAb was added so that the concentration was 40 μg / ml, and the others were performed in the same manner.

リポソームＢの添加による脾細胞からのＩＬ−１２ ｐ４０誘導はａｎｔｉ−ＣＤ１ｄ ｍＡｂ(BD Pharmingen)の添加により有意に低下することが確認された。一方、ｉｓｏｔｙｐｅ ｍＡｂを添加しても、リポソームＢのＩＬ−１２ ｐ４０誘導は阻害されなかった。 (result)
The results are shown in the following table together with comparative examples.

It was confirmed that IL-12 p40 induction from splenocytes by addition of liposome B was significantly reduced by addition of anti-CD1d mAb (BD Pharmingen). On the other hand, addition of isotype mAb did not inhibit IL-12 p40 induction of liposome B.

Example 7 IL-12 production promoting effect by various mice In Example 5, C57BL / 10ScSn mice were replaced with C57BL / 10ScSn (WT) mice and C57BL / 10ScN (TLR4 ^-/- mice), and liposome B was replaced with GSL- Seven amounts were added to give 500 ng / ml, and IL-12 induction from splenocytes was measured in the same manner.

表から明らかなとおり、リポソームＢを用いることにより、著しいＩＬ−１２誘導効果の向上が認められた。 (result)
The results are shown in the following table together with comparative examples.

As is clear from the table, a significant improvement in the IL-12 induction effect was observed by using liposome B.

It was checked for uptake of liposomes by CD11c ⁺ dendritic cells derived from the uptake splenocytes liposomes by CD11c ⁺ dendritic cells from Example 8 splenocytes.
Isolated dendritic cells were prepared from spleen cells of C57BL / 10ScSn mice (8-10 weeks old, male) using anti-CD11c mAb (manufactured by Miltenyi Biotec) bound with magnetic beads, and fluorescently labeled liposome B and Ninety minutes after addition of liposome D, the amount of each liposome taken up by dendritic cells was examined using a flow cytometer (manufactured by Beckman Coulter).

 As shown in FIG. 1, there was no difference in the amount of uptake between liposome B (solid line) and liposome D (dashed line).

Example 9 Dendritic Cell Activation Action (1)
To C57BL / 6 mice (8-10 weeks old, female), 1 μg of GSL-7 (P20) or liposome B (GSL-7 content 1 μg) was administered from the tail vein, and 16 hours later, the spleen was removed. A cell suspension was prepared from the excised spleen. Spleen cells were treated with anti-FcγR antibody (2.4G2), and then PE-labeled anti-CD11c antibody, biotin-labeled anti-CD80 antibody and biotin-labeled anti-CD86 antibody were added. After the antibody was added to the cells, the reaction was performed at 4 ° C. in the dark for 30 minutes. Cells using a biotin-labeled antibody were reacted with Cy-chrome-conjugated streptavidin for 30 minutes at 4 ° C. in the dark. The measurement was performed with EPICS ELITE ESP.

The table below shows the results together with comparative examples.
As shown in the following table, the activation of NKT cells is higher when GSL-7 (P20) is administered than when the control is administered. Furthermore, by administering liposome B containing 1 μg of GSL-7, the higher tree is obtained. Activation of dendritic cells was observed.

Example 10 Dendritic cell activation action (2)
Liposome B (GSL-7 content 1 μg) was administered to C57BL / 6 mice (8-10 weeks old, female) via the tail vein, and 2 hours later, the spleen was removed. Splenocytes were treated with anti-FcγR antibody (2.4G2), and then FITC-labeled anti-CD11c antibody, PE-labeled anti-CD8α antibody and biotin-labeled anti-CD40 antibody were added. After the antibody was added to the cells, the reaction was performed at 4 ° C. in the dark for 30 minutes. Cells using a biotin-labeled antibody were reacted with Cy-chrome-conjugated streptavidin for 30 minutes at 4 ° C. in the dark. The measurement was performed with EPICS ELITE ESP.

The table below shows the results together with comparative examples.
As shown in the table, a significant increase in CD40 expression was observed in CD8α ⁺ cells by addition of GSL-7, and a higher increase in expression was observed by addition of liposome B.

Example 11 In vivo cytokine production induction (measurement of IFN-γ production promotion ability and IL-4 production promotion ability)
GSL-7 (P20) prepared in Example 1 (10 μg) or liposome B (in a GSL-7 content of 10 μg) administered to C57BL / 6 mice (8-10 weeks old, female) from the tail vein Then, serum IFN-γ and IL-4 concentrations after 16 hours were measured using an in vivo capture assay kit (Nippon Becton Dickinson Co., Ltd.).

ＧＳＬ−７（Ｐ２０）またはリポソームＢを投与することにより、ＩＦＮ−γの産生およびＩＬ−４の産生を有意に誘導した。特に、リポソームＢを投与した方が、リポソーム化されていないＧＳＬ−７（Ｐ２０）よりも、顕著に有意に誘導することが確認された。 (result)
The table below shows the results together with comparative examples.

Administration of GSL-7 (P20) or liposome B significantly induced IFN-γ production and IL-4 production. In particular, it was confirmed that administration of liposome B induces significantly more significantly than non-liposomal GSL-7 (P20).

Example 12 NTK cell activation action (1)
(Measurement of NKT cell activation)
GSL-7 (P20) prepared in Example 1 (1 μg) or liposome B (amount that gives GSL-7 content of 1 μg) was administered to C57BL / 6 mice (8-10 weeks old, female) from the tail vein. After 16 hours, the liver and spleen were removed. A cell suspension was prepared from the extracted liver and spleen. In the cell suspension from the liver, mononuclear cells in the liver were obtained by specific gravity centrifugation using 45% percoll and 67.5% percoll. Intrahepatic mononuclear cells and spleen cells were treated with an anti-FcγR antibody (2.4G2) (manufactured by Nippon Becton Dickinson Co., Ltd.), followed by a PE-labeled anti-NK1.1 antibody (manufactured by Nippon Becton Dickinson Co., Ltd.), biotin A labeled anti-TCRαβ antibody (Nippon Becton Dickinson Co., Ltd.) was added. After the antibody was added to the cells, the reaction was performed at 4 ° C. in the dark for 30 minutes. Cells using a biotin-labeled antibody were reacted with Cy-chrome-conjugated streptavidin (manufactured by Zymed) at 4 ° C. in the dark for 30 minutes. The measurement was performed with EPICS ELITE ESP.
Since NTK cells are known to down-regulate NK1.1 molecules on the cell surface when activated, NK1... Is used as an indicator of NKT cell activation by administration of GSL-7 (P20) or liposome B. The expression of one molecule was examined.

リポソームＢを添加することにより、相当量のＧＳＬ−７（Ｐ２０）を投与した場合に比して、著しく高いＮＫＴ細胞の活性化が認められた。
尚、ＮＫＴ細胞の活性化に関し、上記表では、ＧＳＬ−７（Ｐ−２０）の添加した場合、コントロールに対して、有意差が認められていないが、ＧＳＬ−７（Ｐ−２０）の添加量をさらに高くすることにより、コントロールに対して有意差が認められた。 (result)
The table below shows the results together with comparative examples.

By adding liposome B, remarkably high NKT cell activation was observed as compared with the case where a considerable amount of GSL-7 (P20) was administered.
In addition, regarding activation of NKT cells, in the above table, when GSL-7 (P-20) was added, no significant difference was observed with respect to the control, but addition of GSL-7 (P-20) By further increasing the amount, a significant difference was observed with respect to the control.

Example 13 NTK cell activation action (2)
Liposome B (GSL-7 content 1 μg) was administered to C57BL / 6 mice (8-10 weeks old, female) via the tail vein, and 2 hours later, the spleen was removed. Splenocytes were treated with anti-FcγR antibody (2.4G2), and then FITC-labeled anti-TCRβ antibody and biotin-labeled anti-CD40L antibody were added. After the antibody was added to the cells, the reaction was performed at 4 ° C. in the dark for 30 minutes. Cells using a biotin-labeled antibody were reacted with Cy-chrome-conjugated streptavidin for 30 minutes at 4 ° C. in the dark. The measurement was performed with EPICS ELITE ESP.

(result)
The table below shows the results together with comparative examples.
As shown in the table, an increase in CD40L expression in NKT cells was observed by administration of liposome B.

Example 14 Antibody Production Enhancement Action (Preparation of Ovalbumin Encapsulated Liposomes)
Liposomes (liposome F) encapsulating ovalbumin (OVA) (manufactured by SIGMA) were prepared.
The lipid composition was (positive) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (0.1 mol) with respect to 0.1 mol of GSL-7. 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethyleneglycol) -2000] Avanti polar lipids) 0.1 mol, DOTAP 2 mol, DOPE 2 mol.
Lipid F was prepared by adding ovalbumin (OVA) solution (100 μg / ml) to the lipid membrane prepared by the method described in the preparation of liposomes in Example 1 and mixing by vortexing, followed by sonication for 1 minute. Obtained.

(Measurement of antibody production enhancement effect)
C57BL / 6 mice (8-10 weeks old, female) were administered liposome F (25 μg of GSL-7 and 25 μg of OVA) from the tail vein, blood was collected after 14 days, and anti-OVA in serum was collected. The antibody was measured by ELISA. For anti-OVA antibody titer (IgG2a), serially diluted serum was added to a 96-well plate coated with OVA. An anti-mouse IgG2a antibody labeled with alkaline phosphatase (AP) was used as the detection antibody. Color was developed using para-nitrilophenyl phosphate (p-nitrophenyl phosphate) as a substrate, the reaction was stopped with 3N NaOH, and the absorbance at 405 nm was measured with a microplate radar.

(result)
The table below shows the results together with comparative examples.
As shown below, when liposome F was administered, production enhancement of a high anti-OVA antibody was recognized as compared with the case where a mixture of GSL-7 (P20) and OVA that had not been made into liposomes was administered.

Example 15 Induction of antigen-specific IFN-γ production from splenocytes
C57BL / 6 mice (8-10 weeks old, male) were administered with liposome F prepared in Example 14 (amount containing 25 μg of GSL-7) from the tail vein, and one week later, the spleen was removed. . Spleen cells (5 × 10 ⁵ ) Was stimulated with OVA (1 mg / ml), and the amount of IFN-γ in the culture supernatant after 96 hours was measured. The amount of IFN-γ was measured by sandwich ELISA using a purified anti-IFN-γ antibody (R4-6A2) as a capture antibody and a biotin-conjugated anti-IFN-γ antibody (AN-18) as a detection antibody. In the reaction following the biotin-conjugated antibody, alkaline phosphatase-labeled streptavidin was bound, and color was developed using paranitrofukenyl phosphate as a substrate. Absorbance at a wavelength of 405 nm was measured with MICROPLATE READER Model 550. The concentration of IFN-γ in the test sample was calculated from a standard curve with recombinant mouse IFN-γ.

(result)
The table below shows the results together with comparative examples.
As shown in the following table, when liposome F was administered, IFN-γ production from splenocytes was enhanced as compared with the case where a non-liposomal GSL-7 (P20) and OVA mixture was administered.

Example 16 Antitumor immunity enhancing action
Tumor cells expressing OVA as an antigen in C57BL / 6 mice (8-10 weeks old, male); G7-OVA cells were inoculated subcutaneously into the flank of 3 × 10 ⁶ mice, and one week later, when the tumor diameter was 8 to 10 mm, a mixture of GSL-7 (P20) (25 μg) and OVA (25 μg) or Liposome F (GSL-7 content 25 μg and OVA (25 μg)) prepared in Example 14 was administered from the tail vein, and tumor size was measured 20 days later. As a control, a control liposome obtained by removing GSL-7 from the composition of liposome F was used. The tumor volume was calculated using the following formula.
Tumor volume = 0.5 x major axis x (minor axis) ²
The results are shown in the following table. As is clear from the table below, tumor degeneration was observed with the addition of GSL-7 (P20) + OVA, and significant tumor degeneration was observed with the addition of liposome F.

また、これらの写真を図２に示した。
図２中、一列目から順に、コントロール、コントロールリポソームを投与した場合、ＧＳＬ−７（Ｐ２０）とＯＶＡの混合液を投与した場合、リポソームＦを投与した場合を示している。ＧＳＬ−７（Ｐ２０）＋ＯＶＡの添加により、腫瘍の縮退が認められ、リポソームＦの添加により、著しい腫瘍の縮退が認められた。

These photographs are shown in FIG.
In FIG. 2, when control and control liposome are administered sequentially from the first row, a mixture of GSL-7 (P20) and OVA is administered, and liposome F is administered. Tumor degeneration was observed with the addition of GSL-7 (P20) + OVA, and significant tumor degeneration was observed with the addition of liposome F.

In the present invention, it became possible to enhance various immunostimulatory activity abilities of GSL by making GSL into a liposome. For this reason, it is more useful than using conventional GSL as an immunostimulatory activator.
In particular, the present invention is extremely effective in that an immunostimulatory activity effect can be exhibited even with a small amount of GSL that did not show an immunostimulatory activity effect with conventional GSL.
In addition, the liposome of the present invention has an advantage that it also serves as a microencapsulation of GSL. In other words, since the in vivo behavior can be controlled through adjustment of the liposome particle size, lipid composition, charge, etc., GSL can be delivered to the target organ or tissue lesion more efficiently than when administered directly. it can. Therefore, the utility value as a pharmaceutical substance became higher.
Furthermore, since the liposome of the present invention has low toxicity and no endotoxin tolerance is induced, it is useful from the viewpoint of low side effects.

FIG. 1 shows the amount of liposomes taken up by CD11c ⁺ dendritic cells derived from splenocytes. FIG. 2 shows tumor regression with the addition of liposomes.

Claims (18)

1,2-dioleyl-3-lipid transmembrane comprising at least one (trimethylammonium) propane and 3.beta .- [N-(dimethylamino ethane) carbamoyl] cholesterol, is represented by the following formula (1), and, R ¹ is the following R ⁵¹ , R ⁵² , R ⁵³ , R ⁵⁴ , R ⁵⁵ , R ⁵⁶ , R ⁵⁷ , R ⁵⁸ , R ⁵⁹ , R ⁷⁰ , R ⁷¹ , R ⁷² , R ⁷³ , R ⁷⁴ , R ⁷⁵ , R ^76, at least one glycosphingolipids are Ru represented by R ⁷⁷ or R ^78, liposomes containing.
Formula (1)

(In formula (1) , R ² is a hydrogen atom or a group consisting of an α-galactose group, an α-glucose group, an α-mannose group, an α-glucosamine group, a β-glucosamine group, or one or more combinations thereof. is there.)

In the formula (1), R ² consists of a hydrogen atom, an α-galactose group, an α-glucose group, an α-mannose group, an α-glucosamine group, a β-glucosamine group, or a combination of these two or three. The liposome according to claim 1, which is a group. The liposome according to claim 1, wherein, in the formula (1), R ² is a hydrogen atom, the following R ⁶² , R ⁶³ , R ⁶⁴ or R ⁶⁵ .

The liposome according to claim 1, wherein in the formula (1), R ² is a hydrogen atom. The lipid constituting the lipid membrane is such that 1,2-dioleyl-3- (trimethylammonium) propane and / or 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol accounts for 0.4 or more in molar ratio. The liposome according to any one of 1 to 4 . The liposome according to any one of claims 1 to 5, wherein at least one of the lipids constituting the lipid membrane is 1,2-dioleyl-3- (trimethylammonium) propane. The lipids constituting the lipid membrane are 1,2-dioleyl-3- (trimethylammonium) propane, 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphatidyl. The liposome according to any one of claims 1 to 6 , wherein ethanolamine accounts for 0.75 or more in a molar ratio. The liposome according to any one of claims 1 to 7 , further comprising an antigen inside and outside the lipid membrane. The composition for immunostimulatory activity containing the liposome of any one of Claims 1-8 . A composition for promoting IFN-γ production comprising the liposome according to any one of claims 1 to 8 . A composition for promoting IL-4 production comprising the liposome according to any one of claims 1 to 8 . The composition for IL-12 production promotion containing the liposome of any one of Claims 1-8 . An antibody production promoting composition comprising the liposome according to any one of claims 1 to 8 . An antitumor composition comprising the liposome according to any one of claims 1 to 8 . The composition for NKT cell activation containing the liposome of any one of Claims 1-8 . The composition for dendritic cell activation containing the liposome of any one of Claims 1-8 . The antiviral action composition containing the liposome of any one of Claims 1-8 . The antiallergic action composition containing the liposome of any one of Claims 1-8 .

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