Preparations including vaccine adjuvants.

Abstract

Provided is a composition that is useful as a vaccine adjuvant and has excellent storage stability and immunostimulatory activity. Specifically provided is a freeze-dried preparation that has high storage stability, said preparation containing a (4E, 8E, 12E, 16E, 20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexane-3-yl]amino}-6-methylpyrimidine-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide, squalene, a hydrophilic surfactant, and an oleophilic surfactant, and being characterized by containing an ascorbic acid-based antioxidant and an excipient.

Description

Formulations containing vaccine adjuvants

[0001] The present invention relates to emulsion compositions of compounds useful as vaccine adjuvants, and lyophilized formulations of such compositions.

[0002] Compared to vaccines that use the pathogen itself, subunit vaccines, which use a portion of the pathogen's components as antigens, are superior in terms of vaccine safety and manufacturing methodology because they can be produced using chemical synthesis or genetic recombination technology. However, subunit vaccines tend to have lower immunostimulatory efficacy than live or inactivated vaccines that use the pathogen itself. For this reason, preventive or therapeutic methods that use adjuvants in combination with vaccine antigens are being investigated to enhance the immunogenicity of epitopes and improve the immunostimulatory activity of vaccines.

[0003] Recently, (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide (hereinafter referred to as "Compound A") has been reported as an adjuvant with TLR7 agonist activity (Patent Document 1). Although Compound A has excellent vaccine adjuvant activity, it is required to be formulated as a pharmaceutical preparation such as an emulsion in order to actually administer it to mammals as a vaccine adjuvant. Generally, it is known that an ascorbic acid-based antioxidant is used in emulsion preparations to improve the storage stability of the preparation, but it is not known that ascorbic acid-based antioxidants stabilize particle size distribution.

[0004] International Publication No. 2017 / 061532

[0005] The present invention provides a pharmaceutical composition that has excellent storage stability and immunostimulatory activity and is useful as a vaccine adjuvant.

[0006] Compound A has six unsaturated bonds in its molecule, derived from a squalene-like structure. During formulation studies of Compound A, it was found that when Compound A was freeze-dried into a typical emulsion formulation using squalene as the oily composition, the unsaturated bonds in the molecule of Compound A were oxidized, resulting in a decrease in the content of Compound A. Therefore, the inventors conducted extensive research to find a practical vaccine adjuvant formulation with high storage stability. As a result, they found that the use of squalane as the oily composition when preparing an emulsion composition of Compound A increases the stability of Compound A against oxidation. Furthermore, they found that the addition of an ascorbic acid-based antioxidant not only improves storage stability, particularly the stability of Compound A itself against oxidation, but also the stability of particle size distribution, leading to the completion of the present invention.

[0007] That is, the gist of the present invention is as follows: [Item 1] i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide (hereinafter referred to as "Compound A") or a pharmaceutically acceptable salt thereof; ii) squalane; iii) antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate, ascorbic acid palmitate, etc.), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, calcium ascorbate, etc.), and ascorbic acid; and iv) A freeze-dried emulsion formulation containing an excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol).

[0008] [Item 2] The formulation according to Item 1, further comprising v) a hydrophilic surfactant, and vi) a lipophilic surfactant.

[0009] [Item 3] The formulation according to Item 1 or 2, wherein the emulsion is an oil-in-water emulsion.

[0010] [Item 4] The hydrophilic surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, etc.); polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60, etc.); and polyoxyethylene polyoxypropylene glycols (e.g., polyoxyethylene (42) Polyoxypropylene (67) glycol, polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (200) polyoxypropylene (70) glycol, etc.

[0011] [Item 5] The formulation according to Item 2 or 3, wherein the hydrophilic surfactant is polysorbate 20, polysorbate 40, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, or polyoxyethylene (160) polyoxypropylene (30) glycol.

[0012] [Item 6] The formulation according to Item 2 or 3, wherein the hydrophilic surfactant is polysorbate 20, polysorbate 40, or polysorbate 80.

[0013] [Item 7] The formulation according to any one of Items 2 to 6, wherein the lipophilic surfactant is a sorbitan fatty acid ester (e.g., sorbitan fatty acid ester, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, medium-chain triglyceride, etc.); a glycerin fatty acid ester (e.g., glycerin fatty acid ester, glycerin monostearate, glycerin monomyristate, glycerin monooleate, glycerin triisooctanoate, etc.); a sucrose fatty acid ester (e.g., sucrose fatty acid ester, sucrose stearate, sucrose palmitate, etc.); or a propylene glycol fatty acid ester (e.g., propylene glycol fatty acid ester, propylene glycol monostearate, etc.).

[0014] [Item 8] The formulation according to any one of Items 2 to 6, wherein the lipophilic surfactant is a sorbitan fatty acid ester, sorbitan monooleate, sorbitan sesquioleate, or sorbitan trioleate.

[0015] [Item 9] The formulation according to any one of Items 2 to 6, wherein the lipophilic surfactant is sorbitan trioleate.

[0016] [Item 10] The formulation according to any one of Items 1 to 9, further comprising an antioxidant B selected from the group consisting of tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.); tocopherol acetate; and butylhydroxyanisole.

[0017] [Item 11] The formulation according to Item 10, wherein antioxidant B is a tocopherol selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol.

[0018] [Item 12] The formulation according to Item 10, wherein antioxidant B is α-tocopherol.

[0019] [Item 13] The formulation according to any one of Items 1 to 12, wherein antioxidant A is ascorbic acid palmitate, potassium ascorbate, sodium ascorbate, or ascorbic acid.

[0020] [Item 14] The formulation according to any one of Items 1 to 12, wherein antioxidant A is sodium ascorbate or potassium ascorbate.

[0021] [Item 15] The formulation according to any one of Items 1 to 14, wherein excipient A is a non-reducing sugar (e.g., sucrose or trehalose); or a sugar alcohol (e.g., sorbitol, erythritol, xylitol, maltitol, or lactitol).

[0022] [Item 16] The formulation according to any one of Items 1 to 14, wherein excipient A is sucrose, trehalose, sorbitol, or xylitol.

[0023] [Item 17] The formulation according to any one of Items 1 to 14, wherein excipient A is sucrose or trehalose.

[0024] [Item 18] The formulation according to any one of Items 1 to 17, wherein the amount of squalane blended is 50 to 500 times the weight of Compound A.

[0025] [Item 19] The formulation according to any one of Items 1 to 17, wherein the amount of squalane blended is 100 to 400 times the weight of Compound A.

[0026] [Item 20] The formulation according to any one of Items 1 to 17, wherein the amount of squalane blended is 200 to 300 times the weight of Compound A.

[0027] [Item 21] The formulation according to any one of Items 2 to 20, wherein the amount of the hydrophilic surfactant is 0.5 to 250 times the weight of Compound A.

[0028] [Item 22] The formulation according to any one of Items 2 to 20, wherein the amount of the hydrophilic surfactant is 5 to 100 times the weight of Compound A.

[0029] [Item 23] The formulation according to any one of Items 2 to 20, wherein the amount of the hydrophilic surfactant is 10 to 50 times the weight of Compound A.

[0030] [Item 24] The formulation according to any one of Items 2 to 21, wherein the amount of the lipophilic surfactant is 0.5 to 250 times the weight of Compound A.

[0031] [Item 25] The formulation according to any one of Items 2 to 22, wherein the amount of the lipophilic surfactant is 5 to 100 times the weight of Compound A.

[0032] [Item 26] The formulation according to any one of Items 2 to 23, wherein the amount of the lipophilic surfactant is 10 to 50 times the weight of Compound A.

[0033] [Item 27] ​​The formulation according to any one of Items 1 to 26, wherein the amount of antioxidant A incorporated is 0.5 to 500 times the weight of compound A when converted into the weight of sodium ascorbate.

[0034] [Item 28] The formulation according to any one of Items 1 to 26, wherein the amount of antioxidant A incorporated is 2.5 to 250 times the weight of compound A when converted into the weight of sodium ascorbate.

[0035] [Item 29] The formulation according to any one of Items 1 to 26, wherein the amount of antioxidant A incorporated is 5 to 100 times the weight of compound A when converted into the weight of sodium ascorbate.

[0036] [Item 30] The formulation according to any one of Items 1 to 29, wherein the amount of excipient A is 50 to 1000 times the weight of compound A.

[0037] [Item 31] The formulation according to any one of Items 1 to 29, wherein the amount of excipient A is 100 to 750 times the weight of compound A.

[0038] [Item 32] The formulation according to any one of Items 1 to 29, wherein the amount of excipient A is 200 to 625 times the weight of compound A.

[0039] [Item 33] The formulation according to any one of Items 10 to 32, wherein the amount of antioxidant B is 5 to 250 times the weight of compound A.

[0040] [Item 34] The formulation according to any one of Items 10 to 32, wherein the amount of antioxidant B is 12.5 to 125 times the weight of compound A.

[0041] [Item 35] The formulation according to any one of Items 10 to 32, wherein the amount of antioxidant B is 25 to 50 times the weight of compound A.

[0042] [Item 36] The formulation according to any one of Items 1 to 35, wherein the weight of Compound A is 0.0001 to 0.65 times the weight of the lyophilized product not containing Compound A.

[0043] [Item 37] The formulation according to any one of Items 1 to 35, wherein the weight of Compound A is 0.0002 to 0.35 times the weight of the lyophilized product not containing Compound A.

[0044] [Item 38] The formulation according to any one of Items 1 to 35, wherein the weight of Compound A is 0.0005 to 0.065 times the weight of the lyophilized product not containing Compound A.

[0045] [Item 39] Particle diameter D immediately after emulsion preparation ９０ value, and the particle size D of the emulsion reconstituted after 6 months of storage at 25°C as a freeze-dried preparation. ９０ Item 39. The formulation according to any one of Items 1 to 38, wherein all of the values ​​are 1000 nm or less.

[0046] [Item 40] The formulation according to any one of Items 1 to 39, wherein the increase in area percentage value of the impurity UK-1.02 when the lyophilized formulation is stored at 5°C for 6 months is 5.0% or less.

[0047] [Item 41] The formulation according to any one of Items 1 to 39, wherein the increase in area percentage of the impurity UK-1.02 when the lyophilized formulation is stored at 5°C for 6 months is 1.0% or less.

[0048] [Item 42] A vaccine adjuvant comprising the formulation according to any one of Items 1 to 41.

[0049] [Item 43] A vaccine comprising the formulation according to any one of Items 1 to 41 and an antigen.

[0050] [Item 44] The vaccine according to Item 43, wherein the antigen is a substance derived from a pathogen.

[0051] [Item 45] A kit comprising the formulation according to any one of items 1 to 41 and an antigen.

[0052] [Item 46] A freeze-dried emulsion formulation containing: i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide or a pharmaceutically acceptable salt thereof; ii) squalane; iii') tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.); and iv) excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol).

[0053] [Item 47] The formulation according to Item 46, further comprising: v) a hydrophilic surfactant selected from polysorbate 20, polysorbate 40, or polysorbate 80; and vi) a lipophilic surfactant selected from sorbitan fatty acid esters, sorbitan monooleate, sorbitan sesquioleate, or sorbitan trioleate.

[0054] [Item 48] i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide or a pharmaceutically acceptable salt thereof; ii) squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate, ascorbic acid palmitate, etc.), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, calcium ascorbate, etc.), and ascorbic acid; and iv) an excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol).

[0055] [Item 49] The emulsion according to Item 48, further comprising v) a hydrophilic surfactant, and vi) a lipophilic surfactant.

[0056] [Item 50] The emulsion according to Item 48 or 49, further comprising an antioxidant B selected from the group consisting of tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.); tocopherol acetate; and butylhydroxyanisole.

[0057] [Item 51] An emulsion comprising: i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide or a pharmaceutically acceptable salt thereof; ii) squalane; iii') tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.); and iv) excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol).

[0058] According to the present invention, it is possible to provide a vaccine adjuvant formulation that can further enhance the specific immune response to an antigen and has high storage stability.

[0059] The formulation of the present invention is a lyophilized emulsion formulation containing Compound A, squalane, an antioxidant A of ascorbic acid, and excipient A. The present invention also includes emulsion formulations before lyophilization and emulsion formulations obtained by rehydrating the lyophilized formulation. In the formulation of the present invention, Compound A contained as the active ingredient may be in the free form or its pharmaceutically acceptable acid addition salt or base addition salt. Examples of acid addition salts include acid addition salts with inorganic or organic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, citric acid, or maleic acid). Examples of base addition salts include alkali metal salts such as sodium salt or potassium salt, alkaline earth metal salts such as calcium salt, and ammonium salt. Compound A or a pharmaceutically acceptable salt thereof of the present invention may also exist in the form of a hydrate or solvate, and these compounds are also included in Compound A or a pharmaceutically acceptable salt thereof of the present invention. Details and production methods thereof are described in Patent Document 1, and Compound A or a pharmaceutically acceptable salt thereof can be produced, for example, according to the method described in Patent Document 1. In the formulation of the present invention, the content of compound A is stated as the content of the free form of compound A. Therefore, when compound A is used as a pharmaceutically acceptable salt thereof, the content is calculated by converting the weight of compound A into the weight of the salt.

[0060] The emulsion of the present invention refers to an oil-in-water emulsion or a water-in-oil emulsion. An oil-in-water emulsion is preferred. The ratio (weight ratio) of the oily composition to the aqueous solution is preferably 1:99 to 15:85, more preferably 2:98 to 10:90, even more preferably 3:97 to 9:91, and even more preferably 4:96 to 7:93. In the emulsion formulation of the present invention, compound A is present dissolved in the oily composition. The lyophilized formulation of the present invention refers to a formulation obtained by removing water from the emulsion formulation of the present invention under lyophilization conditions. An emulsion formulation can be prepared by reconstituting the lyophilized formulation with water for injection in an amount 2 to 20 times the weight of the lyophilized formulation.

[0061] The hydrophilic surfactants referred to in this specification include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, etc.); polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60, etc.); polyoxyethylene polyoxypropylene glycols (e.g., poly Examples of suitable polyoxyethylene hydroxybenzoates include polyoxyethylene (42) polyoxypropylene (67) glycol, polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, and polyoxyethylene (200) polyoxypropylene (70) glycol. Preferred are polysorbate 20, polysorbate 40, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, and polyoxyethylene (160) polyoxypropylene (30) glycol, more preferred are polysorbate 20, polysorbate 40, and polysorbate 80, and particularly preferred is polysorbate 80. The content of the hydrophilic surfactant in the formulation of the present invention is 0.5 to 250 times, preferably 5 to 100 times, and more preferably 10 to 50 times the weight of Compound A.

[0062] The lipophilic surfactants used herein include sorbitan fatty acid esters (e.g., sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, medium-chain fatty acid triglycerides, etc.); glycerin fatty acid esters (e.g., glycerin fatty acid esters, glycerin monostearate, glycerin monomyristate, glycerin monooleate, glycerin triisooctanoate, etc.); sucrose fatty acid esters (e.g., sucrose fatty acid esters, sucrose stearate, sucrose palmitate, etc.); and propylene glycol fatty acid esters (e.g., propylene glycol fatty acid esters, propylene glycol monostearate, etc.). Preferred are sorbitan fatty acid esters, sorbitan monooleate, sorbitan sesquioleate, and sorbitan trioleate, and more preferred is sorbitan trioleate. The amount of lipophilic surfactant in the formulation of the present invention is 0.5 to 250 times, preferably 5 to 100 times, and more preferably 10 to 50 times the weight of compound A.

[0063] Squalane is used as the oily composition in the formulation of the present invention. In examining the formulation of this formulation, the use of squalane as the oily composition in emulsion formulations increased the stability of Compound A against oxidation compared to the use of squalene, which is commonly used. Therefore, squalane is preferred as the oily composition in this formulation. The amount of squalane to be added is 50 to 500 times, preferably 100 to 400 times, and more preferably 200 to 300 times the weight of Compound A.

[0064] As used herein, antioxidant A includes ascorbic acid esters (e.g., L-ascorbic acid stearate, ascorbic acid palmitate, etc.); inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, calcium ascorbate, etc.); ascorbic acid; etc. Preferred are ascorbic acid palmitate, potassium ascorbate, sodium ascorbate, and ascorbic acid, and more preferred are sodium ascorbate and potassium ascorbate. The content of antioxidant A in the formulation of the present invention, calculated in terms of sodium ascorbate, i.e., the content of ascorbic acid in the ascorbic acid derivative serving as antioxidant A, converted into sodium ascorbate by weight, is 0.5 to 500 times, preferably 2.5 to 250 times, and more preferably 5 to 100 times the weight of compound A.

[0065] The excipient A used herein includes non-reducing sugars or sugar alcohols (excluding mannitol). Preferably, it is a non-reducing sugar (e.g., sucrose, trehalose; or a sugar alcohol (e.g., sorbitol, erythritol, xylitol, maltitol, or lactitol), more preferably sucrose, trehalose, sorbitol, or xylitol, even more preferably sucrose or trehalose, and particularly preferably sucrose. The content of excipient A in the formulation of the present invention is 50 to 1000 times, preferably 100 to 750 times, and more preferably 200 to 625 times the weight of compound A.

[0066] As used herein, antioxidant B includes tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.); tocopherol acetate; butylhydroxyanisole; and the like. α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol are preferred, and α-tocopherol is more preferred. The content of antioxidant B in the formulation of the present invention is 5 to 250 times, preferably 12.5 to 125 times, more preferably 20 to 50 times, and even more preferably 25 to 50 times the weight of compound A.

[0067] The freeze-dried preparation of the present invention can be produced by filling a vial with the emulsion and freeze-drying it using a freeze-dryer under normal production conditions. Although the production conditions are not particularly limited, specific conditions include freezing the emulsion at around −40° C., decompressing the chamber and simultaneously raising the temperature to −20° C. for drying for about 10 to 80 hours, and then raising the temperature to 25° C. for drying for about 10 to 30 hours.

[0068] One embodiment of the formulation of the present invention comprises: i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide (hereinafter referred to as "Compound A") or a pharmaceutically acceptable salt thereof; ii) squalane; iii) antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate, ascorbic acid palmitate, etc.), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, calcium ascorbate, etc.), and ascorbic acid; iv) excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) a lipophilic surfactant.

[0069] Another embodiment of the formulation of the present invention comprises: i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide (hereinafter referred to as "Compound A") or a pharmaceutically acceptable salt thereof; ii) squalane; iii) antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate, ascorbic acid palmitate, etc.), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, calcium ascorbate, etc.), and ascorbic acid; iv) excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, etc.); polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60, etc.); polyoxyethylene polyoxypropylene glycols (e.g., polyoxyethylene (4 2) Hydrophilic surfactants such as polyoxypropylene (67) glycol, polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (200) polyoxypropylene (70) glycol, etc.;vi) lipophilic surfactants such as sorbitan fatty acid esters (e.g., sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, medium-chain fatty acid triglycerides, etc.); glycerin fatty acid esters (e.g., glycerin fatty acid esters, glycerin monostearate, glycerin monomyristate, glycerin monooleate, glycerin triisooctanoate, etc.); sucrose fatty acid esters (e.g., sucrose fatty acid esters, sucrose stearate, sucrose palmitate, etc.); and propylene glycol fatty acid esters (e.g., propylene glycol fatty acid esters, propylene glycol monostearate, etc.); and iii') A freeze-dried emulsion preparation containing an antioxidant B selected from the group consisting of tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.), tocopherol acetate, and butylhydroxyanisole;

[0070] Another embodiment of the formulation of the present invention is a freeze-dried formulation that does not contain iii) antioxidant A as an antioxidant, but contains iii') tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.). The tocopherol is preferably α-tocopherol. The tocopherol content in the formulation is 5 to 250 times, preferably 12.5 to 125 times, more preferably 20 to 50 times, and even more preferably 25 to 50 times the weight of compound A. The formulation may also contain sodium thiosulfate, butylhydroxyanisole, or the like as an additional antioxidant.

[0071] The weight of compound A in the formulation of the present invention is 0.0001 to 0.65 times, preferably 0.0002 to 0.35 times, and more preferably 0.0005 to 0.065 times the weight of the lyophilized product not containing compound A.

[0072] Particle diameter D of oil droplets in the formulation of the present invention ９０ The value is the particle diameter D of the emulsion during the manufacturing process. ９０ The particle diameter D of the oil droplets of the emulsion reconstituted with water after storage as a freeze-dried preparation is 1000 nm or less, preferably 300 nm or less. ９０ The value is preferably the particle diameter D of the oil droplets of the emulsion reconstituted after storage at 5°C or 25°C for 6 months. ９０ The value is 1000 nm or less.

[0073] In the formulation of the present invention, the particle diameter D of the oil droplets ９０ The value is one of the representative values ​​that represents the particle size distribution of oil droplets contained in an emulsion, and means the 90% particle size based on the scattering intensity. ９０ The particle diameter D in this specification is measured and calculated using a dynamic light scattering particle size distribution analyzer, a laser diffraction particle size analyzer, an image processing particle size distribution analyzer, or the like. ９０ The values ​​are those measured using a dynamic light scattering particle size distribution analyzer: Zetasizer Nano ZS (Malvern Instruments).

[0074] In the formulation of the present invention, the impurity UK-1.02 is one of the typical impurities detected in the evaluation of related substances using high-performance liquid chromatography. Using a Phenyl-Hexyl column (Waters Xselect CSH Phenyl-Hexyl XP Column, 4.6 mm x 75 mm, 2.5 μm, model number: 186006134), reversed-phase high-performance liquid chromatography was performed using purified water, acetonitrile, methanol, and trifluoroacetic acid. An amount equivalent to 0.4 to 2 μg of Compound A was injected, and spectroscopic analysis was performed at a wavelength of 220 nm. The impurity UK-1.02 was detected at an elution time 1.02 times that of Compound A. The detailed measurement conditions were as follows: Mobile phase A: 0.1% aqueous trifluoroacetic acid solution; Mobile phase B: acetonitrile / methanol mixture (8:2) containing 0.06% trifluoroacetic acid; Flow conditions: Flow rate: 0.5 mL per minute Column temperature: Constant temperature around 40°C

[0075] Regarding the storage stability of the formulation of the present invention, the increase in the area percentage value of the impurity UK-1.02 after 6 months of storage at 5° C. is 5.0% or less, preferably 1.0% or less, from the value at the start of storage. The area percentage values ​​are compared using actual measured values.

[0076] The formulation of the present invention is prepared by emulsifying the oil-in-water emulsion, then sterile filtering it using a sterile filter, and storing it in a freeze-dried state. ９０ A value of 1000 nm or less is desirable.

[0077] The formulation of the present invention can contain other additives to the extent that the particle size, etc. of the emulsion after reconstitution with water is not changed. Furthermore, at the time of administration, the formulation can be mixed with a formulation containing a vaccine antigen to the extent that the particle size, etc. of the emulsion after reconstitution with water is not changed. The method and mixing ratio of the formulation of the present invention and the vaccine antigen are not particularly limited. For example, the reconstituted emulsion formulation and an equal volume of a formulation containing a vaccine antigen can be mixed by inverting in a vial.

[0078] The vaccine antigen to be administered in combination with the formulation of the present invention is not particularly limited, and examples thereof include antigen proteins or antigen peptides (partial peptides) derived from the antigen proteins, as well as complexes of these with carriers. Specific examples of vaccine antigens include 1) tumor antigen proteins or tumor antigen peptides for cancer immunotherapy, and 2) active ingredients of vaccines for preventing infectious diseases. The carrier is a substance that chemically and / or physically binds the antigen protein or antigen peptide, and examples thereof include proteins and lipids.

[0079] The formulation of the present invention can be provided as a kit containing a lyophilized formulation containing Compound A and a vaccine antigen.

[0080] The formulation of the present invention can be reconstituted with water for injection in an amount 2 to 20 times the weight of the lyophilized formulation prior to administration, and then mixed with a formulation containing a vaccine antigen. The dosage of the formulation of the present invention per dose is 1 ng to 250 mg, preferably 1 ng to 50 mg, of Compound A. Although this varies depending on the type of vaccine antigen to be administered simultaneously and the age of the subject, it may be administered in a single dose, or in one or more booster doses.

[0081] According to the present invention, storage stability was demonstrated as shown in the following test examples.

[0082] The present invention will be explained below with reference to examples, reference examples, comparative examples, test examples, etc., but the present invention is not limited to these examples at all.

[0083] As squalane, "Squalane (manufactured by Wako Pure Chemical Industries)," "Squalane (manufactured by Kishimoto Tokushu Kanyu Kogyosho)," or "Squalane (manufactured by Maruha Nichiro)" was used; as squalene, "Squalene (manufactured by Wako Pure Chemical Industries)," "Squalene (manufactured by Kishimoto Tokushu Kanyu Kogyosho)," or "Squalene (manufactured by Maruha Nichiro)" was used; as sodium ascorbate, "Sodium ascorbate (manufactured by Wako Pure Chemical Industries)" or "Sodium L-ascorbate (manufactured by Kyowa Pharma Chemical)" was used; as α-tocopherol, "α-tocopherol (manufactured by Mitsubishi Chemical Foods)" or "all-rac-α-Tocopherol EMPROVE (registered trademark) ESSENTIAL Ph Eur,BP,USP,E 307 (manufactured by Merck)" was used; as sorbitan trioleate, "Span85 (manufactured by Sigma-Aldrich)" or "Rheodol "SP-O30V (manufactured by Kao)" or "Span85 (manufactured by Croda)" was used; "PS80 (GS) (manufactured by NOF Corp.)," "Polysorbate 80 (HX2) (manufactured by NOF Corp.)," "Tween 80 (manufactured by Merck)," or "Tween 80 HP-LQ-(HM) (manufactured by Croda)" was used as polysorbate 80; "Sucrose (manufactured by Nacalai Tesque)" or "Sucrose low in endotoxins suitable for use as excipient EMPROVE® exp Ph Eur,BP,JP,NF (manufactured by Merck)" was used as sucrose; "Otsuka distilled water for injection (manufactured by Otsuka Pharmaceutical Factory)" was used as water for injection; and ascorbic acid palmitate, butylhydroxyanisole, and sodium thiosulfate were all manufactured by Wako Pure Chemical Industries, Ltd.

[0084] [Preparation of Freeze-Dried Compositions] [Examples 1 to 16, Comparative Examples 1 to 3] Compound A was dissolved in oily components (squalane, sorbitan trioleate, and α-tocopherol in Examples 1 to 9 and 15; squalane and sorbitan trioleate in Examples 10 to 13; squalane, sorbitan trioleate, α-tocopherol, and ascorbic acid palmitate in Example 14; squalane, sorbitan trioleate, α-tocopherol, and butylhydroxyanisole in Example 16; and squalene, sorbitan trioleate, and α-tocopherol in Comparative Examples 1 to 3) to give compositions shown in Tables 1 to 4. Aqueous components (sucrose and polysorbate 80 in Examples 1 to 3, 14, and 16; sucrose, polysorbate 80, and sodium ascorbate in Examples 4 to 13; sucrose, polysorbate 80, and sodium thiosulfate in Example 15) were dissolved in water for injection to obtain the compositions shown in Tables 1 to 4, and the oily composition was then added and premixed, followed by emulsification and dispersion using an ultra-high pressure emulsifying disperser. After filtering through a 0.2 μm sterilizing filter, the solution was filled into glass vials in 1 mL aliquots and freeze-dried. After restoring pressure with nitrogen gas, the solution was sealed with a rubber stopper to obtain the freeze-dried compositions of Examples 1 to 16 and Comparative Examples 1 to 3.

[0085] [Test Example 1] Evaluation of particle size during the manufacturing process In the manufacturing process of the freeze-dried composition, the particle size distribution of oil droplet particles contained in the emulsion after emulsification but before freeze-drying was measured by the following method. The emulsion was diluted 10 times with water for injection, and the 90% particle size (D ) based on scattering intensity was measured using a dynamic light scattering particle size distribution analyzer (Zetasizer Nano ZS). 90 ) was measured. 90 (nm) are shown in Table 5.

[0086] Test Example 2: Stability evaluation (1) The particle size distribution of the prepared freeze-dried compositions was measured by the following method at the start of storage and after 6 months of storage in thermostatic chambers at 5°C and 25°C. 1 mL of water for injection was added to one vial of the freeze-dried compositions prepared in Examples 1 to 16 and Comparative Examples 1 to 3 to reconstitute the composition. 100 μL of the reconstituted drug solution was sampled using a micropipette and mixed with 900 μL of water for injection. The 90% particle size (D ) based on scattering intensity was measured using a dynamic light scattering particle size distribution analyzer (Zetasizer Nano ZS). 90 ) was measured for Examples 1 to 16 and Comparative Examples 1 to 3 before and after storage. 90 (nm) are shown in Table 6. The test results show that in the formulations of Examples 4 to 14 containing ascorbic acid antioxidants, the particle size distribution after storage for 6 months in thermostatic chambers at 5°C and 25°C showed little change from the value at the start of storage, and it can be said that these formulations have highly stable particle size distributions.

[0087] Test Example 3: Stability Evaluation (2) The amount of impurities (area percentage value of Uk-1.02) of the prepared freeze-dried composition was measured at the start of the test and after 6 months of storage in a constant temperature chamber at 5°C using the following method. Using a Phenyl-Hexyl column (Waters Xselect CSH Phenyl-Hexyl XP Column, 4.6 mm x 75 mm, 2.5 μm, model number: 186006134), a reversed-phase high-performance liquid chromatography was performed using pure water, acetonitrile, methanol, and trifluoroacetic acid. 0.4 to 2 μg of Compound A was injected and spectroscopic detection was performed at a wavelength of 220 nm. The measurement conditions were as follows: Mobile phase A: 0.1% aqueous trifluoroacetic acid solution Mobile phase B: acetonitrile / methanol mixture (8:2) containing 0.06% trifluoroacetic acid Flow conditions: Flow rate: 0.5 mL per minute Column temperature: constant temperature around 40°C Using the peak areas and elution times measured by this method, the area percentage of the impurity peak (Uk-1.02) detected at an elution time 1.02 times that of Compound A was calculated according to the following formula: Area percentage (%) of Uk-1.02 = Peak area of ​​Uk-1.02 / Total peak area of ​​related substances and Compound A × 100 The area percentage (%) of the impurity peak (Uk-1.02) of Examples 1 to 16 and Comparative Examples 1 to 3 before and after storage are shown in Table 7. The test results show that the formulations of Examples 4 to 14 containing ascorbic acid antioxidants had low impurity values ​​(area percentage value of Uk-1.02) after 6 months of storage in a constant temperature chamber at 5°C, indicating that the formulations have high storage stability. Furthermore, when the formulations of Examples 1 to 3 containing squalane as an oily component are compared with the formulations of Comparative Examples 1 to 3 containing squalene as an oily component, the formulations containing squalane as an oily component have lower impurity values ​​(area percentage value of Uk-1.02), indicating that squalane contributes to the antioxidant stability of Compound 1, and that the formulations containing squalane have high storage stability.

[0088] These Examples, Reference Examples, Comparative Examples, Test Examples, etc. show that the preparation of the present invention has high storage stability and can therefore be used as a vaccine adjuvant preparation.

Claims

DEPCT641. A lyophilized formulation of an emulsion incorporating: i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexainamide (hereinafter referred to as "Compound A") or its pharmaceutically acceptable salt; ii) squalene; iii) an antioxidant A selected from the group of ascorbate esters( 1. Inorganic salts of ascorbic acid (e.g., L-ascorbic stearate, ascorbyl palmitate, etc.), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, calcium ascorbate, etc.), and ascorbic acid; and iv) selected drug additives A from the group consisting of non-reducing sugars and sugaralcohol, provided that mannitol is not included.

2. Formulations according to claim 1, which include additional v) hydrophilic surfactants and vi) lipophilic surfactants. 3.Formulations under claim 1 or 2, where the emulsion is an oil-in-water emulsion.

4. Formulations under claim 2 or 3, where the hydrophilic surfactant is polyoxyethylene sorbitan fatty acid ester (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, etc.); polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, etc.). Hydrogenated polyoxyethylene castor oil 50, hydrogenated polyoxyethylene castor oil 60, etc.); or polyoxyethylene polyoxypropylene glycol (e.g. polyoxyethylene (42) polyoxypropylene (67) glycol, polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (200) polyoxypropylene (70) glycol, etc.) 5.A formulation under claim 2 or 3, in which the hydrophilic surfactant is polysorbate 20, polysorbate 40, polysorbate 80, castor oil, hydrogenated polyoxyethylene 60, or polyoxyethylene (160), polyoxypropylene (30), glycol 6. A formulation under claim 2 or 3, in which the hydrophilic surfactant is polysorbate 20, polysorbate 40, or polysorbate 80 7. A formulation under any one of claims 2 through 6, in which the lipid surfactant is sorbitan fatty acid ester (e.g., sorbitan fatty acid ester, sorbitan monolorate, sorbitan monopalmite).

8. Glycerin fat acid ester (e.g., glycerin fat acid ester, glyceryl monostearate, glyceryl monomyristate, glyceryl monooleate, glyceryl triisoocthanoate, etc.); sucrose fat acid ester (e.g., sucrose fat acid ester, sucrose stearate, sucrose palmitate, etc.); or propylene glycol fat acid ester (e.g., propylene glycol fat acid ester, propylene glycol monostearate, etc.).A formulation pursuant to any one of claims 2 through 6, in which the lipid-loving surfactant is sorbitan fatty acid ester, sorbitan monooleate, sorbitan sesquioleate, or sorbitan trioleate.

9. A formulation pursuant to any one of claims 2 through 6, in which the lipid-loving surfactant is sorbitan trioleate.

10. A formulation pursuant to any one of claims 1 through 9, which is further incorporated with a selected antioxidant B from the group containing tocopherols (e.g., alpha-tocopherol, beta-tocopherol, gamma-tocopherol).

11. A formulation pursuant to claim 10, in which antioxidant B is a tocopherol selected from a group comprising alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and delta-tocopherol.

12. A formulation pursuant to claim 10, in which antioxidant B is alpha-tocopherol.

13. A formulation pursuant to claims 1 through 12, in which antioxidant A is ascorbyl palmitate, potassium ascorbate, sodium ascorbate, or ascorbic acid. 14.

15. Any formulation under claims 1 through 14, in which the active ingredient A is a non-reducing sugar (e.g., sucrose, and trehalose); or a sugar alcohol (e.g., sorbitol, erythritol, xylitol, maltitol, and lactitol).

16. Any formulation under claims 1 through 14, in which active ingredient A is sucrose, trehalose, sorbitol, or xylitol.

17. Any formulation under claims 1 through 14, in which active ingredient A is sucrose. or trehalose18. A formulation under any one of claims 1 through 17, in which the amount of squalene mixed is 50 to 500 times the weight of compound A.

19. A formulation under any one of claims 1 through 17, in which the amount of squalene mixed is 100 to 400 times the weight of compound A.

20. A formulation under any one of claims 1 through 17, in which the amount of squalene mixed is 200 to 300 times the weight of compound A.

21. A formulation under any one of claims 2 through 20, in which the amount of hydrophilic surfactant mixed is 0.5 to 250 times the weight of compound A.22.A formulation under any one of claims 2 through 20, in which the mixing amount of hydrophilic surfactant is 5 to 100 times the weight of compound A23. A formulation under any one of claims 2 through 20, in which the mixing amount of hydrophilic surfactant is 10 to 50 times the weight of compound A24. A formulation under any one of claims 2 through 21, in which the mixing amount of fatty surfactant is 0.5 to 250 times the weight of compound A25. A formulation under any one of claims 2 through 22, in which the mixing amount of fatty surfactant is 5 to 250 times the weight of compound A23.

26. A formulation under any one of claims 2 through 23, in which the mixing amount of the fatty surfactant is 10 to 50 times the weight of compound A.

27. A formulation under any one of claims 1 through 26, in which the mixing amount of the antioxidant A is 0.5 to 500 times the weight of compound A in terms of the weight of sodium ascorbate.

28. A formulation under any one of claims 1 through 26, in which the mixing amount of the antioxidant A is 2.5 to 250 times the weight of compound A in terms of the weight of sodium ascorbate. 29.

30. A formulation pursuant to any one of claims 1 through 26, in which the mixing amount of antioxidant A is 5 to 100 times the weight of compound A in terms of the weight of sodium ascorbate.

31. A formulation pursuant to any one of claims 1 through 29, in which the mixing amount of pharmaceutical filler A is 50 to 1000 times the weight of compound A.

32. A formulation pursuant to any one of claims 1 through 29, in which the mixing amount of pharmaceutical filler A is 100 to 750 times the weight of compound A.

33. A formulation pursuant to any one of claims 1 through 29, in which the mixing amount of pharmaceutical filler A is 200 to... 625 times the weight of compound A33. A formulation pursuant to any one of claims 10 through 32, in which the amount of antioxidant B mixed is 5 to 250 times the weight of compound A34. A formulation pursuant to any one of claims 10 through 32, in which the amount of antioxidant B mixed is 12.5 to 125 times the weight of compound A35. A formulation pursuant to any one of claims 10 through 32, in which the amount of antioxidant B mixed is 25 to 50 times the weight of compound A36. A formulation pursuant to any one of claims 1 through 35, in which the weight of compound A is 0.0001 to 0.65 times the weight obtained excluding compound A from the freeze-dried formulation.

37. Any formulation under claims 1 through 35, in which the weight of compound A is 0.0002 to 0.35 times the weight obtained excluding compound A from the freeze-dried formulation.

38. Any formulation under claims 1 through 35, in which the weight of compound A is 0.0005 to 0.065 times the weight obtained excluding compound A from the freeze-dried formulation.

39. Any formulation under claims 1 through 38... Any one of the following claims, in which both the D90 value of the particle size of the emulsion immediately after preparation and the reconstituted emulsion after 6 months of storage at 25°C in the form of a freeze-dried formulation is 1000 nm or less:

40. Any one of the following claims, in which the amount of increase in the area percentage values ​​of impurity UK-1.02 after 6 months of storage at 5°C in the form of a freeze-dried formulation is 5.0% or less: 41.

41. A formulation pursuant to any of the claims 1 through 39, in which the percentage increase in the area of ​​impurities UK-1.02 after 6 months of storage at 5°C in the form of a freeze-dried formulation is 1.0% or less; 42. A vaccine adjuvant in which a formulation pursuant to any of the claims 1 through 41 is included; 43. A vaccine in which a formulation pursuant to any of the claims 1 through 41 and an antigen is included; 44. A vaccine pursuant to claim 43, in which the antigen is derived from a pathogen; 45. A kit in which a formulation pursuant to any of the claims 1 through 41 and an antigen is included.