Malaria transmission blocking vaccine

Abstract

The present invention provides a malaria transmission-blocking vaccine that has excellent storage stability and immunostimulating activity. The present invention makes it possible to provide a malaria transmission-blocking vaccine that has excellent storage stability and immunostimulating activity by combining a pharmaceutical composition containing (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-hexanamide or a pharmaceutically acceptable salt thereof as a vaccine adjuvant that can further enhance the specific immune response to an antigen and has high storage stability with a glycosylated, homogeneous, and biologically active malaria vaccine.

Description

[Technical field]

[0001] The present invention relates to the combined use of a pharmaceutical composition comprising a compound, or a pharma- ceutically acceptable salt thereof, useful as a vaccine adjuvant and a malaria vaccine, and to a method for inhibiting the transmission of the malaria parasite. [Background technology]

[0002] Compared with vaccines using the pathogen itself, subunit vaccines using a part of the pathogen's components as antigens can be produced by chemical synthesis and genetic recombination, so that the subunit vaccine is superior in terms of vaccine safety and production method. However, subunit vaccines tend to have lower immunostimulatory activity than live vaccines and inactivated vaccines produced from the pathogen itself. For this reason, in order to enhance the immunogenicity of epitopes and improve the immunostimulatory activity of vaccines, the prevention and treatment of diseases using vaccines and adjuvants in combination have been studied.

[0003] Recently, the following adjuvants have been reported as having TLR7 agonistic activity: [ka] (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-hexanamide (hereinafter referred to as "compound A") represented by the formula (Patent Document 1) has been reported. Compound A has excellent vaccine adjuvant activity, but when actually administered to mammals as a vaccine adjuvant, it is required to be formulated as a formulation such as an emulsion. In general, it is known that antioxidants such as ascorbic acids are used in emulsion formulations to improve the storage stability of the formulation. However, it is not known that antioxidants such as ascorbic acids stabilize particle size distribution.

[0004] Pfs230, a surface protein of gametocytes of the malaria parasite Plasmodium falciparum, is known as a target antigen for a malaria transmission-blocking vaccine consisting of 3,135 amino acids. However, due to its large size, complex domains, and repeated seven-cysteine ​​(7-Cys) motifs with multiple disulfide bonds (Non-Patent Document 3), expression of the full-length protein has been difficult to achieve. Therefore, the generation of single domains containing N-terminal fragments has attracted attention. Among such fragments, several Pfs230 fragments, including Pfs230C1 and Pfs230D1, have been reported (Non-Patent Documents 1 and 2). However, since some of such proteins are known to cause glycosylation, improved antigens that do not cause glycosylation and are homogeneous and biologically active are desired.

[0005] A malaria vaccine that has acquired enhanced immunogenicity by the adjuvant Alhydrogel (registered trademark) is known (Non-Patent Document 2). Alhydrogel is different from compound A. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2017 / 061532 [Non-patent literature]

[0007] [Non-Patent Document 1] Shwu-Maan Lee et al., Clinical and Vaccine Immunology 2017;24:e00140 [Non-Patent Document 2] Camila H. Coelho et al., Vaccine 37 (2019) 1038-1045 [Non-Patent Document 3] Mayumi Tachibana et al., Clinical and Vaccine Immunology v.18(8); 2011, 1343-1350 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a combined use of a pharmaceutical composition containing a compound or a pharma- ceutical acceptable salt thereof, which is useful as a vaccine adjuvant and has excellent storage stability and immunostimulatory activity, and a malaria transmission-blocking vaccine, and a method for blocking the transmission of malaria parasites, which comprises administering the pharmaceutical composition and the malaria transmission-blocking vaccine to a mammal. [Means for solving the problem]

[0009] Compound A has six unsaturated bonds derived from a squalene-like structure in the molecule. In the course of examining the formulation of Compound A, it was found that when Compound A is prepared as a freeze-dried formulation of a general emulsion formulation using squalene as an oily composition, the unsaturated bonds in the molecule of Compound A are oxidized, thereby decreasing the content of Compound A. In order to provide a vaccine adjuvant formulation having practical storage stability, the present inventors have conducted intensive studies and found that the stability of Compound A against oxidation is increased by using squalane as an oily composition when preparing an emulsion composition of Compound A. Furthermore, it was found that the addition of an antioxidant such as ascorbic acid results in a formulation formulation that is excellent not only in storage stability, particularly the stability of Compound A itself against oxidation, but also in the stability of particle size distribution.

[0010] The present inventors also found that an N-terminally modified Pfs230 fragment as a malaria transmission vaccine is homogenous and biologically active without inducing glycosylation.

[0011] The present inventors have also found that the combined use of a pharmaceutical composition containing compound A and a malaria vaccine containing an N-terminally modified Pfs230 fragment is useful as a malaria transmission-blocking vaccine with excellent storage stability and immunostimulatory activity.

[0012] That is, the aspects of the present invention are as follows. [Item 1] A method for preventing the transmission of malaria parasites from humans to mosquitoes, comprising administering to a human living in an area where the prevention of malaria transmission is necessary a combination of a medicamentously effective amount of pharmaceutical composition I) and vaccine II), wherein the pharmaceutical composition I) contains the following components i) to vi): i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide (hereinafter referred to as "Compound A"), or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants and The method, wherein said vaccine II) is a malaria vaccine comprising an antigen having the sequence represented by SEQ ID NO:1 or SEQ ID NO:2.

[0013] [Item 2] i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutical acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants a pharmaceutical composition I comprising: Malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 II) A combination medicine comprising:

[0014] [Item 3] i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutical acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters such as L-ascorbic acid stearate and ascorbic acid palmitate, inorganic salts of ascorbic acid such as potassium ascorbate, sodium ascorbate and calcium ascorbate, and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants a pharmaceutical composition I comprising: Malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 II) 13. A vaccine formulation comprising:

[0015] [Item 4] i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutical acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters such as L-ascorbic acid stearate and ascorbic acid palmitate, inorganic salts of ascorbic acid such as potassium ascorbate, sodium ascorbate and calcium ascorbate, and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants a pharmaceutical composition I comprising: Malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 II) Including the kit.

[0016] [Item 5] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 4, wherein the pharmaceutical composition is an oil-in-water emulsion preparation or a lyophilized preparation thereof.

[0017] [Item 6] The hydrophilic surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80); polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, and polyoxyethylene hydrogenated castor oil 60); and polyoxyethylene polyoxypropylene glycols (e.g., polyoxyethylene (42) polyoxy 6. The method, combination medicine, vaccine formulation, or kit according to any one of items 1 to 5, wherein the polyoxyethylene (124) polyoxypropylene (39) glycol is selected from the group consisting of polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, and polyoxyethylene (200) polyoxypropylene (70) glycol.

[0018] [Item 7] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 5, wherein the hydrophilic surfactant is polysorbate 20, polysorbate 40, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, or polyoxyethylene (160) polyoxypropylene (30) glycol.

[0019] [Item 8] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 5, wherein the hydrophilic surfactant is polysorbate 20, polysorbate 40, or polysorbate 80.

[0020] [Item 9] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 8, wherein the lipophilic surfactant is sorbitan fatty acid esters (e.g., sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, and medium-chain triglycerides); glycerin fatty acid esters (e.g., glycerin fatty acid esters, glycerin monostearate, glycerin monomyristate, glycerin monooleate, and glycerin triisooctanoate); sucrose fatty acid esters (e.g., sucrose fatty acid esters, sucrose stearate, and sucrose palmitate); or propylene glycol fatty acid esters (e.g., propylene glycol fatty acid esters and propylene glycol monostearate).

[0021] [Item 10] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 8, wherein the lipophilic surfactant is a sorbitan fatty acid ester, sorbitan monooleate, sorbitan sesquioleate, or sorbitan trioleate.

[0022] [Item 11] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 8, wherein the lipophilic surfactant is sorbitan trioleate.

[0023] [Item 12] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 11, wherein the pharmaceutical composition further comprises an antioxidant B selected from the group consisting of tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol); tocopherol acetate; and butylhydroxyanisole.

[0024] [Item 13] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 11, wherein the pharmaceutical composition further comprises an antioxidant B selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol.

[0025] [Item 14] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 11, wherein the pharmaceutical composition further comprises antioxidant B which is α-tocopherol.

[0026] [Item 15] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 14, wherein antioxidant A is ascorbic acid palmitate, potassium ascorbate, sodium ascorbate, or ascorbic acid.

[0027] [Item 16] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 14, wherein antioxidant A is sodium ascorbate or potassium ascorbate.

[0028] [Item 17] The method, combination drug, vaccine formulation, or kit according to any one of Items 1 to 16, wherein the excipient is a non-reducing sugar (e.g., sucrose and trehalose) or a sugar alcohol (e.g., sorbitol, erythritol, xylitol, maltitol, and lactitol).

[0029] [Item 18] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 16, wherein the excipient is sucrose, trehalose, sorbitol, or xylitol.

[0030] [Item 19] The method, combination drug, vaccine preparation, or kit according to any one of items 1 to 16, wherein the excipient is sucrose or trehalose.

[0031] [Item 20] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 19, wherein the content of squalane in the pharmaceutical composition is 50 to 500 times the weight of compound A.

[0032] [Item 21] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 19, wherein the content of squalane in the pharmaceutical composition is 100 to 400 times the weight of compound A.

[0033] [Item 22] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 19, wherein the content of squalane in the pharmaceutical composition is 200 to 300 times the weight of compound A.

[0034] [Item 23] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 22, wherein the content of the hydrophilic surfactant in the pharmaceutical composition is 0.5 to 250 times the weight of compound A.

[0035] [Item 24] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 22, wherein the content of the hydrophilic surfactant in the pharmaceutical composition is 5 to 100 times the weight of compound A.

[0036] [Item 25] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 22, wherein the content of the hydrophilic surfactant in the pharmaceutical composition is 10 to 50 times the weight of compound A.

[0037] [Item 26] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 25, wherein the content of the lipophilic surfactant in the pharmaceutical composition is 0.5 to 250 times the weight of compound A.

[0038] [Item 27] ​​The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 25, wherein the content of the lipophilic surfactant in the pharmaceutical composition is 5 to 100 times the weight of compound A.

[0039] [Item 28] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 25, wherein the content of the lipophilic surfactant in the pharmaceutical composition is 10 to 50 times the weight of compound A.

[0040] [Item 29] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 28, wherein the content of antioxidant A in the pharmaceutical composition is 0.5 to 500 times the weight of compound A, calculated in terms of the weight of sodium ascorbate.

[0041] [Item 30] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 28, wherein the content of antioxidant A in the pharmaceutical composition is 2.5 to 250 times the weight of compound A, calculated in terms of the weight of sodium ascorbate.

[0042] [Item 31] The method, combination medicine, vaccine preparation, or kit according to any one of Items 1 to 28, wherein the content of antioxidant A in the pharmaceutical composition is 5 to 100 times the weight of compound A, calculated in terms of the weight of sodium ascorbate.

[0043] [Item 32] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 31, wherein the content of the excipient in the pharmaceutical composition is 50 to 1000 times the weight of compound A.

[0044] [Item 33] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 31, wherein the content of the excipient in the pharmaceutical composition is 100 to 750 times the weight of compound A.

[0045] [Item 34] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 31, wherein the content of the excipient in the pharmaceutical composition is 200 to 625 times the weight of compound A.

[0046] [Item 35] The method, combination medicine, vaccine preparation, or kit according to any one of Items 12 to 34, wherein the content of antioxidant B in the pharmaceutical composition is 5 to 250 times the weight of compound A.

[0047] [Item 36] The method, combination medicine, vaccine preparation, or kit according to any one of Items 12 to 34, wherein the content of antioxidant B in the pharmaceutical composition is 12.5 to 125 times the weight of compound A.

[0048] [Item 37] The method, combination medicine, vaccine preparation, or kit according to any one of Items 12 to 34, wherein the content of antioxidant B in the pharmaceutical composition is 25 to 50 times the weight of compound A.

[0049] [Item 38] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 37, wherein the weight of compound A is 0.0001 to 0.65 times the weight of a lyophilized pharmaceutical composition not containing compound A.

[0050] [Item 39] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 37, wherein the weight of compound A is 0.0002 to 0.35 times the weight of a lyophilized pharmaceutical composition not containing compound A.

[0051] [Item 40] The method, combination drug, vaccine preparation, or kit according to any one of Items 1 to 37, wherein the weight of compound A is 0.0005 to 0.065 times the weight of a lyophilized pharmaceutical composition not containing compound A.

[0052] [Item 41] Particle size D of the emulsion of the pharmaceutical composition immediately after manufacture and of the emulsion of the lyophilized preparation reconstituted after storage at 25 ° C for 6 months 90 Item 41. The method, combination drug, vaccine preparation, or kit according to any one of Items 5 to 40, wherein the value is 1000 nm or less.

[0053] [Item 42] The method, combination drug, vaccine preparation, or kit according to any one of Items 5 to 41, wherein the increase in the area percentage value of the impurity UK-1.02 after storage of a lyophilized formulation of the pharmaceutical composition at 5°C for 6 months is 5.0% or less.

[0054] [Item 43] The method, combination drug, vaccine preparation, or kit according to any one of Items 5 to 41, wherein the increase in the area percentage value of the impurity UK-1.02 after storage of a lyophilized formulation of the pharmaceutical composition at 5°C for 6 months is 1.0% or less.

[0055] [Item 44] A method for preventing the transmission of malaria parasites from humans to mosquitoes, comprising administering to a human living in an area in need of prevention of malaria transmission a medicamentous amount of pharmaceutical composition I) in combination with a medicamentous amount of vaccine II), wherein the pharmaceutical composition I) comprises the following components i) to vi): i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants and The vaccine II) is a malaria vaccine comprising an antigen having the sequence represented by SEQ ID NO:1 or SEQ ID NO:2.

[0056] [Item 45] A pharmaceutical composition for use in blocking the transmission of malaria parasites in combination with a malaria vaccine, the pharmaceutical composition comprising the following components i) to vi): i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants and The malaria vaccine is a pharmaceutical composition comprising an antigen having a sequence represented by SEQ ID NO:1 or SEQ ID NO:2.

[0057] [Item 46] A malaria vaccine for use in combination with a pharmaceutical composition to block transmission of a malaria parasite, the malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2; and The pharmaceutical composition comprises the following components i) to vi): i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants Malaria vaccines, including Effect of the Invention

[0058] According to the present invention, by combining a pharmaceutical composition as a vaccine adjuvant that can further enhance the specific immune response to an antigen and has high storage stability with a malaria vaccine that is homogeneous and biologically active without causing glycosylation, it is possible to provide a malaria transmission-blocking vaccine that has excellent storage stability and immunostimulatory activity. [Brief description of the drawings]

[0059] [Figure 1] Figure 1 shows electrophoresis of purified Pfs230D1+: (a) SDS-PAGE and (b) anti-His Western blot analysis.

[0060] [Diagram 2]Figure 2 shows Pfs230D1 + histidine tag protein induced functional antibodies in rats. (a) Antibody concentration of each serum against the corresponding immunogen was measured using ELISA. Individual (dots) and geometric mean (bar) ELISA units are shown. (b) 250ug / mL of total IgG of each group was tested using SMFA complement. Transmission reduction activity (TRA) of each group is shown along with the standard error of the mean.

[0061] [Diagram 3] FIG. 3 shows SDS-PAGE analysis of purified untagged Pfs230D1+.

[0062] [Figure 4] Figure 4 shows electrophoresis of purified Pfs230D1+ and anti-Pfs230 human monoclonal antibody Western blot analysis. (a) Pfs230D1+ containing a histidine tag reacted with two conformation-dependent anti-Pfs230 monoclonal antibodies under non-reducing conditions. (b) Untagged Pfs230D1+ reacted with two conformation-dependent anti-Pfs230 monoclonal antibodies under non-reducing conditions. (R) indicates reducing conditions, (NR) indicates non-reducing conditions.

[0063] [Diagram 5] Figure 5 shows Pfs230D1 + untagged protein induced functional antibodies in rats. (a) Antibody concentration of each serum against the corresponding immunogen was measured using ELISA. Individual (dots) and geometric mean (bar) ELISA units are shown. (b) 250ug / mL of total IgG from each group was tested using SMFA complement. Transmission reduction activity (TRA) of each group is shown along with the standard error of the mean. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Pharmaceutical Compositions In the present invention, the pharmaceutical composition is a freeze-dried emulsion preparation containing compound A, squalane, antioxidant A which is an ascorbic acid or the like, and excipient A. The present invention also includes emulsion preparations before freeze-drying and emulsion preparations obtained by reconstituting the freeze-dried preparation with water. In the pharmaceutical composition of the present invention, compound A contained as an active ingredient may be in a free form or a pharma- ceutical acceptable acid addition salt or base addition salt thereof. Examples of the acid addition salt include acid addition salts with inorganic or organic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroacetic acid, citric acid, and maleic acid). Examples of the salt addition salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, and ammonium salt. Compound A or a pharma-ceutical acceptable salt thereof in 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 pharma-ceutical acceptable salt thereof in the present invention. Details and preparation methods thereof are described in Patent Document 1, and compound A or a pharma-ceutical acceptable salt thereof can be prepared, for example, according to the method described in Patent Document 1. In the pharmaceutical composition of the present invention, the content of compound A is described as the content of the free form of compound A. Therefore, when compound A is used as a pharma- ceutically acceptable salt, the content is calculated by converting the weight of compound A to the weight of the salt.

[0065] In the present invention, the emulsion or emulsion preparation means an oil-in-water type or water-in-oil type emulsion. The emulsion is preferably an oil-in-water type emulsion. The weight ratio of the oil composition to the aqueous solution is preferably 1:99 to 15:85, more preferably 2:98 to 10:90, even more preferably 3:97 to 9:91, and even more preferably 4:96 to 7:93. In the emulsion preparation of the present invention, compound A is dissolved in the oil composition and exists. The freeze-dried preparation of the present invention means a preparation obtained by removing water from the emulsion preparation of the present invention under freeze-drying conditions. The freeze-dried preparation can be rehydrated with water for injection in an amount of 2 to 20 times the weight of the freeze-dried preparation to obtain an emulsion preparation.

[0066] In the present invention, examples of hydrophilic surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80); polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, and polyoxyethylene hydrogenated castor oil 60); and polyoxyethylene polyoxypropylene glycols (e.g., Examples of the polyoxyethylene (42) polyoxypropylene (67) glycol include polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, and polyoxyethylene (200) polyoxypropylene (70) glycol. Preferred are polysorbate 20, polysorbate 40, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, and polyoxyethylene (160) polyoxypropylene (30) glycol, more preferred are polysorbate 20, polysorbate 40, and polysorbate 80, and particularly preferred is polysorbate 80. In the pharmaceutical composition of the present invention, the content of the hydrophilic surfactant is 0.5 to 250 times, preferably 5 to 100 times, and more preferably 10 to 50 times the weight of compound A.

[0067] In the present invention, examples of lipophilic surfactants include sorbitan fatty acid esters (e.g., sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, and medium-chain triglycerides); glycerin fatty acid esters (e.g., glycerin fatty acid esters, glycerin monostearate, glycerin monomyristate, glycerin monooleate, and glycerin triisooctanoate); sucrose fatty acid esters (e.g., sucrose fatty acid esters, sucrose stearate, and sucrose palmitate); and propylene glycol fatty acid esters (e.g., propylene glycol fatty acid esters and propylene glycol monostearate). Preferred are sorbitan fatty acid esters, sorbitan monooleate, sorbitan sesquioleate, and sorbitan trioleate, and more preferred is sorbitan trioleate. In the pharmaceutical composition of the present invention, the content of the lipophilic surfactant is 0.5 to 250 times, preferably 5 to 100 times, and more preferably 10 to 50 times the weight of compound A.

[0068] In the pharmaceutical composition of the present invention, squalane is used as the oily composition. In the formulation study of the pharmaceutical composition of the present invention, the stability of compound A against oxidation is increased when squalane is used as the oily composition of emulsion formulations compared with the case where squalane is used, which is a general oily composition of emulsion formulations. Therefore, it is preferable to use squalane as the oily composition in the pharmaceutical composition of the present invention. In the pharmaceutical composition of the present invention, the content of squalane is 50 to 500 times, preferably 100 to 400 times, more preferably 200 to 300 times the weight of compound A.

[0069] In the present invention, examples of the antioxidant A include ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate); inorganic acid salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, and calcium ascorbate); and ascorbic acid. Preferred are ascorbic acid palmitate, potassium ascorbate, sodium ascorbate, and ascorbic acid, and more preferred are sodium ascorbate and potassium ascorbate. In the pharmaceutical composition of the present invention, the content of antioxidant A is 0.5 to 500 times, preferably 2.5 to 250 times, and more preferably 5 to 100 times the weight of compound A, and the content is calculated in terms of sodium ascorbate, i.e., by converting the weight of ascorbic acid (ascorbic acid derivative) as antioxidant A into the weight of sodium ascorbate.

[0070] In the present invention, the excipient A includes non-reducing sugars and sugar alcohols (excluding mannitol).Preferably, non-reducing sugars (e.g., sucrose, trehalose) and sugar alcohols (e.g., sorbitol, erythritol, xylitol, maltitol, and lactitol), more preferably, sucrose, trehalose, sorbitol, and xylitol, even more preferably, sucrose and trehalose, and particularly preferably, sucrose. In the pharmaceutical composition of the present invention, the content of excipient A is 50 to 1000 times the weight of compound A, preferably 100 to 750 times, and more preferably 200 to 625 times.

[0071] In the present invention, examples of the antioxidant B include tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol); tocopherol acetate; and butylhydroxyanisole. Preferred are α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, and more preferred is α-tocopherol. In the pharmaceutical composition of the present invention, the content of antioxidant B is 5 to 250 times, preferably 12.5 to 125 times, more preferably 20 to 50 times, and even more preferably 25 to 50 times the weight of compound A.

[0072] In the present invention, the freeze-dried preparation can be produced by filling the emulsion into a vial and freeze-drying it using a freeze-dryer under normal production conditions. The production conditions are not limited, but specifically, for example, after freezing at about -40°C, the inside of the chamber is depressurized to a vacuum, and at the same time, the temperature inside the chamber is raised to -20°C, and the preparation is dried for about 10 to 80 hours, and then the temperature inside the chamber is raised to 25°C, and the preparation is dried for about 10 to 30 hours.

[0073] As one embodiment of the pharmaceutical composition of the present invention, i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) an excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants and lyophilized formulations of emulsions comprising:

[0074] In another embodiment of the pharmaceutical composition of the present invention, i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (e.g., L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (e.g., potassium ascorbate, sodium ascorbate, and calcium ascorbate), and ascorbic acid; iv) an excipient A selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) hydrophilic surfactants, such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80); polyoxyethylene hydrogenated castor oils (e.g., polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, and polyoxyethylene hydrogenated castor oil 60); and polyoxyethylene polyoxypropylene glycols (e.g., For example, polyoxyethylene(42) polyoxypropylene(67) glycol, polyoxyethylene(54) polyoxypropylene(39) glycol, polyoxyethylene(105) polyoxypropylene(5) glycol, polyoxyethylene(124) polyoxypropylene(39) glycol, polyoxyethylene(160) polyoxypropylene(30) glycol, polyoxyethylene(196) polyoxypropylene(67) glycol, and polyoxyethylene(200) polyoxypropylene(70) glycol; vi) lipophilic surfactants, such as sorbitan fatty acid esters (e.g., sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, and medium-chain triglycerides); glycerin fatty acid esters (e.g., glycerin fatty acid esters, glycerin monostearate, glycerin monomyristate, glycerin monooleate, and glycerin triisooctanoate); sucrose fatty acid esters (e.g., sucrose fatty acid esters, sucrose stearate, and sucrose palmitate); propylene glycol fatty acid esters (e.g., propylene glycol fatty acid esters and propylene glycol monostearate); and iii') antioxidants B selected from the group consisting of tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol), tocopherol acetate, and butylhydroxyanisole; and lyophilized formulations of emulsions comprising:

[0075] Another embodiment of the pharmaceutical composition of the present invention is a freeze-dried preparation that contains iii') tocopherols (e.g., α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol) as an antioxidant, but does not contain iii) antioxidant A. The tocopherol is preferably α-tocopherol. In the pharmaceutical composition of the present invention, the content of tocopherol is 5 to 250 times, preferably 12.5 to 125 times, more preferably 20 to 50 times, and even more preferably 25 to 50 times the weight of compound A. The pharmaceutical composition may include additional antioxidants such as sodium thiosulfate or butylated hydroxyanisol.

[0076] In the pharmaceutical composition of the present invention, the weight of compound A is 0.0001 to 0.65 times, preferably 0.0002 to 0.35 times, and more preferably 0.0005 to 0.065 times the weight of the lyophilized pharmaceutical composition not containing compound A.

[0077] In the pharmaceutical composition of the present invention, the particle diameter D 90 The value is the particle size D of the emulsion during or immediately after production. 90 The value of the oil droplet diameter D of the emulsion immediately after production is 1000 nm or less, preferably 300 nm or less. An example of the emulsion immediately after production is an emulsion produced within 30 seconds after production. The oil droplet diameter D of the emulsion reconstituted after storage as a freeze-dried preparation is 90 The value is preferably the particle size D of the oil droplets of the reconstituted emulsion after storage for 6 months at 5°C or 25°C. 90 The value is less than or equal to 1000 nm.

[0078] In the pharmaceutical composition of the present invention, the particle diameter D 90 This value is one of the representative values ​​that indicates the particle size distribution of oil droplets contained in an emulsion, and means the 90% particle size based on the scattering intensity standard. Generally, the particle size D90 The particle diameter D is measured and calculated using a dynamic light scattering particle size distribution analyzer, a laser diffraction particle size distribution analyzer, or an image processing particle size distribution analyzer. 90 The values ​​refer to values ​​measured using a dynamic light scattering particle size distribution analyzer: Zetasizer Nano ZS (Malvern Instruments).

[0079] In the pharmaceutical composition of the present invention, the impurity UK-1.02 is one of the representative impurities detected in the evaluation of related substances using high performance liquid chromatography. In particular, it means an impurity detected at 1.02 times the elution time of compound A when a Phenyl-Hexyl column (Waters Xselect CSH Phenyl-Hexyl XP Column, 4.6mmx75mm, 2.5μm, model number: 186006134) is used, pure water, acetonitrile, methanol, and trifluoroacetic acid are used in a reversed phase high performance liquid chromatography method, and an amount equivalent to 0.4 to 2μg of compound A is injected and spectroscopically measured at a wavelength of 220nm. Details of the measurement conditions are as follows. Mobile phase A: 0.1% aqueous trifluoroacetic acid solution Mobile phase B: Acetonitrile / methanol mixture (8:2) containing 0.06% trifluoroacetic acid Flow conditions: [Table 1]

[0080] The storage stability of the pharmaceutical composition of the present invention means that the increase in the area percentage value of the impurity UK-1.02 after storage of a freeze-dried formulation of the pharmaceutical composition of the present invention for 6 months at 5° C. is 5.0% or less, preferably 1.0% or less, from the start of storage. The area percentages are compared based on actual measured values.

[0081] The pharmaceutical composition of the present invention is prepared by emulsifying the oil-in-water emulsion prepared, then performing sterile filtration using a sterile filtration filter, and storing the composition in a freeze-dried state. 90 It is preferred that the value is 1000 nm or less.

[0082] The pharmaceutical composition of the present invention may further contain other additives to the extent that the particle size of the emulsion after reconstitution does not change. At the time of administration, the pharmaceutical composition of the present invention may be mixed with a preparation containing a vaccine antigen (also referred to as "vaccine" in this specification) to the extent that the particle size of the emulsion after reconstitution does not change. The mixing method and mixing ratio of the pharmaceutical composition of the present invention and the vaccine are not limited, but for example, the reconstituted emulsion preparation and an equal volume of a preparation containing a vaccine antigen can be mixed by inverting in a vial.

[0083] The pharmaceutical composition of the present invention can be provided as a kit containing a lyophilized preparation containing compound A and a vaccine antigen.

[0084] The pharmaceutical composition of the present invention can be administered by reconstituting with water for injection in an amount 2 to 20 times the weight of the lyophilized preparation, and further mixing with a preparation containing a vaccine antigen. The dosage of the pharmaceutical composition of the present invention per administration is 1 ng to 250 mg, preferably 1 ng to 50 mg, in terms of the weight of compound A. Depending on the type of vaccine antigen administered simultaneously or the age of the subject to be administered, the composition may be administered once, or may be administered once or multiple times.

[0085] The storage stability of the pharmaceutical composition of the present invention was evaluated according to the following test examples.

[0086] Vaccine antigens One embodiment of the vaccine antigen of the present invention includes a malaria vaccine comprising an N-terminal Pfs230 antigen. In another embodiment, the vaccine antigen is a vaccine comprising a Pfs230D1+ (also referred to herein as "untagged Pfs230D1+") antigen having the sequence set forth in SEQ ID NO:1. In yet another embodiment, the vaccine antigen is a vaccine comprising a Pfs230D1+ (also referred to herein as "tagged Pfs230D1+") antigen having the sequence set forth in SEQ ID NO:2.

[0087] A scalable baculovirus expression system may be used to express Pfs230D1+ constructs, which are then purified and analyzed. Pfs230D1+ may be engineered to prevent glycosylation and protease digestion, potentially increasing homogeneity and stability. In particular, Pfs230D1+ may eliminate undesired glycosylation, resulting in a two-fold increase in yield and increased stability.

[0088] Combination medicines / combination therapies The malaria transmission blocking vaccine of the present invention exhibits excellent vaccine activity by enhancing the induction of IgG2 antibodies by combining a vaccine antigen with a pharmaceutical composition containing a compound A having TLR7 agonist activity as an adjuvant. The combined use may include administering the vaccine antigen and the pharmaceutical composition simultaneously or separately at a predetermined time interval. In one embodiment, the simultaneous use of such a combination includes a combination medicine containing the vaccine antigen and the pharmaceutical composition. In this specification, the combination medicine may also be referred to as an "adjuvant formulation" or a "vaccine formulation". In another embodiment, the combined use includes a kit containing the vaccine antigen and the pharmaceutical composition. In the present invention, the administration route of the pharmaceutical composition, vaccine antigen, combination drug, and kit can be appropriately selected depending on conditions such as the disease, the condition of the subject, the target site, etc. Examples of the administration route include parenteral administration, specifically, intravascular administration (e.g., intravenous administration), subcutaneous administration, intradermal administration, intramuscular administration, intranasal administration, and transdermal administration. In the present invention, examples of dosage forms of the pharmaceutical composition, vaccine antigen, and combination medicine include injections, for example, pre-filled syringes. In the present invention, the dosage, administration regimen, and time required for each administration of the adjuvant formulation can be appropriately selected depending on conditions such as the age of the subject and the target site. The adjuvant formulation may be administered or inoculated once, or may be administered at a predetermined time period after the first administration, for example, but not limited to, any time period from 20 days to 3 years, preferably 3 months to 2 years, and more preferably 6 months to 1 year. The dose of the malaria transmission-blocking vaccine antigen per administration in the combined use of the present invention may be, but is not limited to, 1 μg to 200 μg, preferably 10 μg to 30 μg, and more preferably 15 μg. The dose of the adjuvant formulation per administration is, for example, 0.5 mL. The dosage of the pharmaceutical composition containing compound A per administration in the combined use of the present invention may be 1 ng to 250 mg, preferably 1 ng to 50 mg, of compound A by weight, but is not limited thereto. EXAMPLES

[0089] The present invention will be described below with reference to Examples, Reference Examples, Comparative Examples, and Test Examples, but the present invention is not limited to these in any way.

[0090] In the present specification, "squalane (manufactured by Wako Pure Chemical Industries)", "squalane (manufactured by Kishimoto Tokushu Kanyu Kogyosho)", or "squalane (manufactured by Maruha Nichiro)" is used as squalane; "squalene (manufactured by Wako Pure Chemical Industries)", "squalene (manufactured by Kishimoto Tokushu Kanyu Kogyosho)", or "squalene (manufactured by Maruha Nichiro)" is used as squalene; "sodium ascorbate (manufactured by Wako Pure Chemical Industries)" or "sodium L-ascorbate (manufactured by Kyowa Pharma Chemical)" is used as sodium ascorbate; "α-tocopherol (manufactured by Mitsubishi Chemical Foods)" or "all-rac-α-tocopherol EMPROVE (registered trademark) ESSENTIAL Ph Eur, BP, USP, E 307 (manufactured by Merck)" is used as α-tocopherol; "Span85 (manufactured by Sigma-Aldrich)" or "Rheodol "SP-O30V (manufactured by Kao Chemical)" or "Span85 (manufactured by Croda)" was used; as polysorbate 80, "PS80 (GS) (manufactured by NOF Corp.)", "Polysorbate 80 (HX2) (manufactured by NOF Corp.)", "Tween80 (manufactured by Merck)", or "Tween80 HP-LQ-(HM) (manufactured by Croda)" was used; as sucrose, "sucrose (manufactured by Nacalai Tesque)" or "sucrose suitable for use as an excipient (low endotoxin) EMPROVE (registered trademark) exp Ph Eur, BP, JP, NF (manufactured by Merck)" was used; as water for injection, "Otsuka distilled water for injection (manufactured by Otsuka Pharmaceutical Factory)" was used; ascorbic acid palmitate, butylated hydroxyanisole, and sodium thiosulfate were all manufactured by Wako Pure Chemical Industries, Ltd.

[0091] Abbreviation ACN: Acetonitrile AAALAC: Association for Assessment and Accreditation of Laboratory Animal Care BCA: Bicinchoninic acid cIEF: Capillary isoelectric focusing CM 1: Cysteine ​​motif 1 CV: column volume Da: Dalton DNA: Deoxyribonucleic acid DTT: Dithiothreitol ELISA: Enzyme-linked immunosorbent assay EPA: Pseudomonas aeruginosa exoprotein A HPLC: High-performance liquid chromatography IACUC: Animal Care and Use Committee kDa: kilodaltons LDS: Lithium dodecyl sulfate MES: 2-(N-morpholino)ethanesulfonic acid MOI: Multiplicity of infection MS: Mass spectrometry MS1: Mass spectrum 1 Ni-NTA: Nickel-nitrilotriacetic acid NIAID: National Institute of Allergy and Infectious Diseases NIH: National Institutes of Health NLS: Noble Life Sciences OLAW: Office of Laboratory Animal Welfare RP: Reverse phase SDS: Sodium dodecyl sulfate SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis SE: Size exclusion SEC: Size Exclusion Chromatography SMFA: Standard Membrane Feeding Assay TBV: Transmission blocking vaccine TFA: Trifluoroacetic acid TRA: Transmission-reducing activity UHPLC: Ultra High Performance Liquid Chromatography USDA: United States Department of Agriculture ZINB: Zero-inflated negative binomial

[0092] Preparation of the Lyophilized Composition Examples 1 to 16, Comparative Examples 1 to 3 Compound A was dissolved in oil components to obtain the compositions shown in Tables 1 to 4. The oil components are as follows: squalane, sorbitan trioleate, and α-tocopherol (Examples 1 to 9 and 15); squalane and sorbitan trioleate (Examples 10 to 13); squalane, sorbitan trioleate, α-tocopherol, and ascorbic acid palmitate (Example 14); squalane, sorbitan trioleate, α-tocopherol, and butylhydroxyanisole (Example 16); squalene, sorbitan trioleate, and α-tocopherol (Comparative Examples 1 to 3). The aqueous components (sucrose and polysorbate 80 in Examples 1 to 3, 14, and 16; sucrose, polysorbate 80, and sodium ascorbate in Examples 4 to 13; sucrose, polysorbate 80, and sodium thiosulfate in Example 15) were dissolved in water for injection to obtain the compositions shown in Tables 1 to 4, and the oily composition was then added thereto. The mixture was premixed and emulsified and dispersed using an ultra-high pressure emulsifying and dispersing machine. The resulting reaction product was filtered through a 0.2 μm sterilized filter, and then filled into glass vials in 1 mL portions and freeze-dried. Each vial was purged with nitrogen gas at normal pressure and then sealed with a rubber stopper to obtain the freeze-dried compositions of Examples 1 to 16 and Comparative Examples 1 to 3. [Table 2] [Table 3] [Table 4] [Table 5]

[0093] [Test Example 1] Evaluation of particle size during manufacturing process In the process of producing the freeze-dried composition, the particle size distribution of the oil droplet particles contained in the emulsion after emulsification and before freeze-drying was measured according to the following method. The emulsion was diluted 10 times with water for injection, and the 90% particle size based on scattering intensity (D 90 ) was measured. 90 (nm) are shown in Table 5. [Table 6]

[0094] [Test Example 2] Stability evaluation (1) According to the method described below, the particle size distribution of the prepared freeze-dried composition was measured at the start of storage and after storage for 6 months in thermostatic chambers at 5°C and 25°C. 1 mL of water for injection was added to each vial of the freeze-dried composition prepared in Examples 1 to 16 and Comparative Examples 1 to 3 to reconstitute the composition. Then, 100 μL of the reconstituted solution was collected using a micropipette and mixed with 900 μL of water for injection. Next, the particle size distribution at 90% of the scattering intensity (D 90 ) was measured for Examples 1 to 16 and Comparative Examples 1 to 3 before and after storage. 90 (nm) are shown in Table 6. [Table 7] The test results showed that the formulations of Examples 4 to 14, which contain ascorbic acids as antioxidants, showed little change in particle size distribution after 6 months of storage in thermostatic chambers at 5°C and 25°C from the value at the start of storage, indicating that the particle size distribution is highly stable.

[0095] [Test Example 3] Stability evaluation (2) The amount of impurities (area percentage value of Uk-1.02) was measured at the start of storage and after 6 months of storage in a thermostatic chamber at 5 ° C and 25 ° C according to the following method. Using a Phenyl-Hexyl column (Waters Xselect CSH Phenyl-Hexyl XP Column, 4.6 mm x 75 mm, 2.5 μm, model number: 186006134), 0.4 to 2 μg equivalent of Compound A was injected by reversed-phase high-performance liquid chromatography using pure water, acetonitrile, methanol, and trifluoroacetic acid, and the amount was detected spectroscopically at a wavelength of 220 nm. Details of the measurement conditions are as follows. Mobile phase A: 0.1% aqueous trifluoroacetic acid solution Mobile phase B: Acetonitrile / methanol mixture (8:2) containing 0.06% trifluoroacetic acid Flow conditions: [Table 8] Using the peak area and elution time measured by this method, the area percentage value of the impurity peak (Uk-1.02) detected at an elution time 1.02 times that of compound A was calculated according to the following formula. Area percentage value of Uk-1.02 (%) = Peak area of ​​Uk-1.02 / Total peak area of ​​related substances and compound A x 100 Table 7 shows the area percentage values ​​(%) of the impurity peak (Uk-1.02) of Examples 1 to 16 and Comparative Examples 1 to 3 before and after storage. [Table 9] The test results showed that the formulations of Examples 4 to 14 containing ascorbic acids as antioxidants had low impurity values ​​(area percentage value of Uk-1.02) after 6 months of storage in a thermostatic chamber at 5° C., and thus had high storage stability. Comparing the formulations of Examples 1 to 3 containing squalane as an oily component with the formulations of Comparative Examples 1 to 3 containing squalene as an oily component, the formulations containing squalane as an oily component had lower impurity values ​​(area percentage value of Uk-1.02), which suggests that squalane contributes to the antioxidant stability of compound A, and that the formulations containing squalane have high reservation stability.

[0096] Vaccine antigen production [Example 17] Production and purification of Pfs230D1+ Baculovirus expression construct (Pfs230D1+) The N-terminal sequence (amino acids 552-731) of Pfs230 (ACCESSION P68874), a surface protein of gametocytes of the 3D7 strain, containing four cysteines as part of the predicted cysteine-rich domain, was cloned. It was designated Pfs230D1+. Codon optimization for baculovirus expression was performed using DNA2.0 (now ATUM). The synthetic deoxynucleic acid (DNA) (552-731) of Pfs230D1+ was fitted with an N-terminal secretion signal (MKFLVNVALVFMVVYISYIYAD from honeybee melittin) and a C-terminal six histidine tag, as described in Non-Patent Document 1. The resulting plasmid was cloned, sequence verified, and recombinant bacmid obtained. The bacmid was again sequence verified and transfected into superSf9 cells (Oxford Expression Technologies) to obtain recombinant baculovirus stocks using Cellfectin® II reagent (Invitrogen) according to the Bac-to-Bac manual.

[0097] Expression and purification of Pfs230D1+ SuperSf9 cells were cultured at 1x10 in SFM4 medium (Hyclone). 6 The cells were seeded at 1000 cells / mL. This MOI (multiplicity of infection) was used to infect 10 L of superSf9 wave cultures. At 96 hours post-infection, the cultures were harvested, concentrated 5-fold, and diluted with 0.5 ml of PBS. 2 The solution was diafiltered against 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 (buffer A) using a tangential flow filtration device (Centrasette LV, Pall) containing an Omega polyethersulfone membrane (Pall). Clarification was performed using a 0.22 μm filter (Stericup-GP vacuum filter, Merck Millipore).

[0098] Two liters of the clarified and concentrated supernatant were loaded onto a 61 mL (2.6x11.5 cm) nickel-nitrilotriacetic acid (Ni-NTA) (His60 Ni Superflow, Clontech) column at 100 cm / h. Washing steps were performed with 5 column volumes (CV) of buffer A, 5 CV of buffer A containing 10 mM imidazole, and then 5 CV of buffer A containing 20 mM imidazole. Proteins were eluted with buffer A containing 50-100 mM imidazole. The pooled eluate of the Ni-NTA column was concentrated 5-fold in an Amicon Ultra-15 centrifugal filter with a 3K regenerated cellulose membrane (Merck Millipore). The protein was further purified and buffer exchanged using size-exclusion chromatography (SEC) on a Superdex 75 (2.6x60cm, 320mL) equilibrated with 20mM HEPES, 150mM NaCl, 5% glycerol, pH 7.2. Multiple runs were performed to hold an injection load limit of 5%CV. Superdex 75 elution fractions were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pooled, and further concentrated using the same Amicon 3K concentrator (Merck Millipore) to a target of 1mg / mL and stored at -80°C.

[0099] Protein concentration measurement To measure the protein concentration of purified Pfs230D1+, i.e., tagged Pfs230D1+, due to its low tryptophan content, a bicinchoninic acid (BCA) assay was performed according to the manufacturer's instructions (Thermo). A protein concentration standard curve for the assay was generated using bovine serum albumin.

[0100] SDS-PAGE Samples were diluted in 4X LDS (lithium dodecyl sulfate, Invitrogen) sample buffer, heated to 90°C for 5 min, and loaded onto SDS-PAGE gels (4-12% NuPAGE Bis-Tris, Invitrogen) in a final volume of 20 μL / well. Gels were run in 1X 2-(N-morpholino)ethanesulfonic acid (MES) sodium dodecyl sulfate (SDS) running buffer at 150-200V for 35-50 min and stained with SimplyBlue™ SafeStain (Invitrogen).

[0101] Western blotting with purified Pfs230D1+ (anti-His) After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and subjected to Western blotting using an anti-penta-His antibody (Qiagen) according to the method described in Non-Patent Document 1. The membrane was developed using ECL Prime (GE Healthcare).

[0102] Western blotting with purified Pfs230D1+ (anti-Pfs230D1+) After SDS-PAGE, proteins were transferred to nitrocellulose membranes and Western blotted with anti-Pfs230 human monoclonal antibodies (non-proprietary). Human monoclonal antibodies were tested at 10 μg / mL for reactivity against 20 ng of Pfs230D1+ (untagged or tagged) loaded per lane. A secondary goat anti-human antibody was used at 1 / 2000 dilution, followed by development with BCIP / NBT substrate.

[0103] N-terminal domain of Pfs230 (Pfs230D1+) An N-terminal construct of Pfs230 containing amino acids 552-731 (denoted as Pfs230D1+) was newly designed based on the Pfs230C1 TBV antigen (amino acids: 443-731) and evaluated in the superSf9 / baculovirus system. The design was chosen to reduce the likelihood of proteolytic cleavage and glycosylation while retaining intact disulfide bond folding predicted to be necessary for the induction of transmission-blocking antibodies after immunization. Pfs230D1+ also contains an additional N585Q mutation to eliminate potential N-glycosylation and, in this initial evaluation, a six histidine C-terminal tag to facilitate purification. Untagged Pfs230D1+ eliminates potential N-glycosylation with the N585Q mutation and does not contain any extraneous C-terminal amino acids. Furthermore, removing the N-terminal amino acids from the original Pfs230C1 protein did not appear to alter the predicted secondary structure of Pfs230D1+ as presented in POLYVIEW-2D.

[0104] A two-step purification approach was used to capture and polish Pfs230D1+, including IMAC (immobilized metal affinity column) and SEC, which gave a yield of 23 mg / L. Furthermore, the resulting purified protein had a predicted molecular weight of approximately 21 kDa (kilodaltons) and was present at a purity of >90% by SDS-PAGE and densitometry (Figure 1a). Reactivity with anti-His antibodies confirmed the presence of a histidine tag at the C-terminus (Figure 1b). A purification approach was used to capture and polish untagged Pfs230D1+, including IEX (ion exchange chromatography) and MMC (mixed mode chromatography). The purified protein was desalted and buffer exchanged to give a final purified yield of 5-10 mg / L with a purity of >90% by SDS-PAGE (Figure 3), with most impurities being product-associated. Reactivity to anti-Pfs230 monoclonal antibodies confirmed the presence of conformational epitopes in both tagged (Figure 4a) and untagged Pfs230D1+ (Figure 4b). Here (Figure 4), both tagged and untagged Pfs230D1+ reacted with two human Pfs230 monoclonal antibodies under non-reducing conditions (disulfide-dependent), indicating that the protein contains the proper disulfide fold.

[0105] Generation of rat anti-Pfs230 antiserum and enzyme-linked immunosorbent assay (ELISA) SD rats (Charles River Japan) were immunized with 50 μL of a vaccine formulation consisting of 20 μg of antigen, tagged Pfs230D1+ or untagged Pfs230D1+, and 8110 μg of the composition of Example 6 as an adjuvant (corresponding to 10 μg of Compound A); 20 μg of tagged Pfs230D1+ alone in FIG. 2 or 20 μg of tagged Pfs230D1+ and 8110 μg of the composition of Example 6 in FIG. 5; or 8110 μg of the composition of Example 6 alone as a control, by intramuscular injection (im) into the thigh muscle of the hind leg. On days 0 and 21 or days 0, 21, and 42, rats were injected twice or three times. On days 42 or 56, rats were sacrificed and blood was collected. After collection of whole blood, the blood was allowed to coagulate by leaving it at room temperature for 4 hours. The clots were removed by centrifugation at 2,000×g for 10 min in a refrigerated centrifuge. For sera from day 42 or day 56, antibody levels against the corresponding proteins were measured individually by ELISA (tagged Pfs230D1+, Fig. 2a; non-tagged Pfs230D1+, Fig. 5a).

[0106] IgG purification and standard membrane feeding assay (SMFA) For each group, equal amounts of serum from each rat were pooled, regardless of ELSA units. Total IgG from pooled serum samples was purified using a Protein G column (GE Healthcare) according to the manufacturer's instructions and adjusted to a final concentration of 4 mg / mL with phosphate-buffered saline. Standardized methods for performing SMFA are known in the art. Briefly, P. falciparum NF54 strain was cultured for 16-18 days to induce mature stage V gametocytes. Stage V gametocytes (approximately 1% stage V gametocytemia) were mixed with 250 μg / mL of test IgG, and the final mixture was immediately fed to approximately 50 female Anopheles stephensi mosquitoes in a membrane feeding apparatus. All feeding experiments were performed with human complement. Mosquitoes were kept for 8 days after feeding, dissected (n=20 per group), and oocysts in the midgut were counted. Only mosquito midguts with eggs in the ovary at the time of dissection were analyzed (tagged Pfs230D1+, Fig. 2b; untagged Pfs230D1+, Fig. 5b). Human serum and red blood cells for gametocyte culture and feeding experiments were purchased from Interstate Blood Bank (Memphis, TN). [Industrial Applicability]

[0107] A pharmaceutical composition containing compound A or a pharma- ceutical acceptable salt thereof has high storage stability and immunostimulatory activity as a vaccine adjuvant, and the combined use of the pharmaceutical composition with a malaria vaccine may be useful in preventing the transmission of malaria parasites. [Sequence List Free Text]

[0108] SEQ ID NO:1 (Pfs230D1+; non-tag) DVGVDELDKI DLSYETTESG DTAVSEDSYD KYASQNTNKE YVCDFTDQLK PTESGPKVKK CEVKVNEPLI KVKIICPLKG SVEKLYDNIE YVPKKSPYVV LTKEETKLKE KLLSKLIYGL LISPTVNEKE NNFKEGVIEF TLPPVVHKAT VFYFICDNSK TEDDNKKGNR GIVEVYVEPY G Sequence number 2 (Pfs230D1+, intermediate; histidine tag) DVGVDELDKI DLSYETTESG DTAVSEDSYD KYASQNTNKE YVCDFTDQLK PTESGPKVKK CEVKVNEPLI KVKIICPLKG SVEKLYDNIE YVPKKSPYVV LTKEETKLKE KLLSKLIYGL LISPTVNEKE NNFKEGVIEF TLPPVVHKAT VFYFICDNSK TEDDNKKGNR GIVEVYVEPY G--HHHHHH

Claims

1. i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (including L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (including potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols (excluding mannitol); v) a hydrophilic surfactant; and vi) Lipophilic surfactants A pharmaceutical composition I) comprising: Malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 II) A combination medicine comprising:

2. i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (including L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (including potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols, excluding mannitol; v) a hydrophilic surfactant; and vi) Lipophilic surfactants A pharmaceutical composition I) comprising: Malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 II) 13. A vaccine formulation comprising:

3. i) (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexanamide or a pharma- ceutically acceptable salt thereof; ii) Squalane; iii) an antioxidant A selected from the group consisting of ascorbic acid esters (including L-ascorbic acid stearate and ascorbic acid palmitate), inorganic salts of ascorbic acid (including potassium ascorbate, sodium ascorbate and calcium ascorbate), and ascorbic acid; iv) excipients selected from the group consisting of non-reducing sugars and sugar alcohols, excluding mannitol; v) a hydrophilic surfactant; and vi) Lipophilic surfactants A pharmaceutical composition I) comprising: Malaria vaccine comprising an antigen having a sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 II) Including the kit.

4. The combination pharmaceutical according to claim 1, wherein the pharmaceutical composition is an oil-in-water emulsion formulation or a lyophilized formulation thereof.

5. The hydrophilic surfactant may be selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (including polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80); polyoxyethylene hydrogenated castor oils (including polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, and polyoxyethylene hydrogenated castor oil 60); and polyoxyethylene polyoxypropylene glycols (polyoxyethylene (42) polyoxyethylene hydrogenated castor oil 50, and polyoxyethylene hydrogenated castor oil 60).

5. The pharmaceutical combination according to claim 1 or 4, wherein the glyceryl ether ester is selected from the group consisting of polyoxyethylene (54) polyoxypropylene (39) glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (124) polyoxypropylene (39) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, and polyoxyethylene (200) polyoxypropylene (70) glycol.

6. 6. The pharmaceutical combination according to claim 1, 4 or 5, wherein the hydrophilic surfactant is polysorbate 20, polysorbate 40 or polysorbate 80.

7. The combination pharmaceutical according to any one of claims 1 and 4 to 6, wherein the lipophilic surfactant is sorbitan fatty acid esters (including sorbitan fatty acid esters, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan tristearate, sorbitan monooleate, sorbitan sesquioleate, sorbitan trioleate, and medium-chain triglycerides); glycerin fatty acid esters (including glycerin fatty acid esters, glycerin monostearate, glycerin monomyristate, glycerin monooleate, and glycerin triisooctanoate); sucrose fatty acid esters (including sucrose fatty acid esters, sucrose stearate, and sucrose palmitate); or propylene glycol fatty acid esters (including propylene glycol fatty acid esters and propylene glycol monostearate).

8. The pharmaceutical combination according to any one of claims 1 and 4 to 7, wherein the lipophilic surfactant is sorbitan trioleate.

9. The pharmaceutical combination according to any one of claims 1 and 4 to 8, wherein the pharmaceutical composition further comprises an antioxidant B selected from the group consisting of tocopherols (including α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol); tocopherol acetate; and butylhydroxyanisole.

10. The pharmaceutical combination according to any one of claims 1 and 4 to 9, wherein the pharmaceutical composition further comprises an antioxidant B which is α-tocopherol.

11. The pharmaceutical combination according to any one of claims 1 and 4 to 10, wherein antioxidant A is ascorbic palmitate, potassium ascorbate, sodium ascorbate, or ascorbic acid.

12. The pharmaceutical combination according to any one of claims 1 and 4 to 11, wherein the antioxidant A is sodium ascorbate or potassium ascorbate.

13. The pharmaceutical combination according to any one of claims 1 and 4 to 12, wherein the excipient is a non-reducing sugar (including sucrose and trehalose) or sugar alcohol (including sorbitol, erythritol, xylitol, maltitol, and lactitol).

14. The pharmaceutical combination according to any one of claims 1 and 4 to 13, wherein the excipient is sucrose or trehalose.

15. The combination pharmaceutical according to any one of claims 1 and 4 to 14, wherein the increase in area percentage of the impurity UK-1.02 after storage of a lyophilized formulation of the pharmaceutical composition at 5°C for 6 months is 5.0% or less.

16. A combination pharmaceutical composition according to any one of claims 1 and 4 to 15 for preventing transmission of malaria parasites from humans to mosquitoes.

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