Liposome compositions containing NKT cell ligands

Abstract

The purpose of the present invention is to provide a preparation containing an NKT cell ligand, which can artificially activate NKT cells in a living body and can be used as an anti-malignant tumor agent. A liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof. Formula (I): [In the formula, each symbol is as defined in the description. ] Formula (II): [In the formula, each symbol is as defined in the description. ] # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a liposome composition containing a NKT cell ligand (glycolipid described below), which can be used as an anti-malignant tumor agent, an anti-infective agent, a NKT cell activator, an IFN-γ production inducer, an activator of innate immune system lymphocytes and acquired immune system lymphocytes, and an inducer of long-term immune memory. Background Art

[0002] Natural killer (hereinafter referred to as NK) T cells are immune cells belonging to novel lymphocyte lineages, showing characteristics different from other lymphocyte lineages (T, B, NK cells). Because there are cytotoxic perforin particles in NKT cells, they are related to NK cells (non-patent literature 1). However, since NKT cells not only express NK cell markers, but also express T cell receptors (TCR), they are a new and completely different cell population (non-patent literature 2). NKT cells have the ability of both Th-1 type cytokines (mainly interferon (IFN)-γ) produced by the auxiliary T (Th)-1 type cells of enhanced immunostimulation and Th-2 type cytokines (mainly interleukin (IL)-4, IL-10, etc.) produced by the Th-2 type cells of enhanced immunosuppression (non-patent literature 3). That is, because NKT cells can induce activation and sedation of the immune system, they may play the balance regulation of the immune system (non-patent literature 4). Therefore, if the function of NKT cells can be controlled, it is possible to treat various diseases caused by immune system imbalance, particularly cancer and infection.

[0003] As a characteristic of NKT cells, the most notable is that the antigen receptor α chain expressed in NKT cells is the same in all individuals of a certain species. This indicates that all NKT cells possessed by organisms of the same species recognize the same substance and are activated. This α chain is Vα24Jα18 in humans and Vα14Jα18 in mice, and there is a very high homology between the two species. In addition, only very limited types of β chains that form pairs with the α chain are known. Therefore, this NKT cell antigen receptor is also called "invariant TCR" and cannot be expressed by normal T cells. In addition, it is also characterized in that the antigen receptor (TCR) of T cells recognizes protein fragments, while the antigen receptor of NKT cells recognizes glycolipids.

[0004] It is known that there are many kinds of glycosphingolipids in living organisms. In the glycosphingolipids commonly found in living organisms, various sugars or sugar chains are linked to ceramide via β-bonds and are present in the cell membranes of various organs.

[0005] On the other hand, glycosphingolipids linked to ceramide by an α bond are known to have strong immunostimulatory effects and anti-tumor activity. α-Galactosylceramide, represented by agelasphins, is a glycolipid isolated from an extract of a sponge Agelasmauritianus, and is reported to strongly activate NKT cells (Non-Patent Document 5). After α-Galactosylceramide is taken up by antigen presenting cells (APCs) represented by dendritic cells (DCs), it is presented on the cell membrane through CD1d molecules similar to major histocompatibility complex (MHC) class I molecules. NKT cells are activated by recognizing the complex of CD1d molecules and α-Galactosylceramide presented by using NKT cell-intrinsic antigen receptors, and various immune responses are induced.

[0006] So far, various analogs have been synthesized and the correlation between their structure and activity has been studied. Among a series of synthetic analogs, KRN7000 (α-GalCer) developed by Kirin Brewery Company, Limited showed very strong antitumor activity, and it is clear that no immunostimulatory activity was found in the corresponding β-form (β-GalCer) (Non-patent Document 6). KRN7000 is a glycosphingolipid formed by galactose combined with ceramide in an α configuration, wherein ceramide is formed by acylation of sphingosine bases with long-chain fatty acids.

[0007]

[0008] In recent years, the functions of such NKT cells have attracted attention, and cancer treatment drugs containing KRN7000 as an active ingredient have been developed. However, NKT cells activated by the administration of KRN7000 produce IFN-γ (a cytokine that can be used in cancer and infectious disease treatment and induces immunostimulatory activity) while also producing IL-4, a cytokine that induces immunosuppressive effects. As a result, the effects of the two cancel each other out, and there is a problem that sufficient effects cannot be obtained for cancer and infectious disease treatment.

[0009] For the treatment of cancer and infectious diseases, many α-GalCer derivatives have been developed, which strongly activate NKT cells and preferentially produce IFN-γ over IL-4 (Patent Documents 1 and 2).

[0010] Patent Document 1 describes a compound represented by the following general formula or a salt thereof.

[0011]

[0012] (wherein, X represents an alkylene group or -NH-;

[0013] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0014] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0015] R4 represents a hydrocarbon group having 1 to 30 carbon atoms).

[0016] Patent Document 2 describes a compound represented by the following general formula or a salt thereof.

[0017]

[0018] [Where R 1 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, and R 2 and R 3 Each independently represents a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-]. However, when R 1 When it is a hydrogen atom, R 2 It represents a substituted or unsubstituted hydrocarbon group having 24 to 28 carbon atoms.]

[0019] NKT cell ligands such as α-GalCer are glycolipids, so if they are directly administered to a living body, they form micelles due to their hydrophobic groups, making them unable to effectively reach target cells and have no effect. However, it is clear that cell therapy that is administered by presenting them on antigen-presenting cells can exert a strong anti-tumor effect.

[0020] Based on the above studies, clinical trials of cancer therapeutic drugs that artificially activate NKT cells in vivo were conducted by administering antigen presenting cell (APC) preparations (cell preparations) after pulsed NKT cell ligands (glycolipids), and extremely effective results were obtained (Non-Patent Documents 7 to 9).

[0021] However, although the above-mentioned cell preparations have been proven to be effective, various problems have also been pointed out (such as the risk of allogeneic cell preparations causing allergic reactions, the complexity of autologous cell cell separation, and the freeze-thaw instability of patient cell preparations, etc.). In fact, in the manufacturing process of cell preparations, extremely much time and complicated operations and high costs are required, such as the need for cell separation and purification, cell differentiation culture, pulse culture, freezing and thawing of cell preparations, and removal of dead cell aggregates before administration. In addition, if allogeneic cell preparations are administered multiple times, there is a risk of allergic reactions. Therefore, it is desirable to develop a preparation containing NKT cell ligands without using a new concept of cells.

[0022] In addition, malignant tumors are cells produced by their own tissues, and unlike pathogens, they do not have adjuvant substances that activate NKT cells, so they cannot activate NKT cells necessary for inducing long-term immune memory, making it difficult to induce and maintain immune memory against malignant tumors. Therefore, it is desirable to develop a preparation that can induce and maintain immune memory against malignant tumors.

[0023] Prior art literature

[0024] Patent Literature

[0025] Patent Document 1: WO2013 / 162016

[0026] Patent Document 2: WO2009 / 119692

[0027] Non-patent literature

[0028] Non-patent document 1: Proc. Natl. Acad. Sci. USA 1998, 95, 5690-5693

[0029] Non-patent document 2: J. Immunol. 1995, 155, 2972-2983

[0030] Non-patent document 3: J. Immunol. 1998, 161, 3271-3281

[0031] Non-patent document 4: Science, 1997, 278, 1623-1626

[0032] Non-patent document 5: Science, 1997, 278, 1626-1629

[0033] Non-patent document 6: J. Med. Chem. 1995, 38, 2176-2187

[0034] Non-patent literature 7: J Immunol 1999; 163: 2387-2391

[0035] Non-patent document 8: Ishikawa et al., Clin Cancer Res. 11: 1910-1917, 2005, 269

[0036] Non-patent document 9: Motohashi et al., J Immunol. 182: 2492-501, 2009 Summary of the invention

[0037] Problems Solved by the Invention

[0038] The object of the present invention is to provide a preparation containing a NKT cell ligand, which can artificially stimulate NKT cells in vivo.

[0039] In addition, the purpose of the present invention is to provide a preparation containing NKT cell ligands, which can induce and maintain long-term immune memory against malignant tumors by artificially activating NKT cells in an organism suffering from malignant tumors, and continuously attack the malignant tumors through the immune memory against the malignant tumors, thereby being able to control the progression, recurrence and metastasis of the malignant tumors.

[0040] Another object of the present invention is to provide a preparation containing a NKT cell ligand that can induce and maintain long-term immune memory against pathogens even during an infection and can be used as an adjuvant to treat and prevent an infection.

[0041] Solutions to the problem

[0042] The inventors of the present invention have conducted in-depth research on the above-mentioned problems and found that by making the following glycolipids as NKT cell ligands in liposomes and administering them into the body, antigen presenting cells in the body can be effectively reached, thereby artificially activating NKT cells, activating and proliferating innate and acquired immune system immune cells through IFN-γ produced by activated NKT cells, and inducing long-term immune memory. Based on this insight, further research was conducted to complete the present invention. That is, the present invention is as follows.

[0043] [1] A liposome composition comprising a glycolipid (NKT cell ligand) selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof.

[0044] Formula (I):

[0045]

[0046] [wherein, X represents an alkylene group or -NH-;

[0047] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0048] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0049] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0050] Formula (II):

[0051]

[0052] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-.]

[0053] [2] The composition according to [1] above, wherein the glycolipid is a compound represented by formula (I) or a salt thereof.

[0054] [3] The composition according to [2] above, wherein the glycolipid is a compound represented by the following formula or a salt thereof:

[0055]

[0056] [4] The composition according to any one of [1] to [3] above, which is an anti-tumor agent or an anti-infective agent.

[0057] [5] The composition according to any one of [1] to [3] above, which is a NKT cell activator.

[0058] [6] The composition according to any one of [1] to [3] above, which is an inducer of IFN-γ production.

[0059] [7] The composition according to any one of [1] to [3] above, which is an activator of lymphocytes of the innate immune system and lymphocytes of the acquired immune system.

[0060] [8] The composition according to any one of [1] to [3] above, which is a long-term immune memory inducer.

[0061] [9] The composition according to any one of [1] to [3] above, which is used for delivering the glycolipid to antigen-presenting cells.

[0062]

[10] A method for delivering a glycolipid to an antigen presenting cell, comprising administering to a subject a liposome composition containing a glycolipid (NKT cell ligand) selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof.

[0063] Formula (I):

[0064]

[0065] [wherein, X represents an alkylene group or -NH-;

[0066] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0067] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0068] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0069] Formula (II):

[0070]

[0071] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-.]

[0072]

[11] A method for treating a malignant tumor or an infectious disease, comprising administering to a subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof.

[0073] Formula (I):

[0074]

[0075] [wherein, X represents an alkylene group or -NH-;

[0076] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0077] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0078] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0079] Formula (II):

[0080]

[0081] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-.]

[0082]

[12] A method for activating NKT cells in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof.

[0083] Formula (I):

[0084]

[0085] [wherein, X represents an alkylene group or -NH-;

[0086] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0087] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0088] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0089] Formula (II):

[0090]

[0091] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-]. ]

[0092]

[13] A method for inducing IFN-γ production in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof.

[0093] Formula (I):

[0094]

[0095] [wherein, X represents an alkylene group or -NH-;

[0096] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0097] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0098] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0099] Formula (II):

[0100]

[0101] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-]. ]

[0102]

[14] A method for activating innate immune system lymphocytes and adaptive immune system lymphocytes in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II) and salts thereof.

[0103] Formula (I):

[0104]

[0105] [wherein, X represents an alkylene group or -NH-;

[0106] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0107] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0108] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0109] Formula (II):

[0110]

[0111] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-.]

[0112]

[15] A method for inducing long-term immune memory in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II) and salts thereof;

[0113] Formula (I):

[0114]

[0115] [wherein, X represents an alkylene group or -NH-;

[0116] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0117] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0118] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0119] Formula (II):

[0120]

[0121] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-.]

[0122]

[16] The composition according to any one of [1] to [3] above, which is used for delivering the glycolipid to antigen-presenting cells.

[0123]

[17] The composition according to any one of [1] to [3] above, which is used for treating a malignant tumor or an infectious disease.

[0124]

[18] The composition according to any one of [1] to [3] above, which is used for activating NKT cells.

[0125]

[19] The composition according to any one of [1] to [3] above, which is used for inducing IFN-γ production.

[0126]

[20] The composition according to any one of [1] to [3] above, which is used for activating lymphocytes of the innate immune system and lymphocytes of the acquired immune system.

[0127]

[21] The composition according to any one of [1] to [3] above, which is used for inducing long-term immune memory.

[0128]

[22] The composition according to any one of [1] to [3] above, which is used as a medicine.

[0129] Effects of the Invention

[0130] The liposome composition of the present invention containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II) and their salts (hereinafter also referred to as the liposome composition of the present invention) can be effectively delivered to antigen-presenting cells in the body by being administered into a living body (particularly a living body suffering from a malignant tumor or a living body carrying an infectious pathogen), thereby artificially activating NKT cells, activating and proliferating immune cells of the innate immune system and the acquired immune system through IFN-γ produced by the activated NKT cells, and inducing long-term immune memory.

[0131] Therefore, the liposome composition of the present invention can be used as an anti-tumor agent, an anti-infective agent, an NKT cell activator, an IFN-γ production inducer, an activator of innate immune system lymphocytes and acquired immune system lymphocytes, and a long-term immune memory inducer.

[0132] Generally speaking, there are two types of cancer cells: cancer cells that express antigens and cancer cells that lose antigen expression. The liposome composition of the present invention can activate both innate immune system lymphocytes and acquired immune system lymphocytes. Therefore, acquired immune system lymphocytes CD8 killer T cells attack the former and innate immune system NK cells attack the latter, thereby eliminating these two types of cancer cells. By activating the innate immune system lymphocytes and acquired immune lymphocytes in the patient's body, it is expected that the anti-tumor effect will be enhanced.

[0133] In addition, it is reported that when tumor cells divide, new cancer cells expressing mutated cancer antigens are generated, which cannot be treated by conventional treatment methods. However, the liposome composition of the present invention can also activate the acquired immune system lymphocytes corresponding to the mutated cancer cells in the cancer tissue, thereby also being able to eliminate the mutated cancer cells.

[0134] Based on the results of the following test examples, the liposome composition of the present invention (liposome containing a NKT cell ligand) can be expected to have the following effects after being administered into a living body.

[0135] 1. By intravenously administering only a very small amount of liposomes containing NKT cell ligands (RK-163, RCAI-61, RCAI-137, etc.) of the present invention (1 ng / 25 g mouse as the amount of RK-163, RCAI-61, RCAI-137: converted to a person weighing 60 kg, the amount of RK-163, RCAI-61 and RCAI-137 is 2.4 mg), NKT cells in the body present outside the tumor tissue are artificially activated (see Test Example 5-1 below). In fact, after administering the liposomes containing NKT cell ligands (RK-163 or RCAI-61 or RCAI-137) of the present invention, the number of artificially activated NKT cells increases, and a large amount of IFN-γ as an adjuvant substance is produced, thereby strongly activating the innate immune system and the acquired immune system immune cell population, and inducing cell proliferation (see Test Examples 5-1, 5-2, 6, 7, 8 and 9 below). Furthermore, it has been demonstrated that the liposomes of the present invention containing the NKT cell ligand (RK-163) can activate human NKT cells.

[0136] 2. Artificially activated NKT cells strongly express the chemokine receptor CXCR6, and therefore effectively infiltrate into cancer tissues by utilizing the concentration gradient of the chemokine CXCL16 produced by tumor tissues. NKT cells that have not been artificially activated also infiltrate into tumor tissues, but no production of IFN-γ was confirmed. This also shows that not only the presence of NKT cells is necessary, but also artificial activation is necessary (see the following experimental example 9). Fig. 9 , left figure).

[0137] 3. The artificially activated NKT cells that reach the tumor tissue release a large amount of adjuvant substance IFN-γ, which strongly activates the innate immune system NK cells and the acquired immune system CD8 killer T cells, CD4 helper T cells, etc. that gather in the cancer tissue to attack tumor cells, induces proliferation, and can attack tumor cells at the same time (see the following test example 9 Fig. 9 (right picture in the figure).

[0138] 4. At the same time, it can induce immune memory cells of CD8 killer T cell groups that are responsible for long-term immune memory and form the main body of attacking tumor cells (see Test Examples 10 and 11 below). This allows sustainable cancer attack. In fact, in the presence of cancer antigens, the liposomes containing NKT cell ligands of the present invention are administered once, and then placed without treatment. A cancer cell transplantation experiment is performed one year later. The proliferation of transplanted cancer cells is extremely strongly inhibited, but the proliferation of cancer cells is not inhibited in liposomes that do not contain NKT cell ligands or in individuals lacking NKT cells (NKT-KO mice) (see Test Example 12 below). Therefore, it can be said that the inhibition of the proliferation of malignant tumors by the liposomes containing NKT cell ligands of the present invention is the result of the induction of long-term immune memory that depends on the artificial activation of NKT cells.

[0139] The present invention relates to "immune memory induction treatment technology for malignant tumors", which includes effectively artificially activating NKT cells in an organism suffering from a malignant tumor, subsequently activating an immune cell population and inducing immune memory.

[0140] More specifically, the present invention relates to a method for producing a "liposome containing a NKT cell ligand" that causes artificial activation of NKT cells in an organism suffering from a malignant tumor, a drug for treating malignant tumors that can induce and maintain long-term immune memory against malignant tumors, which has been difficult to induce and maintain, in an organism suffering from a malignant tumor by administering the liposome, and that can induce sustained anti-tumor activity through immune memory, thereby activating the immune system of an individual suffering from a malignant tumor, thereby controlling the progression, recurrence and metastasis of all malignant tumors regardless of their type, and its use, etc.

[0141] The present invention also relates to drugs (vaccines, etc.) and uses thereof that can induce and maintain long-term immune memory of pathogens in infectious diseases and serve as adjuvants for treating and preventing infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 The results of Test Example 1-1 (left figure) and Test Example 1-2 (right figure) are shown. In the figure, #1 represents a Captisol solution containing RK-163, #2 to #8 represent a liposome administration group containing RK-163, and PBS represents a PBS administration group (control).

[0143] Figure 2 The results of Test Example 2 are shown. In the figure, RK-LIPO represents the group administered with liposomes (#7) containing RK-163, and PBS represents the group administered with PBS (control). The RK-LIPO administration amount (converted to RK amount) in the upper half of the right figure was converted into a curve graph (lower half of the right figure), and the ED50 was calculated.

[0144] Figure 3The results of Test Example 3 are shown. In the figure, RK-LIPO indicates the group administered with liposomes containing RK-163 (#7), RCAI-61-LIPO indicates the group administered with liposomes containing RCAI-61 (#9), and RCAI-137-LIPO indicates the group administered with liposomes containing RCAI-137 (#10). PBS indicates the group administered with PBS (control).

[0145] Figure 4 The results of Test Example 4 are shown. In the figure, RK-LIPO indicates the group administered with liposomes containing RK-163 (#7), LIPO indicates the group administered with liposomes not containing RK-163 (control), and PBS indicates the group administered with PBS (control).

[0146] Figure 5-1 The results of Test Example 5-1 are shown. In the figure, RK-LIPO indicates the group administered with liposomes containing RK-163 (#7), and PBS indicates the group administered with PBS (control).

[0147] Figure 5-2 The results of Test Example 5-2 are shown. In the figure, RK-LIPO indicates the group administered with liposomes (#7) containing RK-163, and medium indicates the group administered with culture fluid (medium) (control).

[0148] Figure 6 The results of Test Example 6 are shown. In the figure, RCAI-61-LIPO indicates the group administered with liposomes containing RCAI-61 (#9), RCAI-137-LIPO indicates the group administered with liposomes containing RCAI-137 (#10), and PBS indicates the group administered with PBS (control).

[0149] Figure 7 The results of Test Example 7 are shown. In the figure, RK-LIPO indicates the group administered with liposomes containing RK-163 (#7), and PBS indicates the group administered with PBS (control).

[0150] Figure 8 The results of Test Example 8 are shown. In the figure, RCAI-61-LIPO on the left represents the group administered with liposomes containing RCAI-61 (#9), and PBS represents the group administered with PBS (control). In the figure, RCAI-137-LIPO on the right represents the group administered with liposomes containing RCAI-137 (#10), and PBS represents the group administered with PBS (control).

[0151] Fig. 9 The results of Test Example 9 are shown. In the figure, RK-LIPO indicates the group administered with liposomes containing RK-163 (#7), and PBS indicates the group administered with PBS (control).

[0152] Fig.10The results of Test Example 10 are shown. In the figure, RK-LIPO indicates the group administered with liposomes containing RK-163 (#7). OVA indicates pseudocancer antigen.

[0153] Fig.11 The results of Test Example 11 are shown. In the figure, RCAI-61-LIPO indicates the group administered with liposomes containing RCAI-61 (#9), and RCAI-137-LIPO indicates the group administered with liposomes containing RCAI-137 (#10).

[0154] Fig.12 The results of Test Example 12 are shown. In the figure, RK-LIPO / WT indicates a group in which liposomes (#7) containing RK-163 were administered to C57BL / 6 mice, RK-LIPO / NKT-KO indicates a group (control) in which liposomes (#7) containing RK-163 were administered to NKT-KO (NKT cell-deficient) mice, and LIPO / WT indicates a group (control) in which liposomes not containing RK-163 were administered to C57BL / 6 mice. DETAILED DESCRIPTION

[0155] The present invention is described in detail below.

[0156] [1. Glycolipid (NKT cell ligand in the present invention)]

[0157] The liposome composition of the present invention contains a glycolipid selected from the group consisting of a compound represented by the following formula (I), a compound represented by the following formula (II), and salts thereof.

[0158] The compound represented by formula (I), the compound represented by formula (II) and their salts are NKT cell ligands. NKT cell ligands refer to compounds that are specifically recognized by the NKT cell-specific antigen recognition receptor (NKT cell receptor) on NKT cells when presented on CD1d molecules, and can specifically activate NKT cells. In this specification, the compound represented by formula (I), the compound represented by formula (II) and their salts are also collectively referred to as "NKT cell ligands in the present invention".

[0159] First, the compound represented by formula (I) (hereinafter also referred to as compound (I)) and a salt thereof are described.

[0160]

[0161] [wherein, X represents an alkylene group or -NH-;

[0162] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom;

[0163] R3 represents a hydrocarbon group having 1 to 20 carbon atoms;

[0164] R4 represents a hydrocarbon group having 1 to 30 carbon atoms.]

[0165] X represents an alkylene group or -NH-. "Alkylene" is, for example, a linear or branched alkylene group having 1 to 8 carbon atoms, and specific examples thereof include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, propylene, ethylethylene, dimethylmethylene, and dimethyltrimethylene.

[0166] R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group which may have a substituent. R1 and R2 may form a 5- to 6-membered ring together with the adjacent nitrogen atom.

[0167] "Alkyl" is, for example, C 1-24 , more preferably C 1-16 , more preferably C 1-10 , particularly preferably C 1-6 The straight chain or branched chain alkyl group of R1 and R2 may be exemplified by methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, etc. As the alkyl group of R1 and R2, preferably C 1-6 Alkyl (eg, methyl, ethyl).

[0168] "Alkoxy" is, for example, C 1-24 , more preferably C 1-16 , more preferably C 1-10 , particularly preferably C 1-6 The linear or branched alkoxy group of R1 and R2 may be exemplified by methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, etc. As the alkoxy group of R1 and R2, preferably C 1-6 Alkoxy (eg, methoxy).

[0169] The "aryl group" in the "aryl group optionally having a substituent" is, for example, C 6-14 , more preferably C 6-12 The monocyclic to tricyclic aryl groups of R1 and R2 are preferably C 6-12Aryl (e.g., phenyl). Examples of substituents that the "aryl" may optionally have include halogen atoms (e.g., chlorine atoms, fluorine atoms, bromine atoms, iodine atoms); alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl); halogenated alkyl groups (e.g., trifluoromethyl); alkoxy groups (e.g., methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy); hydroxyl groups; amino groups; alkylamino groups (e.g., methylamino, dimethylamino, ethylamino, diethylamino); and cycloalkylamino groups. The position and number of substituents are not particularly limited, and at substitutable positions, 1 to the maximum number of substitutable substituents may be optionally present.

[0170] R1 and R2 may form a 5- to 6-membered ring together with the adjacent nitrogen atom, for example, a 5- to 6-membered nitrogen-containing saturated heterocyclic ring, specifically exemplified by pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, etc. Pyrrolidine, piperidine and morpholine are preferred.

[0171] R3 represents a hydrocarbon group having 1 to 20 carbon atoms. 1-20 Alkyl, C 2-20 Alkenyl, C 2-20 Alkynyl, C 3-20 Cycloalkyl, C 3-20 Cycloalkenyl, C 6-20 The concept of aryl group may be straight chain, branched chain or cyclic, or may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, and may optionally have an unsaturated bond in the molecule or at the end. 1-20 Alkyl, C 2-20 Alkenyl and C 2-20 Alkynyl, more preferably C 12-14 As R3, for example, -(CH2) 13 CH3, etc.

[0172] R4 represents a hydrocarbon group having 1 to 30 carbon atoms. The "hydrocarbon group having 1 to 30 carbon atoms" is a hydrocarbon group containing C 1-30 Alkyl, C 2-30 Alkenyl, C 2-30 Alkynyl, C 3-30 Cycloalkyl, C 3-30 Cycloalkenyl, C 6-30 The concept of aryl group may be straight chain, branched chain or cyclic, or may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, and may optionally have an unsaturated bond in the molecule or at the end. 1-30 Alkyl, C 2-30 Alkenyl and C 2-30 Alkynyl, more preferably C 10-30 Alkyl, more preferably C 15-25 As R4, for example, -(CH2) 15 CH3, -(CH2)23 CH3, etc.

[0173] The hydrocarbon group represented by R3 and R4 may optionally have a substituent. When the hydrocarbon group represented by R3 and R4 has a substituent, examples of the substituent include a halogen atom (preferably a chlorine atom or a fluorine atom); an alkoxy group such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, and a tert-butoxy group (preferably having 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, further preferably 1 to 10 carbon atoms, and particularly preferably 1 to 4 carbon atoms); an aryloxy group such as a phenoxy group (preferably having 6 to 14 carbon atoms); a hydroxyl group; an amino group; a methylamino group, a dimethylamino group, an ethylamino group, Electron-donating groups such as alkylamino such as diethylamino; cycloalkylamino; alkylcarbonylamino such as acetylamino; cycloalkylcarbonylamino; arylcarbonylamino such as benzylamino (preferably, arylcarbonylamino, wherein the aryl portion is an aryl group having 6 to 14 carbon atoms), further, carboxyl; alkoxycarbonyl; acyl (as acyl, as described below. Preferably, the alkyl portion is an alkylcarbonyl group of a linear or branched alkyl group having 1 to 24 carbon atoms); carbamoyl; and electron-withdrawing groups such as trifluoromethyl. The position and number of substituents are not particularly limited, and at substitutable positions, there are optionally 1 to the maximum number of substitutable substituents. When there are more than one substituent, they may be the same or different.

[0174] In the present specification, "acyl" refers to, for example, formyl; alkyl-carbonyl (for example, an alkyl-carbonyl whose alkyl portion is a linear or branched alkyl group having 1 to 24 carbon atoms (preferably 1 to 12 carbon atoms) (for example, acetyl, propionyl, butyryl, isobutyryl, valeryl, pivaloyl, hexanoyl)); cycloalkyl-carbonyl (for example, a cycloalkyl-carbonyl whose cycloalkyl portion is a cycloalkyl group having 3 to 10 carbon atoms); alkenyl-carbonyl (for example, an alkenyl-carbonyl whose alkenyl portion is a linear or branched alkenyl group having 2 to 12 carbon atoms (for example, acryloyl, methacryloyl)); aryl-carbonyl (for example, an aryl-carbonyl whose aryl portion is an aryl group having 6 to 14 carbon atoms (for example, benzoyl, naphthoyl)); etc. The aryl in the aryl-carbonyl group represents, for example, a monocyclic to tricyclic aromatic hydrocarbon group, and specific examples thereof include phenyl, naphthyl, anthracenyl, and phenanthryl. Among them, as the acyl group, formyl, acetyl, propionyl, butyryl, isobutyryl, benzoyl, naphthoyl and the like are preferred, and acetyl and benzoyl are more preferred.

[0175] Examples of the alkyl moiety of the above-mentioned alkylamino group and alkylcarbonylamino group include linear or branched alkyl groups (preferably having 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, further preferably 1 to 10 carbon atoms, and particularly preferably 1 to 4 carbon atoms) such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl. Examples of the cycloalkyl moiety of the above-mentioned cycloalkylamino group and cycloalkylcarbonylamino group include cycloalkyl groups (preferably having 3 to 24 carbon atoms, more preferably 3 to 16 carbon atoms, further preferably 3 to 10 carbon atoms, and particularly preferably 3 to 6 carbon atoms) such as cyclopentyl or cyclohexyl.

[0176] As the alkoxy moiety of the above-mentioned alkoxycarbonyl group, those similar to the above-mentioned alkoxy group can be exemplified.

[0177] The above substituents may be further substituted at substitutable positions with at least one of halogen, alkyl, cycloalkyl, alkenyl, alkynyl, phenyl, alkoxy, hydroxy, amino, alkylamino and cycloalkylamino.

[0178] As the halogen, alkoxy group, alkylamino group and cycloalkylamino group, those similar to the above-mentioned alkoxy group can be exemplified.

[0179] Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl and the like (preferably having 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, further preferably 1 to 10 carbon atoms, and particularly preferably 1 to 4 carbon atoms).

[0180] Examples of the cycloalkyl group include cycloalkyl groups such as cyclopentyl and cyclohexyl (preferably having 3 to 24 carbon atoms, more preferably 3 to 16 carbon atoms, further preferably 3 to 10 carbon atoms, and particularly preferably 3 to 6 carbon atoms).

[0181] Examples of the alkenyl group include alkenyl groups (preferably having 2 to 24 carbon atoms, more preferably 2 to 16 carbon atoms, further preferably 2 to 10 carbon atoms, and particularly preferably 2 to 4 carbon atoms) such as vinyl, propenyl and butenyl.

[0182] Examples of the alkynyl group include ethynyl, propargyl, butynyl, pentynyl and the like (preferably having 2 to 24 carbon atoms, more preferably 2 to 16 carbon atoms, further preferably 2 to 10 carbon atoms, particularly preferably 2 to 4 carbon atoms).

[0183] In the present invention, among stereoisomers derived from the ring structure of sugar (galactopyranose), the α-configuration is adopted.

[0184] When compound (I) has a stereoisomer derived from a structure other than the cyclic structure of the sugar (for example, an asymmetric carbon in a portion other than the cyclic structure of the sugar, etc.), any isomer is included in the present invention, and may be a mixture of two or more isomers in any proportion (including a racemic form).

[0185] In particular, in compound (I), there are optical isomers of asymmetric carbons derived from parts other than the cyclic structure of the sugar. In the present invention, it may be a single optically active body or a mixture of two or more optically active bodies in any proportion (including racemates). The asymmetric carbon to which -NHC(=O)X-R4 is bound is preferably in S configuration. The asymmetric carbon to which OH is bound adjacent to the asymmetric carbon to which -NHC(=O)X-R4 is bound is preferably in R configuration. The asymmetric carbon to which R3 is bound is preferably in R configuration.

[0186] The salt of compound (I) is preferably a pharmaceutically acceptable salt, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate; organic acid salts such as succinate, fumarate, acetate, methanesulfonate, toluenesulfonate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salts such as ammonium salt and alkylammonium salt, etc.

[0187] Suitable examples of compound (I) or a salt thereof include the following.

[0188] (a) In formula (I),

[0189] X is -CH2-;

[0190] R1 and R2 are the same or different and are hydrogen atoms, C 1-6 Alkyl, hydroxyl or C 1-6 Alkoxy;

[0191] R3 is C 1-20 alkyl;

[0192] R4 is C 1-30 An alkyl compound or a salt thereof.

[0193] (b) In formula (I),

[0194] X is -CH2-;

[0195] R1 and R2 are the same or different and are hydrogen atoms, C 1-6 Alkyl, hydroxyl or C 1-6 Alkoxy;

[0196] R3 is C 12-14 alkyl;

[0197] R4 is C 15-25 An alkyl compound or a salt thereof.

[0198] (c) In formula (I),

[0199] X is -CH2-;

[0200] R1 and R2 are the same or different and are hydrogen atoms, C 1-6 Alkyl or hydroxyl;

[0201] R3 is C 12-14 alkyl;

[0202] R4 is C 15-25 An alkyl compound or a salt thereof.

[0203] (d) In formula (I),

[0204] X is -CH2-;

[0205] R1 and R2 are the same or different and are hydrogen atoms, methyl or C 1-6 Hydroxyl;

[0206] R3 is -(CH2) 13 CH3;

[0207] R4 is -(CH2) 23 CH3 compound or a salt thereof.

[0208] Specific examples of suitable compound (I) in the present invention are shown in Table 1, but are not limited thereto.

[0209] [Table 1]

[0210]

[0211] In the table, -C 14 H 29 -(CH2) 13 CH3,-C 24 H 49 -(CH2) 23 CH3, and -C 16 H 33 -(CH2) 15 CH3.

[0212] Among them, the following compounds are preferred.

[0213] RCAI-124 (also referred to as RK-163 in this manual)

[0214]

[0215] RCAI-137

[0216]

[0217] The compound represented by formula (I) or a salt thereof can be produced by the method disclosed in WO2013 / 162016A1, US2015 / 152128A1, and the like.

[0218] Next, the compound represented by formula (II) (also referred to as compound (II) in this specification) and a salt thereof are described.

[0219] Formula (II):

[0220]

[0221] [In the formula, R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)-, or -CH=CH-.]

[0222] R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom.

[0223] The alkyl group having 1 to 7 carbon atoms represented by R5 is a substituted or unsubstituted alkyl group which may form a ring. Examples thereof include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, cyclopropylmethyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, and cyclohexylmethyl, and methyl and ethyl are preferred.

[0224] The alkoxy group having 1 to 6 carbon atoms represented by R5 means a group formed by a substituted or unsubstituted alkyl group bonded to an oxygen atom, and the alkyl portion thereof may form a ring. Examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropyloxy, cyclopropylmethoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and cyclohexyloxy, with methoxy, ethoxy, and n-propoxy being preferred.

[0225] Examples of the halogen atom represented by R5 include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, and a fluorine atom and a chlorine atom are preferred.

[0226] R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms. In the present specification, the term "hydrocarbon group" also includes substituted or unsubstituted alkyl groups having 1 to 28 carbon atoms, alkenyl groups having 2 to 28 carbon atoms, alkynyl groups having 2 to 28 carbon atoms, cycloalkyl groups having 3 to 28 carbon atoms, cycloalkenyl groups having 3 to 28 carbon atoms, aryl groups having 6 to 14 carbon atoms, arylalkyl groups having 7 to 28 carbon atoms (e.g., C 6-14Aryl-C 1-14 The concept of alkyl) may be any form of straight chain, branched chain and cyclic, and may be a saturated hydrocarbon group or an unsaturated hydrocarbon group, and may have an unsaturated bond in the molecule or at the end. Among them, as R6 and R7, preferably a substituted or unsubstituted alkyl group having 1 to 28 carbon atoms, or a substituted or unsubstituted arylalkyl group having 7 to 28 carbon atoms.

[0227] However, when R5 is a hydrogen atom, R6 is preferably a substituted or unsubstituted hydrocarbon group having 24 to 28 carbon atoms, and more preferably a substituted or unsubstituted alkyl group having 24 to 28 carbon atoms.

[0228] Examples of the substituent of the hydrocarbon group represented by R6 and R7 include a halogen atom (preferably a chlorine atom or a fluorine atom); an alkoxy group such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, and a tert-butoxy group (preferably having 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, further preferably 1 to 10 carbon atoms, and particularly preferably 1 to 4 carbon atoms); an aryloxy group such as a phenoxy group (preferably having 6 to 14 carbon atoms); a hydroxyl group; an amino group; a methylamino group, a dimethylamino group, an ethylamino group, Electron-donating groups include alkylamino groups such as diethylamino; cycloalkylamino groups; alkylcarbonylamino groups such as acetamide; cycloalkylcarbonylamino groups; arylcarbonylamino groups such as benzylamino (preferably arylcarbonylamino groups having 6 to 14 carbon atoms in the aryl part); and further electron-withdrawing groups such as carboxyl groups; alkoxycarbonyl groups; acyl groups (as acyl groups, as described below. Preferably, the alkyl part is an alkyl-carbonyl group having a linear or branched alkyl group having 1 to 24 carbon atoms); carbamoyl groups; and electron-withdrawing groups such as trifluoromethyl groups.

[0229] In the present specification, the term "acyl" refers to, for example, formyl; alkyl-carbonyl (for example, an alkyl-carbonyl group whose alkyl portion is a linear or branched alkyl group having 1 to 24 carbon atoms (preferably 1 to 12 carbon atoms) (for example, acetyl, propionyl, butyryl, isobutyl, valeryl, pivaloyl, hexanoyl); cycloalkyl-carbonyl (for example, a cycloalkyl-carbonyl group whose cycloalkyl portion is a cycloalkyl group having 3 to 10 carbon atoms); alkenyl-carbonyl (for example, an alkenyl group whose alkenyl portion is a linear or branched alkyl group having 2 to 12 carbon atoms); The aryl group in the aryl-carbonyl group represents, for example, a monocyclic to tricyclic aromatic hydrocarbon group, and specific examples thereof include phenyl, naphthyl, anthracenyl, and phenanthryl. Among them, the acyl group is preferably a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a benzoyl group, a naphthoyl group, and the like, and more preferably an acetyl group and a benzoyl group.

[0230] Examples of the alkyl portion of the alkylamino group and the alkylcarbonylamino group include linear or branched alkyl groups (preferably having 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, further preferably 1 to 10 carbon atoms, and particularly preferably 1 to 4 carbon atoms) such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl and octadecyl.

[0231] Examples of the cycloalkyl moiety of the cycloalkylamino and cycloalkylcarbonylamino groups include cycloalkyl groups (preferably having 3 to 24 carbon atoms, more preferably 3 to 16 carbon atoms, further preferably 3 to 10 carbon atoms, particularly preferably 3 to 6 carbon atoms) such as cyclopentyl and cyclohexyl groups.

[0232] As the alkoxy moiety of the above-mentioned alkoxycarbonyl group, those similar to the above-mentioned alkoxy group can be exemplified.

[0233] The above substituents may be further substituted at the substitutable positions with at least one of halogen, alkyl, cycloalkyl, alkenyl, alkynyl, phenyl, alkoxy, hydroxy, amino, alkylamino and cycloalkylamino.

[0234] As the halogen group, alkoxy group, alkylamino group and cycloalkylamino group, those similar to those mentioned above can be exemplified.

[0235] Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl and the like (preferably having 1 to 24 carbon atoms, more preferably 1 to 16 carbon atoms, furthermore having 1 to 10 carbon atoms, particularly preferably 1 to 4 carbon atoms).

[0236] Examples of the cycloalkyl group include cycloalkyl groups such as cyclopentyl and cyclohexyl (preferably having 3 to 24 carbon atoms, more preferably 3 to 16 carbon atoms, further preferably 3 to 10 carbon atoms, and particularly preferably 3 to 6 carbon atoms).

[0237] Examples of the alkenyl group include alkenyl groups (preferably having 2 to 24 carbon atoms, more preferably 2 to 16 carbon atoms, further preferably 2 to 10 carbon atoms, and particularly preferably 2 to 4 carbon atoms) such as vinyl, propenyl and butenyl.

[0238] Examples of the alkynyl group include ethynyl, propynyl, butynyl, pentynyl and the like (preferably having 2 to 24 carbon atoms, more preferably 2 to 16 carbon atoms, further preferably 2 to 10 carbon atoms, particularly preferably 2 to 4 carbon atoms).

[0239] Among them, as R6, a substituted or unsubstituted alkyl group is preferred, and a linear alkyl group is preferred. As R6, a substituted or unsubstituted arylalkyl group is also preferred. When R5 is an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, the number of carbon atoms of R6 is preferably 18 to 26, and more preferably 24 to 26. When R5 is a hydrogen atom, the number of carbon atoms of R6 is preferably 24 to 26. As R6, for example, -(CH2) 23 -CH3, -(CH2) 24 -CH3, -(CH2) 25 -CH3, 10-(4-fluorophenyl)decyl, etc.

[0240] In addition, R7 is preferably a substituted or unsubstituted alkyl group, and preferably a linear alkyl group. The number of carbon atoms of R7 is preferably 9 to 20, and more preferably 12 to 18. Specific examples of R7 include -(CH2) 11 -CH3, -(CH2) 12 -CH3, -(CH2) 13 -CH3, -(CH2) 14 -CH3, -(CH2) 15 -CH3, -(CH2) 16 -CH3, -(CH2) 17 -CH3, etc.

[0241] Y represents -CH2-, -CH(OH)- or -CH=CH-, among which -CH(OH)- is preferred.

[0242] When compound (II) has stereoisomers, any isomer is included in the present invention, and it may be a mixture of two or more isomers in any ratio (including a racemate).

[0243] In particular, in compound (II), there are optical isomers of asymmetric carbon derived from the lipid part, but in the present invention, it can be a single optically active body or a mixture of two or more optically active bodies in any proportion (including racemates). The asymmetric carbon bonded to -NHCOR6 is preferably in S configuration. The asymmetric carbon adjacent to the asymmetric carbon bonded to -NHCOR6 and having -OH is preferably in trans configuration relative to the asymmetric carbon bonded to -NHCOR6. When Y is -CH(OH)-, the asymmetric carbon in -CH(OH)- represented by Y is preferably in R configuration.

[0244] Examples of the lipid portion of compound (II) include

[0245]

[0246] (In the formula, each symbol has the same meaning as above.) etc.

[0247] The salt of compound (II) is preferably a pharmaceutically acceptable salt, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate; organic acid salts such as succinate, fumarate, acetate, methanesulfonate, toluenesulfonate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salts such as ammonium salt and alkylammonium salt.

[0248] Specific examples of preferred compound (II) in the present invention are shown in Table 2, but are not limited thereto.

[0249] [Table 2]

[0250]

[0251] Among them, the following compounds are preferred.

[0252] The compound represented by Compound No. 2-9' in Table 2, the compound (RCAI-61) having the following stereostructure:

[0253]

[0254] The compound represented by Compound No. 3-4' in Table 2, the compound (RCAI-64) having a stereostructure represented by the following formula:

[0255]

[0256] The compound represented by Compound No. 3-13' in Table 2, the compound (RCAI-85) having a stereostructure represented by the following formula:

[0257]

[0258] The compound represented by Compound No. 165 in Table 2, the compound (RCAI-165) having a stereostructure represented by the following formula:

[0259]

[0260] The compound represented by formula (II) or a salt thereof can be produced by the method disclosed in WO2009 / 119692A1, US Pat. No. 8551959, and the like.

[0261] 2. Liposomes

[0262] In the liposome composition of the present invention, the liposome contains the above-mentioned glycolipid.

[0263] In this specification, a substance having a closed vesicle structure with lipid as the main component (a water-soluble particle obtained by the aggregation of amphiphilic molecules containing a hydrophilic region and a hydrophobic region) is referred to as a liposome. Recently, it is sometimes referred to as a lipid nanoparticle (LNP), but in this specification, the concept of liposome also includes those referred to as lipid nanoparticles as long as they have the above structure.

[0264] In the present invention, as a liposome component, any amphiphilic molecule that can form a lipid-enclosed vesicle structure by a known method can be used, and preferably lipids can be cited. As lipids in the present invention, there are phospholipids such as phosphatidylcholine (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine), phosphatidylglycerol (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and distearoylphosphatidylglycerol), phosphatidylethanolamine (e.g., dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine), phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, glycosphingolipids, glyceroglycolipids, and cationic lipids. (For example, DOTAP, DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-triacontriacontria-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate), ALC-0315 ([(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl))bis(2-hexyldecanoate)), SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino)octanoate)), etc. These can be used alone or in combination with non-polar substances such as cholesterol or lipid derivatives in which water-soluble polymers such as polyethylene glycol are bound to lipids to prepare liposomes.

[0265] In the present invention, the liposome is preferably a liposome having affinity for antigen-presenting cells.

[0266] In the present invention, preferred examples of liposomes (an example of liposomes having affinity for antigen-presenting cells) include the following liposomes (#2) to (#8).

[0267] (#2) Liposomes containing DOTAP, DOPE and cholesterol

[0268] In the liposome (#2), the weight ratio of DOTAP, DOPE, and cholesterol (DOTAP:DOPE:cholesterol) is, for example, 1:0.1-10:0.1-10, and preferably 1:0.3-3:0.2-2.

[0269] When liposome (#2) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of DOTAP, DOPE and cholesterol" is, for example, 1:1 to 10,000, preferably 1:1 to 1,000.

[0270] As one embodiment of the liposome composition of the present invention using the liposome (#2), for example, the following RK-163-containing liposome (#2) can be mentioned.

[0271] (#3) Liposomes containing DOTAP, DOPE, MPEG2000-DMPE, Na salt and cholesterol

[0272] In the liposome (#3), the weight ratio of DOTAP, DOPE, MPEG2000-DMPE, Na salt, and cholesterol (DOTAP:DOPE:MPEG2000-DMPE, Na salt:cholesterol) is, for example, 1:0.1-10:0.1-10:0.1-10, preferably 1:0.3-3:0.2-2:0.2-2.

[0273] When liposome (#3) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of DOTAP, DOPE, MPEG2000-DMPE, Na salt and cholesterol" is, for example, 1:1 to 10,000, preferably 1:1 to 1,000.

[0274] As one embodiment of the liposome composition of the present invention using the liposome (#3), for example, the following liposome (#3) containing RK-163 can be mentioned.

[0275] (#4) Liposomes containing DOTAP, DOPE, MPEG2000-DSPE, Na salt and cholesterol

[0276] In the liposome (#4), the weight ratio of DOTAP, DOPE, MPEG2000-DSPE, Na salt, and cholesterol (DOTAP:DOPE:MPEG2000-DSP, Na salt:cholesterol) is, for example, 1:0.1-10:0.1-10:0.1-10, preferably 1:0.3-3:0.2-2:0.2-2.

[0277] When liposome (#4) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of DOTAP, DOPE, MPEG2000-DSPE, Na salt and cholesterol" is, for example, 1:1 to 10,000, preferably 1:1 to 1,000.

[0278] As one embodiment of the liposome composition of the present invention using the liposome (#4), for example, the following RK-163-containing liposome (#4) can be mentioned.

[0279] (#5) Liposomes containing HSPC, DSPG and cholesterol

[0280] In the liposome (#5), the weight ratio of HSPC, DSPG, and cholesterol (HSPC:DSPG:cholesterol) is, for example, 1:0.1-10:0.1-10, and preferably 1:0.2-2:0.2-2.

[0281] When liposome (#5) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of HSPC, DSPG and cholesterol" is, for example, 1:1 to 10,000, preferably 1:1 to 1,000.

[0282] As one embodiment of the liposome composition of the present invention using the liposome (#5), for example, the following RK-163-containing liposome (#5) can be mentioned.

[0283] (#6) Liposomes containing HSPC, DOPS and cholesterol

[0284] Liposome (#6) and the following liposome (#7) have the same composition, but when used to produce liposomes containing NKT cell ligands, the former is an example of a method in which more NKT cell ligands are mixed, and the latter is an example of a method in which less NKT cell ligands are mixed.

[0285] In the liposome (#6), the weight ratio of HSPC, DOPS, and cholesterol (HSPC:DOPS:cholesterol) is, for example, 1:0.1-10:0.1-10, and preferably 1:0.2-2:0.2-2.

[0286] When liposome (#6) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of HSPC, DOPS and cholesterol" is, for example, 1:1 to 100,000, preferably 1:1 to 10,000.

[0287] As one embodiment of the liposome composition of the present invention using the liposome (#6), for example, the following RK-163-containing liposome (#6) can be mentioned.

[0288] (#7) Liposomes containing HSPC, DOPS and cholesterol

[0289] In the liposome (#7), the weight ratio of HSPC, DOPS, and cholesterol (HSPC:DOPS:cholesterol) is, for example, 1:0.1-10:0.1-10, and preferably 1:0.2-2:0.2-2.

[0290] When liposome (#7) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of HSPC, DOPS and cholesterol" is, for example, 1:1 to 10,000, preferably 1:1 to 1,000.

[0291] As one embodiment of the liposome composition of the present invention using the liposome (#7), for example, there can be mentioned the following liposomes (#7) containing RK-163, liposomes (#9) containing RCAI-61, and liposomes (#10) containing RCAI-137.

[0292] (#8) Liposomes containing HSPC, MPEG2000-DSPE, Na salt and cholesterol In the liposome (#8), the weight ratio of HSPC, MPEG2000-DSPE, Na salt and cholesterol (HSPC:MPEG2000-DSPE, Na salt:cholesterol) is, for example, 1:0.1~10:0.1~10, preferably 1:0.2~2:0.2~2.

[0293] When liposome (#8) is used in the liposome composition of the present invention, the weight ratio of "NKT cell ligand of the present invention" to "the sum of HSPC, MPEG2000-DSPE, Na salt and cholesterol" is, for example, 1:1 to 10,000, preferably 1:1 to 1,000.

[0294] As one embodiment of the liposome composition of the present invention using the liposome (#8), for example, the following liposome (#8) containing RK-163 can be mentioned.

[0295] In the present invention, among the above liposomes, (#5) to (#8) are preferred, (#7) and (#8) are more preferred, and (#7) is further preferred.

[0296] 3. Liposome composition containing glycolipid (NKT cell ligand in the present invention) (liposome composition of the present invention)

[0297] The liposomes constituting the liposome composition of the present invention can be prepared according to known methods. For example, Liposome Technology, Vol. 1, 2nd Edition (Gregory Gregoriadis, CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, Pages 67-80, Chapter 10, Pages 167-184 and Chapter 17, Pages 261-276 (1993) can be used. More specifically, ultrasonic treatment, ethanol injection, French press, ether injection, bile acid method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, etc. can be listed, but they are not limited to these.

[0298] Specifically, as shown in the examples, the liposome components and the NKT cell ligand are dissolved in an organic solvent in a glass test tube, respectively, and the organic solvent is evaporated by spraying nitrogen thereto, and it is completely dried in a desiccator (decompression). An isotonic aqueous solvent (e.g., 9% sucrose aqueous solution, 5% glucose aqueous solution, phosphate buffered saline, etc.) is added to the dried material, and by emulsifying it with a homogenizer, etc., a liposome containing a NKT cell ligand (liposome composition of the present invention) can be obtained. In addition, by using and passing through a polycarbonate membrane filter (pore size: 0.4 μm or 0.2 μm) under heating and pressure, liposomes with uniform particle size can be obtained. Finally, in order to ensure sterility, it can also be passed through a common filter for filtration sterilization (pore size: 0.22 μm; such as a PVDF membrane filter).

[0299] In the liposome composition of the present invention, the ratio of "glycolipids selected from the group consisting of compounds represented by formula (I), compounds represented by formula (II) and their salts (NKT cell ligands in the present invention)" and "the sum of other lipids (other than the main drug NKT cell ligands) constituting the liposome" is, for example, 1:1 to 100,000, preferably 1:1 to 10,000, more preferably 1:1 to 1,000, and further preferably 1:1 to 100.

[0300] The average particle size of the liposome composition of the present invention is not particularly limited, and is, for example, 10 to 1000 nm, preferably 10 to 500 nm, and more preferably 50 to 500 nm. The average particle size can be measured by a conventional quasi-elastic light scattering method.

[0301] The liposome composition of the present invention can be used alone or formulated and used together with common pharmaceutically acceptable additives (eg, solvents, stabilizers, isotonic agents, soothing agents, buffers, pH regulators) by methods known in the art.

[0302] The liposome composition of the present invention is preferably formulated as an intravenous injection. When the liposome composition of the present invention is a liquid preparation such as an injection, it can be stored by freezing or freeze-drying to remove water. The freeze-dried preparation is added with distilled water for injection and then dissolved before use.

[0303] The liposome composition of the present invention can be used as an active ingredient of a drug.

[0304] The liposome composition of the present invention is effective as an anti-tumor agent.

[0305] Examples of the “malignant tumor” include liver cancer, esophageal cancer, gastric cancer, biliary tract cancer, pancreatic cancer, colorectal cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, prostate cancer, bladder cancer, kidney cancer, renal pelvic and ureteral cancer, penile cancer, retroperitoneal tumor, adrenal cancer, head and neck cancer, thyroid cancer, ovarian cancer, uterine cancer, skin cancer, malignant lymphoma, brain tumor, malignant melanoma, retinoblastoma, choroidal melanoma, intraocular lymphoma, eyelid tumor, lacrimal gland cancer, ocular adnexal lymphoma, orbital sarcoma, optic nerve tumor, pilocytic astrocytoma, diffuse astrocytoma, oligodendroglioma, glioblastoma, ependymoma, ganglioglioma, central nervous system cancer, medulloblastoma, embryonal tumor, central nervous system malignant lymphoma, meningioma, schwannoma, pituitary adenoma, craniopharyngioma, chordoma , hemangioblastoma, epidermoid tumor, neuroblastoma, oral cancer, tongue cancer, adenoid cystic carcinoma, ear cancer, head and neck sarcoma, male breast cancer, special breast cancer, malignant pericardial mesothelioma, malignant pleural mesothelioma, thymoma, mediastinal tumor, pulmonary neuroendocrine tumor, malignant peritoneal mesothelioma, anal cancer, gastrointestinal neuroendocrine tumor, small intestine cancer, gastrointestinal membranous tumor, pancreatic / gastrointestinal neuroendocrine tumor, pheochromocytoma, adrenal cortical carcinoma, paraganglioma, malignant testicular sheath mesothelioma, testicular sheath tumor, vulvar cancer, uterine sarcoma, vaginal cancer, urachal carcinoma, peritoneal cancer, retroperitoneal sarcoma, malignant melanoma, basal cell carcinoma, extramammary Paget's disease, cutaneous angiosarcoma, skin tumors, sweat gland carcinoma, sebaceous gland carcinoma, Merkel cell carcinoma, squamous cell carcinoma, uterine sarcoma, osteosarcoma, rhabdomyoma, Ewing's sarcoma, endocardial sarcoma, angiosarcoma, trunk sarcoma, desmoid tumor, head and neck sarcoma, soft tissue sarcoma, bone cyst, zygomatic osteoma, enchondroma, osteochondroma, giant cell tumor of bone, osteosarcoma, chondrosarcoma, chordoma, myxofibrosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor, epithelioid sarcoma, clear cell sarcoma, synovial sarcoma, malignant fibrous histiocytoma, alveolar soft tissue sarcoma, protuberance Cutaneous fibrosarcoma, adult T-cell leukemia lymphoma, extranodal NK / T-cell lymphoma, nasal type, Burkitt's lymphoma, cutaneous lymphoma, Hodgkin's lymphoma, peripheral T-cell lymphoma, chronic lymphocytic leukemia, neuroblastoma, renal tumor, germ cell tumor, fibrosarcoma, medullary carcinoma, clear cell sarcoma, ileal cancer, external auditory canal cancer, thymic cancer, hair follicle cancer, oligoastrocytoma, uveal malignant melanoma, myxofibrosarcoma, soft tissue sarcoma, endochondroma, endometrial stromal sarcoma, internal auditory canal cancer, aneurysmal bone cyst, middle auditory canal cancer, glioma, neurofibroma, lipoma, duodenal tumor, etc.

[0306] The liposome composition of the present invention is effective as an anti-infective agent.

[0307] Examples of “infectious diseases” include viral diseases (e.g., viral hepatitis caused by hepatitis B virus, hepatitis C virus, hepatitis D virus, herpes, pandemic influenza, new coronavirus, Ebola hemorrhagic fever, yellow fever, rabies, Zika virus, SARS, MARS, dengue fever, Japanese encephalitis, poliomyelitis, Lassa fever, chickenpox, pneumococcus, measles), bacterial infections (e.g., drug-resistant tuberculosis, atypical mycobacterial infection, cholera, diphtheria, typhoid), and fungal diseases (e.g., candidiasis, toxoplasmosis, trypanosomiasis).

[0308] The liposome composition of the present invention is effective as an NKT cell activator, an inducer of IFN-γ production from NKT cells, an activator of innate immune system lymphocytes and acquired immune system lymphocytes, and an inducer of long-term immune memory.

[0309] The liposome composition of the present invention can be safely administered to humans and non-human mammals (eg, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, pigs, etc.).

[0310] The administration object of the liposome composition of the present invention is preferably a human or non-human mammal suffering from a malignant tumor, or a human or non-human mammal suffering from an infectious disease or having an initial infection risk. The liposome preparation of the present invention can also be used to treat or prevent the metastasis or recurrence of an infectious disease or a malignant tumor.

[0311] Since NKT cells can activate various immune cells in the body (e.g., cytotoxic T cells, NK cells, etc.) that have the function of directly acting on malignant tumor cells and pathogen-infected cells, the liposome composition of the present invention can be used to treat malignant tumors and infections, regardless of the type of malignant tumor or pathogen.

[0312] As the administration method of the liposome composition of the present invention, as long as it is a method that enables the liposome composition of the present invention to reach the antigen presenting cells present in the affected part or its periphery, there is no particular limitation. For example, intravenous administration can be mainly listed, and intra-arterial administration, intra-mucosal administration, intra-lymph node administration, intra-afflicted tissue administration, etc. can be further listed. Usually, NKT cells are artificially activated by intravenous administration of the liposome composition and its delivery to the lungs and liver where many NKT cells are present. In some cases, the liposome composition can be directly delivered to the patient's diseased part by an injection needle, or the liposome composition can be applied to the artery by a catheter.

[0313] The dosage of the liposome composition of the present invention can be appropriately determined according to the administration route, severity of symptoms, age of the patient, degree of side effects, and the like.

[0314] The dosage of the liposome composition of the present invention is the amount of "glycolipid selected from the group consisting of compounds represented by formula (I), compounds represented by formula (II) and their salts", for example, the first administration is about 12 to 400 ng per 1 kg body weight, preferably about 40 to 400 ng, and the same amount is administered again 4 weeks after the first administration, which constitutes a course of treatment. Thereafter, the number of CD8 immune memory T cells will be tracked by observing the process, and "administered twice every 4 weeks" is repeated, with the goal of maintaining the number of memory cells above the pre-treatment level.

[0315] The liposome composition of the present invention can be effectively delivered to antigen presenting cells (e.g., dendritic cells) in the body by intravenous administration. Therefore, the present invention also relates to "a method for delivering glycolipids to antigen presenting cells, the method comprising administering a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof to a subject".

[0316] Hereinafter, the present invention will be described in more detail based on Examples and Test Examples, but the present invention is not limited thereto.

[0317] Example

[0318] RK-163, RCAI-61 and RCAI-137 used in the examples are compounds represented by the above chemical structural formulas.

[0319] The abbreviations used in this specification have the following meanings.

[0320] HSPC: Hydrogenated Soy Phosphatidylcholine

[0321] DOTAP: 1,2-dioleoyloxy-3-trimethylammonium propane chloride

[0322] DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

[0323] MPEG2000-DMPE, sodium salt: N-(Carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, sodium salt

[0324] MPEG2000-DSPE, sodium salt: N-(Carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt

[0325] DSPG: 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol)

[0326] DOPS: 1,2-dioleoyl-sn-glycero-3-phospho-L-serine

[0327] PEG: polyethylene glycol

[0328] PS: Phosphatidylserine

[0329] OVA: ovalbumin

[0330] PBS: Phosphate buffered saline

[0331] [Preparation of Sample #1 (Captisol Aqueous Solution Containing RK-163 (#1))]

[0332] Using the reagents and ingredients shown in Table 3, sample #1 for safety testing and primary pharmacology testing was prepared according to the following preparation method.

[0333] (Composition of the prepared sample)

[0334] For safety testing

[0335] 15ng / mL RK-163, 177ng / mL Captisol in 9% sucrose aqueous solution

[0336] For major pharmacology tests

[0337] 1.5 ng / mL RK-163, 17.7 ng / mL Captisol in 9% sucrose aqueous solution

[0338] [[Table 3]

[0339]

[0340]

[0341] ※Wako Pure Chemical Industries, Ltd.: Currently Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter the same

[0342] (Preparation method)

[0343] For safety testing

[0344] RK-163 was dissolved in a solution of methanol:chloroform = 1:2 (volume ratio) at 100 ng / mL. 1.5 mL of the solution was placed in a glass bottle, dried by blowing nitrogen gas while heating, and then left to stand in a desiccator. Separately, Captisol was dissolved in a sucrose aqueous solution at 177 ng / mL, and 10 mL was added to the glass bottle containing the dried RK-163 to dissolve RK-163. The solution was filtered through a filter with a pore size of 0.22 μm for sterilization, and stored in a glass bottle with a screw cap at 4°C. This solution was used as sample #1 (for safety testing).

[0345] For major pharmacology tests

[0346] Take 1 mL of sample #1 (for safety test) into another glass bottle, add 9 mL of 9% sucrose aqueous solution to mix and dilute. Then pass the solution through a 0.22 μm pore size PVDF membrane filter for filtration and sterilization, and store it in a glass bottle with a screw cap at 4°C. This solution is used as sample #1 (for main pharmacology test).

[0347] [Preparation of Sample #2 (Liposomes containing RK-163 (#2))]

[0348] Using the reagents and ingredients shown in Table 4, #2 samples for safety testing and primary pharmacology testing were prepared according to the following preparation methods.

[0349] (Composition of the prepared sample)

[0350] For safety testing

[0351] 15 ng / mL RK-163, 203 ng / mL total lipids in 9% sucrose aqueous solution (all lipids except RK-163; the same applies below)

[0352] Lipid composition: DOTAP 91.7ng / mL, DOPE 73.2ng / mL, cholesterol 38.1ng / mL

[0353] For major pharmacology tests

[0354] 1.5 ng / mL RK-163, 20.3 ng / mL total lipids in 9% sucrose aqueous solution

[0355] Lipid composition: DOTAP 9.17ng / mL, DOPE 7.32ng / mL, cholesterol 3.81ng / mL

[0356] Particle size: 141.4nm PDI: 0.1218

[0357] [Table 4]

[0358]

[0359] (Preparation method)

[0360] For safety testing

[0361] 91.7 mg, 73.2 mg and 38.1 mg of DOTAP, DOPE and cholesterol were placed in a flask and dissolved in 100 mL of a solution of methanol:chloroform = 1:2 (volume ratio). 1.0 mL of this solution was mixed with 1.5 mL of a 100 μg / mL solution of RK-163 in methanol:chloroform = 1:2, dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1 piece). The prepared liposome dispersion was further diluted 1000 times with a 9% sucrose aqueous solution, filtered through a PVDF membrane filter with a pore size of 0.22 μm for filtration sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. This solution was designated as sample #2 (for safety testing).

[0362] For major pharmacology tests

[0363] Take 1 mL of sample #2 (for safety test) to another glass bottle, and add 9 mL of 9% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #2 (for main pharmacological test).

[0364] [Preparation of Sample #3 (Liposomes containing RK-163 (#3))]

[0365] Using the reagents and ingredients shown in Table 5, #3 samples for safety testing and primary pharmacology testing were prepared according to the following preparation methods.

[0366] (Composition of the prepared sample)

[0367] For safety testing

[0368] 15ng / mL RK-163, 247.2ng / mL total lipids in 9% sucrose aqueous solution

[0369] Lipid composition: DOTAP 91.7ng / mL, DOPE 73.2ng / mL

[0370] MPEG2000-DMPE, sodium salt 44.2ng / mL, cholesterol 38.1ng / mL

[0371] For major pharmacology tests

[0372] 1.5 ng / mL RK-163, 24.7 ng / mL total lipids in 9% sucrose aqueous solution

[0373] Lipid composition: DOTAP 9.17ng / mL, DOPE 7.32ng / mL, MPEG2000-DMPE, sodium salt 4.42ng / mL, cholesterol 3.81ng / mL

[0374] Particle size: 126.7nm PDI: 0.05407

[0375] [Table 5]

[0376]

[0377] (Preparation method)

[0378] For safety testing

[0379] 9.17 mg, 7.32 mg, 4.42 mg and 3.81 mg of DOTAP, DOPE, MPEG2000-DMPE, sodium salt and cholesterol were weighed into a flask and dissolved in 10 mL of methanol:chloroform = 1:2 (volume ratio). 1.0 mL of this solution was mixed with 1.5 mL of 100 μg / mL RK-163 solution in methanol:chloroform = 1:2, heated and dried with nitrogen, and allowed to stand in a desiccator. 10 mL of 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, 1 polycarbonate membrane filter with a pore size of 0.2 μm). The prepared liposome dispersion was further diluted 1000 times with 9% sucrose aqueous solution, filtered through a 0.22 μm pore size sterilized PVDF membrane filter, placed in a glass bottle with a screw cap, and stored at 4° C. This solution was used as sample #3 (for safety testing).

[0380] For major pharmacology tests

[0381] Take 1 mL of sample #3 (for safety test) to another glass bottle, and add 9 mL of 9% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #3 (for main pharmacological test).

[0382] [Preparation of Sample #4 (Liposomes containing RK-163 (#4))]

[0383] Using the reagents and ingredients shown in Table 6, sample #4 for safety testing and primary pharmacology testing was prepared according to the following preparation method.

[0384] (Composition of the prepared sample)

[0385] For safety testing

[0386] 15ng / mL RK-163, 249ng / mL total lipids in 9% sucrose aqueous solution

[0387] Lipid composition: DOTAP 91.7ng / mL, DOPE 73.2ng / mL MPEG2000-DSPE, sodium salt 46ng / mL, cholesterol 38.1ng / mL

[0388] For major pharmacology tests

[0389] 1.5 ng / mL RK-163, 24.9 ng / mL total lipids in 9% sucrose aqueous solution

[0390] Lipid composition: DOTAP 9.17ng / mL, DOPE 7.32ng / mL, MPEG2000-DSPE, sodium salt 4.6ng / mL, cholesterol 3.81ng / mL

[0391] Particle size: 126.2nm PDI: 0.09744

[0392] [Table 6]

[0393]

[0394]

[0395] (Preparation method)

[0396] For safety testing

[0397] 9.17 mg, 7.32 mg, 4.6 mg and 3.81 mg of DOTAP, DOPE, MPEG2000-DSPE, sodium salt and cholesterol were placed in a flask and dissolved in 10 mL of a solution of methanol:chloroform = 1:2 (volume ratio). 1.0 mL of this solution was mixed with 1.5 mL of a 100 μg / mL solution of RK-163 in methanol:chloroform = 1:2, and dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1 piece). The prepared liposome dispersion was further diluted 1000 times with 9% sucrose aqueous solution, filtered through a 0.22 μm pore size sterilized PVDF membrane filter, placed in a glass bottle with a screw cap, and stored at 4° C. This solution was used as sample #4 (for safety testing).

[0398] For major pharmacology tests

[0399] Take 1 mL of sample #4 (for safety test) to another glass bottle, and add 9 mL of 9% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #4 (for main pharmacological test).

[0400] [Preparation of Sample #5 (Liposomes (#5) containing RK-163)]

[0401] Using the reagents and ingredients shown in Table 7, sample #5 for safety testing and primary pharmacology testing was prepared according to the following preparation method.

[0402] (Composition of the prepared sample)

[0403] For safety testing

[0404] 322 ng / mL RK-163, 3222 ng / mL total lipids in 10% sucrose aqueous solution

[0405] (Each concentration is a value obtained by quantifying cholesterol in the prepared sample and converting it into a total lipid concentration based on the weight ratio at the time of charging.)

[0406] For major pharmacology tests

[0407] 32.2 ng / mL RK-163, 322.2 ng / mL total lipids in 10% sucrose aqueous solution

[0408] Lipid composition (lipid concentration of the samples used in the above safety tests):

[0409] HSPC 1953ng / mL

[0410] DSPG 792ng / mL

[0411] Cholesterol 475 ng / mL

[0412] (Each concentration is a value obtained by quantifying cholesterol in the prepared sample and converting it into each lipid concentration based on the weight ratio at the time of charging.)

[0413] Average particle size: 191.4nm PDI: 0.083

[0414] [Table 7]

[0415]

[0416] (Preparation method)

[0417] For safety testing

[0418] 6.66 mg, 2.7 mg, 1.61 mg of HSPC, DSPG, and cholesterol were placed in a flask and dissolved in 1.5 mL of methanol:chloroform = 1:1 (volume ratio). 1.5 mL of this solution was mixed with 0.22 mL of 5 mg / mL RK-163 in methanol:chloroform = 1:1, and dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 2.42 mL of 10% sucrose aqueous solution was added to the dried product, and granulated with an extruder (heated at 50°C, pore size 400 nm, 1 time, pore size 200 nm, 2 times), filtered through a 0.22 μm PVDF membrane filter, and the particle size was adjusted. The prepared liposome dispersion was placed in a glass bottle with a screw cap.

[0419] The liposome dispersion was further diluted 1000 times with a 10% sucrose aqueous solution, filtered through a 0.22 μm pore size sterilization PVDF membrane filter, placed in a screw cap glass bottle, and stored at 4° C. This solution was used as sample #5 (for safety testing).

[0420] For major pharmacology tests

[0421] Take 1mL of sample #5 (for safety testing) into another glass bottle, and add 9mL of 10% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a PVDF membrane filter with a pore size of 0.22μm for filtration sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. Use this solution as sample #5 (for main pharmacological tests). Since the RK-163 concentration of the sample used for the main pharmacological test is 32.2ng / mL, it is further diluted with PBS during the experiment, and the experiment is carried out with a concentration of 1.5ng / mL as the amount of RK-163.

[0422] [Preparation of Sample #6 (Liposomes containing RK-163 (#6))]

[0423] Using the reagents and ingredients shown in Table 8, sample #6 for safety testing and primary pharmacology testing was prepared according to the following preparation method.

[0424] (Composition of the prepared sample)

[0425] For safety testing

[0426] 15ng / mL RK-163, 221μg / mL total lipids in 9% sucrose aqueous solution

[0427] Lipid composition: HSPC 103μg / mL DOPS 79.7μg / mL Cholesterol 38.1μg / mL

[0428] For major pharmacology tests

[0429] 1.5 ng / mL RK-163, 22.1 μg / mL total lipids in 9% sucrose aqueous solution

[0430] Lipid composition: HSPC 10.3μg / mL DOPS 7.97μg / mL Cholesterol 3.81μg / mL

[0431] Particle size: 168.8nm PDI: 0.1288

[0432] [Table 8]

[0433]

[0434]

[0435] (Preparation method)

[0436] For safety testing

[0437] 10.3 mg, 7.97 mg, 3.81 mg of HSPC, DOPS, and cholesterol were placed in a flask and dissolved in 10 mL of a solution of methanol: chloroform = 1:2 (volume ratio). 1.0 mL of the solution was mixed with 1.5 mL of a 100 ng / mL solution of RK-163 in methanol: chloroform = 1:2, dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1). The prepared liposome dispersion was filtered through a PVDF membrane filter with a pore size of 0.22 μm for filtration sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. The solution was used as sample #6 (for safety testing).

[0438] For major pharmacology tests

[0439] Take 1 mL of sample #6 (for safety test) into another glass bottle, add 9 mL of 9% sucrose aqueous solution, mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #6 (for main pharmacological test).

[0440] [Preparation of Sample #7 (Liposomes (#7) containing RK-163)]

[0441] Using the reagents and ingredients shown in Table 9, sample #7 for safety testing and primary pharmacology testing was prepared according to the following preparation method.

[0442] (Composition of the prepared sample)

[0443] For safety testing

[0444] 15ng / mL RK-163, 220.8ng / mL total lipids in 9% sucrose aqueous solution

[0445] Lipid composition: HSPC 103ng / mL, DOPS 79.7ng / mL, cholesterol 38.1ng / mL

[0446] For major pharmacology tests

[0447] 1.5 ng / mL RK-163, 22.08 ng / mL total lipids in 9% sucrose aqueous solution

[0448] Lipid composition: HSPC 10.3ng / mL DOPS 7.97ng / mL Cholesterol 3.81ng / mL

[0449] Particle size: 145.7nm PDI: 0.06813

[0450] [Table 9]

[0451]

[0452] (Preparation method)

[0453] Safety test

[0454] 10.3 mg, 7.97 mg, 3.81 mg of HSPC, DOPS, and cholesterol were placed in a flask and dissolved in 10 mL of a solution of methanol:chloroform = 1:2 (volume ratio). 1.0 mL of the solution was mixed with 1.5 mL of a 100 μg / mL solution of RK-163 in methanol:chloroform = 1:2, dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1). The prepared liposome dispersion was further diluted 1000 times with a 9% sucrose aqueous solution, filtered through a PVDF membrane filter with a pore size of 0.22 μm for filtration sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. The solution was used as sample #7 (for safety testing).

[0455] For major pharmacology tests

[0456] Take 1 mL of sample #7 (for safety test) to another glass bottle, and add 9 mL of 9% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #7 (for main pharmacological test).

[0457] [Preparation of Sample #8 (Liposomes containing RK-163 (#8))]

[0458] Using the reagents and components shown in Table 10, sample #8 for safety testing and primary pharmacology testing was prepared according to the following preparation method.

[0459] (Composition of the prepared sample)

[0460] For safety testing

[0461] 15ng / mL RK-163, 242.6ng / mL total lipids in 9% sucrose aqueous solution

[0462] Lipid composition: HSPC 145ng / mL, MPEG2000-DSPE, sodium salt 48.8ng / mL, cholesterol 48.8ng / mL

[0463] For major pharmacology tests

[0464] 1.5 ng / mL RK-163, 24.26 ng / mL total lipids in 9% sucrose aqueous solution

[0465] Lipid composition: HSPC 14.5ng / mL, MPEG2000-DSPE, sodium salt 4.88ng / mL, cholesterol 4.88ng / mL

[0466] Particle size: 143.0nm PDI: 0.05755

[0467] [Table 10]

[0468]

[0469]

[0470] (Preparation method)

[0471] For safety testing

[0472] 14.5 mg, 4.88 mg and 4.88 mg of HSPC, MPEG2000-DSPE, Na salt and cholesterol were placed in a flask and dissolved in 10 mL of a solution of methanol:chloroform = 1:2 (volume ratio). 1.0 mL of the solution was mixed with 1.5 mL of a 100 μg / mL solution of RK-163 in methanol:chloroform = 1:2, dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 60°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1). The prepared liposomes were further diluted 1000 times with a 9% sucrose aqueous solution, filtered through a PVDF membrane filter with a pore size of 0.22 μm for filtration sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. This solution was designated as sample #8 (for safety testing).

[0473] For major pharmacology tests

[0474] Take 1 mL of sample #8 (for safety test) to another glass bottle, add 9 mL of 9% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #8 (for main pharmacological test).

[0475] [Preparation of sample #9 (liposome containing RCAI-61 (#9))]

[0476] Sample #9 for safety testing and primary pharmacology testing was prepared according to the following preparation method using the reagents and components shown in Table 11. Sample #9 was prepared using the same preparation method as sample #7 except that RCAI-61 was used instead of RK-163.

[0477] For safety testing

[0478] 15 ng / mL RCAI-61, 220.8 ng / mL total lipids in 9% sucrose aqueous solution

[0479] Lipid composition: HSPC 103ng / mL, DOPS 79.7ng / mL, cholesterol 38.1ng / mL

[0480] For major pharmacology tests

[0481] 1.5 ng / mL RCAI-61, 22.08 ng / mL total lipids in 9% sucrose aqueous solution

[0482] Lipid composition: HSPC 10.3ng / mL DOPS 7.97ng / mL Cholesterol 3.81ng / mL

[0483] Particle size: 145.7nm PDI: 0.06813

[0484] [Table 11]

[0485]

[0486] (Preparation method)

[0487] Safety test

[0488] 10.3 mg, 7.97 mg, and 3.81 mg of HSPC, DOPS, and cholesterol were placed in a flask and dissolved in a 10 mL methanol:chloroform solution of 1:2 (volume ratio). 1.0 mL of the solution was mixed with 1.5 mL of a 100 μg / mL RK-163 solution in methanol:chloroform = 1:2, dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1). The prepared liposome dispersion was further diluted 1000 times with a 9% sucrose aqueous solution, filtered through a PVDF membrane filter with a pore size of 0.22 μm for filtration sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. The solution was used as sample #9 (for safety testing).

[0489] For major pharmacology tests

[0490] Take 1 mL of sample #9 (for safety test) into another glass bottle, add 9 mL of 9% sucrose aqueous solution to mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #9 (for main pharmacological test).

[0491] [Preparation of Sample #10 (Liposomes containing RCAI-137 (#10))]

[0492] Sample #10 for safety testing and primary pharmacology testing was prepared according to the following preparation method using the reagents and components shown in Table 12. Sample #10 was prepared using the same preparation method as sample #7 except that RCAI-137 was used instead of RK-163.

[0493] (Composition of the prepared sample)

[0494] For safety testing

[0495] 15 ng / mL RCAI-137, 220.8 ng / mL total lipids in 9% sucrose aqueous solution

[0496] Lipid composition: HSPC 103ng / mL, DOPS 79.7ng / mL, cholesterol 38.1ng / mL

[0497] For major pharmacology tests

[0498] 1.5 ng / mL RCAI-137, 22.08 ng / mL total lipids in 9% sucrose aqueous solution

[0499] Lipid composition: HSPC 10.3ng / mL DOPS 7.97ng / mL Cholesterol 3.81ng / mL

[0500] Particle size: 145.7nm PDI: 0.06813

[0501] [Table 12]

[0502]

[0503] (Preparation method)

[0504] For safety testing

[0505] 10.3 mg, 7.97 mg, and 3.81 mg of HSPC, DOPS, and cholesterol were placed in a flask and dissolved in 10 mL of a solution of methanol:chloroform = 1:2 (volume ratio). 1.0 mL of this solution was mixed with 1.5 mL of a solution of 100 μg / mL RCAI-137 in methanol:chloroform = 1:2, dried by blowing nitrogen while heating, and allowed to stand in a desiccator. 10 mL of a 9% sucrose aqueous solution was added to the dried product, emulsified with a polytetrafluoroethylene homogenizer, and further granulated with an extruder (heated at 50°C, a polycarbonate membrane filter with a pore size of 0.2 μm, 1). The prepared liposome dispersion was further diluted 1000 times with a 9% sucrose aqueous solution, filtered through a PVDF membrane filter with a pore size of 0.22 μm for filtration sterilization, placed in a glass bottle with a screw cap, and stored at 4°C. This solution was designated as sample #10 (for safety testing).

[0506] For major pharmacology tests

[0507] Take 1 mL of sample #10 (for safety test) to another glass bottle, add 9 mL of 9% sucrose aqueous solution to it, and mix and dilute. Filter the liposome dispersion through a 0.22 μm pore size PVDF membrane filter for sterilization, put it into a glass bottle with a screw cap, and store it at 4°C. This solution is used as sample #10 (for main pharmacological test).

[0508] [Test Example 1] Antitumor effect of liposomes containing NKT cell ligand (RK-163)

[0509] In order to examine the effectiveness of RK-163-containing liposomes having different liposome compositions (samples #2 to #8 prepared by the above-mentioned preparation method), the effectiveness in terms of antitumor activity was studied.

[0510] In Experimental Example 1-1, 0.3 ng (converted to RK-163 amount) of liposomes (#2-#5) samples containing RK-163 (used in the main pharmacological test) and in Experimental Example 1-2, 0.3 ng (converted to RK-163 amount) of liposomes (#5-#8) samples containing RK-163 (used in the main pharmacological test) and 1x10 6 B16 mouse melanoma cells (MO4) expressing pseudotumor antigen OVA were administered via the tail vein of C57BL / 6 mice (10 weeks old). 5 B16 mouse melanoma cells (MO4) were transplanted subcutaneously into the same mice. The tumor diameter was observed on days 7, 10, 14, and 17 after transplantation. It should be noted that all measurements were performed using a digital caliper. Tumor volume (mm 3 )=major axis (mm) x minor axis (mm) x height (mm) for calculation.

[0511] As a comparative example, the same test was performed except that an aqueous solution of Captisol containing RK-163 (sample #1 (for the main pharmacological test)) was used instead of liposomes containing RK-163. As a control group, the same amount of PBS as that of liposomes containing RK-163 was administered.

[0512] The results are as follows Figure 1 shown.

[0513] The results showed that when liposomes containing RK-163 (#2-#5) (Test Example 1-1) and liposomes containing RK-163 (#5-#8) (Test Example 1-2) were compared, liposomes containing RK-163 (#7) and liposomes containing RK-163 (#8) showed significant anti-tumor effects.

[0514] [Test Example 2] Antitumor activity (dose) and ED50 of liposomes containing NKT cell ligand (RK-163)

[0515] Liposomes (#7) containing RK-163 (0.001 ng to 1 ng per 25 g body weight in terms of RK-163) and X-ray-irradiated (50 Gy) pseudotumor antigen OVA-expressing MO4 cells were administered via the tail vein of C57BL / 6 mice (10 weeks old). 5 B16 mouse melanoma cells (MO4) were transplanted subcutaneously into the same mice. The tumor diameter was observed on days 7, 11, 14, and 17 after transplantation. It should be noted that all measurements were performed using a digital caliper. Tumor volume (mm 3)=major axis (mm) x minor axis (mm) x height (mm) The group to which an equal amount of PBS was administered instead of liposomes containing RK-163 (#7) was used as a control.

[0516] The results are as follows Figure 2 shown.

[0517] The results showed that the liposomes containing RK-163 (#7) showed a strong anti-tumor effect and inhibited the proliferation of melanoma tumor cells in proportion to the amount of RK-163 (i.e., in a dose-dependent manner). The effective amount was converted to the amount of RK-163, which was 0.3ng / 25g mouse body weight to 1ng / 25g mouse body weight. The ED50 was 0.01393.

[0518] [Test Example 3] Antitumor effect of liposomes containing NKT cell ligands (RCAI-61 or RCAI-137)

[0519] Liposomes containing RK-163 (#7), RCAI-61 (#9), or RCAI-137 (#10) (0.003 ng to 1 ng per 25 g body weight in terms of RK-163, RCAI-61, or RCAI-137) and X-ray-irradiated (50 Gy) pseudotumor antigen OVA-expressing MO4 cells were intravenously administered to C57BL / 6 mice (10 weeks old), and then 3×10 5 B16 mouse melanoma cells (MO4) were transplanted subcutaneously into the same mice. The tumor diameter was observed on days 7, 11, 14, and 17 after transplantation. It should be noted that all measurements were performed using a digital caliper. Tumor volume (mm 3 )=major axis (mm) x minor axis (mm) x height (mm) The group to which an equal amount of PBS was administered instead of liposomes containing RK-163, RCAI-61 or RCAI-137 was used as a control.

[0520] The results are as follows Figure 3 shown.

[0521] As a result, both the liposomes (#9) containing RCAI-61 and the liposomes (#10) containing RCAI-137 showed strong antitumor effects similar to the liposomes (#7) containing K-163 that showed effectiveness in Test Example 1.

[0522] [Test Example 4] Hepatotoxicity test of liposomes containing NKT cell ligand (RK-163)

[0523] Using the results of Test Example 2 Figure 2A hepatotoxicity test (hepatotoxicity: ALT, AST, leukoplakia) of liposomes (#7) containing RK-163 was conducted with 10 times the effective dose (1 ng / mouse 25 g body weight) of the anti-tumor effect shown in (1 ng / mouse 25 g body weight). 10 ng of liposomes (#7) containing RK-163 was administered through the tail vein of C57BL / 6 mice (10 weeks old) (5 mice per group), and blood was collected on the 3rd, 7th and 17th days after administration, and ALT and AST were measured by ELISA. In addition, the liver was removed from the mouse on the 17th day after administration to confirm the presence of leukoplakia. A group that was administered an equal amount of liposomes (LIPO) without RK-163 and an equal amount of PBS instead of liposomes (#7) containing RK-163 was used as a control group.

[0524] The results are as follows Figure 4 The results showed that even when liposomes containing RK-163 were administered at an amount 10 times the effective amount for antitumor effects, no increase in hepatocyte enzymes AST and ALT and no leukoplakia were observed, confirming safety.

[0525] Figure 4 The "upper limit of the reference value" is calculated by CLEA Japan based on the data of 10-week-old normal C57BL / 6 mice, with ALT (GPT) of 34.4 IU / L and AST (GOT) of 77.4 IU / L. Therefore, the values ​​below the "upper limit of the reference value", 0-34.4 IU / L (ALT) and 0-77.4 IU / L (AST), are normal values.

[0526] [Test Example 5-1] Activation of NKT cells outside tumor tissue by liposomes containing NKT cell ligand (RK-163)

[0527] The effective amount (RK-163 equivalent amount 1 ng / mouse) of liposomes containing RK-163 (#7) and pseudo-tumor antigen (OVA-pulsed C57BL / 6J mouse spleen cells, 2×10 7 Cells) were administered to the tail vein of C57BL / 6J mice (10 weeks old) (5 mice per group), and the liposomes (#7) containing RK-163 were taken up into the antigen presenting cells (Kupffer cells, etc.) in the liver, and the RK-163 ligands on the CD1d molecules were presented by antigens to activate the NKT cells in the liver. As a result, the mouse NKT cells expressed the activation marker KLRG1 on the cell surface and produced the adjuvant substance IFN-γ that activated other immune cell groups. The group that was administered with an equal amount of PBS instead of the liposomes (#7) containing RK-163 was used as the control group.

[0528] The results are as follows Figure 5-1 As shown ( Figure 5-1The data are for the third day after the administration of liposomes containing RK-163 (#7). NKT cells were activated by liposomes containing RK-163, increasing in number and inducing the expression of activation marker KLRG1 and the production of IFN-γ.

[0529] [Test Example 5-2] In vitro activation of human NKT cells by liposomes containing NKT cell ligand (RK-163)

[0530] The activation (IFN-γ production) of human NKT cells by liposomes containing RK-163 (#7) was studied.

[0531] Depletion of CD3 containing NKT cells from splenocytes of C57BL / 6 mice by MACS + After the cell population, 5.0x10 4 Cells / well were seeded in a 96-well plate, and an effective amount (RK-163 equivalent to 1 ng) of liposomes (#7) containing RK-163 was added thereto, and pulse culture was performed at 37°C for 24 hours. Then, after removing the culture supernatant, 5.0×10 4 Human NKT hybridoma cells (Raoult cells) were inoculated into cells / well, cultured for another 24 hours at 37°C, and the culture supernatant was collected and the production of IFN-γ in the culture supernatant was measured using human IFN-γ ELISA. A group to which an equal amount of medium was added instead of liposomes containing RK-163 (#7) was used as a control group.

[0532] The results are as follows Figure 5-2 shown.

[0533] As a result, the production of IFN-γ was significantly increased in the group administered with liposomes containing RK-163 (#7). These results indicate that liposomes containing RK-163 (#7) pulsed to mouse antigen-presenting cells can activate human NKT cells and produce IFN-γ.

[0534] This indicates that (1) liposomes containing RK-163 can activate human NKT cells, and (2) liposomes containing RK-163 that are antigen-presented to mouse antigen-presenting cells can activate human NKT cells, thus NKT cells can be activated across species.

[0535] [Test Example 6] Activation of NKT cells outside tumor tissue by liposomes containing NKT cell ligands (RCAI-61 or RCAI-137)

[0536] By administering liposomes (#9) containing RACI-61 and liposomes (#10) containing RCAI-137 (RCAI-61 or RCAI-137 equivalent to 1 ng / mouse) and spleen cells pulsed with pseudotumor antigen OVA into the tail vein of C57BL / 6 mice, liposomes (#9) containing RACI-61 or liposomes (#10) containing RCAI-137 are taken up into antigen-presenting cells (Kupffer cells, etc.) in the liver, and NKT cells in the liver are activated through antigen presentation to the RCAI-61 or RCAI-137 ligands on the CD1d molecule.

[0537] The results are as follows Figure 6 shown.

[0538] The results showed that NKT cells significantly proliferated by administering liposomes containing RACI-61 (#9) and liposomes containing RCAI-137 (#10). Figure 6 The cells express the activation marker KLRG1 on their surface and produce IFN-γ ( Figure 6 (right image).

[0539] These results indicate that NKT cells artificially activated outside of tumor tissue can infiltrate into tumor tissue and activate immune cell populations within the tumor tissue.

[0540] [Test Example 7] Activation of innate immune system and adaptive immune system cell populations by NKT cells activated by liposomes containing NKT cell ligand (RK-163)

[0541] An effective amount (RK-163 equivalent to 1 ng / mouse) of liposomes (#7) containing RK-163 and 2×10 spleen cells pulsed with OVA were administered via the tail vein of C57BL / 6 mice (10 weeks old) (5 mice per group). 7 After activating NKT cells in the liver, the effect of the auxiliary effect of NKT cell activation on the innate immune system and the acquired immune system cell population was analyzed using PD-1 expression as an indicator. The group that was administered with an equal amount of PBS instead of liposomes containing RK-163 (#7) was used as a control group.

[0542] The results are as follows Figure 7 shown.

[0543] The results showed that when NKT cells were activated by liposomes containing RK-163 (#7) (see Test Example 5-1, Figure 5-1), the acquired immune system CD4T and CD8T cells and the innate immune system NK cells were activated. As the number of cells increased, the expression of PD-1, an activation indicator, was confirmed. By administering RK-163 liposomes to activate NKT cells, not only the acquired immune system CD4T cells and CD8T cells, but also the innate immune system NK cells were activated.

[0544] [Test Example 8] Activation of innate immune system and acquired immune system cell populations by NKT cells activated by liposomes containing NKT cell ligands (RCAI-61 or RCAI-137)

[0545] To verify that the cell populations of the innate and adaptive immune systems are activated through the help of activated NKT cells, liposomes containing RACI-61 (#9) or liposomes containing RCAI-137 (#10) and splenocytes pulsed with the pseudotumor antigen OVA were administered via the tail vein of C57BL / 6 mice, and the cell number and PD-1 expression were analyzed.

[0546] The results are as follows Figure 8 shown.

[0547] As a result, RACI-61-containing liposomes (#9) ( Figure 8 Left image) and liposomes containing RCAI-137 (#10) ( Figure 8 (right figure) After NKT cell activation, the acquired immune system CD4T and CD8T cells and the innate immune system NK cells were activated, and the expression of PD-1, an activation indicator, was confirmed as the number of cells increased.

[0548] [Test Example 9] Activation of innate immune system (NK) and acquired immune system effector (CD8T) cell populations infiltrating and in tumor tissue by NKT cells activated by liposomes containing NKT cell ligand (RK-163)

[0549] An effective amount (RK-163 equivalent to 1 ng / mouse body weight) of liposomes (#7) containing RK-163 and 1×10 6 X-ray irradiated MO4 tumor cells (B16 mouse melanoma cells expressing OVA) were used as pseudotumor antigens, and then 3x10 5 MO4 tumor cells were cultured and the immune cells in the tumor were analyzed 17 days later. A group that was administered with an equal amount of PBS instead of liposomes containing RK-163 (#7) was used as a control group.

[0550] The results are as follows Fig. 9 shown.

[0551] The results showed that in the group administered with liposomes containing RK-163 (#7), artificially activated NKT cells infiltrated into tumor tissues ( Fig. 9 ), IFN-γ is produced in tumor tissue ( Fig. 9 The lower left figure in the figure activates the acquired immune system CD8T cells present in the tumor tissue and the innate immune system NK cells ( Fig. 9 On the other hand, in the PBS administration group, almost the same number of non-artificially activated NKT cells also infiltrated into the tumor tissue ( Fig. 9 ), but because IFN-γ is not produced ( Fig. 9 ), so there is no activation of the innate immune system or the acquired immune cell population, and no expression of PD-1, an activation marker, is observed ( Fig. 9 From this, we can see that artificial activation of NKT cells is very important.

[0552] NKT cells artificially activated outside the tumor tissue infiltrated into the tumor, remained activated within the tumor tissue, and continued to produce large amounts of IFN-γ that activated other immune cell populations. As a result, in the group administered with liposomes containing RK-163 (#7), CD8T cells that attacked antigen-positive cancer cells (including mutated cancer cells) and NK cells that attacked cancer cells that had lost antigen expression were activated, and expression of PD-1, an activation indicator, was confirmed in their immune cell populations, indicating activation of immune cell populations within immunodeficient tumors.

[0553] [Test Example 10] Induction and maintenance of long-term immune memory by liposomes containing NKT cell ligand (RK-163) (after 30 and 90 days)

[0554] Liposomes containing RK-163 (#7) (RK-163 equivalent to 1 ng / mouse body weight) and 2×10 7 C57BL / 6 mice (10 weeks old) simultaneously immunized with spleen cells pulsed with pseudotumor antigen (OVA) (5 mice per group) were sacrificed on days 30 and 90 after administration of liposomes containing RK-163 (#7), and spleen cells were used for analysis.

[0555] The results are as follows Fig.10 shown.

[0556] As a result, the proliferation of OVA-specific CD8 T cell populations and the formation of long-term immune memory CD8 T cell populations (Tcm and Tem scores of OVA-specific CD8 cells were significantly detected) were confirmed by administering liposomes containing RK-163, and long-term memory was confirmed to be formed and maintained 30 days and 90 days after administering liposomes containing RK-163 (#7). This indicates that cancer-specific CD8 T memory cells can be formed and maintained for 30 and 90 days by administering liposomes containing RK-163.

[0557] [Test Example 11] Induction and maintenance of long-term immune memory by liposomes containing NKT cell ligands (RCAI-61 or RCAI-137) (after 30 and 90 days)

[0558] C57BL / 6 mice (5 per group) immunized by intravenous administration of liposomes containing RCAI-61 (#9) or liposomes containing RCAI-137 (#10) and spleen cells pulsed with a pseudotumor antigen (OVA) were sacrificed on days 30 and 90 after administration of liposomes containing RCAI-61 (#9) or liposomes containing RCAI-137 (#10), and spleen cells were used for analysis.

[0559] The results are as follows Fig.11 shown.

[0560] The results showed that administration of liposomes containing RCAI-61 ( Fig.11 ) or liposomes containing RCAI-137 ( Fig.11 After administration of liposomes containing RCAI-61 ( Fig.11 ) or liposomes containing RCAI-137 ( Fig.11 Long-term memory is formed and maintained 30 and 90 days after the onset of the experiment.

[0561] [Test Example 12] Liposomes containing NKT cell ligand (RK-163) establish and maintain long-term immune memory and anti-tumor effect after 1 year

[0562] Liposomes containing RK-163 (#7) and 2x10 7 C57BL / 6 mice (10 weeks old) were simultaneously immunized with spleen cells pulsed with pseudotumor antigen (OVA) and allowed to rest.

[0563] After one year, 3x10 5B16 mouse melanoma cells (MO4) were transplanted subcutaneously into these long-term memory-induced C57BL / 6 mice, and tumor diameters were observed on days 7, 10, 14, 17, and 21. It should be noted that all measurements were performed using a digital caliper. Tumor volume (mm 3 )=major axis (mm) x minor axis (mm) x height (mm) for calculation.

[0564] A similar experiment was performed using NKT-KO mice deficient in NKT cells instead of C57BL / 6 mice. In addition, a group administered with an equal amount of a liposome preparation (LIPO) without RK-163 instead of liposomes (#7) containing RK-163 was used as a control group.

[0565] The results are as follows Fig.12 shown.

[0566] The result was that in the group in which liposomes (#7) containing RK-163 were administered to C57BL / 6 mice, when tumor cells were transplanted into individuals one year after a single administration of liposomes (#7) containing RK-163, a strong anti-tumor effect was exerted and tumor growth was suppressed. That is, a single administration of liposomes (#7) containing RK-163 can induce immune memory for more than one year and suppress tumor growth. On the other hand, in the group in which liposomes not containing RK-163 were administered as a control group and in the group in which liposomes containing RK-163 were administered to NKT-KO mice with NKT cell deficiency, the onset of cancer could not be suppressed. That is, long-term memory could not be induced.

[0567] The liposome containing RK-163 (#7) showed a strong antitumor effect even one year after administration, confirming that it has the effect of establishing long-term cancer memory, which is important for cancer treatment.

[0568] The LIPO-administered group as a control group and the NKT-KO mice deficient in NKT cells failed to induce long-term memory and, as a result, failed to suppress the onset of cancer, thus demonstrating that activated NKT cells are essential cells for long-term immune memory.

[0569] [Industrial Applicability]

[0570] According to the present invention, a preparation containing a NKT cell ligand can be provided, which can artificially activate NKT cells in vivo and is useful as an anti-malignant tumor agent and the like.

[0571] This application is based on patent application No. 2022-020306 filed in Japan, the contents of which are incorporated herein in their entirety.

Claims

1. A liposome composition comprising a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II) and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

2. The composition according to claim 1, wherein The glycolipid is a compound represented by formula (I) or a salt thereof.

3. The composition according to claim 2, wherein The glycolipid is a compound or a salt thereof represented by the following formula: The composition according to any one of claims 1 to 3, which is an anti-tumor agent or an anti-infective agent.

5. The composition according to any one of claims 1 to 3, which is a NKT cell activator. The composition according to any one of claims 1 to 3, which is an inducer of IFN-γ production.

7. The composition according to any one of claims 1 to 3, which is an activator of lymphocytes of the innate immune system and lymphocytes of the acquired immune system. The composition according to any one of claims 1 to 3, which is a long-term immune memory inducer.

9. The composition according to any one of claims 1 to 3, for use in delivering the glycolipid to antigen presenting cells.

10. A method for delivering a glycolipid to an antigen presenting cell, comprising administering to a subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

11. A method for treating a malignant tumor or an infectious disease, comprising administering to a subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

12. A method for activating NKT cells in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

13. A method for inducing IFN-γ production in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

14. A method for activating innate immune system lymphocytes and acquired immune system lymphocytes in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

15. A method for inducing long-term immune memory in a subject, comprising administering to the subject a liposome composition containing a glycolipid selected from the group consisting of a compound represented by formula (I), a compound represented by formula (II), and salts thereof; Formula (I): In the above formula (I), X represents an alkylene group or -NH-; R1 and R2 are the same or different and represent a hydrogen atom, an alkyl group, a hydroxyl group, an alkoxy group or an aryl group optionally having a substituent, and R1 and R2 optionally form a 5-6 membered ring together with the adjacent nitrogen atom; R3 represents a hydrocarbon group having 1 to 20 carbon atoms; R4 represents a hydrocarbon group having 1 to 30 carbon atoms; Formula (II): In the above formula (II), R5 represents a hydrogen atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or a halogen atom, R6 and R7 each independently represent a substituted or unsubstituted hydrocarbon group having 1 to 28 carbon atoms, and Y represents -CH2-, -CH(OH)- or -CH=CH-.

16. The composition according to any one of claims 1 to 3, for use in the delivery of the glycolipid to antigen presenting cells.

17. The composition according to any one of claims 1 to 3, for use in treating a malignant tumor or an infectious disease.

18. The composition according to any one of claims 1 to 3, for use in activating NKT cells.

19. The composition according to any one of claims 1 to 3, for use in inducing IFN-γ production.

20. The composition according to any one of claims 1 to 3, for use in activating innate immune system lymphocytes and acquired immune system lymphocytes.

21. The composition according to any one of claims 1 to 3, for use in inducing long-term immunological memory.

22. A composition according to any one of claims 1 to 3 for use as a medicament.

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