Liposome preparation containing vaccine adjuvant

Abstract

This disclosure relates to a liposome preparation excellent in storage stability for a compound useful as a vaccine adjuvant, and to a lyophilized preparation of the liposome preparation.

Description

Liposome formulations containing vaccine adjuvants

[0001] The present invention relates to a liposome preparation of a compound useful as a vaccine adjuvant, which has excellent storage stability, and to an aqueous liquid formulation and a freeze-dried formulation of said liposome preparation.

[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 formulate it into a liposome, emulsion, or other formulation in order to actually administer it to mammals as a vaccine adjuvant. Furthermore, in order to stockpile adjuvants not only for infectious diseases such as seasonal influenza but also for future pandemics, it is required to prepare a formulation that can withstand long-term storage. Highly storage-stable emulsion formulations of Compound A containing an ascorbic acid-based antioxidant have been reported (Patent Document 2). It has also been reported that liposome formulations of Compound A can be prepared (Patent Documents 3 and 4). However, no liposome formulations of Compound A with high storage stability are known. When combining a vaccine with an adjuvant, the formulation of the adjuvant that is compatible varies depending on the type of vaccine, so a highly storage-stable liposome formulation of Compound A has been desired.

[0004] International Publication No. WO 2017 / 061532 International Publication No. WO 2020 / 138217 International Publication No. WO 2018 / 181420 International Publication No. WO 2020 / 022272

[0005] Visudyne: EPAR-Product Information (ANNEXI SUMMARY OF PRODUCT CHARACTERISTICS)

[0006] The present invention provides a liposome formulation composition of Compound A which has excellent storage stability and immunostimulatory activity and is useful as a vaccine adjuvant.

[0007] In the liposome formulation of Compound A known to date, the weight ratio of Compound A to lipid components is 1:10, and it has been found that when this liposome formulation is freeze-dried and stored at 25°C for 6 months, it has low stability (the lipid content and Compound A content decrease, and the liposome particle size increases). Therefore, Visudyne, a commercially available freeze-dried liposome formulation, ＴＭ

[0003] Based on the formulation of an intravenous injection (Cheplapharm Co., Ltd.) (Non-Patent Document 1; ascorbic acid palmitate and dibutylhydroxytoluene as antioxidants), the addition of ascorbic acid palmitate and butylhydroxyanisole as antioxidants to a liposome formulation of Compound A was investigated. Although the addition of ascorbic acid palmitate and butylhydroxyanisole to a liposome formulation of Compound A somewhat inhibited the decrease in lipid content and Compound A content after 6 months of storage at 25°C, the effect was not sufficient, and it was also found that ascorbic acid palmitate was rapidly degraded immediately after liposome preparation. To solve these problems, the present inventors conducted extensive research and found that, although generally, decreasing the content of a drug substance in a formulation accelerates the decomposition of the drug substance, decreasing the content of Compound A in the formulation (to a weight ratio of Compound A to lipid component of 1:50) unexpectedly improved the stability of Compound A. Furthermore, the inventors have found that the addition of the buffering agent L-histidine not only suppresses pH fluctuations in the solution state but also improves the stability of Compound A and the lipid component during freeze-drying and storage without the addition of the antioxidant ascorbic acid palmitate. Furthermore, the inventors have found that in freeze-dried formulations containing L-histidine, pH stability, lipid component and Compound A stability, and particle size distribution stability are maintained during freeze-drying and long-term storage, even without the addition of the antioxidant butylhydroxyanisole. Furthermore, the inventors have found that in solution formulations containing L-histidine, both formulations containing butylhydroxyanisole and formulations without butylhydroxyanisole, pH stability, lipid component and Compound A stability, and particle size distribution stability are maintained after storage under accelerated conditions, leading to the completion of the present invention. Compound A has six unsaturated bonds derived from a squalene-like structure in its molecule, and these unsaturated bonds are susceptible to oxidation. The inventors have found that the addition of L-histidine maintains the antioxidant stability of Compound A without the addition of an antioxidant. It was also found that the addition of L-histidine contributes to the stability of the particle size distribution of Compound A.Furthermore, freeze-dried preparations of Compound A have a problem in that the pH of the aqueous liquid preparation after reconstitution with water is unstable. However, it has been found that by adding L-histidine, trishydroxymethylaminomethane or a phosphoric acid as a buffer, it is possible to prepare a freeze-dried preparation that maintains stability in the lipid content, the Compound A content, and the particle size distribution of liposomes after reconstitution with water, and that has a stable pH after reconstitution with water.

[0008] That is, the gist of the present invention is as follows.

[0009] [Item 1] i) A lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) one or more isotonicity agents selected from the group consisting of sucrose and trehalose.

[0010] [Item 2] i) a lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) sucrose.

[0011] [Item 3] i) a lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) trehalose.

[0012] [Item 4] The formulation according to any one of Items 1 to 3, wherein the buffering agent is L-histidine and / or L-histidine hydrochloride.

[0013] [Item 5] The formulation according to any one of Items 1 to 3, wherein the buffering agent is selected from the group consisting of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol).

[0014] [Item 6] The formulation according to any one of Items 1 to 5, wherein the liposome formulation is an aqueous formulation.

[0015] [Item 7] The liposome preparation according to any one of Items 1 to 5, wherein the liposome preparation is a freeze-dried preparation.

[0016] [Item 8] The formulation according to any one of Items 1 to 7, comprising one or more antioxidants selected from the group consisting of butylhydroxyanisole, L-methionine, L-cysteine, L-cysteine ​​hydrochloride hydrate, tocopherols (natural vitamin E, d-δ-tocopherol, alpha-tocopherol, tocopherol acetate), sodium edetate hydrate (EDTA), sodium thioglycolate, sodium pyrosulfite, sodium nitrite, sodium bisulfite, and sodium sulfite hydrate.

[0017] [Item 9] The formulation according to Item 8, wherein the antioxidant comprises one or more antioxidants selected from the group consisting of butylhydroxyanisole, alpha-tocopherol, and edetate sodium hydrate (EDTA).

[0018] [Item 10] The formulation according to Item 8, wherein the antioxidant is butylhydroxyanisole.

[0019] [Item 11] The formulation according to Item 8, wherein the antioxidant is alpha-tocopherol.

[0020] [Item 12] The formulation according to Item 8, wherein the antioxidant is edetate sodium hydrate (EDTA).

[0021] [Item 13] The formulation according to any one of Items 1 to 12, wherein the weight ratio of compound A or a pharmaceutically acceptable salt thereof to the lipid component is 1:40 to 1:100.

[0022] [Item 14] The formulation according to any one of Items 1 to 12, wherein the weight ratio of compound A or a pharmaceutically acceptable salt thereof to the lipid component is 1:40 to 1:70.

[0023] [Item 15] The formulation according to any one of Items 1 to 12, wherein the weight ratio of compound A or a pharmaceutically acceptable salt thereof to the lipid component is 1:50.

[0024] [Item 16] The formulation according to any one of Items 1 to 15, wherein the weight ratio of the lipid components dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG) is 1:1 to 2:1.

[0025] [Item 17] The formulation according to any one of Items 1 to 16, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, is 5 mmol / L to 50 mmol / L.

[0026] [Item 18] The formulation according to any one of Items 1 to 16, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, is 10 mmol / L to 40 mmol / L.

[0027] [Item 19] The formulation according to any one of Items 1 to 16, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 5 mmol / L to 50 mmol / L.

[0028] [Item 20] The formulation according to any one of Items 1 to 16, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 10 mmol / L to 40 mmol / L.

[0029] [Item 21] The formulation according to any one of Items 1 to 16, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, is 5 mmol / L to 50 mmol / L.

[0030] [Item 22] The formulation according to any one of Items 1 to 16, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, is 10 mmol / L to 40 mmol / L.

[0031] [Item 23] The formulation according to any one of Items 1 to 18, wherein the amount of sucrose and / or trehalose blended is 50 mg / mL to 110 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0032] [Item 24] The formulation according to any one of Items 1 to 18, wherein the amount of sucrose and / or trehalose blended is 80 mg / mL to 100 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0033] [Item 25] The formulation according to any one of Items 1 to 16, 19, and 20, wherein the amount of sucrose and / or trehalose blended when the aqueous liquid preparation is prepared so that Compound A is 0.2 mg / mL is 50 mg / mL to 110 mg / mL.

[0034] [Item 26] The formulation according to any one of Items 1 to 16, 19, and 20, wherein the amount of sucrose and / or trehalose blended when the aqueous liquid preparation is prepared so that Compound A is 0.2 mg / mL is 80 mg / mL to 100 mg / mL.

[0035] [Item 27] ​​The formulation according to any one of Items 1 to 16, 21, and 22, wherein the amount of sucrose and / or trehalose blended when the aqueous liquid preparation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 50 mg / mL to 110 mg / mL.

[0036] [Item 28] The formulation according to any one of Items 1 to 16, 21, and 22, wherein the amount of sucrose and / or trehalose blended when the aqueous liquid preparation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 80 mg / mL to 100 mg / mL.

[0037] [Item 29] The formulation according to any one of Items 1, 2, and 4 to 18, wherein the amount of sucrose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL is 50 mg / mL to 110 mg / mL.

[0038] [Item 30] The formulation according to any one of Items 1, 2, and 4 to 18, wherein the amount of sucrose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL is 80 mg / mL to 100 mg / mL.

[0039] [Item 31] The formulation according to any one of Items 1, 2, 4 to 16, 19, and 20, wherein the amount of sucrose blended when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 50 mg / mL to 110 mg / mL.

[0040] [Item 32] The formulation according to any one of Items 1, 2, 4 to 16, 19, and 20, wherein the amount of sucrose blended when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 80 mg / mL to 100 mg / mL.

[0041] [Item 33] The formulation according to any one of Items 1, 2, 4 to 16, 21, and 22, wherein the amount of sucrose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 50 mg / mL to 110 mg / mL.

[0042] [Item 34] The formulation according to any one of Items 1, 2, and 4 to 22, wherein the amount of sucrose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 80 mg / mL to 100 mg / mL.

[0043] [Item 35] The formulation according to any one of Items 1 and 3 to 18, wherein the amount of trehalose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, is 50 mg / mL to 110 mg / mL.

[0044] [Item 36] The formulation according to any one of Items 1, 3 to 18, wherein the amount of trehalose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, is 80 mg / mL to 100 mg / mL.

[0045] [Item 37] The formulation according to any one of Items 1, 3 to 16, 19, and 20, wherein the amount of trehalose blended when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 50 mg / mL to 110 mg / mL.

[0046] [Item 38] The formulation according to any one of Items 1, 3 to 16, 19, and 20, wherein the amount of trehalose blended when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 80 mg / mL to 100 mg / mL.

[0047] [Item 39] The formulation according to any one of Items 1, 3 to 16, 21, and 22, wherein the amount of trehalose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, is 50 mg / mL to 110 mg / mL.

[0048] [Item 40] The formulation according to any one of Items 1, 3 to 16, 21, and 22, wherein the amount of trehalose blended when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 80 mg / mL to 100 mg / mL.

[0049] [Item 41] The formulation according to any one of Items 8 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of the antioxidant is 0.05 μg / mL to 5 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0050] [Item 42] The formulation according to any one of Items 8 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of antioxidant blended is 0.25 μg / mL to 500 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0051] [Item 43] The formulation according to any one of Items 8 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of antioxidant blended is 0.5 μg / mL to 10 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0052] [Item 44] The formulation according to any one of Items 8 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of antioxidant blended is 1 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0053] [Item 45] The formulation according to any one of Items 8 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of antioxidant blended is 0.5 μg / mL to 10 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL.

[0054] [Item 46] The formulation according to any one of Items 8 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of antioxidant blended is 1 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL.

[0055] [Item 47] The formulation according to any one of Items 8 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of antioxidant blended is 0.5 μg / mL to 10 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0056] [Item 48] The formulation according to any one of Items 8 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of antioxidant is 1 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0057] [Item 49] The formulation according to any one of Items 8 to 10, 13 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of butylhydroxyanisole blended is 0.05 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0058] [Item 50] The formulation according to any one of Items 8 to 10, 13 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of butylhydroxyanisole blended is 0.25 μg / mL to 2 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0059] [Item 51] The formulation according to any one of Items 8 to 10, 13 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of butylhydroxyanisole blended when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 0.05 μg / mL to 5 μg / mL.

[0060] [Item 52] The formulation according to any one of Items 8 to 10, 13 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of butylhydroxyanisole blended when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL is 0.25 μg / mL to 2 μg / mL.

[0061] [Item 53] The formulation according to any one of Items 8 to 10, 13 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of butylhydroxyanisole blended is 0.05 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0062] [Item 54] The formulation according to any one of Items 8 to 10, 13 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of butylhydroxyanisole blended is 0.25 μg / mL to 2 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0063] [Item 55] The formulation according to any one of Items 8, 9, 11, 13 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of alpha-tocopherol blended when the aqueous liquid preparation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL is 0.5 μg / mL to 50 μg / mL.

[0064] [Item 56] The formulation according to any one of Items 8, 9, 11, 13 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of alpha-tocopherol blended when the aqueous liquid preparation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL is 2 μg / mL to 40 μg / mL.

[0065] [Item 57] The formulation according to any one of Items 8, 9, 11, 13 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of alpha-tocopherol blended when the aqueous liquid preparation is prepared so that Compound A is 0.2 mg / mL is 0.5 μg / mL to 50 μg / mL.

[0066] [Item 58] The formulation according to any one of Items 8, 9, 11, 13 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of alpha-tocopherol blended when the aqueous liquid preparation is prepared so that Compound A is 0.2 mg / mL is 2 μg / mL to 40 μg / mL.

[0067] [Item 59] The formulation according to any one of Items 8, 9, 11, 13 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of alpha-tocopherol blended when the aqueous liquid preparation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 0.5 μg / mL to 50 μg / mL.

[0068] [Item 60] The formulation according to any one of Items 8, 9, 11, 13 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of alpha-tocopherol blended when the aqueous liquid preparation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL is 2 μg / mL to 40 μg / mL.

[0069] [Item 61] The formulation according to any one of Items 8, 9, 12 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of edetate sodium hydrate (EDTA) is 1 μg / mL to 5 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0070] [Item 62] The formulation according to any one of Items 8, 9, 12 to 18, 23, 24, 29, 30, 35, and 36, wherein the amount of edetate sodium hydrate (EDTA) is 5 μg / mL to 2 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0071] [Item 63] The formulation according to any one of Items 8, 9, 12 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of edetate sodium hydrate (EDTA) is 1 μg / mL to 5 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL.

[0072] [Item 64] The formulation according to any one of Items 8, 9, 12 to 16, 19, 20, 25, 26, 31, 32, 37, and 38, wherein the amount of edetate sodium hydrate (EDTA) is 5 μg / mL to 2 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL.

[0073] [Item 65] The formulation according to any one of Items 8, 9, 12 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of edetate sodium hydrate (EDTA) is 1 μg / mL to 5 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0074] [Item 66] The formulation according to any one of Items 8, 9, 12 to 16, 21, 22, 27, 28, 33, 34, 39, and 40, wherein the amount of edetate sodium hydrate (EDTA) is 5 μg / mL to 2 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0075] [Item 67] The formulation according to any one of Items 1 to 18, 23, 24, 29, 30, 35, 36, 41 to 44, 49, 50, 55, 56, 61, and 62, wherein the aqueous solution has a pH of 6.0 to 8.0 when prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0076] [Item 68] The formulation according to any one of Items 1 to 18, 23, 24, 29, 30, 35, 36, 41 to 44, 49, 50, 55, 56, 61, and 62, wherein the aqueous solution has a pH of 6.5 to 7.5 when prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

[0077] [Item 69] The formulation according to any one of Items 1 to 16, 19, 20, 25, 26, 31, 32, 37, 38, 45, 46, 51, 52, 57, 58, 63, and 64, wherein the aqueous solution has a pH of 6.0 to 8.0 when prepared so that Compound A is 0.2 mg / mL.

[0078] [Item 70] The formulation according to any one of Items 1 to 16, 19, 20, 25, 26, 31, 32, 37, 38, 45, 46, 51, 52, 57, 58, 63, and 64, wherein the aqueous solution has a pH of 6.5 to 7.5 when prepared so that Compound A is 0.2 mg / mL.

[0079] [Item 71] The formulation according to any one of Items 1 to 16, 21, 22, 27, 28, 33, 34, 39, 40, 47, 48, 53, 54, 59, 60, 65, and 66, wherein the aqueous solution has a pH of 6.0 to 8.0 when prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0080] [Item 72] The formulation according to any one of Items 1 to 16, 21, 22, 27, 28, 33, 34, 39, 40, 47, 48, 53, 54, 59, 60, 65, and 66, wherein the aqueous solution has a pH of 6.5 to 7.5 when prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL.

[0081] [Item 73] The formulation according to any one of Items 1 to 18, 23, 24, 29, 30, 35, 36, 41 to 44, 49, 50, 55, 56, 61, 62, 67, and 68, wherein when an aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, the liposomes have an average particle size of 60 nm to 200 nm and a polydispersity index (PDI) of 0.01 to 0.2.

[0082] [Item 74] The formulation according to any one of Items 1 to 18, 23, 24, 29, 30, 35, 36, 41 to 44, 49, 50, 55, 56, 61, 62, 67, and 68, wherein when an aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, the liposomes have an average particle size of 80 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.15.

[0083] [Item 75] The formulation according to any one of Items 1 to 16, 19, 20, 25, 26, 31, 32, 37, 38, 45, 46, 51, 52, 57, 58, 63, 64, 69, and 70, wherein the liposomes have an average particle size of 60 nm to 200 nm and a polydispersity index (PDI) of 0.01 to 0.2 when an aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL.

[0084] [Item 76] The formulation according to any one of Items 1 to 16, 19, 20, 25, 26, 31, 32, 37, 38, 45, 46, 51, 52, 57, 58, 63, 64, 69, and 70, wherein the liposomes have an average particle size of 80 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.15 when an aqueous liquid formulation is prepared so that Compound A is 0.2 mg / mL.

[0085] [Item 77] The formulation according to any one of Items 1 to 16, 21, 22, 27, 28, 33, 34, 39, 40, 47, 48, 53, 54, 59, 60, 65, 66, 71, and 72, wherein when an aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, the liposomes have an average particle size of 60 nm to 200 nm and a polydispersity index (PDI) of 0.01 to 0.2.

[0086] [Item 78] The formulation according to any one of Items 1 to 16, 21, 22, 27, 28, 33, 34, 39, 40, 47, 48, 53, 54, 59, 60, 65, 66, 71, and 72, wherein when an aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, the liposomes have an average particle size of 80 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.15.

[0087] [Item 79] The formulation according to any one of Items 1 to 78, which is a freeze-dried formulation.

[0088] [Item 80] The formulation according to any one of Items 1 to 78, which is an aqueous liquid formulation.

[0089] [Item 81] The formulation according to any one of Items 1 to 80, wherein the lipid multilayer is a lipid bilayer.

[0090] [Item 82] A vaccine adjuvant comprising the formulation according to any one of Items 1 to 81.

[0091] [Item 83] A vaccine comprising the formulation according to any one of Items 1 to 81 and an antigen.

[0092] [Item 84] The vaccine of Item 83, wherein the antigen is a substance derived from a pathogen.

[0093] [Item 85] A kit comprising the formulation according to any one of Items 1 to 81 and an antigen.

[0094] According to the present invention, it is possible to provide a liposome formulation of Compound A, which is a vaccine adjuvant, that can further enhance the specific immune response to an antigen and has high storage stability.

[0095] FIG. 1 shows the results of Test Example 3-1 and is a graph showing the results of quantification by ELISA of OVA-specific IgG2c in the serum of immunized mice that were intramuscularly administered with the liposome preparation of Example 7. The vertical axis shows the antibody titer of OVA-specific IgG2c in the serum. The horizontal axis shows the administered sample (the dose of Example 7 administered is shown in parentheses). 1: Negative control (a mixture of equal volumes of ovalbumin (OVA) (2 mg / mL) and phosphate-buffered saline (100 μL / mouse)), 2: Liposome preparation of Example 7 (10 μg / mouse in terms of Compound A). FIG. 2 shows the results of Test Example 3-2 and is a graph showing the proportion of type 1 helper T cells in the spleen cells of mice that were intramuscularly administered with the liposome preparation of Example 7. The horizontal axis is the same as in FIG. 1. Figure 3 shows the results of Test Example 3-2 and is a graph showing the proportion of OVA tetramer-positive CD8 T cells in spleen cells of mice administered intramuscularly with the liposome preparation of Example 7. The horizontal axis is the same as in Figure 1. Figure 4 shows the results of Test Example 3-2 and is a graph showing the proportion of effector memory CD8 T cells in spleen cells of mice administered intramuscularly with the liposome preparation of Example 7. The horizontal axis is the same as in Figure 1.

[0096] The formulation of the present invention is a liposome formulation containing dimyristoyl phosphatidylcholine (DMPC), egg yolk phosphatidylglycerol (EPG), Compound A, a buffer (L-histidine, L-histidine hydrochloride, etc.), and sucrose. The liposome formulation of the present invention also includes aqueous formulations containing the above-mentioned composition that have not been subjected to lyophilization, lyophilized formulations, and aqueous formulations obtained by reconstituting lyophilized formulations. Another embodiment of the present invention is a liposome formulation containing dimyristoyl phosphatidylcholine (DMPC), egg yolk phosphatidylglycerol (EPG), Compound A, a buffer (L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, trishydroxymethylaminomethane (trometamol), etc.), and an isotonic agent (sucrose and / or trehalose).

[0097] In the formulations of the present invention, Compound A contained as an active ingredient may be in its 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 preparation methods thereof are described in Patent Document 1, and Compound A or a pharmaceutically acceptable salt thereof can be prepared, for example, according to the method described in Patent Document 1. In the formulations of the present invention, the weight of Compound A refers to the weight of the free form of Compound A unless otherwise specified. Therefore, when Compound A is used as its pharmaceutically acceptable salt, the weight of Compound A plus the weight of the salt must be used. Conversely, when the weight ratio of a pharmaceutically acceptable salt of Compound A to other components is shown, it represents the weight ratio of Compound A in its free form.

[0098] The liposome formulation of the present invention refers to an aqueous formulation that does not undergo a lyophilization process, a lyophilized formulation, or an aqueous formulation obtained by reconstituting a lyophilized formulation. The liposome formulation of the present invention is obtained by mixing dimyristoylphosphatidylcholine (DMPC), egg yolk phosphatidylglycerol (EPG), Compound A, and optionally an antioxidant in an organic solvent, evaporating the solvent under reduced pressure, and then vacuum-drying the mixture. The mixture is then hydrated with an aqueous solution containing a buffer (e.g., L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, trishydroxymethylaminomethane (trometamol)) and an isotonic agent (sucrose and / or trehalose), followed by sizing using an extruder or the like and filtering. The lyophilized formulation can be obtained by freezing the aqueous formulation obtained above, removing the solvent at -5°C to -30°C under reduced pressure, and then drying at 20°C to 30°C under reduced pressure. The freeze-dried preparation is administered after reconstitution with water for injection or the like so that the concentration of compound A is 0.01 to 0.2 mg / mL.

[0099] In the liposome formulation of the present invention, Compound A is prepared so that its concentration when made into an aqueous liquid is 0.01 mg / mL to 0.2 mg / mL. In one embodiment, the content of Compound A in the formulation of the present invention is 0.2 mg / mL. In another embodiment, the content of Compound A in the formulation of the present invention is 0.01 mg / mL to 0.04 mg / mL.

[0100] The lipid component in the liposome preparation of the present invention may be a combination of dimyristoyl phosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG). The weight ratio of Compound A to the lipid component (total weight of dimyristoyl phosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG)) in the liposome preparation of the present invention may be 1:40 to 1:100, preferably 1:40 to 1:70, and more preferably 1:50. The weight ratio of dimyristoyl phosphatidylcholine (DMPC) to egg yolk phosphatidylglycerol (EPG) in the liposome preparation of the present invention may be 1:1 to 2:1, preferably 1:1 to 3:2.

[0101] Examples of buffers in the liposome formulation of the present invention include L-histidine, L-histidine hydrochloride, phosphates (disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate), or trishydroxymethylaminomethane (trometamol), and may be a combination of two or more of these. Preferred are L-histidine or L-histidine hydrochloride, and more preferred is L-histidine hydrochloride. Note that L-histidine hydrochloride in the buffer exists as L-histidine hydrochloride monohydrate in the solid state and as L-histidine hydrochloride in the solution state, and both forms are included in L-histidine hydrochloride. The content of the buffer in the formulation of the present invention is, regardless of the content of Compound A, added within a concentration range that exhibits buffering capacity when prepared as an aqueous formulation. In one embodiment of the content of the buffering agent in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, the blending concentration of the buffering agent is 5 mmol / L to 50 mmol / L, preferably 10 mmol / L to 40 mmol / L. In another embodiment of the content of the buffering agent in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.2 mg / mL, the blending concentration of the buffering agent is 5 mmol / L to 50 mmol / L, preferably 10 mmol / L to 40 mmol / L. In another embodiment of the content of the buffering agent in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, the blending concentration of the buffering agent is 5 mmol / L to 50 mmol / L, preferably 10 mmol / L to 40 mmol / L.

[0102] Sucrose and / or trehalose are used as the isotonic agent in the liposome formulation of the present invention. The content of the isotonic agent (sucrose and / or trehalose) in the formulation of the present invention is added in a concentration range such that the osmotic pressure of the aqueous formulation becomes equivalent to that of physiological saline, regardless of the content of Compound A. When the formulation of the present invention is a lyophilized formulation, the isotonic agent (sucrose and / or trehalose) is added in an amount necessary not only to adjust the osmotic pressure when the lyophilized formulation is rehydrated to form an aqueous formulation, but also to form a lyophilized cake. The content of the isotonic agent (sucrose and / or trehalose) in the formulation of the present invention is 50 mg / mL to 110 mg / mL, preferably 80 mg / mL to 100 mg / mL, and more preferably 90 mg / mL, when the aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL. In another embodiment of the content of the isotonic agent (sucrose and / or trehalose) in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.2 mg / mL, the amount of the isotonic agent (sucrose and / or trehalose) is 50 mg / mL to 110 mg / mL, preferably 80 mg / mL to 100 mg / mL, and more preferably 90 mg / mL. In another embodiment of the content of the isotonic agent (sucrose and / or trehalose) in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of the isotonic agent (sucrose and / or trehalose) is 50 mg / mL to 110 mg / mL, preferably 80 mg / mL to 100 mg / mL, and more preferably 90 mg / mL.

[0103] Examples of antioxidants in the liposome formulation of the present invention include butylhydroxyanisole, L-methionine, L-cysteine, tocopherols (natural vitamin E, d-δ-tocopherol, alpha-tocopherol, tocopherol acetate), edetate sodium hydrate (EDTA), sodium thioglycolate, sodium pyrosulfite, sodium nitrite, sodium bisulfite, and sodium sulfite hydrate, and combinations of two or more of these are also acceptable. Butylhydroxyanisole, alpha-tocopherol, and edetate sodium hydrate (EDTA) are preferred. Alpha-tocopherol and edetate sodium hydrate (EDTA) are more preferred. The antioxidant content in the formulation of the present invention is within a concentration range that allows the antioxidant to exert its antioxidant activity when prepared as an aqueous formulation.

[0104] In one embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of antioxidant is 0.5 μg / mL to 10 μg / mL, preferably 1 μg / mL to 5 μg / mL. In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of antioxidant is 0.01 μg / mL to 10 μg / mL, preferably 1 μg / mL to 5 μg / mL. In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.2 mg / mL, the amount of antioxidant is 0.5 μg / mL to 10 μg / mL, preferably 1 μg / mL to 5 μg / mL. In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of antioxidant is 0.5 μg / mL to 10 μg / mL, preferably 1 μg / mL to 5 μg / mL. In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of antioxidant is 10 μg / mL to 5 mg / mL, preferably 100 μg / mL to 2 mg / mL. In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.2 mg / mL, the amount of antioxidant is 10 μg / mL to 5 mg / mL, preferably 100 μg / mL to 2 mg / mL. In another embodiment of the content of the antioxidant in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of the antioxidant is 10 μg / mL to 5 mg / mL, preferably 100 μg / mL to 2 mg / mL. In another embodiment of the content of the antioxidant in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of the antioxidant is 1 μg / mL to 5 mg / mL, preferably 5 μg / mL to 2 mg / mL, more preferably 10 μg / mL to 1 mg / mL, and even more preferably 50 μg / mL to 500 μg / mL.In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.2 mg / mL, the amount of antioxidant is 1 μg / mL to 2 mg / mL, preferably 5 μg / mL to 2 mg / mL, more preferably 10 μg / mL to 1 mg / mL, and even more preferably 50 μg / mL to 500 μg / mL. In another embodiment of the antioxidant content in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of antioxidant is 1 μg / mL to 2 mg / mL, preferably 5 μg / mL to 2 mg / mL, more preferably 10 μg / mL to 1 mg / mL, and even more preferably 50 μg / mL to 500 μg / mL.

[0105] In one embodiment of the content of butylhydroxyanisole in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of butylhydroxyanisole is 0.05 μg / mL to 5 μg / mL, preferably 0.25 μg / mL to 2 μg / mL. In another embodiment of the content of butylhydroxyanisole in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.2 mg / mL, the amount of butylhydroxyanisole is 0.05 μg / mL to 5 μg / mL, preferably 0.25 μg / mL to 2 μg / mL. In another embodiment of the content of butylhydroxyanisole in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of butylhydroxyanisole is 0.05 μg / mL to 5 μg / mL, preferably 0.25 μg / mL to 2 μg / mL.

[0106] In one embodiment of the alpha-tocopherol content in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of alpha-tocopherol blended is 0.5 μg / mL to 50 μg / mL, preferably 2 μg / mL to 40 μg / mL, and more preferably 5 μg / mL to 20 μg / mL. In another embodiment of the alpha-tocopherol content in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.2 mg / mL, the amount of alpha-tocopherol blended is 0.5 μg / mL to 50 μg / mL, preferably 2 μg / mL to 40 μg / mL, and more preferably 5 μg / mL to 20 μg / mL. In another embodiment of the alpha-tocopherol content in the formulation of the present invention, when an aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of alpha-tocopherol blended is 0.5 μg / mL to 50 μg / mL, preferably 2 μg / mL to 40 μg / mL, and more preferably 5 μg / mL to 20 μg / mL.

[0107] In one embodiment of the content of sodium edetate hydrate (EDTA) in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.2 mg / mL, the amount of sodium edetate hydrate (EDTA) is 1 μg / mL to 5 mg / mL, preferably 5 μg / mL to 2 mg / mL, more preferably 10 μg / mL to 1 mg / mL, and even more preferably 50 μg / mL to 500 μg / mL. In another embodiment of the content of sodium edetate hydrate (EDTA) in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.2 mg / mL, the amount of sodium edetate hydrate (EDTA) is 1 μg / mL to 5 mg / mL, preferably 5 μg / mL to 2 mg / mL, more preferably 10 μg / mL to 1 mg / mL, and even more preferably 50 μg / mL to 500 μg / mL. In another embodiment of the content of sodium edetate hydrate (EDTA) in the formulation of the present invention, when an aqueous formulation is prepared so that compound A is 0.01 mg / mL to 0.04 mg / mL, the amount of sodium edetate hydrate (EDTA) blended is 1 μg / mL to 5 mg / mL, preferably 5 μg / mL to 2 mg / mL, more preferably 10 μg / mL to 1 mg / mL, and even more preferably 50 μg / mL to 500 μg / mL.

[0108] The pH of the aqueous solution of the liposome formulation of the present invention is 6.0 to 8.0, preferably 6.5 to 8.0, and more preferably 6.5 to 7.5, regardless of the content of Compound A. Hydrochloric acid or sodium hydroxide may be added as appropriate to adjust the pH to an appropriate range. The pH of the formulation of the present invention is 6.0 to 8.0, preferably 6.5 to 8.0, and more preferably 6.5 to 7.5, when the aqueous formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL. The pH of the formulation of the present invention is 6.0 to 8.0, preferably 6.5 to 8.0, and more preferably 6.5 to 7.5, when the aqueous formulation is prepared so that Compound A is 0.2 mg / mL. The pH of the formulation of the present invention, when prepared so that the concentration of compound A is 0.01 mg / mL to 0.04 mg / mL, is 6.0 to 8.0, preferably 6.5 to 8.0, and more preferably 6.5 to 7.5.

[0109] The liposome particles in the liposome preparation of the present invention preferably have an average particle size of 60 nm to 160 nm and a polydispersity index (PDI) of 0.01 to 0.2. More preferably, the average particle size is 80 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.15, and even more preferably, the average particle size is 100 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.15. In the liposome particles of the present invention, the average particle size of the liposomes during the production process (before lyophilization) is preferably 60 nm to 160 nm and a polydispersity index (PDI) of 0.01 to 0.2. Preferably, the average particle size is 80 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.15. Furthermore, the average particle size of liposomes stored as a lyophilized formulation and then rehydrated is preferably 80 nm to 140 nm and the polydispersity index (PDI) is 0.01 to 0.15 after 6 months of storage at 5°C or 25°C. Furthermore, the average particle size of liposomes stored as an aqueous formulation is preferably 80 nm to 140 nm and the polydispersity index (PDI) is 0.01 to 0.15 after 6 months of storage at 5°C or 1 month at 25°C. Generally, particle size distribution (particle size and polydispersity index (PDI)) is measured and calculated using a dynamic light scattering particle size analyzer, a laser diffraction particle size analyzer, an image processing particle size analyzer, or the like. The particle size and polydispersity index (PDI) referred to herein refer to values ​​measured using a dynamic light scattering particle size distribution analyzer (Zetasizer Nano ZS (Malvern Instruments) or Zetasizer Ultra (Malvern Instruments)) after diluting a sample with a 9% wt sucrose aqueous solution, an L-histidine / 9% wt sucrose buffer solution having each L-histidine concentration, a buffer / 9% wt sucrose buffer solution, or an L-histidine / 9% wt trehalose buffer solution.

[0110] The aqueous preparation of the present invention refers to an aqueous preparation that has not undergone a freeze-drying process, or an aqueous preparation obtained by rehydrating a freeze-dried preparation. In the present application, the terms aqueous preparation and aqueous liquid preparation are used interchangeably. The aqueous preparation or aqueous liquid preparation is preferably an injectable preparation. The freeze-dried preparation of the present invention can be produced by filling an aqueous liposome preparation into a vial and freeze-drying it using a freeze-dryer under standard production conditions. While the production conditions are not particularly specified, specific examples include freezing at -40°C or below, depressurizing the chamber, simultaneously drying at a temperature between -30°C and 5°C for approximately 10 to 80 hours, and then raising the temperature to 20°C to 30°C and drying for approximately 10 to 30 hours. In the present application, the terms freeze-dried preparation and freeze-dried composition are used interchangeably. The freeze-dried preparation or freeze-dried composition is preferably an injectable preparation.

[0111] One embodiment of the liposome preparation of the present invention is: i) a lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) one or more isotonicity agents selected from the group consisting of sucrose and trehalose.

[0112] Another embodiment of the liposome preparation of the present invention is a liposome forming a lipid multilayer comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) sucrose.

[0113] Another embodiment of the liposome preparation of the present invention is a liposome forming a lipid multilayer comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) sucrose.

[0114] Another embodiment of the liposome preparation of the present invention is a liposome forming a lipid multilayer comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); iii) sucrose; iv) one or more antioxidants selected from the group consisting of butylated hydroxyanisole, L-methionine, L-cysteine, alpha-tocopherol, sodium edetate hydrate (EDTA), sodium thioglycolate, sodium pyrosulfite, sodium nitrite, sodium bisulfite, and sodium sulfite hydrate.

[0115] Another embodiment of the liposome preparation of the present invention includes a liposome preparation containing: i) a lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) a buffering agent selected from L-histidine or L-histidine hydrochloride; and iii) sucrose.

[0116] Another embodiment of the liposome preparation of the present invention includes a liposome preparation containing: i) a lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) a buffer selected from L-histidine and L-histidine hydrochloride; iii) sucrose; and iv) butylhydroxyanisole.

[0117] Another embodiment of the liposome preparation of the present invention is a lipid multilayer-forming liposome comprising: i) lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) a buffer selected from L-histidine and L-histidine hydrochloride; iii) one or more isotonicity agents selected from the group consisting of sucrose and trehalose; iv) A liposome preparation containing one or more antioxidants selected from the group consisting of butylhydroxyanisole, edetate sodium hydrate (EDTA), and alpha-tocopherol.

[0118] Another embodiment of the liposome preparation of the present invention is a lipid multilayer-forming liposome comprising: i) lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) a buffer selected from L-histidine and L-histidine hydrochloride; iii) one or more isotonicity agents selected from the group consisting of sucrose and trehalose; iv) Liposome preparations containing edetate sodium hydrate (EDTA).

[0119] Another embodiment of the liposome preparation of the present invention is a lipid multilayer-forming liposome comprising: i) lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) a buffer selected from L-histidine and L-histidine hydrochloride; iii) one or more isotonicity agents selected from the group consisting of sucrose and trehalose; iv) Liposomal preparations containing alpha tocopherol.

[0120] The liposome formulation of the present invention can contain other additives to the extent that the particle size of the liposomes after reconstitution does not change. Furthermore, at the time of administration, the liposome formulation can be mixed with a formulation containing a vaccine antigen to the extent that the particle size of the liposomes after reconstitution does not change. The method and mixing ratio of the formulation of the present invention and the vaccine antigen are not particularly limited. For example, the reconstituted liposome formulation can be mixed with an equal volume of a formulation containing a vaccine antigen by inverting the vial.

[0121] The vaccine antigen to be administered in combination with the liposome preparation of the present invention is not particularly limited, and examples thereof include antigen proteins, antigen peptides (partial peptides) derived from such antigen proteins, and complexes of these with carriers. Specific examples of vaccine antigens include 1) active ingredients of vaccines for preventing infectious diseases, and 2) tumor antigen proteins or tumor antigen peptides for cancer immunotherapy. The carrier is a substance that chemically and / or physically binds the antigen protein or antigen peptide, and examples thereof include proteins and lipids.

[0122] The formulation of the present invention can be provided as a kit containing a lyophilized formulation containing Compound A and a vaccine antigen.

[0123] The liposome formulation of the present invention, if a lyophilized formulation, can be reconstituted with water for injection so that the compound A concentration is 0.01 mg / mL to 0.2 mg / mL at the time of administration, and can be further mixed with a formulation containing a vaccine antigen before administration. If the formulation is an aqueous formulation, it can be prepared so that the compound A concentration is 0.01 mg / mL to 0.2 mg / mL, and can be further mixed with a formulation containing a vaccine antigen before administration. The dosage of the formulation of the present invention per dose is 1 ng to 250 μg, preferably 2.5 μg to 10 μg, in terms of the weight of compound A. Although the dosage varies depending on the type of vaccine antigen to be administered simultaneously and the age of the subject, it may be administered once, or it may be administered once or multiple times as booster doses.

[0124] According to the present invention, storage stability was demonstrated as shown in the following test examples.

[0125] 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.

[0126] In the following Examples, Reference Examples, and Comparative Examples, the following dimyristoyl phosphatidylcholine, egg yolk phosphatidylglycerol, butylhydroxyanisole, ascorbic acid palmitate, sucrose, trehalose, L-histidine hydrochloride monohydrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, trishydroxymethylaminomethane, sodium edetate hydrate, alpha-tocopherol, sodium hydroxide, water for injection, cyclohexane, and ethanol were used, but the present invention is not limited to these. Dimyristoyl phosphatidylcholine (DMPC) (Nippon Fine Chemical Co., Ltd.) Egg yolk phosphatidylglycerol (EPG) (Nippon Fine Chemical Co., Ltd.) Butylhydroxyanisole (BHA) (Tokyo Chemical Industry Co., Ltd.) Ascorbic acid palmitate (AP) (Tokyo Chemical Industry Co., Ltd.) Sucrose (Merck & Co. or Nacalai Tesque, Inc.) Trehalose (Hayashibara Co., Ltd.) L-histidine hydrochloride monohydrate: L-histidine monohydrochloride monohydrate (Nacalai Tesque, Inc.) Disodium hydrogen phosphate dihydrate (Merck & Co.) Sodium dihydrogen phosphate dihydrate (Merck & Co.) Trishydroxymethylaminomethane (Nacalai Tesque, Inc.) Sodium edetate hydrate: Disodium dihydrogen ethylenediaminetetraacetate dihydrate (EDTA) (Nacalai Tesque, Inc.) Alpha-tocopherol: tocopherol (Merck) Sodium hydroxide (Nacalai Tesque, Inc.) Water for injection: distilled water for injection (Otsuka Pharmaceutical Factory, Inc.) Cyclohexane (Japan Energy Corporation or Fujifilm Wako Pure Chemical Industries, Ltd.) Ethanol (Nacalai Tesque, Inc.) The ultrapure water used for preparing each formulation was Milli-Q water (ultrapure water) produced using an ultrapure water production system (Merck).

[0127] Preparation of Liposomes of Comparative Examples 1 to 3 Comparative Example 1 The lipid components (dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG)) and (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 (Compound A) in the amounts shown in Table 1 were weighed into a screw tube, and 20.0 g of cyclohexane and 0.40 g of ethanol were added, followed by dissolution in a water bath at 60°C. The mixture was filtered through a 0.2 μm membrane filter (Advantec), frozen with dry ice and acetone, and then vacuum-dried using a freeze dryer (Tokyo Rikakikai Co., Ltd.) to remove the solvent and obtain a premix. 4.8 g of the resulting premix was rehydrated by adding 431 mL of 9% sucrose aqueous solution and stirred in a 60°C water bath until no aggregates remained. The solution was sieved three times using an extruder equipped with a 100 nm polycarbonate filter at an extrusion pressure of 1.5 MPa, and then filtered through a 0.22 μm sterile filter (Stericup; Thermo Scientific) to obtain the liposome bulk solution of Comparative Example 1. 5 mL of this solution was filled into 20 mL amber glass vials and freeze-dried using a shelf-type freeze dryer (DRC-1000, square dry chamber for freeze dryer, FDU-2100; Tokyo Rikakikai Co., Ltd.). Thereafter, the pressure was restored with nitrogen gas, and the container was sealed with a rubber stopper and an aluminum cap to obtain a freeze-dried composition of Comparative Example 1.

[0128] Comparative Examples 2 and 3 Lipid components (DMPC and EPG) and Compound A were weighed into screw tubes in the amounts shown in Table 1, and a BHA solution prepared by dissolving butylhydroxyanisole (BHA) in cyclohexane and an AP solution prepared by dissolving ascorbic acid palmitate (AP) in cyclohexane / ethanol (9:1) were separately added to the tubes so that the BHA and AP amounts were as shown in Table 1. Further addition was made so that the final amounts were 20.0 g of cyclohexane and 0.40 g of ethanol. The same preparation method as in Comparative Example 1 was used to obtain the liposome bulk solutions and freeze-dried compositions of Comparative Examples 2 and 3.

[0129] Preparation of liposomes in Examples 1 to 8 and Comparative Examples 4 and 5 (Reference Examples 1 and 2) Preparation of preliminary mixture (lipid / compound A / BHA mixed preparation) The lipid components (dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG)), (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 (Compound A), and butylhydroxyanisole (BHA) were weighed into a recovery flask in the amounts shown in Table 2, and the amounts of cyclohexane and ethanol shown in Table 2 were added, followed by dissolution in a water bath at 50°C. The mixture was filtered through a 0.2 μm membrane filter (ADVANTEC), frozen with dry ice-acetone, and then the solvent was removed by vacuum drying using a freeze dryer (Tokyo Rikakikai Co., Ltd.) to obtain pre-mixtures of Reference Examples 1 and 2. The mixture was placed in a glass container, sealed with nitrogen, and stored frozen until use.

[0130] (Reference Examples 3 to 7) Preparation of L-histidine / 9 wt% sucrose buffer solutions L-histidine hydrochloride monohydrate and sucrose were dissolved in water for injection in the amounts shown in Table 3, and 1 mol / L aqueous sodium hydroxide solution and water for injection were added thereto to adjust the volume after preparation to 600 mL and the pH to 6.5 to 7.0, thereby preparing L-histidine / 9% wt sucrose buffer solutions with various L-histidine concentrations (Reference Examples 3 to 7).

[0131] (Examples 1 to 8, Comparative Examples 4 to 5) For the combinations of premixes and L-histidine / 9% wt sucrose buffer solutions listed in Table 4, 2.87 g of the premix and 258.04 g of L-histidine / 9% wt sucrose buffer solutions with the respective L-histidine concentrations were weighed into an Erlenmeyer flask and stirred and dispersed at room temperature using a magnetic stirrer until no aggregates were observed. Using an extruder equipped with a 100 nm polycarbonate filter, the solution was sieved 4 to 6 times until the average particle size was 150 nm or less and the PDI was less than 0.1. Sterile filtration was performed through a 0.22 μm sterilizing filter (Stericup; Thermo Scientific) to obtain the liposome bulk solutions of Comparative Example 4, Examples 1 to 4, Comparative Example 5, and Examples 5 to 8, formulated in the ratios listed in Table 5. Each of these solutions was filled into a glass vial (ISO 2R vial: manufactured by SCHOTT) in an amount of 1 mL, and freeze-dried using a freeze dryer (LyoStar3; manufactured by SP Scientific). The vial was then re-pressurized with nitrogen gas, and sealed with a rubber stopper and an aluminum cap to obtain the freeze-dried compositions of Comparative Example 4, Examples 1 to 4, Comparative Example 5, and Examples 5 to 8.

[0132] Comparative Example 6: 18.71 g of DMPC, 12.9 g of EPG, 0.7119 g of Compound A, 0.004 g of BHA, and 0.0398 g of AP were weighed into a recovery flask, and 129.46 g of cyclohexane and 2.59 g of ethanol were added. The mixture was dissolved in a water bath at 50°C. The mixture was filtered through a 0.2 μm membrane filter (Advantec), frozen with dry ice and acetone, and then the solvent was removed by vacuum drying using a freeze dryer (Tokyo Rikakikai Co., Ltd.) to obtain a premix. 24.57 g of the resulting premix was hydrated by adding 2375.4 g of 9% sucrose aqueous solution and stirred at room temperature until no clumps remained. This solution was sized three times using an extruder equipped with a 100 nm polycarbonate filter at an extrusion pressure of 1 MPa, and then filtered through a 0.22 μm sterilizing filter (Stericup; Thermo Scientific) to obtain the liposome bulk solution of Comparative Example 6.

[0133] Preparation of Liposomes of Comparative Examples 7 to 10 and Examples 9 to 15 (Reference Example 8) Preparation of Premixture (Lipid / Compound A Mixture Preparation) 214.63 g of dimyristoylphosphatidylcholine (DMPC), 147.88 g of egg yolk phosphatidylglycerol (EPG), and 7.49771 g of (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexane-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 (Compound A) were weighed into an Erlenmeyer flask, and 1485.27 g of cyclohexane was added. 37.04 g of ethanol was added and dissolved in a water bath at 50°C. The mixture was filtered through a 0.2 µm membrane filter (ADVANTEC), frozen in a freeze dryer (Tokyo Rikakikai Co., Ltd.), and the solvent was removed by vacuum drying to obtain a premix of Reference Example 8. The mixture was placed in a plastic container, sealed with nitrogen, and stored frozen until use.

[0134] (Reference Examples 9 to 15) Preparation of each buffer / 9% wt sucrose buffer solution and L-histidine / 9% wt trehalose buffer solution Each buffer (disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, or trishydroxymethylaminomethane, or L-histidine hydrochloride monohydrate) and an isotonic agent (sucrose or trehalose) were dissolved in ultrapure water in the amounts shown in Table 6, and 1 mol / L aqueous sodium hydroxide solution and ultrapure water were added to adjust the volume after preparation to 500 mL and the pH to 6.5 to 7.1, to prepare each buffer / 9% wt sucrose buffer solution (Reference Examples 9 to 14) and L-histidine / 9% wt trehalose buffer solution (Reference Example 15).

[0135] (Examples 9-15, Comparative Examples 7-10) The premixes listed in Table 7 were combined with each buffer / 9% wt sucrose buffer or L-histidine / 9% wt trehalose buffer to give a Compound A concentration of 0.2 mg / mL. The premixes and each buffer / 9% wt sucrose buffer or L-histidine / 9% wt trehalose buffer were weighed into glass containers and stirred and dispersed at room temperature using a magnetic stirrer until no aggregates were observed. Using an extruder equipped with a 100 nm polycarbonate filter, the solution was sized to an average particle size of 150 nm or less and a PDI of less than 0.15. Sterile filtration was performed through a 0.22 μm sterilizing filter (Stericup; Thermo Scientific) to obtain the liposome bulk solutions formulated in the ratios listed in Comparative Examples 7-9, Examples 9-11, and Example 15 in Table 8. Of these liposome bulk solutions, 0.5 mL of each of the liposome bulk solutions of Comparative Examples 7 to 9, Example 10, and Example 15 was filled into glass vials (2 mL vials: manufactured by Fuji Glass Co., Ltd.) and freeze-dried using a freeze-dryer (LyoStar4; manufactured by SP Scientific Co., Ltd.). The pressure was then restored with nitrogen gas, and the vials were sealed with rubber stoppers and aluminum caps to obtain the freeze-dried compositions of Comparative Example 10 and Examples 12 to 15.

[0136] Preparation of Liposomes of Examples 16 to 25 (Reference Example 16) Preparation of L-histidine / 9% wt sucrose buffer solution (for Examples 16 to 20) 2.10 g of L-histidine hydrochloride monohydrate and 45 g of sucrose were dissolved in ultrapure water, and a 1 mol / L aqueous sodium hydroxide solution and ultrapure water were added to adjust the pH to approximately 6.8. Ultrapure water was then added to bring the volume to 500 mL, yielding an L-histidine / 9% wt sucrose buffer solution with a pH of 6.8 (Reference Example 16).

[0137] Reference Example 17 Preparation of L-histidine / 9% wt sucrose buffer solution (for Examples 21 to 25) 2.10 g of L-histidine hydrochloride monohydrate and 45 g of sucrose were dissolved in ultrapure water, and a 1 mol / L aqueous solution of sodium hydroxide and ultrapure water were added to adjust the pH to approximately 7.3. Ultrapure water was then added to bring the volume to 500 mL, yielding an L-histidine / 9% wt sucrose buffer solution with a pH of 7.3 (Reference Example 17).

[0138] Examples 16 to 20: The premix of Reference Example 8 and the L-histidine / 9% wt sucrose buffer solution of Reference Example 16 were combined to a Compound A concentration of 0.2 mg / mL. The premix and the L-histidine / 9% wt sucrose buffer solution were weighed into a glass container and stirred and dispersed at room temperature using a magnetic stirrer until no aggregates were observed. Using an extruder equipped with a 100 nm polycarbonate filter, the solution was sized to an average particle size of 150 nm or less and a PDI of less than 0.15. The solution was diluted with the L-histidine / 9% wt sucrose buffer solution of Reference Example 16 to the Compound A concentration listed in Table 9. Sterile filtration was performed through a 0.22 μm sterilizing filter (Stericup; Thermo Scientific) to obtain the liposome bulk solutions of Examples 16 to 20 formulated in the ratios listed in Table 9.

[0139] Examples 21 to 25: The premix of Reference Example 8 and the L-histidine / 9% wt sucrose buffer solution of Reference Example 17 were combined to a compound A concentration of 0.2 mg / mL. The premix and the L-histidine / 9% wt sucrose buffer solution were weighed into a glass container and stirred and dispersed at room temperature using a magnetic stirrer until no aggregates were observed. Using an extruder equipped with a 100 nm polycarbonate filter, the solution was sized to an average particle size of 150 nm or less and a PDI of less than 0.15. The solution was diluted with the L-histidine / 9% wt sucrose buffer solution of Reference Example 17 to the compound A concentrations listed in Table 9. Sterile filtration was performed through a 0.22 μm sterilizing filter (Stericup; Thermo Scientific) to obtain the liposome bulk solutions of Examples 21 to 25 formulated in the ratios listed in Table 9. Each of these solutions was filled into a glass vial (2 mL vial: manufactured by Fuji Glass Co., Ltd.) in an amount of 1 mL, and freeze-dried using a freeze dryer (LyoStar4; manufactured by SP Scientific Co., Ltd.). The pressure was then restored with nitrogen gas, and the vial was sealed with a rubber stopper and an aluminum cap, yielding the freeze-dried compositions of Examples 21 to 25.

[0140] Preparation of Liposomes of Examples 26 to 37 (Reference Examples 18 and 19) - Preparation of Premix (Lipid / Compound A / Antioxidant Mixture Preparation) 3.0 g of the premix of Reference Example 8 was weighed into an Erlenmeyer flask, and a BHA solution prepared separately by dissolving butylhydroxyanisole (BHA) in cyclohexane to a BHA concentration of 0.37 mg / mL was added to the flask so that the BHA amount was 0.37 mg. Further, cyclohexane and ethanol were added to the mixture so that the final amounts were 12 g of cyclohexane and 0.24 g of ethanol, and the mixture was dissolved in a water bath at 50°C. The mixture was filtered through a 0.2 μm membrane filter (ADVANTEC), frozen in a freeze dryer (LyoStar4; SP Scientific), and the solvent was removed by vacuum drying to obtain the premix of Reference Example 18. The premixes of Reference Example 19 were obtained in the same manner as in Reference Example 18, except that an alpha-tocopherol solution prepared by dissolving alpha-tocopherol in cyclohexane to give an alpha-tocopherol concentration of 5.88 mg / mL was added instead of the BHA solution, so that the amount of alpha-tocopherol was 5.88 mg. These premixes were each placed in a glass container, sealed with nitrogen, and stored frozen until use.

[0141] (Reference Example 20) Preparation of L-histidine / 9% wt sucrose buffer solution 2.10 g of L-histidine hydrochloride monohydrate and 45 g of sucrose were dissolved in ultrapure water, and a 1 mol / L aqueous sodium hydroxide solution and ultrapure water were added to adjust the pH to approximately 6.9. Ultrapure water was then added to adjust the volume after preparation to 500 mL, thereby preparing an L-histidine / 9% wt sucrose buffer solution with a pH of 6.9 (Reference Example 20).

[0142] (Reference Example 21) - Preparation of L-histidine / EDTA / 9% wt sucrose buffer solution An L-histidine / EDTA / 9% wt sucrose buffer solution (Reference Example 21) having a pH of 6.9 was obtained in the same manner as in Reference Example 20, except that 50 mg of edetate disodium edetate hydrate (EDTA) was additionally added and dissolved in ultrapure water.

[0143] (Examples 26 to 37) The premix and buffer solution combinations shown in Table 10 were weighed into glass containers so as to achieve the blending ratios shown in Examples 26 to 29 in Table 10, and the mixtures were stirred and dispersed at room temperature using a magnetic stirrer until no aggregates were observed. Using an extruder equipped with a 100 nm polycarbonate filter, the solution was sized to an average particle size of 150 nm or less and a PDI of less than 0.15. This solution was diluted with each buffer solution to achieve the Compound A concentrations shown in Examples 30 to 37 in Table 10. Sterile filtration was performed through a 0.22 μm sterilizing filter (Stericup; Thermo Scientific) to obtain the liposome bulk solutions of Examples 26 to 37 formulated at the ratios shown in Table 10. Of these liposome bulk solutions, 1 mL of each of the liposome bulk solutions of Examples 26 to 29 and Examples 34 to 37 was filled into glass vials (2 mL vials: manufactured by Fuji Glass Co., Ltd.) and freeze-dried using a freeze dryer (LyoStar4; manufactured by SP Scientific Co., Ltd.). The pressure was then restored with nitrogen gas, and the vials were sealed with rubber stoppers and aluminum caps to obtain the freeze-dried compositions of Examples 26 to 29 and Examples 34 to 37.

[0144] (Test Methods) Measurement of particle size distribution (average particle size and polydispersity index (PDI)), quantification of the content of lipid components, quantification of the content of Compound A and related substances, measurement of pH, measurement of water content, and measurement of the encapsulation rate of Compound A in liposomes were carried out according to the following test methods.

[0145] Test Method 1-1 (Particle Size Distribution Measurement: Evaluation of Mean Particle Diameter and Polydispersity Index (PDI)) For each of the liposome bulk solutions of Comparative Examples 1 to 3 and the reconstituted products of each of the freeze-dried compositions of Comparative Examples 1 to 3, particle size distribution (mean particle diameter and PDI) was measured by dynamic light scattering using a Zetasizer Nano (MALVERN) (see Tables 12 to 14). Specifically, the liposome bulk solution was used as is, and the freeze-dried composition was reconstituted with 4.5 mL of water for injection to prepare a pre-dilution solution. 30 μL of the pre-dilution solution was injected into 1.5 mL of 9% wt sucrose solution filtered through a 0.2 μm filter to prepare a measurement solution, which was then placed in a polystyrene measurement cell and measured.

[0146] Test Method 1-2 (Particle Size Distribution Measurement: Evaluation of Mean Particle Diameter and Polydispersity Index (PDI)) For each of the liposome bulk solutions of Comparative Examples 4, 5, 7 to 10 and Examples 1 to 37, and the reconstituted products of each freeze-dried composition, particle size distribution (mean particle diameter and PDI) was measured by dynamic light scattering using a Zetasizer Nano (MALVERN) (see Tables 16, 17, 20, 23, 26, 29, 32, 35, and 39). Specifically, the liposome bulk solutions were used as they were, and the freeze-dried compositions were reconstituted with 0.9 mL (Comparative Examples 4 and 5, Examples 1 to 8) or 0.45 mL (Comparative Example 10, Examples 12 to 15) of water for injection or ultrapure water to prepare pre-dilution solutions. In Comparative Examples 4, 5, 7 to 10 and Examples 1 to 16, 21, 26 to 29, L-histidine / 9% wt sucrose buffer solution (Reference Examples 3 to 7) having each L-histidine concentration listed in Table 3, or each buffer / 9% wt sucrose buffer solution (Reference Examples 9 to 14) or L-histidine / 9% wt trehalose buffer solution (Reference Example 15) listed in Table 6 was filtered through a 0.2 μm filter to prepare a dilution solution, and 30 μL of the pre-dilution solution was injected into 1.5 mL of the dilution solution corresponding to each pre-dilution solution (see Table 4 or Table 7) to prepare a measurement solution. In Examples 17 to 20, 22 to 25, and 30 to 37, the pre-dilution solution was diluted with a buffer corresponding to each pre-dilution solution so that the total lipid concentration was 0.2 mg / mL to prepare a measurement solution, which was then placed in a polystyrene measurement cell and measured.

[0147] Test Method 2-1 (Lipid Content: Initial Samples of Comparative Examples 1 to 3 and Samples Stored for One Month) The lipid content of each initial sample (stored at -20°C) of each freeze-dried composition of Comparative Examples 1 to 3 and samples stored for one month under each storage condition was quantified using high-performance liquid chromatography. Specifically, the freeze-dried sample was dissolved by accurately adding 9.5 mL of the diluent shown below to prepare a test solution, which was then measured using high-performance liquid chromatography under the following conditions. Separately, approximately 81.6 mg of EPG and approximately 118.4 mg of DMPC were precisely weighed and the diluent shown below was added to make exactly 20 mL (standard stock solution). Exactly 4 mL, 5 mL, and 6 mL of this standard stock solution were weighed into a volumetric flask, and the diluent was added to make exactly 10 mL each to prepare standard solutions. These were then measured using high-performance liquid chromatography under the following conditions, and a calibration curve was created for the concentration and area of ​​each lipid. The lipid content was evaluated using this calibration curve. <Dilution solution> Methanol / water mixture (9:1) <High-performance liquid chromatography measurement conditions> Mobile phase A: Chloroform / methanol / 28% aqueous ammonia = 160 / 39 / 1 Mobile phase B: Chloroform / methanol / water / 28% aqueous ammonia = 120 / 68 / 11 / 1 Gradient conditions: Standard solution: B Conc. 0% → 100% (14-25 min) → 0% (25.1 min) Test solution: B Conc. 0% → 100% (14-45 min) → 0% (45.1 min) Detector: Evaporative light scattering detector ELSD (Gain: 7) Column: Astec Diol (5 μm, 4.6 × 250 mm) Column temperature: 25°C Autosampler temperature: 25°C Syringe cleaning solution: Methanol / water mixture (9:1) Flow rate: 1 mL / min Injection volume: 2 μL

[0148] Test method 2-2 (Lipid content: 3-month and 6-month stored products of Comparative Examples 1 to 3, Comparative Examples 4 and 5, and Examples 1 to 8) The lipid content of each of the 3-month and 6-month stored products of each of the freeze-dried compositions of Comparative Examples 1 to 3, and each of the liposome bulk solutions of Comparative Examples 4 and 5, and Examples 1 to 8 was quantified using high performance liquid chromatography. Specifically, the measurement and evaluation were carried out by the method of Test Example 2, except that the high performance liquid chromatography measurements were as follows. <High-performance liquid chromatography measurement conditions> Mobile phase A: Isopropanol / hexane mixture (7:3) Mobile phase B: Methanol / water / acetic acid / triethylamine mixture (1700:300:9:1) Gradient conditions: B Conc. 10% (0-5 min) → 95% (20-25 min) → 10% (25.1 min) Detector: Evaporative light scattering detector ELSD (gain: 7) Column: Alltima Silica (5 μm, 4.6 x 250 mm) Column temperature: 40°C Autosampler temperature: 25°C Syringe cleaning solution: Methanol / water mixture (9:1) Flow rate: 1 mL / min Injection volume: 2 μL Adjust the ELSD gain and injection volume appropriately so that the peak of the substance to be measured does not plateau.

[0149] Test Method 3-1 (Quantitative Analysis of Compound A and Related Substances: Comparative Examples 1 to 3) The liposome bulk solutions and lyophilized compositions of Comparative Examples 1 to 3 were evaluated for the content of Compound A and related substances using high-performance liquid chromatography. Specifically, for the liposome bulk solutions, 1 mL of each liposome bulk solution was accurately measured and diluted to exactly 5 mL to prepare test solutions. For the lyophilized compositions, 9.5 mL of the diluent shown below was accurately added to each lyophilized composition to prepare test solutions, which were then measured using high-performance liquid chromatography under the following conditions. Separately, approximately 14 mg of Compound A was accurately measured and dissolved in methanol. 1 mL of water was added, followed by methanol to make exactly 20 mL (standard solution 1). Exactly 1 mL and 3 mL of this standard solution 1 were weighed into volumetric flasks, and the diluent was added to make exactly 10 mL each to prepare standard solutions 2 and 3. These were measured using high-performance liquid chromatography under the following conditions, and calibration curves for the concentration and area of ​​Compound A were prepared. The content of Compound A was evaluated using this calibration curve. Related substances were calculated by the area percentage method. <Dilution> Methanol / water mixture (9:1) <High-performance liquid chromatography measurement conditions> Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: Acetonitrile / methanol mixture (4:1) containing 0.06% trifluoroacetic acid Gradient conditions: B Conc. 40% (0-0.5 min) → 90% (50.5-70 min) → 40% (70.1 min) Detector: UV (220 nm) Column: Waters Xselect CSH Phenyl-Hexyl (2.5 μm, 4.6 × 75 mm) Column temperature: 40°C Autosampler temperature: 25°C Syringe cleaning solution: Methanol / water mixture (9:1) or acetonitrile / methanol (4:1) Flow rate: 0.5 mL / min Injection volume: 2 μL

[0150] Test Method 3-2 (Quantitative Determination of Compound A: Comparative Examples 4, 5, 7-10 and Examples 1-37) For the freeze-dried compositions of Comparative Examples 4 and 5 and Examples 1-8, the freeze-dried composition was transferred to a volumetric flask with the addition of Diluent 1 shown below and dissolved to make exactly 10 mL to prepare a test solution, and the content was measured under the high performance liquid chromatography measurement condition 1 shown below. For the liposome bulk solutions of Comparative Examples 4 and 5 and Examples 1-8, Diluent 1 was added to 1 mL of the liposome bulk solution, transferred to a volumetric flask and dissolved to make exactly 10 mL to prepare a test solution, and the content was measured under the high performance liquid chromatography measurement condition 1 shown below. For the liposome bulk solutions of Comparative Examples 7-9 and Examples 9-11, methanol was added to 0.5 mL of the liposome bulk solution, transferred to a volumetric flask and dissolved to make exactly 5 mL to prepare a test solution, and the content was measured under the high performance liquid chromatography measurement condition 1 shown below. For the freeze-dried compositions of Comparative Example 10 and Examples 12 to 15, the freeze-dried compositions were transferred to a volumetric flask with the addition of Diluent 1 shown below and dissolved to make exactly 5 mL of test solution, and the content was measured under the following high performance liquid chromatography measurement condition 1. For the freeze-dried compositions of Examples 21 to 29 and 34 to 37, the freeze-dried compositions were transferred to a volumetric flask with the addition of Diluent 2 shown below and dissolved to make exactly 25 mL of test solution for Examples 21 and 26 to 29, exactly 10 mL of test solution for Example 22, and exactly 5 mL of test solution for Examples 23 to 25 and 34 to 37, and the content was measured under the following high performance liquid chromatography measurement condition 2. For the liposome bulk solutions of Examples 16 and 26 to 29, diluent 2 was added to 0.4 mL of the liposome bulk solution, transferred to a volumetric flask, and dissolved to make exactly 10 mL to prepare the test solution; for the liposome bulk solution of Example 17, diluent 2 was added to 0.5 mL of the liposome bulk solution, transferred to a volumetric flask, and dissolved to make exactly 5 mL to prepare the test solution; for the liposome bulk solutions of Examples 18 to 20 and Examples 30 to 37, methanol was added to 0.4 mL of the liposome bulk solution, transferred to a volumetric flask, and dissolved to make exactly 2 mL to prepare the test solution, and the content was measured using the high-performance liquid chromatography measurement condition 2 described below. Separately, approximately 50 mg of compound A was accurately weighed, and the solution was dissolved in diluent 1 shown below to make exactly 50 mL, which was used as the standard mother solution (approximately 1.0 mg / mL).In Comparative Examples 4, 5, 7 to 9 and Examples 9 to 11, this standard mother solution was accurately measured into a 2 mL volumetric flask, and Diluent 1 was added to make exactly 20 mL. This solution was then accurately measured into a 2 mL volumetric flask, and Diluent 1 was added to make exactly 10 mL, creating Standard Solution 1 (approximately 20 μg / mL). Standard Solution 1 was measured under the following high-performance liquid chromatography measurement condition 1, and the content of Compound A was evaluated from the peak areas of Compound A in Standard Solution 1 and each test solution. In Examples 16 to 18, Examples 21 to 23, and Examples 26 to 33, the standard mother solution was accurately measured into a 2 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL. This solution was then accurately measured into a 4 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL. This solution was then accurately measured into a 4 mL volumetric flask, and Diluent 2 was added to make exactly 10 mL, creating Standard Solution 2 (approximately 8 μg / mL). Standard solution 2 was measured under the following high-performance liquid chromatography measurement condition 2, and the content of Compound A was evaluated from the peak area of ​​Compound A in Standard solution 2 and each test solution. In Examples 19 and 24, the standard mother solution was accurately measured into a 2 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL. This solution was then accurately measured into a 4 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL. This solution was then accurately measured into a 2 mL volumetric flask, and Diluent 2 was added to make exactly 10 mL, to prepare Standard solution 3 (approximately 4 μg / mL). Standard solution 3 was measured under the following high-performance liquid chromatography measurement condition 2, and the content of Compound A was evaluated from the peak area of ​​Compound A in Standard solution 3 and each test solution. In Examples 20, 25, and 34 to 37, the standard mother solution was accurately measured into a 2 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL. This solution was then accurately measured into a 4 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL. This solution was then accurately measured into a 2 mL volumetric flask, and Diluent 2 was added to make exactly 20 mL to prepare Standard Solution 4 (approximately 2 μg / mL). Standard Solution 4 was measured under the following high performance liquid chromatography measurement conditions 2, and the content of Compound A was evaluated from the peak area of ​​Compound A in Standard Solution 4 and each test solution.<Dilution solution 1> Methanol / water mixture (9:1) <Dilution solution 2> Methanol / water mixture (8:2) <High-performance liquid chromatography measurement condition 1> Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: Acetonitrile / methanol mixture (4:1) containing 0.06% trifluoroacetic acid Gradient conditions: B Conc. 60% (0-10 min) → 95% (11-20 min) → 60% (20.1 min) Detector: UV (220 nm) Column: Waters Xselect CSH Phenyl-Hexyl (2.5 μm, 4.6 mm × 75 mm) Column temperature: 40°C Autosampler temperature: 25°C Syringe cleaning solution: Methanol / water mixture (9:1) Flow rate: 1.0 mL / min Injection volume: 5 μL <High-performance liquid chromatography measurement condition 2> Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: Acetonitrile / methanol mixture (4:1) containing 0.06% trifluoroacetic acid. Gradient conditions: B Conc. 60% (0-10 min) → 95% (11-20 min) → 60% (20.1 min-). Detector: UV (220 nm). Column: Waters Xselect CSH Phenyl-Hexyl (2.5 μm, 4.6 mm × 75 mm). Column temperature: 40°C. Autosampler temperature: 25°C. Syringe cleaning solution: Methanol / water mixture (9:1). Flow rate: 1.0 mL / min. Injection volume: 10 μL.

[0151] Test Method 3-3 (Related Substances of Compound A: Examples 26 to 37) The liposome bulk solutions of Examples 26 to 37 were evaluated for related substances of Compound A using high performance liquid chromatography. Specifically, for the liposome bulk solutions of Examples 26 to 33, methanol was added to each liposome bulk solution to dilute them exactly two-fold to prepare test solutions, and for the liposome bulk solutions of Examples 34 to 37, each liposome bulk solution was used as is as the test solution. The injection volume was 5 μL for Examples 26 to 29, 25 μL for Examples 30 to 33, and 50 μL for Examples 34 to 37, and measurements were performed using high performance liquid chromatography under the following conditions. The amount of related substances was calculated using the area percentage method. <High-performance liquid chromatography measurement conditions> Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: Acetonitrile / methanol mixture (4:1) containing 0.06% trifluoroacetic acid Gradient conditions: B Conc. 40% (0-0.5 min) → 90% (50.5-75 min) → 40% (75.1 min) Detector: UV (220 nm) Column: Waters Xselect CSH Phenyl-Hexyl (2.5 μm, 4.6 × 75 mm) Column temperature: 40°C Autosampler temperature: 25°C Syringe cleaning solution: Methanol / water mixture (9:1) Flow rate: 0.5 mL / min

[0152] Test Method 4-1 (pH Measurement: Initial Samples and Samples Stored for One Month in Comparative Examples 1 to 3) The pH after reconstitution with water was evaluated for the initial samples of each freeze-dried composition of Comparative Examples 1 to 3 and the samples stored for one month under each storage condition. Specifically, the freeze-dried composition was reconstituted with 4.5 mL of water for injection, and the pH was measured using a pH meter (portable pH meter D-71: Horiba, Ltd.).

[0153] Test method 4-2 (pH measurement: 3-month and 6-month stored products of Comparative Examples 1 to 3) The pH after reconstitution was evaluated for each of the 3-month and 6-month stored freeze-dried compositions of Comparative Examples 1 to 3. Specifically, two tubes of each freeze-dried composition were reconstituted with 4.5 mL of water for injection and combined into one screw tube, and 30 μL of saturated potassium chloride aqueous solution was added and the pH was measured using a pH meter (portable pH meter D-71: Horiba, Ltd.).

[0154] Test Method 4-3 (pH Measurement: Comparative Examples 4, 5, 7-10, and Examples 1-37) The pH of the liposome bulk solutions and lyophilized compositions of Comparative Examples 4, 5, 7-10, and Examples 1-37 after reconstitution was evaluated. Specifically, approximately 10 mL of the liposome bulk solution was placed in a screw tube, and 30 μL of saturated potassium chloride aqueous solution was added in Comparative Examples 4, 5, and 7. The pH of the liposome bulk solution was measured directly in Comparative Examples 8 and 9, Examples 1-11, Examples 16-20, and Examples 26-37 using a pH meter (Portable pH Meter D-71, Horiba, Ltd.). The lyophilized compositions were reconstituted with 0.9 mL (Comparative Examples 4 and 5, Examples 1-8, Examples 21-29, and Examples 34-37) or 0.45 mL (Comparative Example 10 and Examples 12-15) of water for injection or ultrapure water per tube. If necessary, multiple tubes were combined into a single screw tube. In Comparative Examples 4, 5, and 10, a saturated potassium chloride aqueous solution was added at a ratio of 30 μL of saturated potassium chloride aqueous solution to 10 mL of the condensate, and in Examples 1 to 8, 12 to 15, 21 to 29, and 34 to 37, the pH was measured directly using a pH meter (portable pH meter D-71: Horiba, Ltd.).

[0155] Test Method 5-1 (Moisture content: initial samples and samples stored for one month in Comparative Examples 1 to 3) For the freeze-dried compositions of Comparative Examples 1 to 3, the moisture content in the initial samples and samples stored for one month under each storage condition was measured by the Karl Fischer method. Specifically, 0.05 to 0.1 g of each pulverized freeze-dried composition was placed in a trace moisture analyzer (CA-200: Mitsubishi Chemical) set under the following conditions, and the moisture content was measured. <Measurement conditions> Anolyte: Aquamicron AX Catholyte: Aquamicron CXU

[0156] Test Method 5-2 (Moisture Content: Comparative Examples 1 to 3, Stored for 3 and 6 Months) The moisture content of the freeze-dried compositions stored for 3 and 6 months in Comparative Examples 1 to 3 was measured by the Karl Fischer method. Specifically, 2 mL of chloroform / methanol = 1 / 1 (v / v) was injected into an unopened vial containing the freeze-dried composition using a syringe and uniformly dispersed using an ultrasonic irradiator to prepare a test solution. 500 μL of this test solution was placed in a trace moisture analyzer (CA-200: Mitsubishi Chemical) set under the following conditions, and moisture content was measured. Separately, moisture content was measured in the same manner for the chloroform / methanol = 1 / 1 (v / v) solution used to disperse the freeze-dried composition as a blank. The moisture content of the freeze-dried composition was calculated from the moisture content of the test solution, the moisture content of the blank, and the solid content in the vial. <Measurement Conditions> Anolyte: Aquamicron AX Catholyte: Aquamicron CXU

[0157] Test Method 6 (Liposome Encapsulation Rate of Compound A (hereinafter, Compound A Encapsulation Rate): Comparative Examples 1 to 3) The lyophilized composition was reconstituted with 4.5 mL of water for injection, and 1 mL of this solution was mixed with 1 mL of water for injection. The liposome fraction was sedimented using an ultracentrifuge (ultracentrifuge CS100FX, rotor S100AT5: Hitachi Koki) at a mean acceleration of 406,000 × g for 15 min at 5°C. 1 mL of the supernatant after ultracentrifugation was collected and used as the test solution. Separately, approximately 14 mg of Compound A was precisely weighed and dissolved in methanol. 1 mL of water was added, and the solution was adjusted to exactly 20 mL (approximately 0.700 mg / mL) with methanol. Exactly 3 mL and 1 mL of this solution were measured, and dilution solution was added to make exactly 10 mL each, creating standard solution 1 (approximately 0.210 mg / mL) and standard solution 2 (approximately 0.070 mg / mL). Furthermore, exactly 1 mL of each solution was measured, and dilution solution was added to make exactly 10 mL each, creating standard solution 3 (approximately 0.021 mg / mL) and standard solution 4 (approximately 0.007 mg / mL). Exactly 1 mL of standard solution 3 was measured, and dilution solution was added to make exactly 20 mL, creating standard solution 5 (approximately 0.001 mg / mL). Standard solutions 3 to 5 and the test solution were measured by high-performance liquid chromatography under the conditions below, and calibration curves were created for the concentration and area of ​​compound A. The content of compound A in the test solution was calculated, and this was taken as the amount of compound A not encapsulated in the liposomes (measured value (1)). Using the quantitative value (2) of compound A obtained separately by Test Method 3-1, the compound A encapsulation rate was calculated according to the following formula.<Dilution solution> Methanol / water mixture (9:1) <High-performance liquid chromatography measurement conditions> Mobile phase A: 0.1% trifluoroacetic acid aqueous solution Mobile phase B: Acetonitrile / methanol mixture (4:1) containing 0.06% trifluoroacetic acid Gradient conditions: B Conc. 40% (0-0.5 min) → 90% (50.5-70 min) → 40% (70.1 min) Detector: UV (220 nm) Column: Waters Xselect CSH Phenyl-Hexyl (2.5 μm, 4.6 × 75 mm) Column temperature: 40 °C Autosampler temperature: 25 °C Syringe cleaning solution: Methanol / water mixture (9:1) or acetonitrile / methanol (4:1) Flow rate: 0.5 mL / min Injection volume: 2 μL <Formula> {Quantitative value (2) - Measured value (1)} / Quantitative value (2) × 100 = Compound A encapsulation rate (%).

[0158] Test Example 1-1 The average particle size and polydispersity index were measured before and after freeze-drying for Comparative Examples 1 to 3. The results are shown in the table below. From the results of Test Example 1-1, there was no significant problem in the polydispersity index of the liposome bulk liquid (before freeze-drying) and the rehydrated product (after freeze-drying), but in Comparative Examples 1 and 2, the average particle size of the rehydrated product tended to be smaller than 100 nm.

[0159] Test Example 1-2-1 The freeze-dried product of Comparative Example 1 was stored at 5°C or 25°C, and then the content of each component and the particle state were measured. The results are shown in the table below. The lipid content decreased, the Compound A content decreased, and the particle size distribution became unstable (increased average particle diameter and increased PDI value).

[0160] Test Example 1-2-2 The freeze-dried product of Comparative Example 2 was stored at 5°C or 25°C, and then the content of each component and the particle state were measured. The results are shown in the table below. The lipid content decreased, the Compound A content decreased, and the particle size distribution became unstable (increased average particle diameter and increased PDI value), but there was some improvement over Comparative Example 1.

[0161] Test Example 1-2-3 The freeze-dried product of Comparative Example 3 was stored at 5°C or 25°C, and then the content of each component and the particle state were measured. The results are shown in the table below. Improvements in lipid content, Compound A content, and particle size distribution stability (small fluctuations in average particle size and PDI value) were observed.

[0162] Test Examples 1-3 The content of each component and particle state after preparation of the liposomes of Comparative Example 6 were measured. The results are shown in the table below. The stability of the lipid components and Compound A was improved compared to Comparative Examples 1 and 2, but ascorbic palmitate was decomposed immediately after preparation and was not detected.

[0163] Test Example 2-1 The average particle size and polydispersity index were measured for the solutions reconstituted with water before and after freeze-drying for Examples 1 to 8 and Comparative Examples 4 and 5. The results are shown in the table below. In Examples 1 to 8 and Comparative Examples 4 and 5, no significant change was observed in the particle size distribution before and after freeze-drying.

[0164] Test Example 2-2-1 The freeze-dried products of Examples 1 to 8 and Comparative Examples 4 and 5 were stored at 5°C or 25°C, reconstituted with water, and the particle state was measured. The results are shown in the table below. In Examples 1 to 8 and Comparative Examples 4 and 5, no significant change was observed in the particle size distribution even after storage in the freeze-dried state.

[0165] Test Example 2-2-2 The freeze-dried products of Examples 1 to 8 and Comparative Examples 4 and 5 were stored at 5°C or 25°C, reconstituted with water, and the pH was measured. The results are shown in the table below. The pH fluctuated in the Comparative Example containing no histidine, but no pH fluctuation occurred in Examples 1 to 8 containing histidine.

[0166] Test Example 2-2-3 The lyophilized products of Examples 1 to 8 and Comparative Examples 4 and 5 were stored at 5°C or 25°C, and the compound A content (%) was measured. The results are shown in the table below. In Examples 1 to 8 and Comparative Examples 4 and 5, no significant change in the compound A content (%) was observed even after storage in the lyophilized state.

[0167] Test Example 2-3-1 The solutions of Examples 1 to 8 and Comparative Examples 4 and 5 were stored at 5°C, 25°C, or 40°C, and the particle state was measured. The results are shown in the table below. In Comparative Example 5, which did not contain histidine or an antioxidant, destabilization of the particle size distribution (increase in polydispersity index) was observed, but in Examples 1 to 8 and Comparative Example 4, no significant change was observed in the particle size distribution.

[0168] Test Example 2-3-2 The solutions of Examples 1 to 8 and Comparative Examples 4 and 5 were stored at 25°C or 40°C and the pH was measured. The results are shown in the table below. The pH fluctuated in the comparative example containing no histidine, but the pH was stable in Examples 1 to 8.

[0169] Test Example 2-3-3 The solutions of Examples 1 to 8 and Comparative Examples 4 and 5 were stored at 5°C, 25°C, or 40°C, and the compound A content (%) was measured. The results are shown in the table below. The compound A content decreased in the formulation of Comparative Example 5, which did not contain histidine or an antioxidant, but there was no significant change in the compound A content in Examples 1 to 8 and Comparative Example 4.

[0170] Test Example 2-4-1 Examples 9 to 11 and Comparative Examples 7 to 9 were stored at 5°C or 25°C, and the particle state was measured. The results are shown in the table below. Comparative Example 7, which did not contain a buffer, showed instability in the particle size distribution (increase in polydispersity index, decrease in average particle diameter) when stored at 25°C, but Examples 9 to 11 and Comparative Examples 8 and 9 showed no significant change in particle size distribution.

[0171] Test Example 2-4-2 Examples 9 to 11 and Comparative Examples 7 to 9 were stored at 5°C or 25°C and the pH was measured. The results are shown in the table below. pH fluctuated in Comparative Example 7, which contained no buffering agent, and Comparative Example 9, which used trishydroxymethylaminomethane as a buffering agent, but the pH was stable in Examples 9 to 11 and Comparative Example 8.

[0172] Test Example 2-4-3 Examples 9 to 11 and Comparative Examples 7 to 9 were stored at 5°C or 25°C, and the compound A content (%) was measured. The results of the residual rate of compound A, assuming the initial compound A content to be 100%, are shown in the table below. The compound A content decreased in Comparative Example 7, which contained no buffer, and in Comparative Examples 8 and 9, which used sodium phosphate or trishydroxymethylaminomethane as the buffer, but there was no significant change in the compound A content in Examples 9 and 10.

[0173] Test Example 2-5-1 For Examples 12 to 15 and Comparative Example 10, the particles were stored at 5°C or 25°C, reconstituted with water, and the particle state was measured. The results are shown in the table below. No significant changes in particle size distribution were observed in Examples 12 to 15 and Comparative Example 10.

[0174] Test Example 2-5-2 Examples 12 to 15 and Comparative Example 10 were stored at 5°C or 25°C, reconstituted with water, and the pH was measured. The results are shown in the table below. pH fluctuated in Comparative Example 10, which did not contain a buffering agent, but the pH was stable in Examples 12 to 15.

[0175] Test Example 2-5-3 For Examples 12 to 15 and Comparative Example 10, the compound A content (%) was measured after storage at 5°C or 25°C. The results are shown in the table below, assuming the initial compound A content to be 100%. In Examples 12 to 15 and Comparative Example 10, there was no significant change in the compound A content (%) even after storage in a freeze-dried state, and no difference in the compound A content (%) was observed depending on the type of buffer and isotonic agent.

[0176] Test Example 2-6-1 The solutions of Examples 16 to 20 were stored at 5°C or 25°C, and the particle state was measured. The results are shown in the table below. In Examples 16 to 20, after long-term storage at 25°C, the particle size distribution became more unstable (a decrease in average particle size) as the compound concentration decreased, but after long-term storage at 5°C, no significant change in particle size distribution was observed regardless of the compound A concentration.

[0177] Test Example 2-6-2 The solutions of Examples 16 to 20 were stored at 5°C or 25°C and the pH was measured. The results are shown in the table below. In Examples 16 to 20, no change in pH occurred after long-term storage, regardless of the compound A concentration.

[0178] Test Example 2-6-3 The solutions of Examples 16 to 20 were stored at 5°C or 25°C, and the compound A content (%) was measured. The results are shown in the table below, assuming the initial compound A content to be 100%. In Examples 16 to 20, after long-term storage at 25°C, the compound A content decreased more as the compound concentration decreased, but after long-term storage at 5°C, no significant change in compound A content was observed regardless of the compound A concentration.

[0179] Test Example 2-7-1 The freeze-dried products of Examples 21 to 25 were stored at 5°C or 25°C, reconstituted with water, and the particle state was measured. The results are shown in the table below. In Examples 21 to 25, no significant change was observed in particle size distribution even after storage in a freeze-dried state, regardless of the compound A concentration.

[0180] Test Example 2-7-2 The lyophilized products of Examples 21 to 25 were stored at 5°C or 25°C, reconstituted with water, and the pH was measured. The results are shown in the table below. In Examples 21 to 25, no change in pH occurred even after storage in the lyophilized state, regardless of the compound A concentration.

[0181] Test Example 2-7-3 The lyophilized products of Examples 21 to 25 were stored at 5°C or 25°C, and the compound A content (%) was measured. The results are shown in the table below, assuming the initial compound A content to be 100%. In Examples 21 to 25, no significant change in the compound A content (%) was observed even after storage in a lyophilized state, regardless of the compound A concentration.

[0182] Test Example 2-8-1 The particle state of the solutions of Examples 26 to 37 was measured after storage at 5° C. or 25° C. In Examples 26, 30 to 32, and 34 to 36, instability in particle size distribution (increase in polydispersity index, decrease in average particle diameter) was observed, but no significant change in particle size distribution was observed in Examples 27 to 31, 33, and 37.

[0183] Test Example 2-8-2 The solutions of Examples 26 to 37 were stored at 5° C. or 25° C. and the pH was measured. In Examples 26 to 37, no change in pH occurred after storage.

[0184] Test Example 2-8-3 The solutions of Examples 26 to 37 were stored at 5°C or 25°C, and the compound A content (%) was measured. The results are shown in the table below. In Examples 26, 30, and 34, which did not contain an antioxidant, and in Examples 35 and 36, in which the antioxidants were BHA and alpha-tocopherol and the compound A concentration was 0.01 mg / mL, a significant decrease in the compound A content (%) was observed after storage at 25°C. In Examples 27 to 29, 31 to 33, and 37, which contained an antioxidant, no significant change in the compound A content (%) was observed even after storage at 25°C.

[0185] Test Example 2-8-4 The solutions of Examples 26 to 37 were stored at 5°C or 25°C, and the total amount (%) of Compound A related substances was measured. The results are shown in the table below. In Examples 26, 30, and 34, which did not contain an antioxidant, and Examples 31, 32, 35, and 36, in which the antioxidants were BHA and alpha-tocopherol and the Compound A concentrations were 0.04 mg / mL and 0.01 mg / mL, respectively, an increase in the total amount (%) of related substances was observed after storage at 25°C. In Examples 27 to 29, 33, and 37, which contained an antioxidant, no significant change was observed in the total amount (%) of related substances even after storage at 25°C.

[0186] Test Example 2-9-1 The freeze-dried products of Examples 26 to 29 and Examples 34 to 37 were stored at 5°C or 25°C, reconstituted with water, and the particle state was measured. In Examples 26 to 29 and Examples 34 to 37, regardless of the presence or absence of an antioxidant, no significant change in particle size distribution was observed after storage, even after freeze-drying.

[0187] Test Example 2-9-2 The freeze-dried products of Examples 26 to 29 and Examples 34 to 37 were stored at 5°C or 25°C, reconstituted with water, and the pH was measured. In Examples 26 to 29 and Examples 34 to 37, regardless of the presence or absence of an antioxidant, no change in pH occurred after storage even in the freeze-dried state.

[0188] Test Example 2-9-3 The lyophilized products of Examples 26 to 29 and Examples 34 to 37 were stored at 5°C or 25°C, and the compound A content (%) was measured. The results are shown in the table below, assuming the initial compound A content to be 100%. In Examples 26 to 29 and Examples 34 to 37, regardless of the presence or absence of an antioxidant, no significant change in the compound A content after storage was observed, even in the lyophilized state.

[0189] Test Example 3-1 Seven-week-old C57BL / 6 male mice were given an initial immunization by intramuscularly administering an equal volume mixture (100 μL / mouse) of ovalbumin (OVA) (2 mg / mL) and the liposome of Example 7 (0.2 mg / mL in terms of Compound A) to the gastrocnemius muscle. Two weeks later, an equal volume of the same mixture was administered intramuscularly to the gastrocnemius muscle as a booster immunization. One week after the booster immunization, blood was collected from the heart under maintenance isoflurane inhalation anesthesia, and serum was collected by centrifugation. The OVA-specific IgG2c level in the serum was quantified by the following ELISA method. Specifically, an OVA solution (SIGMA) was added to a 96-well plate, followed by blocking with 1% skim milk (Wako). After adding a serum sample diluted with phosphate buffer, a secondary antibody, goat anti-mouse IgG2c (Southern Bio), was added, and the plate was then incubated with SureBlue TMAfter adding TMB Microwell Peroxidase Substrate (KPL), the product of the enzymatic reaction was measured and quantified using a microplate reader (see Figure 1). The liposome preparation of Example 7 showed a stronger induction of OVA-specific IgG2c than the negative control group.

[0190] Test Example 3-2 Spleen cells from the mice in Test Example 3-1 were prepared, and then cultured overnight after adding OVA and brefeldin A (Thermo Fisher). The cells were then collected and analyzed by immunoblotting using an APC-labeled anti-mouse CD3 antibody (Invitrogen), a PerCP-labeled anti-mouse CD4 antibody (BioLegend), and Fixable Viability Dye eFluor. TM After staining with 520 (Invitrogen), the cells were fixed with Fixation / Permeabilization buffer (Invitrogen). After treating the cells with Permeabilization buffer (Invitrogen), they were stained with the antibody cocktail BV421-labeled anti-IFN-γ antibody (BioLegend), PE-Cy7-labeled anti-IL-2 antibody (eBioscience), and PE-labeled TNF-α (BioLegend). Data acquisition and analysis were performed using a FACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). The results are shown in Figure 2. The above spleen cells were also stained with V450-labeled anti-mouse CD3 antibody (Invitrogen), Alexa Fluor 520 (Invitrogen), and PE-labeled TNF-α (BioLegend). TMThe cells were stained with 647-labeled anti-mouse CD8 antibody (MBL), PE-labeled H-2Kb OVA Tetramer-SIINFEKL (MBL), and Fixable Viability Dye eFluor 520 (Invitrogen). Data acquisition and analysis were performed using a FACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). The results are shown in Figure 3. Furthermore, the above splenocytes were stained with the antibody cocktail V450-labeled anti-mouse CD3 antibody (Invitrogen), Alexa Fluor 647-labeled anti-mouse CD8 antibody (MBL), PE-Cy7-labeled anti-mouse CD44 antibody, PerCP-Cy5.5-labeled anti-mouse CD62L antibody, and Fixable Viability Dye 520 (Invitrogen). Data acquisition and analysis were performed using a FACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). The results are shown in Figure 4. The liposomes of Example 7 increased the percentage of OVA-specific type 1 helper T cells, particularly OVA-specific multifunctional CD4-positive T lymphocytes, the percentage of MHC-restricted OVA-specific CD8-positive T lymphocytes (OVA tetramer-positive CD8 T cells in Figure 2), and the percentage of CD8-positive effector memory T lymphocytes compared to the negative control group.

[0191] These Examples, Reference Examples, Comparative Examples, Test Examples, etc. show that the liposome preparation of the present invention has high storage stability and can therefore be used as a vaccine adjuvant preparation.

Claims

1. i) A lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) one or more isotonicity agents selected from the group consisting of sucrose and trehalose.

2. i) A lipid multilayer-forming liposome comprising lipid components including dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG), and (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) one or more buffering agents selected from the group consisting of L-histidine, L-histidine hydrochloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, dipotassium phosphate, and trishydroxymethylaminomethane (trometamol); and iii) sucrose.

3. The formulation according to claim 1 or 2, wherein the buffering agent is L-histidine and / or L-histidine hydrochloride.

4. The preparation according to any one of claims 1 to 3, wherein the liposome preparation is an aqueous preparation.

5. The preparation according to any one of claims 1 to 3, wherein the liposome preparation is a freeze-dried preparation.

6. The formulation according to any one of claims 1 to 5, comprising one or more antioxidants selected from the group consisting of butylhydroxyanisole, L-methionine, L-cysteine, L-cysteine ​​hydrochloride hydrate, tocopherols (natural vitamin E, d-δ-tocopherol, alpha-tocopherol, tocopherol acetate), sodium edetate hydrate (EDTA), sodium thioglycolate, sodium metabisulfite, sodium nitrite, sodium bisulfite, and sodium sulfite hydrate.

7. The formulation of claim 6, wherein the antioxidant comprises one or more antioxidants selected from the group consisting of butylhydroxyanisole, alpha tocopherol, and edetate sodium hydrate (EDTA).

8. The formulation of claim 6, wherein the antioxidant is butylhydroxyanisole.

9. The formulation of claim 6, wherein the antioxidant is alpha tocopherol.

10. The formulation of claim 6, wherein the antioxidant is edetate sodium hydrate (EDTA).

11. The formulation according to any one of claims 1 to 10, wherein the weight ratio of compound A or a pharmaceutically acceptable salt thereof to the lipid component is 1:40 to 1:

100.

12. The formulation according to any one of claims 1 to 10, wherein the weight ratio of compound A or a pharmaceutically acceptable salt thereof to the lipid component is 1:40 to 1:

70.

13. The formulation according to any one of claims 1 to 10, wherein the weight ratio of compound A or a pharmaceutically acceptable salt thereof to the lipid component is 1:

50.

14. A formulation according to any one of claims 1 to 13, wherein the weight ratio of the lipid components dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylglycerol (EPG) is 1:1 to 2:

1.

15. The formulation according to any one of claims 1 to 14, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, is 5 mmol / L to 50 mmol / L.

16. The formulation according to any one of claims 1 to 14, wherein the amount of buffering agent added when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL, is 10 mmol / L to 40 mmol / L.

17. The formulation according to any one of claims 1 and 3 to 16, wherein the amount of sucrose and / or trehalose blended is 50 mg / mL to 110 mg / mL when the aqueous liquid formulation is prepared so that Compound A is at a concentration of 0.01 mg / mL to 0.2 mg / mL.

18. The formulation according to any one of claims 1 and 3 to 16, wherein the amount of sucrose and / or trehalose blended is 80 mg / mL to 100 mg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

19. The formulation according to any one of claims 1 to 16, wherein the amount of sucrose blended when the aqueous liquid preparation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL is 50 mg / mL to 110 mg / mL.

20. The formulation according to any one of claims 1 to 16, wherein the amount of sucrose blended when the aqueous liquid preparation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL is 80 mg / mL to 100 mg / mL.

21. The formulation according to any one of claims 6 to 20, wherein the amount of antioxidant incorporated is 0.05 μg / mL to 5 mg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

22. The formulation according to any one of claims 6 to 20, wherein the amount of antioxidant blended is 0.25 μg / mL to 500 μg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

23. The formulation according to any one of claims 6 to 20, wherein the amount of antioxidant incorporated is 0.5 μg / mL to 10 μg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

24. The formulation according to any one of claims 6 to 20, wherein the amount of antioxidant incorporated is 1 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

25. The formulation according to any one of claims 6 to 8 and 11 to 20, wherein the amount of butylhydroxyanisole blended is 0.05 μg / mL to 5 μg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

26. The formulation according to any one of claims 6 to 8 and 11 to 20, wherein the amount of butylhydroxyanisole blended is 0.25 μg / mL to 2 μg / mL when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

27. The formulation of any one of claims 6, 7, 9, 11 to 20, wherein the amount of alpha-tocopherol blended is 0.5 μg / mL to 50 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

28. The formulation of any one of claims 6, 7, 9, 11 to 20, wherein the amount of alpha-tocopherol blended is 2 μg / mL to 40 μg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

29. The formulation of any one of claims 6, 7, and 10 to 20, wherein the amount of edetate sodium hydrate (EDTA) blended is 1 μg / mL to 5 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

30. The formulation of any one of claims 6, 7, and 10 to 20, wherein the amount of edetate sodium hydrate (EDTA) blended is 5 μg / mL to 2 mg / mL when the aqueous liquid formulation is prepared so that Compound A is 0.01 mg / mL to 0.2 mg / mL.

31. The formulation according to any one of claims 1 to 30, wherein the aqueous solution has a pH of 6.0 to 8.0 when prepared so that compound A has a concentration of 0.01 mg / mL to 0.2 mg / mL.

32. The formulation according to any one of claims 1 to 30, wherein the aqueous solution has a pH of 6.5 to 7.5 when prepared so that compound A is present at a concentration of 0.01 mg / mL to 0.2 mg / mL.

33. The formulation of any one of claims 1 to 32, wherein the liposomes have an average particle size of 60 nm to 200 nm and a polydispersity index (PDI) of 0.01 to 0.2 when the aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL.

34. The formulation of any one of claims 1 to 32, wherein when an aqueous liquid formulation is prepared so that the concentration of compound A is 0.01 mg / mL to 0.2 mg / mL, the liposomes have an average particle size of 80 nm to 140 nm and a polydispersity index (PDI) of 0.01 to 0.

15.

35. The formulation according to any one of claims 1 to 34, which is a freeze-dried formulation.

36. A formulation according to any one of claims 1 to 34, which is an aqueous liquid formulation.

37. A formulation according to any one of claims 1 to 36, wherein the lipid multilayer is a lipid bilayer.

38. A vaccine adjuvant comprising a formulation according to any one of claims 1 to 37.

39. A vaccine comprising a formulation according to any one of claims 1 to 37 and an antigen.

40. The vaccine of claim 39, wherein the antigen is a substance derived from a pathogen.

41. A kit comprising a formulation according to any one of claims 1 to 37 and an antigen.

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