JPH05229960A - New influenza vaccine - Google Patents

Abstract

PURPOSE:To obtain an influenza vaccine having excellent antibody-forming ability and safety, comprising a complex of a glutamamide derivative and an influenza virus. CONSTITUTION:A compound of the formula (R is 2-6C acyl; X is L-alanine residue, L-serine residue, L-valine residue or glycine residue; A is 10-60C fatty acid residue; R1 and R2 are H, lower alkyl, aryl, aralkyl, amino, etc.) or its salt is blended with an influenza antigen and optionally a phospholipid in a proper buffer solution such as a phosphoric acid buffer solution, solubilized with a surfactant and the surfactant is removed by dialysis to give the objective substance. This influenza vaccine has the same or more excellent antibody- forming ability than the conventional virosome vaccine, is extremely excellent in terms of safety, flare and fever, has excellent stability in a preserved state and can be kept in cold storage and freeze-dried.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an influenza vaccine comprising a complex of a compound represented by the general formula (I) described below or a salt thereof and an antigen of influenza virus. The vaccine of the present invention has excellent antibody-producing ability and safety as a vaccine.

[0002]

2. Description of the Related Art Influenza HA currently used
Since the effectiveness of the vaccine is not stable due to mutation on the hemagglutinin molecule of the epidemic virus, the development of a more effective vaccine is strongly desired. As an attempt to do so, a component vaccine containing HA (hemagglutinin) and NA (neuraminidase) as main components, and 6-0- (2
-Tetradecylhexadecanoyl) -N-acetylmuramoyl-L-alanyl-D-isoglutamine, an influenza virus particle-like artificial membrane vaccine, so-called virosome vaccine (see Japanese Patent Laid-Open No. 61-282321).
Are known. In particular, the latter is known to have an excellent effect in terms of improving the antibody titer in blood, and was expected to be useful.

[0003] However, this vaccine may cause redness as an undesirable side effect due to local irritation at the administration site, and was not necessarily satisfactory as a pharmaceutical product.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an influenza vaccine having excellent antibody-producing ability and safety as a vaccine.

[0005]

The present inventors have completed the present invention as a result of intensive studies to solve the above problems. That is, the present invention has the general formula (I)

[0006]

[Chemical 2]

[In the formula, R represents an acyl group having 2 to 6 carbon atoms,
X represents an L-alanine residue, L-serine residue, L-valine residue or glycine residue, A represents a fatty acid residue having 10 to 60 carbon atoms, and R ₁ and R ₂ independently represent hydrogen. Atom, lower alkyl group optionally having substituent (s), cycloalkyl group optionally having substituent (s), aryl group optionally having substituent (s), aralkyl group optionally having substituent (s), hydroxyl group , A lower alkoxy group, an amino group, a lower alkylamino group, or a cyclic amino group together with a nitrogen atom to be bonded, and the cyclic amino group is selected from an oxygen atom, a sulfur atom and a nitrogen atom as a ring-constituting atom. It may have one or more heteroatoms, and may have a substituent. ] An influenza vaccine comprising a complex of the compound represented by the formula or a salt thereof and an influenza virus antigen is provided.

The substituents in formula (I) will be described below. Examples of the acyl group having 2 to 6 carbon atoms include acetyl, propionyl, butyryl and the like.
Examples of the fatty acid residue include linear or branched ones having 20 to 60 carbon atoms, which may have one or more unsaturated bonds, and specific examples thereof include:
Decanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, eicosanoyl, docosanoyl, tetracosanoyl, hexacosanoyl, triacontanoyl, tetracontanoyl, 9-hexadecenoyl, 9-octadecenoyl, 1
1,14-Eicosadienoyl, 11-Eicosenoyl, 11,14,17-Eicosatrienoyl, 2-Dodecylhexadecanoyl, 2-Tetradecylhexadecanoyl, 2-Dodecyltetradecanoyl, 2-Tetradecene Nylhexadecanoyl, 2-tetradecenylhexadecanoyl, 2-hexadecyloctadecanoyl and the like, preferably branched ones having 20 to 40 carbon atoms can be mentioned.

The lower alkyl group has a carbon number of 1 such as methyl, ethyl, propyl and isopropyl tertiary butyl.
To 6 can be mentioned, and examples of the substituent which the lower alkyl group may have include a halogen atom, a hydroxyl group, an amino group and the like, and the number thereof is usually preferably 1 to 3.

Examples of the cycloalkyl group include 5- to 7-membered ones such as cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl, and the substituent which the cycloalkyl group may have is a lower alkyl group. , Halogen atom, hydroxyl group, amino group and the like, and the number thereof is usually preferably 1 to 3. Examples of the halogen atom include chlorine, bromine, fluorine and iodine.

As the aryl group, phenyl, naphthyl,
Biphenyl etc. can be mentioned. Examples of the substituent that the aryl group may have include a lower alkyl group, a halogen atom, a hydroxyl group, an amino group and the like, and the number thereof is usually preferably 1 to 3. As an aralkyl group,
Examples thereof include phenylmethyl, phenylethyl, naphthylmethyl and the like. Examples of the substituent that the aralkyl group may have in the aryl portion thereof include lower alkyl, halogen atom, hydroxyl group, amino group and the like, and the number thereof is usually preferably 1 to 3.

Examples of the cyclic amino group include 5- to 7-membered groups such as pyrrolidinyl, piperidinyl and homopiperidinyl. The cyclic amino group may contain one or more heteroatoms selected from oxygen, sulfur and nitrogen as ring-constituting atoms, preferably one heteroatom, and examples thereof include 3-oxazolidinyl, 3-thiazolidinyl, morpholino and thio. Examples thereof include morpholino, 1-pyrazolidinyl, 1-imidazolidinyl and the like. Examples of the substituent that the cyclic amino group may have include lower alkyl, halogen atom, hydroxyl group, amino group and the like, and the number thereof is usually preferably 1 to 3. As the lower alkoxy group, methoxy, ethoxy, propoxy, isopropoxy and the like can be mentioned. The lower alkylamino group means a mono-lower alkylamino group and a di-lower alkylamino group, and examples thereof include monomethylamino, monoethylamino, dimethylamino, dipropylamino, monobutylamino and the like.

Partial Structure of the Compound of Formula (I) --NH (CONH ₂ ) C
Since HCH ₂ CH ₂ CO- has one asymmetric carbon, D
Form and L form isomers exist, but D form is generally preferred. Further, at the 1-position of the sugar moiety of the compound of formula (I), there are α and β isomers, both of which can be used in the vaccine of the present invention.

In the present specification, when both isomers are represented at the same time, they are represented by the following partial structural formulas for convenience.

[0015]

[Chemical 3]

Next, a method for producing the compound of formula (I) will be explained.

[0017]

[Chemical 4]

That is, a compound of formula (II) is reacted with a compound of formula (III) by a condensation method commonly used in peptide synthesis such as a carbodiimide method, an eintop method or an active ester method. Compounds of (I) can be prepared. For example, when the active ester method is used, the compound of formula (II) is dissolved in dimethylformamide, tetrahydrofuran, dioxane, acetonitrile or a mixture thereof to prepare N, N-disuccinimidyl carbonate, N, N-carbonyl. Imidazole, N, N
A reagent such as disuccinimidyl oxalate and an organic base such as triethylamine, N-methylmorpholine,
In the presence of 4-dimethylaminopyridine, usually 0 to about 60
The reaction is carried out at about 0 ° C for about 30 minutes to several hours to give an active ester form, and then, in the presence of a base as in the above reaction, about -15 ° C to 60 ° C, preferably 0 ° C to 25 ° C at the formula (III). The compound of formula (I) can be produced by adding the amine compound of (1) and reacting for several tens of minutes to 1 day. Here, the above reagent can be used in the same amount as the compound of formula (II), and the organic base can also be used in the same amount or excess amount as the compound of formula (II). Then, the product is purified by a means such as silica gel column chromatography to obtain the compound of formula (I).

The starting material for the compound of formula (II) can be prepared by the method described in JP-B-63-11359. Regarding the complex relating to the vaccine of the present invention, if the compound of formula (I) or a salt thereof is optionally assembled with a lipid to form a closed package, and an influenza virus antigen is embedded on the surface thereof. Well, a representative example thereof is a virus particle-like vaccine, so-called virosomes.

Next, the antigen in the vaccine of the present invention will be described. The antigens used in the present invention include HA (hemagglutinin) antigens of influenza virus and NA.
(Neuraminidase) antigen and the like can be mentioned, and it is generally preferable to use a mixture thereof, so-called HANA antigen. The HANA antigen is purified from influenza virus-infected allantoic fluid by low-speed centrifugation or chemical treatment, and the purified virus obtained is solubilized with a surfactant such as Triton X-100 or sodium cholate. Alternatively, the virus can be separated with an organic solvent such as ether, and then further purified and isolated by a sucrose density gradient centrifugation method, an affinity chromatography method, or the like. The antigen thus obtained is usually 1/300 to 1 with respect to the compound of formula (I) or a salt thereof.
It is used 10 times by weight, preferably 1/100 to 1 times by weight.

The vaccine of the present invention can be produced by various methods such as a usual method for producing liposomes, and typical examples thereof are shown below. First, an influenza virus antigen, a compound of the formula (I) or a salt thereof is mixed, preferably with a phospholipid, in a suitable buffer such as a phosphate buffer, and the mixture is mixed with sodium cholate and octyl. An effective amount of a surfactant such as glycoside (preferably 0.1 to 20 w
/ v%) and solubilize. Next, the influenza vaccine of the present invention can be produced by dialysis to remove the surfactant. In the thus obtained vaccine of the present invention, sugars such as glucose, maltose and lactose, and salts such as sodium chloride may be added as isotonic agents.

Examples of the phospholipid include phosphatidylglycerol such as dimyristoylphosphatidylglycerol and dipalmitoylphosphatidylglycerol,
Examples include phosphatidylcholine such as phosphatidylserine and dipalmitoylphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and the like, and the origins thereof may be natural products such as egg yolk and soybean and synthetic products. These may be used alone or as a mixture, and the amount thereof is usually 1/20 to 20 relative to the compound of the formula (I) or a salt thereof.
It is 20 times by weight, preferably 1/4 to 3 times by weight.

In the above production method, cholesterol,
α-tocopherol, dicetyl phosphate, stearylamine and the like may be used in combination with the phospholipid, and the amount thereof is usually not more than 2 parts by weight based on the compound of the formula (I) or a salt thereof, preferably 1 / 10 to 1/2 times by weight. The thus obtained vaccine of the present invention generally comprises a compound of formula (I) or a salt thereof and a phospholipid forming particles, and a form in which an antigen is embedded on the surface thereof, that is, a so-called virosome is formed. ing. The average particle size of the virosomes and the zeta potential of the surface can be adjusted by changing the composition ratio of the compound of formula (I) or a salt thereof and the phospholipid, and generally, the particle size of the virosomes is the average particle size. The diameter is 40 to 300 nm, and the zeta potential is -5 to
-70 mV is suitable. The vaccine of the present invention is usually subcutaneously administered, and the amount of antigen protein in a single administration is 350 to 12
μg may be administered once to several times in one season.

[0024]

INDUSTRIAL APPLICABILITY The vaccine of the present invention shows antibody production ability equal to or higher than that of the conventional virosome vaccine, and is far superior in terms of safety in terms of redness and fever. Moreover, the vaccine of the present invention was excellent in stability in a storage state and could be stored in a frozen state or a freeze-dried state.

The present invention will be further described below with reference to reference examples and examples, but the present invention is not limited thereto.

Reference Example 1 (6-0- (2-tetradecylhexadecanoyl) -N
-Acetylmuramoyl) -L-alanyl-D-isoglutamine (1.0 g) was dissolved in tetrahydrofuran (100 ml), N, N-disuccinimidyl carbonate (0.3 g) and triethylamine (0.15 ml) were added, and the mixture was stirred at room temperature for 1.5 hours. Add 0.22 ml of 28% aqueous ammonia, and add another 30 at room temperature.
Stir for a minute.

The reaction solution was concentrated under reduced pressure and the residue was subjected to silica gel column chromatography. Purification by eluting with chloroform-methanol and lyophilization from water-dioxane gave (6-0- (2-tetradecylhexadecanoyl).
0.44 g of -N-acetylmuramoyl) -L-alanyl-D-glutamamide was obtained.

Melting point 155-165 ° C. Molecular weight; 926 (C ₄₉ H ₉₁ N ₅ O ₁₁ ): FAB Mas m / z 927 (M + 1) ¹ H-NMR (DMSO-d ₆ ) δ: 0.85 (6H, t , J = 7 Hz), 1.2 ~
1.3, 1.39, 1.50 (58 H, m) 1.72 (1H, m), 1.80 (3H, s) 1.93 (1H, m) 2.08 (2H, t, J = 8 Hz), 2.30 (1H, m), 3.28 (1H, t, J =
9 Hz), 3.46 (1H, t, J = 9 Hz) 3.67 (1H, m), 3.81 (1H, m) 4.02 (1H, dd, J = 12 H
z, 5 Hz), 4.12 (1H, dq, J = 9 Hz, 5 Hz) 4.31 (2H, m), 4.34 (1H, d, J = 10 Hz) 4.44 and 4.98 (1H) 5.43 (1H, d, J = 4 Hz), 6.67 (1H, d, J = 4) 6.74 (1H, s) 7.01 (1H, s), 7.27 (1H, s) 7.30 (1H, s) 7.65 (1H, d, J = 7 Hz) 8.08 (1H, d, J = 8 Hz), 8.17 (1H, d, J = 8 Hz)

Reference Example 2 (6-0- (2-tetradecylhexadecanoyl) -N
-Acetylmuramoyl) -L-alanyl-D-isoglutamine (1.0 g) was dissolved in tetrahydrofuran (100 ml), N, N-disuccinimidyl carbonate (0.3 g) and triethylamine (0.15 ml) were added, and the mixture was stirred at room temperature for 1.5 hours. 0.2 ml of 40% methylamine aqueous solution is added, and the mixture is stirred at room temperature for another 30 minutes.

The reaction solution was concentrated under reduced pressure and the residue was subjected to silica gel column chromatography. Purification by eluting with chloroform-methanol and lyophilization from water-dioxane gave (6-0- (2-tetradecylhexadecanoyl).
-N- acetylmuramoyl) -L- alanyl -N ^r - methyl -D- Gurutamuamido 0.5g was obtained.

Melting point 95-100 ° C. Molecular weight; 939 (C ₅₀ H ₉₃ N ₅ O ₁₁ ): FAB Mas m / z 940 (M + 1) ¹ H-NMR (DMSO-d ₆ ) δ: 0.85 (6H, t , J = 7 Hz), 1.2 ~
1.3, 1.39, 1.50 (58 H, m) 1.73 (1H, m), 1.79 (3H, s) 1.93 (1H, m) 2.07 (2H, t, J = 8 Hz), 2.30 (1H, m), 2.55 (3H, d, J =
5 Hz) 3.26 (1H, t, J = 9 Hz), 3.46 (1H, t, J = 9 Hz) 3.68 (1H, m), 3.82 (1H, m) 4.03 (1H, dd, J = 12 H)
z, 5 Hz), 4.12 (1H, dq, J = 9 Hz, 5 Hz) 4.30 (2H, m), 4.36 (1H, d, J = 10 Hz) 4.98 (1H, t, J = 3.5 Hz) 5.43 (1H, d, J = 7 Hz), 6.67 (1H, d, J = 4 Hz) 7.01 (1H, s), 7.31 (1H, s) 7.64 (1H, d, J = 7 Hz) 7.71 (1H, d, J = 5 Hz) 8.07 (1H, d, J = 8 Hz), 8.17 (1H, d, J = 8 Hz)

Reference Example 3 [6-0- (2-Tetradecylhexadecanoyl) -N-acetylmuramoyl] -L-alanyl-N ^r -ethyl-D-glutamamide was produced in the same manner as in Reference Example 2. .. Molecular weight; 953 (C ₅₁ H ₉₅ N ₅ O ₁₁ ): FAB Mas m / z 954 (M + 1) ¹ H-NMR (DMSO-d ₆ ) δ: 0.85 (6H, t, J = 7 Hz), 0.99 (3
H, t, J = 7 Hz) 1.2 to 1.3, 1.39, 1.50 (58 H, m) 1.71 (1H, m), 1.79 (3H, s) 1.93 (1H, m) 2.06 (2H, t, J = 8 Hz), 2.29 (1H, m), 3.04 (2H, dq,
J = 3.5 Hz, 7 Hz) 3.28 (1H, t, J = 9 Hz), 3.45 (1H, t, J = 9 Hz) 3.68 (1H, m), 3.81 (1H, m) 4.03 (1H, dd, J = 12 Hz, 5 Hz) 4.12 (1H, dq, J = 9 Hz, 5 Hz) 4.29 (2H, m), 4.35 (1H, d, J = 10 Hz) 4.97 (1H, t, J = 3.5 Hz ) 5.44 (1H, d, J = 7 Hz), 6.68 (1H, d, J = 4 Hz) 7.01 (1H, s), 7.30 (1H, s), 7.64 (1H, d, J = 7 Hz),
7.76 (1H, d, J = 5.5 Hz) 8.07 (1H, d, J = 8 Hz), 8.16 (1H, d, J = 8 Hz)

Example 1 Preparation of Influenza HANA Antigen From influenza A / Yamagata / 120/86 strain infected allantoic fluid, high speed centrifugation (23000 rpm, 90 minutes) low speed centrifugation (6000 rpm, 60 minutes) followed by sucrose density Gradient centrifugation (30000 rpm, 3 hours) was performed to obtain a purified virus. Further, Triton X-100 was added to this virus solution so as to have a concentration of 1% and sufficiently stirred to solubilize the virus, and a purified HANA antigen solution was obtained by a sucrose density gradient equilibrium method.

Preparation of Virosome Vaccine Using the purified HANA antigen solution prepared above, four kinds of vaccine samples having the formulations shown in Table 1 were prepared as follows.
First, after mixing the ingredients, add 4 octyl glucosides.
The solubilization was carried out by adding so that the concentration became%, and dialysis was carried out by a usual method with a phosphate buffer (pH 7.4) containing 5% glucose. Each of the obtained samples was adjusted to have a HANA antigen concentration of 70 μg / ml. The obtained sample formed virosomes. Sample No. of the present invention
The virosomes 1 and 2 are shown in FIGS.

The guinea pigs in each group were subcutaneously inoculated on the dorsal part of the back with 15 times diluted solutions of the virosomal vaccine (No. 1 and 2) of the present invention and the control vaccine at a dose of 0.5 ml / animal. Three weeks after the inoculation, the same amount was additionally inoculated. Blood was collected at the third and fifth weeks, and hemagglutinin inhibition test was performed according to the WHO method to measure the antibody-producing ability. The results are shown in Table 1.

Table 1 Sun antibody producing ability Pull composition (HI value) No. 3 weeks 5 weeks HANA 70 μg 1 Compound of Reference Example 1 300 μg <80 5120 Phosphatidylcholine 225 μg Phosphatidylglycerol 150 μg HANA 70 μg 2 Compound of Reference Example 2 300 μg <80 2560 Phosphatidylcholine 225 μg Phosphatidylglycerol 150 μg HANA 70 μg 3 Compound of Reference Example 1 600 μg <80 2560 Dimyristoylphosphatidylcholine 450 μg Dimyristoylphosphatidylglycerol 300 μg HANA 70 μg 4 Compound of Reference Example 1 600 μg <80 2560 Dimyristoylphosphatidylglycerol 300 μg Cholesterol 600 μg 70 μg 1 Control compound ^* 300 μg <80 2560 Cholesterol 300 μg Control 2 HANA 70 μg <80 1280 Control 3 HANA vaccine 70 μg <80 1280 Control Formalin inactivated 70 μg <80 1280 4 Whole particle vaccine

As is clear from the above table, the vaccine of the present invention showed antibody production ability equivalent to or better than that of the control vaccine. * Control compound: 6-0- (2-tetradecylhexadecanoyl) -N-acetylmuramoyl-L-alanyl-D
-Isoglutamine redness reaction test The redness reaction test was performed on the rabbits (5 animals) with a sample of 0.
Inoculation of 5 ml, the surface area of redness was measured daily, the total area of 5 animals was determined, and then the average redness surface area per animal was calculated. The results are shown in Table 2.

Table 2 Sun redness reaction test pull composition ( the surface area of positive redness (cm ² ) No.) Day 1 Day 2 Day 3 HANA 70 μg 1 Compound of Reference Example 1 300 μg 1/5 animal 1.1 0 0 Phosphatidylcholine 225 μg Phosphatidylglycerol 150 μg HANA 70 μg 2 Compound of Reference Example 2 300 μg 1/5 1.00 0 Phosphatidylcholine 225 μg Phosphatidylglycerol 150 μg Control HANA 70 μg 1 Control compound 300 μg 5/5 5.7 4.2 8.5 Cholesterol 300 μg Control 2 HANA vaccine 70 μg 3 / 5 animals 4.5 4.0 1.6

As is apparent from the above table, the vaccine of the present invention showed lower redness than the control vaccine. Fever test For the fever test, 1 ml of each sample was intravenously administered to a rabbit using a general test method based on biological products. When the sum of the three exothermic reactions was 1.3 ° C or lower, the exothermic test was negative, and when it was 2.5 ° C or higher, the exothermic test was positive. The results are shown in Table 3.

Table 3rd item Composition heat generation test sample No. (total elevated body temperature of 3 animals) HANA 70 μg 1 Compound of Reference Example 1 300 μg 0.81 ° C. Phosphatidylcholine 225 μg Judgment: Negative phosphatidylglycerol 150 μg HANA 70 μg 2 Reference Example 2 Compound 300 μg 0.14 ° C Phosphatidylcholine 225 μg Judgment: Negative phosphatidylglycerol 150 μg HANA 70 μg 2.66 ° C Control 1 Control compound 300 μg Judgment: Positive cholesterol 300 μg As is clear from the above table, the vaccine of the present invention showed lower pyrogenicity than the control vaccine. ..

[Brief description of drawings]

FIG. 1 95,000 of the sample No. 1 vaccine of the present invention
Double electron micrograph.

FIG. 2 95,000 of the sample No. 2 vaccine of the present invention
Double electron micrograph.

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[Procedure amendment]

[Submission date] March 11, 1993

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 shows a sample No. 1 of the present invention. It is a 95,000 times electron micrograph showing the particle structure of the vaccine of No. 1.

FIG. 2 shows a sample No. of the present invention. It is a 95,000 times electron micrograph showing the particle structure of the vaccine of No. 2.

[Procedure 3]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Figure 1]

[Fig. 2]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kunio Okuma 313-20 Okubo, Shimizu-cho, Kumamoto-shi, Kumamoto Prefecture (72) Inventor Tetsuya Oka 2-44-2, Koto, Kumamoto-shi, Kumamoto

Claims (3)

[Claims] 1. A general formula: [In the formula, R is an acyl group having 2 to 6 carbon atoms, X is an L-alanine residue, L-serine residue, L-valine residue or glycine residue, and A is a fatty acid having 10 to 60 carbon atoms. A residue is meant, and R ₁ and R ₂ are independently a hydrogen atom, a lower alkyl group which may have a substituent, a cycloalkyl group which may have a substituent, or a substituent. An aryl group, an aralkyl group which may have a substituent, a hydroxyl group, a lower alkoxy group, an amino group or a lower alkylamino group, or a cyclic amino group together with a nitrogen atom to be bonded, and the cyclic amino group May have one or more heteroatoms selected from an oxygen atom, a sulfur atom and a nitrogen atom as a ring-constituting atom, or may have a substituent. ] An influenza vaccine comprising a complex of the compound represented by the formula or a salt thereof and an antigen of influenza virus. 2. The influenza vaccine according to claim 1, wherein the vaccine is a virosome vaccine. 3. The influenza vaccine according to claim 1, wherein the influenza virus antigen is an influenza HANA antigen.