JPH09202735A - Liposome-type allergy treating agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject treating agent composed of a liposome containing an antigen immobilized exclusively in the outer water-phase of a specific liposome, containing a sugar in the inner and outer water phases, effective for mitigating allergic diseases, having excellent stability and preservable over a long period. SOLUTION: This treating agent is composed of a liposome containing an antigen immobilized exclusively in the outer water-phase of a liposome containing an amino group-containing phospholipid as a membrane-constituting component and having particle diameter of 0.1-3μm and contains a sugar in the inner and outer water-phases. This liposome-type allergy-treating agent can suppress the production of IgE, increase the formation of IgG and mitigate the allergic symptoms. The ratio of the increment in IgG production to the increment in IgE production is preferably 5-100 times the ratio of the increment in IgG production to the increment in IgE production in the case of single use of the antigen. The allergy antigen is preferably selected from mite antigen, ragweed antigen, orchard grass antigen and cedar pollen antigen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a liposome-type allergy therapeutic agent comprising an antigen-immobilized liposome in which an antigen is immobilized only on the outer aqueous phase side of the liposome, and more particularly to a liposome-type allergy therapeutic agent for hyposensitization therapy. .

[0002]

2. Description of the Related Art Mite antigens, ragweed antigens, Dactylis japonicum antigens, cedar pollen antigens, etc. are known as substances that cause so-called allergic reactions. Desensitization of small doses of aqueous solutions of these antigens to allergic patients. Clinic therapy is given. However, simply administering an aqueous solution of an antigen often causes frequent problems such as frequent administration, and a significant effect cannot always be obtained.

The allergic reaction is a type of antibody, IgE
It is said that the production of IgG is suppressed by suppressing the production of IgE as a therapeutic drug for allergy, although it has been developed. For example, modifying the antigen protein with polyethylene glycol (Int. Arc
h. Allergy Appl. Immunol., 56. p159-170, 1978),
Antigen is polymerized with glutaraldehyde (Int. Arch.
Allergy Appl. Immunol., 74, p332-340, 1984) has been attempted, but there are problems such as insufficient production of IgG against the antigen. In addition, a vaccine in which albumin is reacted with the polysaccharide pullulan is known (Int. Arch. Allergy Appl. Immunol., 102, p276-27.
8, 1993), but still insufficient IgG production.

A method of encapsulating an antigen in a liposome has also been tried (Clinical and Experimental Allerg).
y, Vol. 22 p35-42, 1992), IgE production can be suppressed to about a fraction of that in the case where the antigen is not encapsulated. And the treatment method of allergic patients using this technology is known (DE 3412793). Ie DE34
12793 is a method of orally administering inhaled antigens that are known to cause allergic symptoms by encapsulating them in liposomes, which are absorbed from Peyer's patches of the intestine and enter the bloodstream via lymphatic vessels. In this case, the amount of IgE produced is about 1/30 in the liposome type compared to the aluminum hydroxide type.
Has fallen to. However, there is a problem that the antigen may leak from the liposome depending on the liposome composition, and the leaked antigen binds to IgE and causes an allergic symptom.

Further, USP5049390 describes an immunotherapeutic agent in which an allergen is bound to liposomes, and it is described that the immunotherapeutic agent can suppress the production of IgE and increase the production of IgG. However, in the above publication, I
The quantitative production amount of gG and IgE is not described, and the production of IgG and IgE in vitro is only qualitatively shown, and the therapeutic effect on allergy is insufficient.

[0006]

SUMMARY OF THE INVENTION The object of the present invention is to improve
Suppresses the production of E, increases the production of IgG, can alleviate allergic symptoms, and has excellent stability,
It is intended to provide a liposome-type therapeutic agent for allergy which can be stably stored for a long time by freeze-drying.

[0007]

Means for Solving the Problems The present invention is the following liposome type allergy therapeutic agent. (1) An allergy therapeutic agent comprising a liposome having a phospholipid having an amino group as a membrane constituent and having an antigen immobilized only on the outer aqueous phase side of a liposome having a particle size of 0.1 to 3 μm, A liposome-type therapeutic agent for allergy, which comprises a sugar in its internal and external aqueous phases. (2) The ratio of the IgG production enhancing amount to the IgE production enhancing amount is 5 to 100 times the ratio of the IgG production enhancing amount to the IgE production enhancing amount of the antigen alone, and the above (1) is described. Liposomal allergy remedy. (3) The liposome-type therapeutic agent for allergy according to the above (1) or (2), wherein the antigen is an allergic antigen that causes allergy. (4) The liposome-type therapeutic agent for allergy according to the above (3), wherein the allergic antigen is an allergic antigen selected from the group consisting of mite antigen, ragweed antigen, Dactylis antigen and cedar pollen antigen.

Examples of the phospholipid having an amino group which can be used in the present invention include phosphatidylethanolamine, phosphatidylserine, phosphatidylthreonine and the like, and they can be used alone or in combination of two or more kinds. In these phospholipids, two fatty acid residues at the 1st and 2nd positions can be arbitrarily selected, and the fatty acid residue may be derived from a natural product or a synthetic product, mixed fatty acid, saturated fatty acid, Examples thereof include fatty acid residues having 4 to 30 carbon atoms, which are derived from unsaturated fatty acids and polymerizable fatty acids.

The saturated fatty acid has 12 to 24 carbon atoms.
Those of, for example, lauric acid, myristic acid,
Examples thereof include palmitic acid and stearic acid. The unsaturated fatty acid preferably has 14 to 22 carbon atoms and 1 to 6 unsaturated bonds, and examples thereof include oleic acid, linoleic acid, linolenic acid and arachidonic acid. Among such phospholipids having an amino group, egg yolk- or soybean-derived phospholipids are preferable as natural products.

As the membrane-constituting component (membrane-forming component) of the liposome, other compounds capable of forming liposomes can be used in addition to the phospholipid having an amino group, and examples thereof include soybean lecithin, egg yolk lecithin, phosphatidylglycerol, and others. Phospholipids, cholesterol, fatty acids, fatty acid salts and the like which have been conventionally used as a membrane constituent component of liposomes can be used.

The proportion of the amino group-containing phospholipid in the entire membrane constituents is 0.01 to 100 mol%, preferably 0.1 to 30 mol%. The larger the proportion of the phospholipid having an amino group, the larger the amount of the antigen that can be immobilized on the outer aqueous phase side of the liposome. Therefore, by adjusting the ratio of the phospholipid having an amino group, the amount of the antigen immobilized on the outer aqueous phase side can be adjusted.

As the antigen immobilized only on the outer aqueous phase side of the liposome, an antigen causing allergies can be used. Specifically, house dust; mite antigens; ragweed antigens, duckweed antigens, cedar pollen antigens, pollens such as mugwort, cannabis, himejima; grains such as rice, flour, buckwheat flour; foods such as milk, egg yolk, and egg white Epidermis such as dog hair, cat hair, and feathers; allergen antigens such as fungi such as Candida and Aspergillus. Among them, the mite antigen, ragweed antigen, Dactylis rumen antigen, or cedar pollen antigen can be preferably used. The above-mentioned antigens can be used alone or in combination of two or more.

The liposomes before immobilization of the antigen are exudation method, vortex mixer method, ultrasonic method, surfactant removal method, reverse phase evaporation method, ethanol injection method, prevesicle method, French press method, W / It can be produced by various known methods such as an O / W emulsion method, an annealing method, and a freeze-thaw method. In addition, by selecting these production methods, multilamellar liposomes,
It is possible to produce liposomes having various sizes and morphologies such as small unilamellar vesicles and large unilamellar vesicles. Any form of liposomes can be used in the present invention. By the above method, a liposome having amino groups on the inner and outer aqueous phase sides (outer surface, inner surface and inner surface) of the liposome can be obtained. The particle size of the liposome is 0.
It is 1 to 3 μm, preferably 0.2 to 2.5 μm. If the particle size of the liposome exceeds the above upper limit, it will gel and cannot be used as the therapeutic agent of the present invention.

To immobilize the antigen only on the outer aqueous phase side of the liposome having the amino group obtained as described above, the amino group of the phospholipid such as phosphatidylethanolamine and the antigen are used with glutaraldehyde. A known method such as a method of directly cross-linking with the amino group of 1), a method of chemically bonding the amino group of the phospholipid such as phosphatidylethanolamine and the amino group of the antigen using a reaction activator can be employed.

As the above-mentioned reaction activating reagent, N-hydroxysuccinimidyl 3- (2-pyridythio) propionate is used.
ionate), m-maleimidobenzoyl-N-hydro
xysuccinimide ester), dithiobis (succinimidylpropiona)
te), bis (sulfosuccinimidyl) suberate (Bi
s (sulfosuccinimidyl) suberate), disuccinimidyl suberate and the like.

The antigen immobilization reaction using glutaraldehyde is represented by the following formula (1).

Embedded image

The immobilization reaction of the antigen is carried out in an aqueous solvent such as distilled water; physiological saline; various buffer solutions such as phosphate buffer solution, carbonate buffer solution, Tris buffer solution and acetate buffer solution; , A mixed solvent with an organic solvent such as ethanol, methanol, and 1,4-dioxane, pH 1 to 12, preferably pH 7 to 10, reaction temperature 0 to 100 ° C., preferably 0 to 60 ° C., reaction time 30 minutes ~ 200 hours, preferably 1 to 24 hours.

By carrying out the reaction in this manner, the amino group on the outer aqueous phase side of the liposome and the amino group of the antigen are covalently bound via the residue of glutaraldehyde or the active reagent, and An antigen-bound liposome in which the antigen is immobilized only on the aqueous phase side can be obtained. After completion of the reaction, a compound having an amino group such as glycine is added to deactivate unreacted glutaraldehyde or an active reagent, and then it is easily separated by a method such as gel filtration, dialysis, ultrafiltration or centrifugation. It can be separated and purified. Further, it can be dried and stored by a method such as freeze-drying, and can be regenerated as necessary.

The liposome-type therapeutic drug for allergies of the present invention is an allergy consisting of liposomes in which an antigen is immobilized only on the external water phase side, and a liposome is contained in the internal and external aqueous phases of the liposome, that is, the internal aqueous phase and the external aqueous phase. It is a therapeutic drug. Examples of sugars contained in the aqueous phase of the liposome include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; disaccharides such as saccharose, lactose, cellobiose, trehalose and maltose; and raffinose and melezitose. Examples include trisaccharides; oligosaccharides such as cyclodextrin; polysaccharides such as dextrin; sugar alcohols such as xylitol, sorbitol, mannitol and maltitol.
Of these, monosaccharides or disaccharides are preferable, and glucose or saccharose is particularly preferable.

The concentration of sugar contained in the internal and external aqueous phases of the liposome is 0.05 to 0.5M, preferably 0.15 to 0.35.
It is preferably M. In this case, as the solvent for dissolving the sugar, an aqueous solvent such as distilled water; physiological saline; a buffer solution such as a phosphate buffer solution, a carbonate buffer solution, a Tris buffer solution, an acetate buffer solution, or the like can be used. It is desirable that the pH of such an aqueous solvent is 5 to 10, preferably 6 to 8.

The method of incorporating the sugar into the aqueous phase of the liposome is not particularly limited, and known methods can be employed. For example, a method in which the sugar is dissolved in advance at the time of liposome preparation is used, or the antigen is immobilized on the liposome to form the antigen. After preparing the bound liposome, a method of incorporating the sugar added to the outer aqueous phase also into the inner aqueous phase by using an osmotic pressure gradient method or the like can be adopted. Among these, the method of using a solution in which sugar is dissolved in advance at the time of liposome preparation is preferable.

Since the antigen for allergy treatment of the present invention has an antigen bound only to the outer aqueous phase side of the liposome, the use of such a treatment drug causes IgE that exacerbates allergic symptoms.
Production is suppressed, and IgG production is enhanced.
Therefore, the IgG production enhancement rate (the ratio of the secondary immunity to the primary immunity) to the IgE production enhancement rate (the ratio of the secondary immunity to the primary immunity) (2 to the primary immunity)
Ratio of secondary immunity), ie, IgG production enhancement rate / IgE
The production enhancement rate ratio is large, and the titer ratio is 5 to 100, as compared with the case of using an aqueous solution of the antigen alone.
It is doubled and is suitably used as an allergy therapeutic drug for hyposensitization therapy of allergic patients. In addition, the therapeutic agent for allergy of the present invention has excellent stability because it contains sugar in the inner and outer aqueous phases of liposomes having a specific particle size, and it is aggregated even if the liposomes are lyophilized and then regenerated with an aqueous solvent. Does not occur. Therefore, freeze-drying enables stable storage for a long period of time.

The titer ratio value is the production ratio when blood IgG and IgE are measured by the following ELISA method. ELISA method (IgG antibody detection): 1) Coating of plate with antigen The antigen was dissolved in 0.05M carbonate buffer (pH 9.0) at a concentration of 1 mg / ml, and 50 μl / well was divided into 96-well assay plates. Pour and leave at room temperature for 1 hour. 2) Plate blocking 0.2M bovine serum albumin (hereinafter referred to as BSA)
Dissolve it in a phosphate buffer (pH 7.2: PBS) at a concentration of 1 mg / ml, dispense 100 μl / well into the plate of 1) above, and leave at room temperature for 1 hour. 3) Dilution and addition of serum sample (primary antibody) PBS containing BSA at a concentration of 1 mg / ml (hereinafter, PB
Mouse serum immunized with the aluminum hydroxide-antigen or the allergy therapeutic agent of the present invention in SA) is diluted 11 times in a 2-fold step starting from a 10-fold dilution, and 2) above.
Dispense 50 μl / well into each plate and leave at room temperature for 1 hour. 4) Addition of peroxidase-labeled rabbit anti-mouse IgG antibody solution (secondary antibody) After washing the plate of 3) above three times with PBS, 50 μl / well of PBSA solution of peroxidase-labeled rabbit anti-mouse IgG antibody was dispensed. Leave at room temperature for 1 hour. 5) Addition of enzyme substrate solution After washing the plate of 4) above three times with PBS, 100 μl / well of o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed. Let stand for 15 minutes at room temperature to develop color. After coloring
The reaction is stopped by dispensing 50 μl / well of 2M sulfuric acid. 6) Measurement using an absorptiometer Using an ELISA plate reader, measurement is performed at 490 nm absorbance, the color development end point is determined, and the average dilution ratio of the sample serum at that point is used for EL measurement.
Use as ISA titer (ELISA titer is used as the quantitative value of IgG).

ELISA method (IgE antibody detection): 1) Coating of plate with rat monoclonal antibody mouse IgE antibody Rat monoclonal antibody against mouse IgE antibody was added at 4 μg / ml in 0.05M carbonate buffer (pH 9.0).
96-well assay plate adjusted to the concentration of 1
Dispense by 00 μl / well and leave at 37 ° C. for 3 hours. 2) Blocking of plate Bovine serum albumin (BSA) was dissolved in 0.2 M phosphate buffer (pH 7.2: PBS) at a concentration of 1 mg / ml, and 100 μl / well was dispensed to the plate of 1) above. Leave at room temperature for 1 hour. 3) Dilution and addition of serum sample (primary antibody) PBS containing BSA at a concentration of 1 mg / ml (PBSA)
In this, mouse sera immunized with aluminum hydroxide-antigen or the therapeutic agent of the present invention was diluted 11 times in 2-fold steps starting from 10-fold dilution, and 50 μl / well was added to the plate of 2) above.
Dispense wells by well and leave at room temperature for 1 hour. 4) Addition of biotinylated antigen After washing the plate of 3) above with PBS three times, a PBSA solution (1 μg / ml) of the biotinylated antigen solution was added to the plate.
Dispense by 00 μl / well and leave at room temperature for 1 hour. 5) Addition of peroxidase-labeled streptavidin solution After washing the plate of 4) above three times with PBS, 100 μl of peroxidase-labeled streptavidin solution was added.
Dispense wells by well and leave at room temperature for 1 hour. 6) Addition of enzyme substrate solution After washing the plate of 5) above with PBS three times, 100 μl / well of o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed. Let stand for 15 minutes at room temperature to develop color. After coloring
The reaction is stopped by dispensing 50 μl / well of 2M sulfuric acid. 7) Measurement using an absorptiometer Using an ELISA plate reader, measurement is performed at 490 nm absorbance, the color development end point is determined, and the average dilution ratio of the sample serum at that point is used to calculate the EL.
ISA titer (ELISA titer is used as the quantitative value of IgE).

[0025]

INDUSTRIAL APPLICABILITY Since the liposome type allergy therapeutic agent of the present invention has the antigen immobilized only on the outer aqueous phase side of the liposome, it can suppress the production of IgE and increase the production of IgG. Therefore, allergic symptoms can be relieved, and it can be suitably used as an allergy therapeutic agent for hyposensitization therapy. Further, since it has a specific particle size and contains sugar in the inner and outer aqueous phases, it is highly stable even by freeze-drying and can be stably stored for a long period of time.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION Next, embodiments of the present invention will be described. Reference Example 1 (Preparation of liposome) Dipalmitoylphosphatidylcholine 0.9175 g
(1.25 mmol), dipalmitoylphosphatidylethanolamine 0.6490 g (0.938 mmo)
l), cholesterol 0.8445 g (2.19 mmo)
1) and 0.4305 g (0.625 mmol) of Na salt of dimyristoylphosphatidylglycerol are placed in an eggplant-shaped flask, and chloroform / methanol / water (65
/ 25/4, volume ratio) Add 50 ml of mixed solvent, 40 ℃
Was dissolved. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Further, 30 ml of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen with liquid nitrogen and dried with a freeze dryer for 24 hours.

Next, a separately prepared buffer solution (0.12 mM
Na ₂ HPO ₄ , 0.88 mM KH ₂ PO ₄ , 0.25
60 ml of M sucrose, pH 6.5) was placed in the eggplant-shaped flask, and the lipid was hydrated while stirring at 40 ° C. to obtain a liposome. Next, the particle size of the liposome was adjusted using an Extruder. First pass 8μm polycarbonate filter, then 5μm, 3μ
m, 1 μm, 0.65 μm, 0.22 μm in this order. 1 μm, 0.65 μm, 0.22 μ
A sample at the time of passage of m was taken and subjected to the test of the example. In addition, when the particle size of the liposome was measured by a dynamic light scattering particle size distribution meter (NICOMP model 370HPL, manufactured by Pacific Scientific, trademark), the average particle size was 1 μm, 0.65 μm, 0.22 μm sample. At 1.428 μm, 0.664 μm, 0.
It was 167 μm.

Example 1 An antibody production test was carried out as follows using a mite antigen. Preparation of aluminum hydroxide-mite antigen vaccine Aluminum hydroxide 15 mg / ml in PBS [A
l (OH) ₃ precipitate was applied to a homogenizer to form an emulsion], and the final concentration of mite antigen was 10 μg / m 2.
It mixed so that it might become 1 and the aluminum hydroxide mixed vaccine was prepared. This vaccine was immunized by intraperitoneal injection of 200 μl per mouse.

Preparation of mite antigen-binding liposome 1 μm, 0.65 μm, 0.2 with the liposome of Reference Example 1
2 ml of each 2 μm-passed product was collected in a test tube,
5 ml of mite antigen solution (10 mg / ml) was added. Next, 0.5 ml of a 2.4% glutaraldehyde solution was added dropwise, and the mixture was gently mixed on a warm bath at 37 ° C. for 1 hour.
The mite antigen was immobilized on the outer aqueous phase side of the liposome. Then 2
0.5 g of M glycine-NaOH buffer (pH 7.2)
1 was added and the solution was left at 4 ° C. overnight to deactivate unreacted glutaraldehyde. In addition, Sepharose CL-4B (Ph
This solution was passed through a column packed with armacia Biotech (trademark), and liposomes having mite antigen bound to the outer aqueous phase side via glutaraldehyde were fractionated to obtain antigen-bound liposomes.

Antibody Production Test The vaccine-administered mice prepared as described above were divided into three groups, and after 4 weeks, a group as it was (control group) and a group in which 100 μg of mite antigen solution was intraperitoneally injected as a secondary immunization group. (Mite antigen single administration group), or the mite antigen-bound liposomes prepared above in a range of 0.5 to 2.0 mg as the lipid amount of liposome
A group injected intraperitoneally in an amount of μl (administration group of mite antigen-binding liposome-type allergy therapeutic agent, hereinafter sometimes abbreviated as liposome agent administration group).

Then, antigen-specific IgG in mouse serum
Antibodies and IgE antibodies were quantified by ELISA as described below. Then, with respect to the IgG and IgE values of the control group, the IgG and IgE values changed in the secondary immunization group were calculated as the enhancement rate from the following mathematical formula (1) or (2). In addition, the ratio of the IgG enhancement rate to the IgE enhancement rate, which is an index of desensitization, was calculated and shown in Table 1 together with all the values.

[0032]

## EQU00001 ## IgG enhancement rate = IgG value after secondary immunization / IgG value of control ... (1) IgE enhancement rate = IgE value after secondary immunization / IgE value of control ... (2)

IgG antibody detection IgG antibody detection was carried out by the ELISA method as follows. 1) Coating of plate with antigen Mite antigen was dissolved in 0.05M carbonate buffer solution (pH 9.0) at a concentration of 10 μg / ml, and 50 μl / well was dispensed in a 96-well assay plate, and the temperature was adjusted to 1 at room temperature. Left for hours. 2) Blocking of plate Bovine serum albumin (BSA) was dissolved in 0.2 M phosphate buffer (pH 7.2: PBS) at a concentration of 1 mg / ml, and 100 μl / well was dispensed to the plate of 1) above. It was left at room temperature for 1 hour. 3) Dilution and addition of serum sample (primary antibody) In PBS (PBSA) containing BSA at a concentration of 1 mg / ml, with mouse serum of control group, mouse serum enhanced with mite antigen solution, or antigen-bound liposome The enhanced mouse sera were diluted 11 times in 2-fold steps starting from 10-fold and added to the plate in 2) above at 50 μl /
Each well was dispensed and left at room temperature for 1 hour. 4) Addition of peroxidase-labeled rabbit anti-mouse IgG antibody solution (secondary antibody) After washing the plate of 3) above three times with PBS, 50 μl / well of PBSA solution of peroxidase-labeled rabbit anti-mouse IgG antibody was dispensed. It was left at room temperature for 1 hour. 5) Addition of enzyme substrate solution After washing the plate of 4) above with PBS three times, 100 μl / well of o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed. The sample was left at room temperature for 15 minutes to develop color. After coloring
The reaction was stopped by dispensing 50 μl / well of 2M sulfuric acid. 6) Measurement using an absorptiometer Using a plate reader for ELISA, measurement is performed at an absorbance of 490 nm, the endpoint of color development is determined, and the ELISA of the dilution ratio of the sample serum at that point is determined.
A titer (ELISA titer was used as a quantitative value for IgG).

Detection of IgE antibody IgE antibody was detected by the ELISA method as follows. 1) Coating of plate with rat monoclonal antibody mouse IgE antibody 0.0.
The concentration adjusted to 4 μg / ml with 5 M carbonate buffer solution (pH 9.0) was added to a 96-well assay plate to give 100 μm.
Each 1 / well was dispensed and left at 37 ° C. for 3 hours. 2) Blocking of plate Bovine serum albumin (BSA) was dissolved in 0.2 M phosphate buffer (pH 7.2: PBS) at a concentration of 1 mg / ml, and 100 μl / well was dispensed to the plate of 1) above. It was left at room temperature for 1 hour. 3) Dilution and addition of serum sample (primary antibody) In PBS (PBSA) containing BSA at a concentration of 1 mg / ml, the mouse serum of the control group and the mouse serum enhanced with the mite antigen solution were diluted 10-fold to 2 11 in double stage
Diluted once, dispensed 50 μl / well to the plate of 2) above, and left at room temperature for 1 hour. 4) Addition of biotinylated antigen After washing the plate of 3) above with PBS three times, a PBSA solution (1 μg / ml) of the biotinylated antigen solution was added to the plate.
It was dispensed by 00 μl / well and left at room temperature for 1 hour. 5) Addition of peroxidase-labeled streptavidin solution After washing the plate of 4) above three times with PBS, 100 μl of peroxidase-labeled streptavidin solution was added.
Each well was dispensed and left at room temperature for 1 hour. 6) Addition of enzyme substrate solution After washing the plate of 5) above with PBS three times, 100 μl / well of o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed. The sample was left at room temperature for 15 minutes to develop color. After coloring
The reaction was stopped by dispensing 50 μl / well of 2M sulfuric acid. 7) Measurement using an absorptiometer Using an ELISA plate reader, measurement is performed at 490 nm absorbance, the color development end point is determined, and the average dilution ratio of the sample serum at that point is used for EL measurement.
It was used as an ISA titer (ELISA titer was used as a quantitative value of IgE).

[0035]

[Table 1]

Note to Table 1 * 2 Ratio of enhancement rate: IgG enhancement rate / IgE enhancement rate * 3 C / B: The value of IgG enhancement rate / IgE enhancement rate of liposome administration group (column C) is the mite antigen alone administration group (column B).
Value divided by the value of IgG enhancement rate / IgE enhancement rate. * 4 Mite antigen-binding liposome type allergy therapeutic drug administration group

Example 2 The procedure of Example 1 was repeated, except that the cedar pollen antigen was used in place of the mite antigen, and the preparation method and test method were changed as follows. Table 2 shows the results.

Preparation of aluminum hydroxide cedar pollen vaccine The preparation was carried out in the same manner as in Example 1 except that cedar pollen antigen (SBP) was used in place of the mite antigen. Preparation of cedar pollen antigen-binding liposomes The concentration of the antigen solution was 10 mg / ml to 2.9 mg / ml.
Was prepared in the same manner as in Example 1 except that

Antibody Production Test The vaccine-administered mice prepared above were divided into 3 groups, and 4 weeks later, the same group (control group) was injected intraperitoneally with 100 μg of a cedar pollen antigen solution as a secondary immunization group. Group (cedar pollen antigen alone administration group) or a group in which the cedar pollen antigen-binding liposomes prepared above were intraperitoneally injected in an amount of 0.5 to 2.0 mg per animal intraperitoneally (200 μl / mouse). The combined liposome-type allergy therapeutic agent administration group, hereinafter may be abbreviated as the liposome agent administration group). Thereafter, the antigen-specific IgG antibody and IgE antibody in the mouse serum were quantified by the ELISA method. Then, with respect to the IgG and IgE values of the control group, the IgG and IgE values changed in the secondary immunization group were calculated as the enhancement rate from the above formula (1) or (2).

Detection of IgG antibody 1) The concentration of the antigen used in the operation of coating the plate with the antigen of 10 μg / ml to 5 μg / m
The same procedure as in Example 1 was repeated except that the amount was changed to 1. Detection of IgE antibody The same procedure as in Example 1 was performed.

[0041]

[Table 2]

Note to Table 2 * 2 Ratio of enhancement rate: IgG enhancement rate / IgE enhancement rate * 3 C / B: The value of IgG enhancement rate / IgE enhancement rate of the liposome agent administration group (column C) is the cedar pollen antigen alone administration group (B
Value divided by the value of IgG enhancement rate / IgE enhancement rate in the column). * 4 Japanese cedar pollen antigen-binding liposome type allergy therapeutic drug administration group

Comparative Example 1 For comparison, an example of a mixed antibody production test in which a mite antigen is encapsulated in liposomes is shown. 0.321 g (0.437 mmol) of dipalmitoylphosphatidylcholine, 0.1689 g (0.438 mmol) of cholesterol and 0.0861 g (0.125 mmol) of dimyristoylphosphatidylglycerol Na salt were placed in an eggplant-shaped flask, and chloroform / methanol (2/1 , Volume ratio) 30 ml of a mixed solvent was added and dissolved at 40 ° C. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Further, 30 ml of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen with liquid nitrogen and then freeze-dried with a freeze dryer.
It was dried for a time to obtain a powder.

0.1440 g of this powder was placed in a round bottom flask and 5 ml of an aqueous solution of mite antigen (12.5 mg / ml) was added.
Was added, and the lipid was hydrated while stirring at 40 ° C. Next, the particle size of the liposome was adjusted using an extruder. First, a 5 μm polycarbonate filter was passed, and then 3 μm and 1 μm were passed in this order. A sample having a passage of 1 μm was passed through a column packed with Sepharose CL-4B (trademark, manufactured by Pharmacia Biotech), and mite antigen-encapsulating liposomes were divided and collected. When the particle size of the liposome having a particle size of 1 μm was measured in the same manner as in Reference Example 1, the average particle size was 1.382 μm.

An antibody production test was conducted in the same manner as in Example 1 except that the mite antigen-encapsulating liposome was used in place of the mite antigen-bound liposome type allergy therapeutic agent in Example 1. The results are shown in Table 3.

[0046]

[Table 3]

Note to Table 3 * 1 to * 3 See Table 1 * 4 Mite antigen-encapsulating liposome administration group

From the results of Tables 1 to 3, inclusion of the antigen in the liposome promotes the production of IgE,
It can be seen that the difference from Examples 1 and 2 in which the IgE value is not increased is clear.

Comparative Example 2 For comparison, an example of the storage stability test of a sugar-free liposome is shown. Preparation of liposomes Dipalmitoylphosphatidylcholine 0.9175g
(1.25 mmol), dipalmitoylphosphatidylethanolamine 0.6490 g (0.938 mmo)
l), cholesterol 0.8445 g (2.19 mmo)
1) and 0.4305 g (0.625 mmol) of Na salt of dimyristoylphosphatidylglycerol are placed in an eggplant-shaped flask, and chloroform / methanol / water (65
/ 25/4, volume ratio) Put 50ml mixed solvent at 40 ℃
Was dissolved. Next, phosphate buffer (pH: 7.2) 10
ml was added and the solvent was distilled off under reduced pressure using a rotary evaporator to prepare a gel. After shaking for 1 minute with a vortex mixer, phosphate buffer solution 50m
1 was put in the flask and stirred at 40 ° C. for 10 minutes.
Next, the particle size of the liposome was adjusted using an extruder. First, it was passed through a polycarbonate filter of 8 μm, and then passed through 5 μm, 3 μm, and 1 μm in this order.

Preparation of Antigen-Binding Liposomes 2 ml each of 8 μm, 5 μm, and 3 μm-passed products of the above liposomes were collected in a test tube, and 0.5 ml of mite antigen solution (12 mg / ml) was added. Next, 0.5 ml of a 2.4% glutaraldehyde solution was added dropwise, and then 3
Mix gently for 1 hour on a 7 ° C warm bath. As a result, each liposome aggregated. When sugar was included, only some liposomes aggregated, but when sugar was not added, 8 μm
All were aggregated with liposomes from m to 3 μm.

Next, a mite antigen-binding liposome was prepared using a small size 1 μm-passing product. That is, 2 ml of the above liposome that passed through 1 μm was collected in a test tube, and 0.5 ml of a mite antigen solution (12 mg / ml) was put therein. Then 0.5% of 2.4% glutaraldehyde solution
After dropwise adding ml, the mixture was gently mixed on a warm bath at 37 ° C. for 1 hour. In this case as well, aggregation of liposomes was slightly observed, but the reaction proceeded as it was. That is, 0.5 ml of 2 M glycine-NaOH buffer (pH 7.2) was added and the solution was left at 4 ° C. overnight to deactivate unreacted glutaraldehyde. In addition, Sepharose CL-4B (Pharmacia
This solution was passed through a column packed with Biotech (trademark), and liposomes having mite antigen bound to the liposome surface on the outer aqueous phase side via glutaraldehyde were fractionated to obtain antigen-bound liposomes.

Storage stability test 5 ml each of the antigen-bound liposomes (product using phosphate buffer solution) prepared above and the antigen-bound liposomes (product using sugar-containing solution) prepared in Example 1 were sampled and frozen in liquid nitrogen. After that, it was freeze-dried for 6 hours. Next, 5 ml of distilled water for injection was added, and the shape of the liposome was observed.
For Example 1 in which no aggregation occurred after the condensate, the particle size was measured in the same manner as in Example 1. Table 4 shows the results
Shown in

[0053]

[Table 4] Evaluation criteria ◎: No aggregation of liposomes ×: Some aggregation of liposomes

As can be seen from the results shown in Table 4, the liposome of Comparative Example 2 which did not use sugar easily aggregated, which caused difficulty in preparation. Further, it can be seen that the liposome aggregates due to the interaction of the proteins through the freeze-drying treatment. On the other hand, in the liposome of Example 1, addition of sugar can prevent aggregation (3 μm or less) at the time of preparation, and the liposome containing sugar in the internal and external aqueous phases does not aggregate even if freeze-dried. It does not occur, and it can be seen that the stability is excellent.

Comparative Example 3 For comparison, an example of a storage stability test of a liposome in which an antigen is immobilized on both the inner and outer phases and which does not contain sugar is shown. Preparation of Antigen-Binding Phospholipid Dipalmitoylphosphatidylethanolamine 0.0
649 g (0.0938 mmol) was placed in an eggplant-shaped flask, and chloroform / methanol / water (65/25 /
(4, volume ratio) 20 ml of a mixed solvent was added and dissolved at 40 ° C. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to prepare a lipid thin film. Further 1.
5 ml of mite antigen solution (12 mg / ml) was added, and then 1.5 ml of 2.4% glutaraldehyde solution was added dropwise, followed by gentle mixing for 1 hour in a 37 ° C warm bath. Then, 1.5 ml of 2 M glycine-NaOH buffer (pH 7.2) was added, and the solution was left at 4 ° C. overnight to deactivate unreacted glutaraldehyde. Furthermore Sepharose
This solution was passed through a column packed with CL-4B (trademark, manufactured by Pharmacia Biotech) to fractionate a fraction to which a mite antigen was bound via glutaraldehyde.

Preparation of liposome having mite antigen bound to internal and external phases Dipalmitoylphosphatidylcholine 0.09175 g
(0.125mmmol), cholesterol 0.084
45 g (0.219 mmol) and dimyristoyl phosphatidyl glycerol NaOH salt 0.04305 g
(0.0625 mmol) was placed in an eggplant-shaped flask, and 20 ml of a mixed solvent of chloroform / methanol / water (65/25/4, volume ratio) was added and dissolved at 40 ° C.
Next, the solvent was distilled off under reduced pressure using a rotary evaporator to prepare a lipid thin film. Further, the glutaraldehyde-mediated mite antigen-binding phospholipid solution prepared above was added. Stir with a vortex mixer (3
After 0 seconds), the particle size of the liposome was adjusted using an extruder. First, a 5 μm polycarbonate filter was passed, and then 3 μm, 1 μm, and 0.65 μm were passed in this order. The particle size of these liposomes was measured in the same manner as in Reference Example 1. Table 5 shows the results.

Storage stability test About 2 ml of liposomes containing mite antigen bound to the inner and outer phases and containing no sugar and having a particle size of each were taken, frozen in liquid nitrogen and dried for 6 hours. Next, 2 ml each of distilled water for injection was added, and the shape of liposomes was observed. Table 5 shows the results.

[0058]

[Table 5] Evaluation criteria ◎: No aggregation of liposomes ×: Some aggregation of liposomes

From the results in Table 5, it can be seen that the stability of the liposome is impaired by freeze-drying storage even if the antigen is bound to the inner and outer phases when the sugar is not contained.

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Claims (4)

[Claims] 1. An allergy therapeutic agent comprising a liposome having an amino group-containing phospholipid as a membrane constituent and having an antigen immobilized only on the outer aqueous phase side of a liposome having a particle size of 0.1 to 3 μm. And a liposome-type therapeutic agent for allergy, characterized in that the internal and external aqueous phases contain sugar. 2. The ratio of the amount of enhanced IgG production to the amount of enhanced IgE production is the Ig to the enhanced amount of IgE production of the antigen alone.
The liposome-type therapeutic agent for allergy according to claim 1, which is 5 to 100 times the ratio of the G production enhancing amount. 3. The liposome-type therapeutic agent for allergy according to claim 1, wherein the antigen is an allergic antigen that causes allergy. 4. The allergic antigen is an allergic antigen selected from the group consisting of mite antigen, ragweed antigen, Dactylis antigen and cedar pollen antigen.
The liposome-type allergy therapeutic agent described.