Liposome formulation containing CD1d ligand compound with improved pharmacokinetics

Abstract

The present invention provides a liposome preparation that includes a population of liposomes containing a CD1d ligand compound, the average particle size of the population of liposomes being 90-110 nm, and the polydispersity of the particle size distribution being 0.2 or less. The present invention also provides the use of the aforementioned liposome preparation in the prevention or treatment of graft-versus-host disease or organ transplant rejection.

Description

[Technical Field]

[0001] The present invention relates to a liposome preparation containing a CD1d ligand compound with improved pharmacokinetics. More specifically, the present invention relates to a liposome preparation containing a CD1d ligand compound with improved pharmacokinetics by adjusting the particle size to a uniform size. [Background technology]

[0002] Graft-versus-host disease (GVHD) is one of the complications associated with allogeneic hematopoietic stem cell transplantation, in which donor-derived lymphocytes attack the recipient's organs, viewing them as foreign bodies. The immune system of the transplanted blood donor attacks and destroys the recipient's systemic tissues, and there are two types of GVHD: acute GVHD in the pre-transplant period and chronic GVHD in the post-transplant period. GVHD is also known to develop after blood transfusions.

[0003] CD4 + CD25 + Foxp3 + Regulatory T cells (Tregs) are considered key players in maintaining central and peripheral immune tolerance. Animal studies have shown that these cells can prevent or ameliorate T cell-mediated diseases, such as autoimmune diseases and graft rejection, by restoring immune tolerance to alloantigens as well as self-antigens. Studies in both humans and mice have demonstrated the crucial role of Tregs in controlling GVHD. However, the small population size of this cell population has hindered the development of Treg-based therapeutic modalities. The discovery of molecules that efficiently expand functional Tregs in vivo could contribute to the development of novel therapies for treating not only GVHD but also other immune disorders. Various strategies are emerging to activate and expand Tregs in situ.

[0004] α-Galactosylceramide (α-GalCer) functions as a ligand for CD1d molecules expressed on antigen-presenting cells (APCs). CD1d molecules are non-polymorphic tumor histocompatibility complex (MHC) class I-like antigen-presenting molecules with an antigen-binding groove specifically designed for lipid antigen presentation. When presented on various cell types, including dendritic cells (DCs), macrophages, and B cells, α-GalCer is recognized by the invariant T cell receptor (TCR) expressed on invariant NKT (iNKT) cells, resulting in CD1d-restricted activation of iNKT cells. CD1d-restricted iNKT cell activation results in the rapid and massive release of both Th1 and Th2 cytokines, a unique feature that distinguishes iNKT cells from conventional T cells and suggests important immunoregulatory functions in both innate and adaptive immunity. KRN7000 ((2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol) is a synthetic derivative of α-GalCer, originally discovered in a marine sponge.

[0005] While KRN7000 has been shown to act as an immunostimulator in aqueous form, liposomal formulations of KRN7000 have been found to induce antigen-specific immunosuppression or tolerance (Patent Document 1, Non-Patent Documents 1 and 2). Previous studies have suggested that targeting different cells with liposomal KRN7000 results in different immunomodulatory responses. Specifically, aqueous KRN7000 was presented primarily to DCs, resulting in stimulation of iNKT cells in the presence of IL-12 secreted by DCs. In contrast, liposomal KRN7000 was presented primarily on B cells, and its interaction with iNKT cells induced the production of IL-10 from both iNKT cells and B cells, leading to the proliferation of tolerogenic DCs (Non-Patent Document 1). Subsequently, antigen-specific CD4 + CD25 + Foxp3 +The lipid composition of the liposomes was thought to enhance the uptake of liposomal KRN7000 by B cells, thereby skewing the immune response toward tolerance.

[0006] The present inventors have demonstrated that liposomal KRN7000 (RGI-2001) can induce alloantigen-specific tolerance through Treg induction (Non-Patent Document 3). In a mouse acute GVHD model, a single administration of RGI-2001 significantly extended mouse survival. Enhanced proliferation of donor-derived CD4+Foxp3+ Tregs was identified as the key mechanism. Host alloantigen-specific immunosuppression was induced early after bone marrow transplantation (BMT), but responses to third-party alloantigens or white blood cells were not suppressed. Furthermore, RGI-2001 was shown to alleviate symptoms in a mouse chronic GVHD model. These results suggest that RGI-2001 could be a novel therapeutic approach for preventing both acute and chronic GVHD (Non-Patent Document 4).

[0007] The potential application of RGI-2001 to GVHD has also been confirmed in human clinical trials (Non-Patent Document 5). A phase 1 / 2a clinical trial was conducted in which 29 patients undergoing allogeneic hematopoietic stem cell transplantation were treated with a single intravenous administration of RGI-2001 on day 0. In some patients treated with RGI-2001, the number of Tregs (CD4+CD25+CD127loFoxp3+) increased significantly within 1 to 3 weeks after transplantation. Patients who responded with an increased number of Tregs showed a stronger reduction in GVHD compared to non-responders.

[0008] The applicability of RGI-2001 to organ transplantation has been confirmed in a mouse heart transplant model (Non-Patent Documents 6 and 7). After sublethal irradiation of recipient mice, RGI-2001 and a CD40-CD40L blocking antibody were co-administered during transplantation of donor mouse spleen and bone marrow cells. This resulted in the establishment of bone marrow chimeras in the recipient mice, and the suppression of donor-derived heart and skin rejection was maintained for a long period of time.

[0009] Patent Document 2 discloses a liposome production technique that can easily control the concentration of a dialysate (e.g., a liposome solution) after dialysis in a step of dialysis using a hollow fiber dialysis column, thereby obtaining a dialysate (e.g., a liposome solution) having a desired concentration.

[0010] Patent Document 3 discloses a lipid particle production technology in which an alcohol-containing solution in which lipids have been dissolved is subjected to a first dilution at an alcohol concentration that destabilizes the lipid particles, and then a second dilution is performed to obtain stabilized particles, and by adjusting the retention time between the first and second dilutions, it is possible to control the particle size of the lipid particles while maintaining a uniform particle size distribution.

[0011] Non-Patent Document 8 discloses the results of analyzing the pharmacokinetics of liposomes with various particle sizes using PET technology. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] WO2005 / 120574 A1 [Patent Document 2] WO2016 / 024510 A1 [Patent Document 3] WO2019 / 088193 A1 [Non-patent literature]

[0013] [Non-Patent Document 1] Tamura Y et al., Biochem Biophys Res Commun 369: 485-492, 2008 [Non-patent document 2] Ishii Y et al., Front Biosci 13: 6214-6228, 2008 [Non-patent document 3] Duramad O et al., Biol Blood Marrow Transplant 17: 1154-1168, 2011 [Non-patent document 4] Du Jing et al.,Blood 129: 3121-3125, 2017 [Non-patent document 5] Chen YB et al., Biol Blood Marrow Transplant 23: 625-634, 2017 [Non-patent document 6] Hirai et al., Am J Transplant 14: 1154-1168, 2014 [Non-Patent Document 7] Hirai et al., Am J Transplant 16: 426-439, 2016 [Non-patent document 8] Oku, N et al., Biochim. Biophys. Acta, 1238:86-90, 1995 Summary of the Invention [Problem to be solved by the invention]

[0014] As mentioned above, in the Phase 1 / 2a clinical trial of liposomal KRN7000 for the prevention of GVHD, while some patients showed significant therapeutic responses, others showed insufficient responses. Investigation into the cause of this finding revealed unstable pharmacokinetics, with plasma KRN7000 concentrations being low immediately after administration, rising temporarily, and then declining again. These results suggest that for liposomal KRN7000 to fully exert its therapeutic effect, it is important for the drug to remain in the blood for a long period of time immediately after administration. The formulation used in the Phase 1 / 2a clinical trial (RGI-2001-001) may not have met this requirement.

[0015] An object of the present invention is to provide a CD1d ligand-containing liposome preparation that can be retained in the blood for a long period of time and can maintain a high blood CD1d ligand concentration for a long period of time. [Means for solving the problem]

[0016] The present inventors conducted extensive research to solve the above-mentioned problems and discovered that the cause of the instability of blood kinetics was the particle size of the liposomes used in the Phase 1 / 2a clinical trial. The mean particle size of the liposomes in the liposomal KRN7000 formulation (RGI-2001-001) used in the Phase 1 / 2a clinical trial was approximately 120 nm. However, a new liposome formulation (RGI-2001-003) was prepared with the same KRN7000 content and lipid composition as RGI-2001-001, but with a mean particle size of approximately 100 nm. When this new formulation was administered intravenously to patients after hematopoietic stem cell transplantation, surprisingly, the pharmacokinetics were improved, the maximum blood concentration of KRN7000 was increased, and the blood half-life was extended. These formulations were administered intravenously to mice, and plasma IFN-γ and IL-4 concentrations were measured. IL-4 levels were higher in the RGI-2001-003 group than in the RGI-2001-001 group, but IFN-γ levels were lower in the RGI-2001-003 group than in the RGI-2001-001 group, suggesting that RGI-2001-003 may have the ability to more effectively induce Tregs. Based on these findings, the present inventors conducted further studies and completed the present invention.

[0017] That is, the present invention relates to the following. [1] A liposome preparation comprising a population of liposomes containing a CD1d ligand compound, wherein the liposome population has an average particle size of 90 to 110 nm and a polydispersity index of particle size distribution of 0.2 or less. [2] The liposome formulation of [1], having an average particle size of 92.9 to 101.0 nm. [3] The liposome formulation of [1] or [2], wherein the polydispersity index of the particle size distribution is 0.133 or less. [4] The liposome formulation according to any one of [1] to [3], wherein the number of liposomes having a particle diameter of less than 50 nm is 10% or less of the entire population of liposomes. [5] The liposome formulation according to any one of [1] to [4], wherein the number of liposomes having a particle diameter of more than 450 nm is 10% or less of the total population of liposomes. [6] A liposome formulation according to any one of [1] to [5], which maintains an average particle size of 90 to 110 nm for at least one month under conditions of a temperature of 25°C and a relative humidity of 60%RH. [7] The liposome formulation according to any one of [1] to [6], wherein the CD1d ligand compound is α-galactosylceramide. [8] The liposome preparation of [7], wherein the α-galactosylceramide is (2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol. [9] The liposome formulation according to any one of [1] to [8], wherein the population of liposomes is contained as a liposome suspension.

[10] The liposome formulation of [9], wherein the pH of the liposome suspension is 5.8 to 6.8.

[11] The liposome formulation according to any one of [1] to [8], wherein the population of liposomes is contained as a lyophilized product.

[12] A liposome formulation according to any one of [1] to

[11] , which is for injection administration.

[13] A liposome formulation according to any one of [1] to

[12] for the prevention or treatment of graft-versus-host disease.

[14] Graft-versus-host disease caused by allogeneic hematopoietic stem cell transplantation,

[13] a liposomal formulation.

[15] A liposome preparation according to any one of [1] to

[12] for the prevention or treatment of organ transplant rejection.

[16] The liposome formulation according to

[15] , wherein the organ transplant is an allogeneic organ or cell transplant.

[17] The liposome preparation according to

[15] , wherein the organ transplant is a transplant of a heterologous organ or cell.

[18] A method for reducing the risk of developing graft-versus-host disease in a subject at risk of developing graft-versus-host disease, comprising administering to the subject an effective amount of a liposome preparation according to any one of [1] to

[12] .

[19] The method of

[18] , wherein the subject at risk of developing graft-versus-host disease has received an allogeneic tissue or cell transplant or is scheduled to receive an allogeneic tissue or cell transplant.

[20] A method for treating graft-versus-host disease in a subject who has developed graft-versus-host disease, comprising administering to the subject an effective amount of a liposome preparation according to any one of [1] to

[12] .

[21] Any of the methods described in

[18] -

[20] , in which graft-versus-host disease is caused by allogeneic hematopoietic stem cell transplantation.

[22] A method for reducing the risk of organ transplant rejection in a subject at risk of developing organ transplant rejection, comprising administering to the subject an effective amount of a liposome preparation according to any one of [1] to

[12] .

[23] The method of

[22] , wherein the subject at risk of developing organ transplant rejection has received an allogeneic or xenogeneic organ or cell transplant, or is scheduled to receive an allogeneic or xenogeneic organ or cell transplant.

[24] A method for treating organ transplant rejection in a subject who has developed organ transplant rejection, comprising administering to the subject an effective amount of a liposome preparation according to any one of [1] to

[12] .

[25] Any of the methods

[22] to

[24] , wherein the organ transplant is an allogeneic organ or cell transplant.

[26] Any of the methods

[22] to

[24] , wherein the organ transplantation is a transplantation of a heterologous organ or cell.

[27] A liposome formulation according to any one of [1] to

[12] for use in the prevention or treatment of graft-versus-host disease.

[28] A liposomal formulation of

[27] in which graft-versus-host disease was caused by allogeneic hematopoietic stem cell transplantation.

[29] A liposome formulation according to any one of [1] to

[12] for use in the prevention or treatment of organ transplant rejection.

[30] The liposome preparation of

[29] , wherein the organ transplant is an allogeneic organ or cell transplant.

[31] The liposome preparation of

[29] , wherein the organ transplant is a transplant of a heterologous organ or cell.

[32] Use of a liposome preparation according to any one of [1] to

[12] in the manufacture of a medicament for the prevention or treatment of graft-versus-host disease.

[33] The use of graft-versus-host disease caused by allogeneic hematopoietic stem cell transplantation.

[32]

[34] Use of any of the liposome preparations [1] to

[12] in the manufacture of a medicament for the prevention or treatment of organ transplant rejection.

[35] The use of

[34] , wherein the organ transplant is an allogeneic organ or cell transplant.

[36] The use of

[34] , wherein the organ transplant is a transplant of a heterologous organ or cell. [Effects of the Invention]

[0018] The present invention provides a CD1d ligand-containing liposome preparation that can be retained in the blood for a long period of time and maintain a high blood CD1d ligand concentration for a long period of time. The liposome preparation of the present invention can effectively induce Tregs, and is therefore expected to have excellent preventive or therapeutic effects against GVHD, organ transplant rejection, autoimmune diseases, etc. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows the time course of blood KRN7000 concentrations in patients administered RGI-2001 after hematopoietic stem cell transplantation. [Figure 2] FIG. 2 shows the blood IFN-γ and IL-4 concentrations in mice administered RGI-2001. [Figure 3] FIG. 3 shows the blood IL-10 concentration in mice administered RGI-2001. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a liposome preparation (the liposome preparation of the present invention) comprising a population of liposomes containing a CD1d ligand compound.

[0021] The term "liposome formulation" refers to a pharmaceutical composition containing an active ingredient encapsulated in a liposome.

[0022] The term "liposome population" refers to a collection of multiple liposomes. The number of liposomes constituting the liposome population of the present invention is usually 10 3 or more (e.g., 10 4 That's it, 10 5 That's it, 10 6 That's it, 10 7 That's it, 10 8 That's it, 10 9 That's it, 10 10 That's it, 10 11 That's it, 10 12 The upper limit of the number of liposomes constituting the liposome population of the present invention is not particularly limited, but may be, for example, 10 21 Below, 10 20 Below, 10 19 Below, 10 18 Below, 10 17 Below, 10 16 It may be the following:

[0023] The term "CD1d ligand compound" refers to a compound that, when presented on CD1d molecules expressed on antigen-presenting cells (dendritic cells (DCs), macrophages, B cells, etc.), is recognized by the invariant T cell receptor (NKT cell receptor) on invariant NKT (iNKT) cells and activates iNKT cells in a CD1d-restricted manner. Examples of CD1d ligand compounds that can be used in the present invention include, but are not limited to, α-glycosylceramide, isoglobotrihexosylceramide (Science, 306, pp. 1786-1789, 2004), and OCH (Nature, 413:531, 2001). α-Glycosylceramide is a glycosphingolipid in which a sugar such as galactose or glucose is bound to ceramide in an α-position. α-Glycosylceramide in which the sugar moiety is galactose is called α-galactosylceramide. Examples of α-glycosylceramides include, but are not limited to, those disclosed in WO93 / 05055, WO94 / 02168, WO94 / 09020, WO94 / 24142, WO98 / 44928, Science, 278, pp. 1626-1629, 1997, etc.

[0024] Examples of α-glycosylceramides include compounds of the following formula (I) or salts or solvates thereof:

[0025] [ka]

[0026] (In the above formula, R 1 is H or OH, X is an integer between 7 and 27, R 2 is a substituent selected from the group consisting of the following (a) to (e) (wherein Y is an integer of 5 to 17), (a)-CH2(CH2) Y CH3, (b) -CH(OH)(CH2) Y CH3, (c) -CH(OH)(CH2) Y CH(CH3)2, (d) -CH=CH(CH2) Y CH3, (e) -CH(OH)(CH2) Y CH(CH3)CH2CH3, and R 3 ~R 9 is a substituent defined in i) or ii) below: i)R 3 , R 6 , and R 8 When is H R 4 is H, OH, NH2, NHCOCH3, or one of the following groups (A) to (D):

[0027] [ka]

[0028] is a substituent selected from the group consisting of R 5 is OH, or the following groups (E) and (F):

[0029] [ka]

[0030] is a substituent selected from the group consisting of R 7 is OH or one of the following groups (A) to (D):

[0031] [ka]

[0032] is a substituent selected from the group consisting of R 9 is H, CH3, CH2OH, or one of the following groups (A') to (D'):

[0033] [ka]

[0034] is a substituent selected from the group consisting of: ii)R 3 , R 6 and R 7 When is H R 4 is H, OH, NH2, NHCOCH3, or one of the following groups (A) to (D):

[0035] [ka]

[0036] is a substituent selected from the group consisting of R 5 is OH, or the following groups (E) and (F):

[0037] [ka]

[0038] is a substituent selected from the group consisting of R 8 is OH, or the following groups (A) to (D):

[0039] [ka]

[0040] is a substituent selected from the group consisting of R 9 is H, CH3, CH2OH or one of the following groups (A') to (D'):

[0041] [ka]

[0042] is a substituent selected from the group consisting of

[0043] α-Galactosylceramides include R 3 , R 6 and R 8 is H and R 4 , R 5 and R 7 is OH and R 9 is CH2OH, or a salt or solvate thereof.

[0044] The α-galactosylceramide is preferably (2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol (also referred to as (2S,3S,4R)-1-O-(α-D-galactosyl)-N-hexacosanoyl-2-amino-1,3,4-octadecanetriol or KRN7000). KRN7000 has the following chemical structure:

[0045] [ka]

[0046] The liposome population contained in the liposome formulation of the present invention is characterized by having a mean particle size of 90 to 110 nm and a polydispersity index (PdI) of particle size distribution of 0.2 or less. The present invention is based on the finding that converging the particle size of KRN7000-containing liposomes to around 100 nm improves the pharmacokinetics of liposomes administered to patients, increases the maximum blood concentration of KRN7000, and extends the blood half-life. The average particle size of the population of liposomes of the present invention can be 90 nm or more (preferably 91.0 nm or more, 92.0 nm or more, 92.5 nm or more, 92.9 nm or more, 93.0 nm or more, 93.1 nm or more, 93.5 nm or more, 94.0 nm or more, 94.5 nm or more, 94.6 nm or more, 95.0 nm or more, 95.5 nm or more, 95.7 nm or more, 95.8 nm or more, 95.9 nm or more, 96.0 nm or more, or 96.2 nm or more). The average particle size of the population of liposomes of the present invention can be 110 nm or less (preferably 109.0 nm or less, 108.5 nm or less, 108.0 nm or less, 107.5 nm or less, 107.0 nm or less, 106.5 nm or less, 106.0 nm or less, 105.5 nm or less, 105.0 nm or less, 104.5 nm or less, 104.0 nm or less, 103.5 nm or less, 103.0 nm or less, 102.5 nm or less, 102.0 nm or less, 101.5 nm or less, 101.0 nm or less). In one embodiment, the average particle size of the liposome population of the present invention can be 92.9 to 101.0 nm, 93.1 to 101.0 nm, 93.5 to 101.0 nm, 94.6 to 101.0 nm, 95.7 to 101.0 nm, 95.8 to 101.0 nm, or 95.9 to 101.0 nm.

[0047] The population of liposomes contained in the liposome preparation of the present invention is homogeneous with respect to particle size, and the polydispersity of the particle size distribution can be 0.2 or less (preferably 0.190 or less, 0.180 or less, 0.170 or less, 0.160 or less, 0.150 or less, 0.140 or less, 0.135 or less, 0.133 or less, 0.130 or less, 0.127 or less, 0.125 or less, 0.124 or less, 0.120 or less, 0.116 or less, 0.115 or less, 0.112 or less, 0.110 or less, 0.105 or less, or 0.102 or less).

[0048] As used herein, "particle size" is used in the usual sense in the art to indicate the size of a particle, and is a convenient value equivalent to the diameter of the particle when it is assumed to be a perfect sphere. As used herein, "average particle size" can refer to either the number-average particle size or the Z-average particle size, but unless otherwise specified, refers to the Z-average particle size calculated from measured particle sizes. As used herein, "particle size distribution" is used in the usual sense in the art to indicate the spread of particle sizes. Polydispersity index (PDI) is used as a measure of particle size distribution. The average particle size of a liposome population and the polydispersity index of the particle size distribution can be measured by DLS (dynamic light scattering, backscattering) using a Malvern ZetaSizer Nano ZS (for example, Condition 1 in Test Example 5). If necessary, the formulation can be dissolved in PBS (Ca 2+ It may be diluted with (not containing).

[0049] In a preferred embodiment, in the population of liposomes contained in the liposome preparation of the present invention, the number of liposomes having a particle size of less than 50 nm remains 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less) of the total.

[0050] In a preferred embodiment, in the population of liposomes contained in the liposome preparation of the present invention, the number of liposomes having a particle size of more than 450 nm is 10% or less of the total (e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less).

[0051] The liposomes constituting the liposome population contained in the liposome formulation of the present invention contain a CD1d ligand compound (e.g., KRN7000) as an essential component. The content of the CD1d ligand compound is not particularly limited, as long as it is an amount that activates iNKT cells in a subject mammal when the liposome formulation of the present invention is administered to the subject mammal. The content (weight) of the CD1d ligand compound (e.g., KRN7000) in the liposomes constituting the liposome population of the present invention can be, for example, 1 to 21% (w / w), preferably 5 to 15% (w / w), more preferably 7 to 13% (w / w), and even more preferably 9 to 11% (w / w) (10±1% (w / w)) of the total weight of components other than the CD1d ligand compound that form the liposomes (i.e., lipids). In this specification, unless otherwise specified, the term "components forming a liposome" means the components of the lipid bilayer membrane that forms the liposome and the CD1d ligand compound encapsulated in the liposome, and does not include other components in the internal and external aqueous phases of the liposome.

[0052] Components other than the CD1d ligand compound that form the liposomes may be any amphiphilic molecule capable of forming micelles, preferably lipids. Examples of lipids include phospholipids such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG-Na), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG-Na), glycosphingolipids, and glyceroglycolipids. These may be used alone or in combination with other lipid derivatives in which a nonpolar substance such as cholesterol or a water-soluble polymer such as polyethylene glycol is bound to the lipid to form the liposomes. In one embodiment, the liposomes that constitute the liposome population of the present invention contain, in addition to the CD1d ligand compound, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG-Na), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG-Na), and Cholesterol (Cho) Includes.

[0053] When liposomes constituting the liposome population contained in the liposome preparation of the present invention contain DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho, the composition ratio thereof is not particularly limited, but the molar ratio of DOPC:DOPG-Na:DPPC:DPPG-Na:Cho is preferably Preferably, 15±6:15±6:15±6:15±6:40±16, More preferably, 15±3:15±3:15±3:15±3:40±8; More preferably, the ratios are 15±1.5:15±1.5:15±1.5:15±1.5:40±4.

[0054] In one embodiment, liposomes constituting the population of liposomes contained in the liposome formulation of the present invention contain, as lipid bilayer membrane-forming components, a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In one embodiment, the lipid bilayer membrane-forming components of liposomes constituting the population of liposomes of the present invention consist of a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In these embodiments, the relative content (by weight) of each component is preferably as follows: KRN7000:5.0±0.5 DOPC: 9.6±2.4 DOPG-Na: 9.7±2.4 DPPC: 9.0±2.3 DPPG-Na: 9.1±2.3 Cho: 12.6±3.2

[0055] The structure of the liposome is not particularly limited as long as it is a small vesicle having a lipid bilayer membrane structure, and may be any liposome such as a unilamellar or multilamellar liposome.

[0056] The solution encapsulated inside the liposome (internal solution) can be water, buffer solution, physiological saline, etc. These can also be used by adding an appropriate amount of a water-soluble organic solvent (e.g., glycerin, etc.). The internal solution of the liposome may contain additives such as an osmotic pressure adjuster, a stabilizer, an antioxidant, and a pH adjuster.

[0057] Examples of osmotic pressure adjusters include, but are not limited to, inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol; and sugars such as glucose, fructose, lactose, maltose, and sucrose.

[0058] Examples of stabilizers include, but are not limited to, sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose, and sterols such as cholesterol.

[0059] The antioxidant is not particularly limited, but examples thereof include ascorbic acid, uric acid, and tocopherol homologues (e.g., vitamin E). Tocopherol has four isomers, α, β, γ, and δ, and any of these can be used in the present invention.

[0060] The pH adjuster may be any basic or acidic compound, such as sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, L-histidine and its hydrochloride.

[0061] The internal solution of the liposome is preferably an aqueous buffer solution containing an osmotic pressure adjuster and a pH adjuster. The osmotic pressure adjuster is preferably sucrose or maltose, more preferably sucrose. The pH adjuster is preferably L-histidine or its hydrochloride. The internal solution of the liposome is preferably adjusted to be isotonic or nearly isotonic with human body fluid (plasma) (e.g., 285±50 mOsm / L). When sucrose is used as the osmotic pressure adjuster, the sucrose concentration in the internal solution of the liposome is, for example, about 9.0 to 11.0 (10.0±1.0)% (w / v). When maltose is used as the osmotic pressure adjuster, the maltose concentration in the internal solution of the liposome is, for example, about 9.0 to 11.0 (10.0±1.0)% (w / v). The pH of the internal solution of the liposome is adjusted to, for example, 5.3 to 7.3 (6.3±1.0), preferably 5.8 to 6.8 (6.3±0.5). In one embodiment, the internal solution of the liposome is an aqueous buffer solution containing sucrose, L-histidine, and L-histidine hydrochloride, which is isotonic or nearly isotonic with human body fluid (plasma) (e.g., 285±50 mOsm / L) and contains sucrose at a concentration of preferably 9.0 to 11.0 (10.0±1.0)% (w / v). In one embodiment, the internal solution of the liposome is an aqueous buffer solution containing maltose, L-histidine, and L-histidine hydrochloride, which is isotonic or nearly isotonic (e.g., 285±50 mOsm / L) with human body fluid (plasma) and has a pH of 5.8 to 6.8 (6.3±0.5), and the maltose concentration is preferably 9.0 to 11.0 (10.0±1.0)% (w / v).

[0062] The liposome formulations of the present invention can be prepared using known liposome production techniques. For example, methods such as those described in Liposome Technology, vol. 1, 2nd edition (by Gregory Gregoriadis (CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp. 67-80, Chapter 10, pp. 167-184, and Chapter 17, pp. 261-276 (1993)) can be used. More specifically, examples of such methods include, but are not limited to, sonication, ethanol injection, French press, ether injection, cholate, calcium fusion, freeze-thaw, and reverse-phase evaporation. A population of liposomes of the present invention can also be continuously prepared within a closed capillary tube using in-line liposome production techniques such as those described in WO 2016 / 024510 and WO 2019 / 088193.

[0063] From the viewpoint of producing a population of liposomes with a uniform particle size, the liposome preparation of the present invention is preferably produced according to the method described in WO 2019 / 088193. The present invention also provides a method for producing such a liposome preparation of the present invention.

[0064] The production method of the present invention includes, for example, the following steps. A) preparing a primary dilution solution by mixing a first solution containing a CD1d ligand (e.g., KRN7000), a lipid, and an alcohol with a second solution containing water in a first mixing region; B) transferring the primary dilution solution from the first mixing region to the second mixing region through a transfer tube for a predetermined time; C) mixing the primary dilution solution with a third solution comprising water in the second mixing area to prepare a secondary dilution solution; D) transferring the secondary dilution solution from the second mixing area to the third mixing area through the transfer pipe at a predetermined time; and E) In a third mixing region, mixing the second dilution solution with a fourth solution (which may be defined similarly to any of the first, second, and third solutions) containing water to prepare a third dilution solution. Here, the average particle size of the liposome population produced is controlled to fall within the desired range (typically, an average particle size of 90 to 110 nm and a polydispersity index (PdI) of the particle size distribution of 0.2 or less) by adjusting at least one condition selected from the group consisting of the alcohol concentration in the primary dilution solution, the lipid concentration, the specified time, and the temperature during mixing. In one embodiment, preparation of a solution containing a CD1d ligand (e.g., KRN7000), a lipid, and an alcohol may include a step of dissolving the CD1d ligand (e.g., KRN7000) and the lipid in alcohol. The dissolution may be performed under heating.

[0065] The average particle size of the liposome population can be adjusted by adjusting at least one of the alcohol concentration, lipid concentration, the specified time of solution delivery, and the temperature during mixing in the primary dilution solution. The average particle size of the liposome population can be finely adjusted by gradually adjusting the alcohol concentration. Furthermore, by gradually adjusting the alcohol concentration and then allowing the specified time of solution delivery, a liposome population with a desired average particle size can be produced. In this case, it is possible to achieve a particle size distribution narrow enough for pharmaceutical use. Furthermore, by adjusting the temperature during mixing, the average particle size of the liposome population can be adjusted even more precisely.

[0066] The lipids contained in the first solution can be any of those mentioned above as components other than the CD1d ligand compound that form the liposomes that make up the population of liposomes contained in the liposome preparation of the present invention. In one embodiment, the lipids contained in the first solution include DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In one embodiment, the lipids contained in the first solution consist of DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. When the first solution contains DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho, the composition ratio thereof is not particularly limited, but the molar ratio of DOPC:DOPG-Na:DPPC:DPPG-Na:Cho is Preferably, 15±6:15±6:15±6:15±6:40±16, More preferably, 15±3:15±3:15±3:15±3:40±8; More preferably, the ratios are 15±1.5:15±1.5:15±1.5:15±1.5:40±4. The content (weight) of the CD1d ligand compound (e.g., KRN7000) in the first solution can be, for example, 1 to 21% (w / w), preferably 5 to 15% (w / w), more preferably 7 to 13% (w / w), and even more preferably 9 to 11% (w / w) (10±1% (w / w)) of the total weight of components (i.e., lipids) other than the CD1d ligand compound (e.g., KRN7000) that form the liposomes.

[0067] In one embodiment, the first solution contains, as components other than alcohol, a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In one embodiment, the components other than alcohol of the first solution consist of a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In these embodiments, the relative contents (by weight) of each component are preferably as follows: CD1d ligand compounds (e.g., KRN7000): 5.0±0.5 DOPC: 9.6±2.4 DOPG-Na: 9.7±2.4 DPPC: 9.0±2.3 DPPG-Na: 9.1±2.3 Cho: 12.6±3.2

[0068] The alcohol contained in the first solution comprises a monohydric or dihydric alcohol containing 1 to 6 carbon atoms. Alternatively, the alcohol contained in the first solution comprises a monohydric or dihydric alcohol. In another embodiment, the alcohol contained in the first solution comprises a monohydric alcohol. In certain embodiments, the alcohol contained in the first solution comprises a monohydric alcohol containing 1 to 3 carbon atoms. In specific embodiments, the alcohol contained in the first solution comprises methanol, ethanol, or isopropyl alcohol, or a combination thereof. Preferably, the alcohol contained in the first solution is ethanol.

[0069] In one embodiment, the second solution and / or the third solution may contain a lower concentration of alcohol than the first solution.

[0070] The solutions used in the production method of the present invention, including the first solution, the second solution, and the third solution, may contain solvents other than water and alcohol. Examples of such solvents are as described in WO 2019 / 088193.

[0071] Any of the solutions used in the production method of the present invention, including the first solution, the second solution, and the third solution, may contain additives such as an osmotic pressure adjuster, a stabilizer, an antioxidant, a pH adjuster, etc. Examples of additives such as an osmotic pressure adjuster, a stabilizer, an antioxidant, and a pH adjuster are as described above.

[0072] The second and third solutions are preferably aqueous buffer solutions. The second and third solutions preferably contain an osmotic pressure adjuster and a pH adjuster. The osmotic pressure adjuster is preferably sucrose. The pH adjuster is preferably L-histidine and its hydrochloride. The second and third solutions are preferably adjusted to be isotonic or nearly isotonic with human body fluid (plasma) (e.g., 285±50 mOsm / L). When sucrose is used as the osmotic pressure adjuster, the sucrose concentration in the internal solution of the liposomes is, for example, about 9.0 to 11.0 (10.0±1.0)% (w / v). When maltose is used as the osmotic pressure adjuster, the maltose concentration in the internal solution of the liposomes is, for example, about 9.0 to 11.0 (10.0±1.0)% (w / v). In one embodiment, the second and third solutions are buffered aqueous solutions that are isotonic or nearly isotonic (e.g., 285±50 mOsm / L) with human body fluid (plasma) and contain sucrose, L-histidine, and L-histidine hydrochloride, and the sucrose concentration is preferably 9.0 to 11.0 (10.0±1.0)% (w / v). In another embodiment, the second and third solutions are buffered aqueous solutions that are isotonic or nearly isotonic (e.g., 285±50 mOsm / L) with human body fluid (plasma) and contain maltose, L-histidine, and L-histidine hydrochloride, and the maltose concentration is preferably 9.0 to 11.0 (10.0±1.0)% (w / v). In one embodiment, the pH of the second solution and the third solution is, for example, 5.3 to 7.3 (6.3±1.0), preferably 5.8 to 6.8 (6.3±0.5). In one embodiment, the pH of any solution containing water used in the production method of the present invention, including the first solution, the second solution, and the third solution, is, for example, 5.3 to 7.3 (6.3±1.0), preferably 5.8 to 6.8 (6.3±0.5). In this manner, the pH of the final suspension of the liposome population of the present invention or the internal solution of the liposomes can be adjusted to, for example, 5.3 to 7.3 (6.3±1.0), preferably 5.8 to 6.8 (6.3±0.5).

[0073] When the alcohol concentration (wt%) in the primary dilution solution is adjusted to a specific value (also referred to as the "fluidity fluctuation point") or higher, the average particle size of the liposome population changes over time. Conversely, at alcohol concentrations below the fluidity fluctuation point, the liposome particle size changes very little. In one embodiment, the fluidity fluctuation point may vary depending on the liposome composition, temperature, and pressure. In one embodiment, the fluidity fluctuation point may vary depending on the type of alcohol in the primary dilution solution. In one embodiment, the fluidity fluctuation point may vary depending on the liposome composition, temperature, and pressure. In one embodiment, the fluidity fluctuation point does not vary depending on the liposome composition and / or the presence or absence of a drug to be loaded into the liposomes, provided that the type of alcohol in the primary dilution solution is the same.

[0074] In one embodiment, in the step of transferring the primary diluted solution from the first mixing region to the second mixing region through the liquid transfer tube for a predetermined time, the alcohol concentration and temperature are adjusted so that the alcohol concentration in the primary diluted solution is equal to or higher than the fluidity fluctuation point. For example, the liquid transfer tube is heated to 85±5°C. Furthermore, in the step of transferring the secondary diluted solution from the second mixing region to the third mixing region through the liquid transfer tube for a predetermined time, the alcohol concentration and temperature are adjusted so that the alcohol concentration in the secondary diluted solution is equal to or lower than the fluidity fluctuation point. For example, the liquid transfer tube is set to 20±5°C. That is, the step of transferring the primary diluted solution from the first mixing region to the second mixing region through the liquid transfer tube for a predetermined time is performed at a temperature equal to or higher than the phase transition temperature of the lipid in the primary diluted solution, and the step of transferring the secondary diluted solution from the second mixing region to the third mixing region through the liquid transfer tube for a predetermined time is performed at a temperature lower than the phase transition temperature of the secondary diluted solution. By controlling the reaction conditions in this way, liposomes or their membranes become unstable in the primary dilution solution, and heating above the phase transition temperature increases the fluidity of lipids, increasing the frequency of fusion when they come into contact with each other due to Brownian motion, etc. As a result, fusion of the generated liposomes progresses uniformly over time, increasing the particle size while maintaining a constant particle size distribution. Meanwhile, in the secondary dilution solution, by adjusting the temperature below the phase transition temperature, the particle size distribution of the liposomes controlled in the primary dilution solution is fixed and does not change.

[0075] A stainless steel capillary tube (SSCT) can be used as the liquid transfer tube in steps B and D, and the reaction liquid may be mixed using an in-line mixer.

[0076] In one embodiment, the mean particle size of the liposome population can be controlled within the desired range (typically, a mean particle size of 90 to 110 nm and a polydispersity index (PdI) of the particle size distribution of 0.2 or less) by adjusting the predetermined time (retention time) for the primary diluted solution to reach the second mixing region from the first mixing region (or, in some cases, the predetermined time for the secondary diluted solution to reach the third mixing region from the second mixing region). In one embodiment, the predetermined time for the primary diluted solution to be retained in the liquid transfer tube from the first mixing region to the second mixing region (or, in some cases, the time for the secondary diluted solution to be retained in the liquid transfer tube from the second mixing region to the third mixing region) is controlled by at least one of the length of the flow path between the mixing regions and the flow rate. In one embodiment, the time for the primary diluted solution to be retained in the liquid transfer tube from the first mixing region to the second mixing region is controlled by the flow rate between the first mixing region and the second mixing region.

[0077] In addition, the average particle size of the liposome population can be controlled within the desired range described above (typically, an average particle size of 90 to 110 nm and a polydispersity index (PdI) of the particle size distribution of 0.2 or less) by controlling the temperature in each step of the production method of the present invention, the lipid concentration in the primary dilution solution, the pressure in each step, and the like.

[0078] In one embodiment, the production method of the present invention includes a step of adjusting the composition of a solution after preparing a liposome population. In one embodiment, the production method of the present invention includes a step of adjusting the liposome concentration after preparing a liposome population. The step of adjusting the solution composition and the step of adjusting the liposome concentration may be performed simultaneously or separately. For example, as disclosed in WO 2016 / 024510, the step of adjusting the solution composition and the step of adjusting the liposome concentration can be performed simultaneously by using a hollow fiber membrane column. Methods for adjusting the liposome concentration and solution composition in a solution containing the prepared liposome population include, but are not limited to, ultrafiltration and dialysis. For example, this step adjusts the pH of the solution containing the prepared liposome population (liposome suspension) to 5.3 to 7.3 (6.3±1.0), preferably 5.8 to 6.8 (6.3±0.5).

[0079] In one embodiment, the surface of liposomes constituting the liposome population contained in the liposome formulation of the present invention may be modified with a modifying agent. Examples of modifying agents include, but are not limited to, polyethylene glycol (PEG), Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinylpyrrolidone, polyvinyl methyl ether, polyvinylmethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, polyaspartamide, synthetic polyamino acids, and derivatives thereof. Modification of liposomes with PEG or PEG derivatives can facilitate prolonged circulation. Furthermore, modification of liposomes with targeting molecules (e.g., antibodies) that have affinity for specific tissues can facilitate delivery of liposomes to target tissues. In one embodiment, the surfaces of liposomes constituting the population of liposomes contained in the liposome preparation of the present invention are not modified with a modifying agent.

[0080] In one aspect, the liposome formulation of the present invention is stable, and the average particle size of the population of liposomes contained in the formulation is maintained at 90 to 110 nm (preferably 92.9 to 101.0 nm, 93.1 to 101.0 nm, 93.5 to 101.0 nm, 94.6 to 101.0 nm, 95.7 to 101.0 nm, 95.8 to 101.0 nm, or 95.9 to 101.0 nm) for at least one month (e.g., two months or more, three months or more, or six months or more) under conditions of a temperature of 25°C and a relative humidity of 60% RH (accelerated test conditions).

[0081] The liposome formulation of the present invention may be provided in any form, for example, as a liposome suspension (i.e., a suspension) in which a population of liposomes is suspended in an aqueous solvent, or as a lyophilized solid (i.e., a lyophilized formulation).

[0082] In one embodiment, the liposome population is contained in the liposome formulation of the present invention as a liposome suspension. That is, the liposome formulation of the present invention can be provided as a suspension formulation (referred to as the liposome suspension formulation of the present invention). The liposome suspension formulation of the present invention comprises the above-described liposome population suspended in an aqueous solvent. The liposome suspension formulation of the present invention is suitable for administering the CD1d ligand-containing liposome population to a subject such as a human by injection. The aqueous solvent is not particularly limited, but includes water and a mixed solvent of water and a water-soluble organic solvent. The water-soluble organic solvent is not particularly limited, but includes alcohol. The alcohol is preferably a monohydric or dihydric alcohol containing 1 to 6 (preferably 1 to 3) carbon atoms. Examples of the alcohol include methanol, ethanol, isopropyl alcohol, or a combination thereof. The alcohol is preferably ethanol. The aqueous solvent is preferably water.

[0083] The aqueous solvent may contain pharmaceutically acceptable additives such as an osmotic pressure adjusting agent, a stabilizer, an antioxidant, a pH adjusting agent, and the like, as needed.

[0084] Examples of osmotic pressure adjusters include, but are not limited to, inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol; and sugars such as glucose, fructose, lactose, maltose, and sucrose.

[0085] Examples of stabilizers include, but are not limited to, sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose, and sterols such as cholesterol.

[0086] The antioxidant is not particularly limited, but examples thereof include ascorbic acid, uric acid, and tocopherol homologues (e.g., vitamin E). Tocopherol has four isomers, α, β, γ, and δ, and any of these can be used in the present invention.

[0087] The pH adjuster may be any basic or acidic compound, such as sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, L-histidine and its hydrochloride.

[0088] The aqueous solvent is preferably a buffered aqueous solution containing an osmotic pressure adjusting agent and a pH adjusting agent. The osmotic pressure adjusting agent is preferably sucrose or maltose, more preferably sucrose. The pH adjusting agent is preferably L-histidine and its hydrochloride.

[0089] In addition to the above-mentioned additives, additives such as a soothing agent and a preservative may be added to the aqueous solvent as needed.

[0090] Examples of the soothing agent include glucose, benzyl alcohol, mepivacaine hydrochloride, xylocaine hydrochloride, procaine hydrochloride, carbocaine hydrochloride, and the like.

[0091] Examples of preservatives include parahydroxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0092] When preparing a lyophilized formulation by lyophilizing the liposome suspension formulation of the present invention, it is preferable to add a cryoprotectant to the aqueous solvent to prevent liposome aggregation or fusion or lipid membrane collapse. Examples of cryoprotectants include sugars such as lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, glucosamine, galactosamine, N-methylglucosamine, mannitol, sorbitol, trehalose, and sucrose; amino acids such as glycine, alanine, lysine, and arginine; glycerin, polyethylene glycol, polyvinylpyrrolidone, and dextran.

[0093] The aqueous solvent preferably contains a sugar such as sucrose, since a single component of the sugar has multiple functions including an osmotic pressure adjusting agent, a stabilizer, and a cryoprotectant.

[0094] The aqueous solvent (i.e., liposome suspension) is preferably adjusted to be isotonic or nearly isotonic with human body fluid (plasma) (e.g., 285±100 mOsm / L). When sucrose is used as an osmotic pressure adjuster, the sucrose concentration in the aqueous solvent is preferably about 9.0 to 11.0 (10.0±1.0)% (w / v).

[0095] The pH of the aqueous solvent (i.e., liposome suspension) is adjusted to, for example, 5.3 to 7.3 (6.3±1.0), preferably 5.8 to 6.8 (6.3±0.5). In one embodiment, the aqueous solvent is a buffer solution containing sucrose, L-histidine, and L-histidine hydrochloride, which is isotonic or nearly isotonic with human body fluid (plasma) (e.g., 285±50 mOsm / L) at a pH of 5.8 to 6.8 (6.3±0.5), and the sucrose concentration is preferably 9.0 to 11.0 (10.0±1.0)% (w / v). For example, the theoretical osmotic pressure of a buffer solution containing L-histidine (15 mM), L-histidine hydrochloride (5 mM), and sucrose (10.0% (w / v)) is 311.6 mOsm / L.

[0096] The content of CD1d ligand compound-containing liposomes contained in the liposome suspension formulation of the present invention is not particularly limited, but may be, for example, 0.01 to 100 mg / ml, preferably 0.5 to 50 mg / ml, and more preferably 1.0 to 10 mg / ml (e.g., 4.5 to 5.5 (5.0±0.5) mg / ml) in terms of the concentration of the CD1d ligand compound (e.g., KRN7000).

[0097] In one embodiment, the liposome population is contained in the liposome formulation of the present invention as a lyophilized product. That is, the liposome formulation of the present invention can be provided as a lyophilized preparation (referred to as the liposome freeze-dried formulation of the present invention). The lyophilized product of the present invention can be obtained by subjecting the above-mentioned liposome population suspended in an aqueous solvent (i.e., the above-mentioned liposome suspension formulation of the present invention) to a freeze-drying process.

[0098] For example, the liposome suspension of the present invention can be divided into small portions and filled into containers such as vials, and then frozen at a temperature of about -20 to -80°C to obtain a frozen composition, which can then be subjected to reduced pressure (e.g., 10 Pa or less) to sublimate the water, thereby obtaining the liposome freeze-dried preparation of the present invention. In order to prevent aggregation or fusion of liposomes or collapse of the lipid membrane during freeze-drying, it is preferable to add the above-mentioned cryoprotectant to the liposome suspension.

[0099] The lyophilized liposomal formulation of the present invention can be dispersed in an aqueous solvent (e.g., water) to reconstitute the liposomal suspension formulation of the present invention that satisfies the above-mentioned conditions. The reconstituted liposomal suspension formulation of the present invention is then administered.

[0100] As used herein, the average particle size and polydispersity of particle size distribution of the liposome population contained in a freeze-dried formulation refer to the average particle size and polydispersity of particle size distribution of the liposome population contained in a suspension formulation obtained by dispersing and reconstituting the freeze-dried formulation in an aqueous solvent (e.g., water).

[0101] The liposome preparation of the present invention can be administered either orally or parenterally, but is particularly suitable for use in injection administration because it has improved in vivo pharmacokinetics when administered by injection, resulting in a higher maximum blood concentration of the CD1d ligand compound (e.g., KRN7000) and a longer blood half-life compared to conventional preparations. The liposome preparation of the present invention can be used, for example, for intravenous injection administration, intramuscular injection administration, intradermal injection administration, subcutaneous injection administration, or intraorgan injection administration, and is preferably used for intravenous injection administration.

[0102] When the liposome formulation of the present invention is used as a formulation for injection, it can be stored and used in a container filled with it. The container is preferably a sealed container. Examples of the sealed container include ampoules, vials, and bags. Materials for the container include glass and plastic. When the liposome formulation of the present invention is filled into a container such as an ampule or vial, the gas phase in the container space may be replaced with an inert gas. A preferred example of the inert gas is nitrogen. For example, each container is filled with liposomes containing a single injection dose of a CD1d ligand compound (e.g., KRN7000). For example, when KRN7000 is used as the CD1d ligand compound, the liposome formulation of the present invention is filled into the container so that each container contains 0.5 to 100 mg (e.g., 5.0±0.5 mg, 11.5±0.5 mg) of KRN7000.

[0103] The liposome preparation of the present invention has improved in vivo pharmacokinetics, resulting in a higher maximum blood concentration and a longer blood half-life of the CD1d ligand compound (e.g., KRN7000) compared to conventional preparations. Furthermore, the liposome preparation of the present invention has high IL-4 induction potential but low IFN-γ induction potential, and can effectively induce Tregs, so it is expected to have excellent immunosuppressive or immune tolerance-inducing effects. Therefore, the liposome preparation of the present invention is useful for the prevention or treatment of graft-versus-host disease (GVHD), organ transplant rejection, autoimmune diseases, and the like. Examples of autoimmune diseases include, but are not limited to, systemic lupus erythematosus, scleroderma, polyarteritis, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis, type 1 diabetes, rheumatoid arthritis, Sjogren's syndrome, ANCA-associated vasculitis, Takayasu's disease, Behçet's disease, adult Still's disease, relapsing polychondritis, IgA-associated vasculitis, polymyalgia rheumatica, antiphospholipid syndrome, ankylosing spondylitis, Kawasaki disease, Crohn's disease, ulcerative colitis, psoriasis vulgaris, pemphigoid, primary biliary cirrhosis, primary sclerosing cholangitis, and idiopathic interstitial pneumonia.

[0104] By administering an effective amount of the liposome preparation of the present invention to a subject (e.g., human) in need thereof, it is possible to prevent the onset of GVHD, organ transplant rejection, autoimmune disease, etc. in the subject, or to treat GVHD, organ transplant rejection, autoimmune disease, etc. In the case of preventing or treating GVHD, subjects in need thereof include people who have already received allogeneic tissue (bone marrow, blood, etc.) or cell (hematopoietic stem cell, etc.) transplantation and have not yet developed GVHD but are at high risk of developing GVHD in the future, people who have received allogeneic tissue (bone marrow, blood, etc.) or cell (hematopoietic stem cell, etc.) transplantation and have developed GVHD, and people who are scheduled to receive allogeneic tissue (bone marrow, blood, etc.) or cell (hematopoietic stem cell, etc.) transplantation. When preventing or treating organ transplant rejection, subjects in need thereof include people who have already received allogeneic or xenogeneic organ or cell transplants and have not yet developed rejection but are at high risk of developing rejection in the future, people who have received allogeneic or xenogeneic tissue or cell transplants and have developed rejection, and people who are scheduled to receive allogeneic or xenogeneic tissue or cell transplants. The present invention provides the liposome preparation of the present invention for use in the prevention or treatment of GVHD, organ transplant rejection, autoimmune diseases, etc. In one embodiment, GVHD can be caused by allogeneic hematopoietic stem cell transplantation.

[0105] As used herein, the term "effective amount" refers to an amount that produces a desired effect (e.g., a therapeutic effect) in a subject, and means, for example, that the symptoms or condition of a disease are alleviated, mitigated, or eliminated, or the progression of the disease is delayed or inhibited in a subject to whom this amount is administered, compared to a subject to whom this amount is not administered. The effective amount can be determined appropriately by a physician depending on the age, weight, sex, severity of symptoms, etc. of the subject.

[0106] As used herein, the term "prevention" refers to preventing a disease or disorder (e.g., GVHD, organ transplant rejection) from occurring before the condition develops, reducing the risk of developing such a condition, or alleviating or mitigating such a condition.

[0107] All references cited herein, including publications, patent documents, and the like, are incorporated herein by reference to the same extent as if each was individually and specifically incorporated by reference and the contents thereof were specifically set forth in their entirety.

[0108] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited thereto. [Example]

[0109] [Test Example 1] Production of a comparative formulation (hereinafter referred to as RGI-2001-001) and evaluation of its stability The manufacturing process for RGI-2001-001 is described below. Step 1: RGI-2001-001 intermediate (lyophilized product of KRN7000 / lipid mixture) Step 1a) Preparation of 90% (wt / wt) tert-butanol solution Water for injection was placed in a beaker and heated to 55-60°C. tert-Butanol was dissolved while stirring on a hot plate. The tert-butanol was poured into a pyrogen-free glass container (Pyrex), and water for injection was added to a final concentration of 90% (wt / wt). The mixture was heated to 55-60°C and stirred for 10±5 minutes.

[0110] Step 1b) Dissolving the lipid mixture The following lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG-Na), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG-Na), and cholesterol To a lipid mixture containing the above (molar ratio 15:15:15:15:40), tert-butanol was added to a final concentration of 100 g / L, and the mixture was stirred at 45±5°C until it became clear.

[0111] Step 1c) Preparation of KRN7000 / cyclohexane solution KRN7000 ((2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol) (manufactured by ReguImmune) powder was weighed into a pyrogen-free glass container (Pyrex), and cyclohexane was added to a final concentration of 10 g / L. The solution was stirred at 47±1°C for 20±5 minutes.

[0112] Step 1d) Preparation of KRN7000 / lipid mixture solution The lipid mixture solution was added to the KRN7000 / cyclohexane solution and stirred at 47±1° C. until it became transparent, thereby obtaining a KRN7000 / lipid mixture solution.

[0113] Step 1e) Freeze drying The glass container containing the KRN7000 / lipid mixture solution was carefully rotated in a dry ice / acetone bath to freeze evenly onto the inner wall of the container. After freezing, the glass container was left to stand on dry ice for at least 1 hour (maximum 24 hours). The glass container containing the frozen KRN7000 / lipid mixture was placed in a freeze dryer, and the following steps were carried out. After the product's temperature sensor reaches -40°C or below, it is frozen for at least 2±0.5 hours. After freezing was completed, the temperature of the frozen product was maintained below -40°C. The vacuum level was set to 250 microns or less. The shelf temperature was raised from -40°C or below to -35°C ±3°C over 6±0.5 hours. The shelf temperature was maintained at -35±3°C for 12±0.5 hours. The shelf temperature was raised from -35±3°C to 25±3°C over 4±0.5 hours. The product temperature sensor was maintained at 25±3°C for 12±0.5 hours until the solenoid bleed valve was disabled. After the maximum vacuum was reached, the product was kept at 25±3°C for a minimum of 83 hours. After freeze-drying was completed and the pressure inside the chamber reached atmospheric pressure, the glass container containing the product was quickly sealed with a cap and stored at -20°C. If the total weight of the freeze-dried product was 102% or more of the total weight of the KRN7000 / lipid mixture, it was further freeze-dried at 25±3°C for 24 hours.

[0114] Step 2: Manufacturing of RGI-2001-001 drug substance Step 2a) Preparation of formulation buffer Sucrose, L-histidine, and L-histidine hydrochloride hydrate were added to water for injection to a final concentration of 10%, 15 mM, and 5 mM, respectively, and the mixture was stirred for 15 minutes and adjusted to a final pH of 6.5±0.2. The resulting buffer solution was passed through a sterile filter (pore size 0.2 μm) before the next step.

[0115] Step 2b) Hydration of the RGI-2001-001 intermediate The frozen RGI-2001-001 intermediate (KRN7000 / lipid mixture) was returned to room temperature and left for 30 minutes. The amount of formulation buffer used was calculated based on the buffer density (1.04 g / mL) and the final solution density (0.055 g / mL). The formulation buffer was added to the glass container containing the RGI-2001-001 intermediate under aseptic conditions and stirred at 30-45°C for approximately 60 minutes until complete hydration.

[0116] Step 2c) High-pressure extrusion The hydrated RGI-2001-001 intermediate solution was passed five times through an extruder equipped with a polycarbonate membrane filter (0.2 μm pore size) under aseptic conditions at a target pressure of 200-300 psi (not exceeding 600 psi). The resulting filtrate was then passed once through an extruder equipped with a polycarbonate membrane filter (0.1 μm pore size). As a final step, the resulting filtrate was passed ten times through an extruder equipped with two polycarbonate membrane filters (0.1 μm pore size). The final recovered product was passed through a sterile filter (0.2 μm pore size) and stored at 2-8°C as RGI-2001-001 drug substance.

[0117] The results of accelerated (25°C) testing of RGI-2001-001 are set forth in the table below.

[0118] [Table 1]

[0119] The mean particle size of RGI-2001-001 was 124 nm immediately after manufacturing and remained at approximately 120 nm during the subsequent 6-month accelerated testing period. Furthermore, the DOPG-Na content after 6 months deviated from the specification. These results suggest that the comparative formulation (RGI-2001-001) had a uniform and stable mean particle size of approximately 120 nm.

[0120] [Test Example 2] Manufacturing method and stability evaluation of the example formulation (hereinafter referred to as RGI-2001-003) The manufacturing process for RGI-2001-003 is described below. Step 1) Preparation of formulation buffer Sucrose, L-histidine, L-histidine hydrochloride hydrate, and water for injection were added to a single-use sterile bag and the mixture was completely dissolved at room temperature. Water for injection was added to a final sucrose concentration (wt / v) of 10% and a final sucrose density of 1.038 g / mL. The mixture was then passed through a sterile filter (pore size 0.2 μm) and collected in a sterilized plastic container.

[0121] Step 2a) Dissolving RGI-7000 and lipids KRN7000 (manufactured by RegImmune) (final concentration 5 mg / mL), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (final concentration 15 mol %), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG-Na) (final concentration 15 mol %), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (final concentration 15 mol %), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DPPG-Na) (final concentration 15 mol %), and Cholesterol (final concentration 40 mol %) was added to ethanol and dissolved at 85±5°C, and ultrasonic treatment was continued until the solution became clear.

[0122] Step 2b) Preparation of RGI-2001-003 drug substance solution An ethanol solution containing KRN7000 and lipids and a formulation buffer solution were passed through a stainless steel capillary tube (SSCT) set at 85±5°C and mixed in an in-line mixer to obtain a crude RGI-2001-003 drug substance solution. The crude product was mixed with the formulation buffer solution in another in-line mixer and then cooled by passing it through an SSCT set at 20±5°C. The liposome particle size of the RGI-2001-003 drug substance was fine-tuned to an average particle size of approximately 100 nm by changing the pump speed.

[0123] Step 2c) Concentration and diafiltration The RGI-2001-003 drug substance was concentrated to 60% using a polyethersulfone resin hollow fiber membrane module (less than 500 kDa exclusion) and diafiltered with 10 times its weight of formulation buffer.

[0124] Step 2d) Adjustment of RGI-2001-003 drug substance concentration The weight of KRN7000 in the RGI-2001-003 drug substance was confirmed by high-performance liquid chromatography, and then the final concentration was adjusted to 5.0±0.5 mg / mL by dilution with water for injection and concentration by diafiltration. The mean particle size was adjusted to approximately 100 nm by dynamic light scattering.

[0125] Step 2e) Sterilization of RGI-2001-003 API The product was passed through a sterilizing filter (0.2 μm) and collected in a sterilized plastic bag. The RGI-2001-003 drug substance was stored in a refrigerator (5±3°C) protected from light until filling into vials.

[0126] The results of the accelerated (25°C) testing of RGI-2001-003 are set forth in the table below.

[0127] [Table 2]

[0128] RGI-2001-003 was manufactured according to the techniques described in Patent Documents 2 and 3, with the liposome mean particle size set to 100 nm. The KRN7000 content and lipid composition of RGI-2001-003 were identical to those of RGI-2001-001. The mean particle size was approximately 96 nm immediately after manufacture, very close to the set value, and remained at around 100 nm until the end of the accelerated testing. No deviation from the specification was observed. These results suggest that the RGI-2001-003 liposome formulation has the target set mean particle size (100 nm) and excellent long-term stability.

[0129] [Test Example 3] Pharmacokinetic analysis in human clinical trials In a phase 1 clinical trial, RGI-2001-001 was administered intravenously at a dose of 100 μg / kg (as KRN7000) within 30 minutes after transplantation to six patients undergoing hematopoietic stem cell transplantation. Two milliliters of peripheral blood was collected before transplantation and at 0.5, 1, 2, 4, 6, 8, 24, 48, 72, and 96 hours after transplantation, and plasma components were recovered. KRN7000 in each plasma sample was separated and identified using liquid chromatography-mass spectrometry (LC-MS / MS). Similarly, RGI-2001-003 was administered intravenously at a dose of 100 μg / kg (as KRN7000) within 30 minutes after hematopoietic stem cell transplantation to seven patients. Two mL of peripheral blood was collected before transplantation and 0.5, 2, 4, 6, 8, 24, and 48 hours after transplantation. Plasma components were recovered, and KRN7000 in the plasma was separated and identified by LC-MS / MS. These patients received 100 μg / kg of RGI-2001-003 intravenously once a week for five to six consecutive days. On Day 14, 2 mL of peripheral blood was collected before administration and 0.5 and 4 hours after administration. Plasma components were recovered, and KRN7000 in the plasma was separated and identified by LC-MS / MS. In addition, NKT responses and activated Treg counts (Ki-67+%) were monitored over time in patients receiving RGI-2001-003.

[0130] The maximum blood concentration (Cmax) and blood half-life (t1 / 2) were 187.6 ng / mL and 23.4 hours in the RGI-2001-001 group, respectively, while those in the RGI-2001-003 group were 881 ng / mL and 35.8 hours. These results indicate that the RGI-2001-003 group had a significantly higher maximum blood concentration (Cmax) and a significantly longer blood half-life (t1 / 2) than the RGI-2001-001 group (Table 3). Furthermore, the pharmacokinetics over time showed a typical decay curve for RGI-2001-003, whereas RGI-2001-001 showed an irregular progression (Figure 1). Plasma KRN7000 levels were similar between the first and multiple doses of RGI-2001-003, and patients' exposure to KRN7000 was similar on days 0 and 14 when receiving repeated weekly doses of RGI-2001-003 (Table 4, Figure 1). RGI-2001-003 administration induced NKT cell responses and elevated numbers of regulatory T cells (Table 5).

[0131] [Table 3]

[0132] [Table 4]

[0133] [Table 5]

[0134] [Test Example 4] Drug efficacy evaluation in mice It has been reported that RGI-2001, after intravenous administration, is taken up by splenic marginal zone B cells, releases KRN7000 intracellularly, and after binding to CD1d molecules, is presented on the cell surface, inducing IL-4 production in iNKT cells and inducing regulatory T cells (Tregs) (Non-Patent Document 1). On the other hand, when dendritic cells take up RGI-2001, it acts to induce IFN-γ production in iNKT cells.

[0135] C57BL / 6 mice (female, 8 weeks old; Claire) were injected intravenously with 2 μg of RGI-2001-001 or RGI-2001-003 (KRN7000 dose) and retro-orbital blood was collected 2 and 24 hours later. Plasma IFN-γ, IL-4, and IL-10 concentrations were measured using a Cytometric Bead Array (BD Biosciences). IFN-γ levels were lower in the RGI-2001-003 group than in the RGI-2001-001 group, whereas IL-4 levels were higher in the RGI-2001-003 group than in the RGI-2001-001 group (Figure 2). IL-10 production was similar between the two groups, or slightly higher in the RGI-2001-003 group than in the RGI-2001-001 group (Figure 3). These results suggest that RGI-2001-003, with its average particle size adjusted to approximately 100 nm, is more easily taken up by marginal zone B cells than RGI-2001-001, and as a result, RGI-2001-003 more potently induces IL-4 production from iNKT cells and more effectively induces Tregs. However, in contrast to IL-4, RGI-2001-003's ability to induce IFN-γ production from iNKT cells was lower than that of RGI-2001-001.

[0136] [Test Example 5] Measurement of particle size The particle size and polydispersity of the liposome preparation produced in Test Example 2 were measured by dynamic light scattering. <Condition 1> The liposome preparation prepared in Test Example 2 was dissolved in PBS (Ca 2+ After diluting 1000 times with 100% ethanol (containing no ethanol), the particle size and polydispersity were analyzed by DLS (dynamic light scattering, backscattering) using a Malvern ZetaSizer Nano ZS at 25° C. The results are shown in Table 6.

[0137] [Table 6]

[0138] <Condition 2> The liposome preparation prepared in Test Example 2 was dissolved in PBS (Ca 2+ The particle size and polydispersity were analyzed by DLS (dynamic light scattering, 90°C scattering). The results are shown in Table 7.

[0139] [Table 7]

[0140] From the results of these tests, the average particle size of the RGI-2001-003 liposome preparation when Test Examples 3 and 4 were carried out was estimated to be within the range of 92.9 to 101.0 nm.

[0141] [Test Example 7] In Test Example 3, seven patients administered RGI-2001-003 were followed up. One of the seven patients transiently developed grade II acute GVHD, and one developed grade I acute GVHD (not counted as GVHD). However, the other five patients did not develop acute GVHD. The patient who developed grade II acute GVHD had mild, transient skin lesions, and no recurrence of GVHD was observed for more than one year after hematopoietic stem cell transplantation. These results suggest that RGI-2001-003 has an excellent inhibitory effect on the development of GVHD.

[0142] [Table 8] [Industrial Applicability]

[0143] The present invention provides a liposome preparation containing KRN7000 that can be retained in the blood for a long period of time and maintain a high blood KRN7000 concentration for a long period of time. The liposome preparation of the present invention is expected to have excellent preventive or therapeutic effects against GVHD.

[0144] This application is based on patent application No. 2020-201802 filed in Japan (filing date: December 4, 2020), the contents of which are incorporated in their entirety herein.

Claims

1. A liposome preparation comprising a population of liposomes containing (2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol, wherein the liposome population has an average particle size of 92.9 to 101.0 nm and a polydispersity index of particle size distribution of 0.133 or less.

2. The liposome preparation according to claim 1, wherein the number of liposomes having a particle size of less than 50 nm is 10% or less of the total population of liposomes.

3. 3. The liposome preparation according to claim 1, wherein the number of liposomes having a particle diameter of more than 450 nm is 10% or less of the total population of liposomes.

4. 4. The liposome preparation according to claim 1, wherein the liposome preparation maintains an average particle size of 90 to 110 nm for at least one month under conditions of a temperature of 25°C and a relative humidity of 60% RH.

5. 5. The liposome formulation of claim 1, wherein the population of liposomes is contained as a liposome suspension.

6. 6. The liposome preparation according to claim 5, wherein the pH of the liposome suspension is 5.8 to 6.

8.

7. 5. The liposome formulation of claim 1, wherein the population of liposomes is contained as a lyophilizate.

8. The liposome preparation according to any one of claims 1 to 7, which is for administration by injection.

9. The liposome preparation according to any one of claims 1 to 8, which is for the prevention or treatment of graft-versus-host disease.

10. The liposome preparation according to claim 9, wherein the graft-versus-host disease is caused by allogeneic hematopoietic stem cell transplantation.

11. The liposome preparation according to any one of claims 1 to 8, which is for the prevention or treatment of organ transplant rejection.

12. 12. The liposome preparation according to claim 11, wherein the organ transplant is an allogeneic organ or cell transplant.

13. 12. The liposome preparation according to claim 11, wherein the organ transplant is a transplant of a heterologous organ or cell.

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