Liposome formulation containing CD1d ligand compound with improved pharmacokinetics

Abstract

It can remain in the bloodstream for a long period, allowing for the maintenance of high blood concentrations of the active ingredient over extended periods. To provide a liposome formulation containing a CD1d ligand compound that can perform the necessary functions. [Solution] The present invention provides a liposome formulation comprising a group of liposomes containing a CD1d ligand compound, wherein the average particle size of the group of liposomes is 90 to 110 nm, and the polydispersity of the particle size distribution is 0.2 or less. The present invention also provides a method for producing the formulation.

Description

[Technical Field]

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

[0002] Graft-versus-host disease (GVHD) is a complication associated with allogeneic hematopoietic stem cell transplantation, in which donor-derived lymphocytes attack the recipient's organs, mistaking them for foreign bodies. It is a condition in which the immune system of the blood donor attacks and destroys the recipient's tissues throughout the body. There are two types: acute GVHD in the early stages of transplantation and chronic GVHD in the later stages. GVHD is also known to develop as a result of 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 prevent or improve T-cell-mediated diseases such as autoimmune diseases and graft rejection by restoring immune tolerance to both autoantigens and alloantigens. The crucial role of Tregs in controlling GVHD has been demonstrated in both human and mouse studies. However, the small size of this cell population has hindered the development of Treg-based therapies. The discovery of molecules that efficiently increase functional Tregs in vivo could contribute to the development of novel therapies not only for GVHD but also for other immune diseases. A variety of strategies for activating and increasing Tregs in situ are emerging.

[0004] α-galactosylceramide (α-GalCer) functions as a ligand for the CD1d molecule expressed on antigen-presenting cells. The CD1d molecule is a non-polymorphic tumor histocompatibility complex (MHC) class I-like antigen-presenting molecule with an antigen-binding groove adopted for lipid antigen presentation. When α-GalCer is presented to CD1d molecules expressed on various cell types such as dendritic cells (DCs), macrophages, and B cells, it is recognized by invariant T cells (TCRs) expressed on invariant NKT (iNKT) cells, and CD1d-restricted activation of iNKT cells occurs. CD1d-restricted iNKT cell activation results in the rapid and massive release of both Th1 and Th2 cytokines, a unique characteristic 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, which was originally discovered in sponges.

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

[0006] The inventors demonstrated that liposomal KRN7000 (RGI-2001) can induce alloantigen-specific tolerance through Treg induction (Non-Patent Literature 3). In a mouse acute GVHD model, a single dose of RGI-2001 significantly extended mouse survival. Enhanced proliferation of donor-derived CD4+Foxp3+ Tregs was found to be the key mechanism. Host alloantigen-specific immunosuppression was induced early after bone marrow transplantation (BMT), but responses to third-party alloantigens and leukocytes were not suppressed. Furthermore, RGI-2001 also showed a symptom-reducing effect in a mouse chronic GVHD model. These results suggest that RGI-2001 could be a novel treatment for preventing both acute and chronic GVHD (Non-Patent Literature 4).

[0007] The applicability of RGI-2001 to GVHD has also been confirmed in human clinical trials (Non-Patent Literature 5). A Phase 1 / 2a clinical trial was conducted in which 29 patients who underwent allogeneic hematopoietic stem cell transplantation received a single intravenous dose of RGI-2001 on day 0. In some patients treated with RGI-2001, the number of Tregs (CD4+CD25+CD127loFoxp3+) increased significantly within 1–3 weeks post-transplant. Patients with a response accompanied by an increase in Treg count showed a more pronounced reduction in GVHD compared to non-responders.

[0008] The applicability of RGI-2001 to organ transplantation has been confirmed in a mouse heart transplantation model (Non-Patent Literature 6, 7). When recipient mice were irradiated with a sublethal dose of radiation and then transplanted with spleen cells and bone marrow cells from a donor mouse, RGI-2001 and a CD40-CD40L blocking antibody were administered simultaneously. This resulted in the formation of bone marrow chimeras in the recipient mice, and the rejection of donor-derived hearts and skin was suppressed for a long period of time.

[0009] Patent Document 2 discloses a liposome manufacturing technique capable of easily controlling the concentration of a dialysate (for example, liposome solution) after dialysis and obtaining a dialysate (for example, liposome solution) having a desired concentration in the step of dialysis using a hollow fiber dialysis column.

[0010] Patent Document 3 discloses a manufacturing technique for lipid particles, in which an alcohol-containing solution in which lipids are dissolved is first diluted at an alcohol concentration at which lipid particles are destabilized, and then further diluted to obtain stabilized particles. Here, by adjusting the retention time from the first dilution to the second dilution, it is possible to control the particle diameter of the lipid particles while maintaining a uniform particle size distribution.

[0011] Non-Patent Document 8 discloses the results of analyzing the in vivo behavior of liposomes with various particle diameters using the PET technique.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0014] As mentioned above, in the Phase 1 / 2a clinical trial of liposomal KRN7000 for the prevention of GVHD, some patients showed a significant therapeutic response, while others did not show a sufficient response. Upon investigating the cause, it was found that the pharmacokinetics were unstable, with plasma KRN7000 concentrations being low immediately after administration, then rising temporarily, but then decreasing again. This result suggests that long-term retention in the bloodstream immediately after administration is important for liposomal KRN7000 to exert its full efficacy, and that the formulation used in the Phase 1 / 2a clinical trial (RGI-2001-001) may not have met this condition.

[0015] The present invention aims to provide a CD1d ligand-containing liposome formulation that can remain in the bloodstream for a long period of time and maintain a high blood CD1d ligand concentration for an extended period. [Means for solving the problem]

[0016] The inventors diligently investigated the above problem and discovered that the factor causing the instability in blood pharmacokinetics was the particle size of the liposomes used in the Phase 1 / 2a clinical trial. The average 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, without changing the KRN7000 content and lipid composition from RGI-2001-001, a new liposomal formulation (RGI-2001-003) was prepared with an average particle size of around 100 nm. When this was administered intravenously to patients after hematopoietic stem cell transplantation, surprisingly, the pharmacokinetics improved, the peak blood concentration of KRN7000 increased, and the blood half-life was extended. When these formulations were administered intravenously to mice and plasma concentrations of IFN-γ and IL-4 were measured, IL-4 was higher in the RGI-2001-003 administration group than in the RGI-2001-001 administration group, but IFN-γ was lower in the RGI-2001-003 administration group than in the RGI-2001-001 administration group, suggesting that RGI-2001-003 may have properties that more effectively induce Treg. Based on these findings, the inventors conducted further investigations and completed the present invention.

[0017] In other words, the present invention relates to the following: [1] A liposome formulation comprising a group of liposomes containing a CD1d ligand compound, wherein the average particle size of the group of liposomes is 90 to 110 nm, and the polydispersity of the particle size distribution is 0.2 or less. [2] A liposome formulation of [1] having an average particle size of 92.9–101.0 nm. [3] A liposome formulation of [1] or [2], wherein the polydispersity of the particle size distribution is 0.133 or less. [4] A liposome formulation according to any of [1] to [3], wherein the number of liposomes with a particle size of less than 50 nm is 10% or less of the total population of liposomes. [5] A liposome formulation according to any of [1] to [4], wherein the number of liposomes with a particle size greater than 450 nm is 10% or less of the total population of liposomes. [6] A liposome formulation of any of [1] to [5], wherein the average particle size is maintained at 90–110 nm for at least one month under conditions of 25°C and 60% RH relative humidity. [7] A liposomal formulation of any of [1] to [6], wherein the CD1d ligand compound is α-galactosylceramide. [8] A liposomal formulation of [7] in which α-galactosylceramide is (2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecantriol. [9] A liposome formulation comprising the group of liposomes as a liposome suspension, any of [1] to [8].

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

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

[12] A liposomal formulation for injectable administration, one of the formulations [1] to

[11] .

[13] A liposomal formulation of any of [1] to

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

[14] A liposomal formulation of graft-versus-host disease caused by allogeneic hematopoietic stem cell transplantation,

[13] .

[15] A liposomal formulation of any of [1] to

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

[16] A liposomal preparation of an organ transplant in which an organ transplant is the transplantation of allogeneic organs or cells,

[15] .

[17] A liposomal preparation of an organ transplant in which a different organ or cell is transplanted,

[15]

[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 an effective amount of any of the liposomal formulations [1] to

[12] to the subject.

[19] The method of

[18] , wherein the subject at risk of developing graft-versus-host disease is a subject who has received 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 an effective amount of any of the liposomal formulations [1] to

[12] to the subject.

[21] Graft-versus-host disease caused by allogeneic hematopoietic stem cell transplantation, by any of the methods described in

[18] to

[20] .

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

[12] to the subject.

[23] The method of

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

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

[12] to the subject.

[25] Organ transplantation is the transplantation of allogeneic organs or cells, by any of the methods described in

[22] to

[24] .

[26] Organ transplantation is the transplantation of a different organ or cells, by any of the methods described in

[22] to

[24] .

[27] A liposomal formulation of any of [1] to

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

[28] A liposomal formulation of graft-versus-host disease caused by allogeneic hematopoietic stem cell transplantation,

[27]

[29] Any liposomal formulation of [1] to

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

[30] A liposomal preparation of an organ transplant in which an organ transplant is the transplantation of an allogeneic organ or cell,

[29]

[31] A liposomal preparation of an organ transplant in which a different organ or cell is transplanted.

[29]

[32] Use of any of the liposome formulations [1] to

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

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

[32] use.

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

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

[35] The use of organ transplantation as the transplantation of allogeneic organs or cells,

[34] .

[36] The use of organ transplantation as the transplantation of xenoorgans or cells,

[34] .

[0018] A liposome formulation comprising a group of liposomes containing [1A](2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol, wherein the average particle size of the group of liposomes is 92.9 to 101.0 nm, and the polydispersity of the particle size distribution is 0.133 or less. [2A] The liposome formulation of [1A], wherein the number of liposomes with a particle size of less than 50 nm is 10% or less of the total population of liposomes. [3A] A liposome formulation of [1A] or [2A], wherein the number of liposomes with a particle size greater than 450 nm is 10% or less of the total population of liposomes. [4A] A liposome formulation of any of [1A] to [3A], wherein the average particle size is maintained at 90-110 nm for at least one month under conditions of 25°C and 60% RH relative humidity. [5A] A liposome formulation according to any of [1A] to [4A], wherein the group of liposomes is contained as a liposome suspension. [6A] A liposome formulation of [5A], wherein the pH of the liposome suspension is 5.8 to 6.8. [7A] A liposome formulation according to any of [1A] to [4A], wherein the group of liposomes is contained as a lyophilized product. [8A] A liposomal formulation of any of [1A] to [7A] for injectable administration. [9A] A liposomal formulation of any of [1A] to [8A] for the prevention or treatment of graft-versus-host disease. [10A] A liposomal formulation of [9A] in which graft-versus-host disease is caused by allogeneic hematopoietic stem cell transplantation. [11A] A liposomal formulation of any of [1A] to [8A] for the prevention or treatment of organ transplant rejection. [12A] A liposomal preparation of [11A], in which organ transplantation is the transplantation of allogeneic organs or cells. [13A] A liposomal preparation of [11A] an organ transplant in which a different organ or cell is transplanted. [Effects of the Invention]

[0019] The present invention provides a CD1d ligand-containing liposomal formulation that can remain in the bloodstream for a long period and maintain high blood CD1d ligand concentrations for an extended period. Since the liposomal formulation of the present invention can effectively induce Tregs, excellent preventive or therapeutic effects against GVHD, organ transplant rejection, autoimmune diseases, and the like can be expected. [Brief explanation of the drawing]

[0020] [Figure 1] Figure 1 shows the changes in serum KRN7000 concentration in patients who received RGI-2001 after hematopoietic stem cell transplantation. [Figure 2] Figure 2 shows the blood IFN-γ and IL-4 concentrations in mice administered RGI-2001. [Figure 3] Figure 3 shows the blood IL-10 concentration in mice administered RGI-2001. [Modes for carrying out the invention]

[0021] The present invention provides a liposome formulation (the liposome formulation of the present invention) comprising a group of liposomes containing a CD1d ligand compound.

[0022] The "liposome preparation" means a pharmaceutical composition containing an active ingredient encapsulated in liposomes.

[0023] The "population of liposomes" refers to an aggregate of a plurality of liposomes. The number of liposomes constituting the population of liposomes of the present invention is usually 10 3 or more (for example, 10 4 or more, 10 5 or more, 10 6 or more, 10 7 or more, 10 8 or more, 10 9 or more, 10 10 or more, 10 11 or more, 10 12 or more). The upper limit value of the number of liposomes constituting the population of liposomes of the present invention is not particularly limited, but for example, it may be 10 21 or less, 10 20 or less, 10 19 or less, 10 18 or less, 10 17 or less, 10 16 or less.

[0024] A "CD1d ligand compound" refers to a compound that, when presented on a CD1d molecule 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-restrictive 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, p.1786-1789, 2004), and OCH (Nature 413:531, 2001). α-glycosylceramide is a sphingoglycolipid in which ceramide is linked to a sugar such as galactose or glucose in an α-coordinate. α-glycosylceramide in which the sugar portion is galactose is called α-galactosylceramide. Examples of α-glycosylceramides include those disclosed in WO93 / 05055, WO94 / 02168, WO94 / 09020, WO94 / 24142, WO98 / 44928, Science, 278, pp. 1626-1629, 1997, etc., but are not limited to these.

[0025] Examples of α-glycosylceramides include the compound of formula (I) below, or its salt or solvate.

[0026] [ka]

[0027] (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 (a) to (e) below (where Y is an integer from 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 H R 4 is H, OH, NH2, NHCOCH3, or the following groups (A)~(D):

[0028] [ka]

[0029] A substituent selected from the group consisting of, R 5 is OH, or the following groups (E) and (F):

[0030] [ka]

[0031] A substituent selected from the group consisting of, R 7 is OH or the following groups (A)~(D):

[0032] [ka]

[0033] A substituent selected from the group consisting of, R 9 is H, CH3, CH2OH, or the following groups (A')~(D'):

[0034] [ka]

[0035] A substituent selected from the group consisting of; ii)R 3 , R 6 and R 7 When H R 4 is H, OH, NH2, NHCOCH3, or the following groups (A)~(D):

[0036] [ka]

[0037] A substituent selected from the group consisting of, R 5 is OH, or the following groups (E) and (F):

[0038] [ka]

[0039] A substituent selected from the group consisting of, R 8 is OH, or the following groups (A)~(D):

[0040] [ka]

[0041] A substituent selected from the group consisting of, R 9 is H, CH3, CH2OH or the following groups (A')~(D'):

[0042] [ka]

[0043] (A substituent selected from the group consisting of the following.)

[0044] As for α-galactosylceramide, R 3 , R 6 and R 8 H is R 4 , R 5 and R 7 is OH, and R 9 Examples include the compound of formula (I) above, or its salt or solvate, where CH2OH is present.

[0045] α-galactosylceramide is preferably (2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol ((2S,3S,4R)-1-O-(α-D-galactosyl)-N-hexecosanoyl-2-amino-1,3,4-octadecanetriol, also known as KRN7000). KRN7000 has the following chemical structure.

[0046] [ka]

[0047] The liposomes contained in the liposome formulation of the present invention are characterized by having an average particle size of 90 to 110 nm and a polydispersity (PdI) of 0.2 or less in the particle size distribution. 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 peak blood concentration of KRN7000, and extends the blood half-life. The average particle size of the liposomes of the present invention may be 90 nm or larger (preferably 91.0 nm or larger, 92.0 nm or larger, 92.5 nm or larger, 92.9 nm or larger, 93.0 nm or larger, 93.1 nm or larger, 93.5 nm or larger, 94.0 nm or larger, 94.5 nm or larger, 94.6 nm or larger, 95.0 nm or larger, 95.5 nm or larger, 95.7 nm or larger, 95.8 nm or larger, 95.9 nm or larger, 96.0 nm or larger, or 96.2 nm or larger). The average particle size of the liposome population of the present invention may 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, and 101.0 nm or less). In one embodiment, the average particle size of the liposome population of the present invention may be 92.9-101.0 nm, 93.1-101.0 nm, 93.5-101.0 nm, 94.6-101.0 nm, 95.7-101.0 nm, 95.8-101.0 nm, or 95.9-101.0 nm.

[0048] The group of liposomes contained in the liposome formulation of the present invention is homogeneous with respect to its particle size, and the polydispersity of the particle size distribution may 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, 0.102 or less).

[0049] In this specification, "particle size" is a measure used to represent the size of a particle, as is commonly used in this field, and is a convenient value corresponding to the diameter of a particle assuming it is a perfect sphere. In this specification, "average particle size" may refer to either the number-average particle size or the Z-average particle size, but unless otherwise specified, it refers to the Z-average particle size calculated from the measured particle size. In this specification, "particle size distribution" is used in the common sense used in this field and refers to the spread of particle size. Polydispersion (PDI) is used as a measure to represent particle size distribution. The average particle size of a liposome population and the polydispersity of the particle size distribution can be measured by DLS (dynamic light scattering, backscattering) using Malvern ZetaSizer Nano ZS (e.g., condition 1 in Test Example 5). If necessary, the formulation is PBS (Ca 2+ It may be diluted (without containing any other ingredients).

[0050] In a preferred embodiment, in the group of liposomes contained in the liposome formulation of the present invention, the number of liposomes with a particle size of less than 50 nm is limited to 10% or less of the total (for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less).

[0051] In a preferred embodiment, in the group of liposomes contained in the liposome formulation of the present invention, the number of liposomes with a particle size greater than 450 nm is limited to 10% or less of the total (for example, 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).

[0052] The liposomes constituting the group of liposomes 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 target mammal when the liposome formulation of the present invention is administered to the target mammal. The content (by weight) of the CD1d ligand compound (e.g., KRN7000) in the liposomes constituting the group of liposomes of the present invention may 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 (i.e., lipids) that form the liposome. In this specification, unless otherwise specified, "components forming liposomes" refers to the components of the lipid bilayer that forms liposomes and the CD1d ligand compounds encapsulated within the liposomes, and does not include any other components in the inner or outer aqueous phases of the liposomes.

[0053] Other components besides the CD1d ligand compound that forms the liposomes can be any amphiphilic molecule capable of forming micelles, and lipids are preferred. 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), as well as sphingoglycolipids and glyceroglycolipids, which are used to produce liposomes, either alone, in combination of two or more, or in combination with nonpolar substances such as cholesterol or lipid derivatives in which water-soluble polymers such as polyethylene glycol are bound to lipids. In one embodiment, the liposomes constituting the group of liposomes of the present invention, 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.

[0054] When the liposomes constituting the group of liposomes contained in the liposome formulation of the present invention include DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho, the composition ratio 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, Furthermore, 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.

[0055] In one embodiment, the liposomes constituting the group of liposomes contained in the liposome formulation of the present invention include a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho as lipid bilayer forming components. In one embodiment, the lipid bilayer forming components of the liposomes constituting the group 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 (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

[0056] The structure of liposomes is not particularly limited as long as they consist of small vesicles with a lipid bilayer membrane structure; they may be unilamellar, multilamellar, or any other type of liposome.

[0057] The solution encapsulated inside the liposome (internal solution) can be water, buffer solution, physiological saline, or other solutions. It is also possible to add an appropriate amount of a water-soluble organic solvent (such as glycerin) to these solutions. The internal solution of the liposome may also contain additives such as osmotic pressure regulators, stabilizers, antioxidants, and pH adjusters.

[0058] Osmotic pressure regulators are not particularly limited, but examples include 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, or sucrose.

[0059] Stabilizers are not particularly limited, but examples include sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose, and sterols such as cholesterol.

[0060] While not particularly limited, antioxidants include, for example, ascorbic acid, uric acid, and tocopherol congeners (e.g., vitamin E). Note that tocopherol has four isomers, α, β, γ, and δ, and any of them can be used in this invention.

[0061] Examples of pH adjusting agents include any basic or acidic compound, such as sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, L-histidine, and their hydrochloride salts.

[0062] The internal solution of the liposome is preferably a buffered aqueous solution containing an osmotic pressure regulator and a pH adjuster. The osmotic pressure regulator is preferably sucrose or maltose, and more preferably sucrose. The pH adjuster is preferably L-histidine and its hydrochloride salt. The internal solution of the liposome is preferably adjusted to be isotonic or nearly isotonic (e.g., 285 ± 50 mOsm / L) with human body fluid (plasma). When sucrose is used as the osmotic pressure regulator, the sucrose concentration of 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 regulator, the maltose concentration of 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, for example, 5.3 to 7.3 (6.3 ± 1.0), and is preferably adjusted to 5.8 to 6.8 (6.3 ± 0.5). In one embodiment, the internal solution of the liposome is an isotonic or nearly isotonic (e.g., 285 ± 50 mOsm / L) buffer aqueous solution with a pH of 5.8 to 6.8 (6.3 ± 0.5) that contains 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 one embodiment, the internal solution of the liposome is an isotonic or nearly isotonic (e.g., 285±50 mOsm / L) buffer aqueous solution with a pH of 5.8 to 6.8 (6.3±0.5) human body fluid (plasma) containing maltose, L-histidine, and L-histidine hydrochloride, and the maltose concentration is preferably 9.0 to 11.0 (10.0±1.0)% (w / v).

[0063] The liposome formulation of the present invention can be prepared using known liposome manufacturing techniques. For example, methods described in Liposome Technology, vol. 1, 2nd edition (by Gregory Gregoriadis (CRC Press, Boca Raton, Ann Arbor, London, Tokyo), Chapter 4, pp67-80, Chapter 10, pp167-184 and Chapter 17, pp261-276 (1993)) can be used. More specifically, methods such as sonication, ethanol injection, French press, ether injection, cholic acid, calcium fusion, freeze-thaw, and reverse-phase evaporation can be used, but are not limited thereto. By using in-line liposome manufacturing techniques described in WO 2016 / 024510 and WO 2019 / 088193, etc., a population of liposomes of the present invention can be continuously prepared in a closed capillary tube.

[0064] From the viewpoint of producing a population of liposomes with uniform particle size, the liposome formulation of the present invention is preferably manufactured according to the method described in WO 2019 / 088193. The present invention also provides a method for manufacturing such a liposome formulation of the present invention.

[0065] The manufacturing method of the present invention includes, for example, the following steps. A) A step of preparing a primary dilution solution by mixing a first solution containing a CD1d ligand (e.g., KRN7000), lipids, and alcohols with a second solution containing water in a first mixing area; B) A step of supplying the primary diluted solution from the first mixing area to the second mixing area through a liquid supply pipe at a predetermined time; C) A step of preparing a secondary dilution solution by mixing the primary dilution solution with a third solution containing water in the second mixing area; D) A step of supplying the secondary diluted solution from the second mixing region to the third mixing region through a liquid supply pipe at a predetermined time; and E) A step of preparing a tertiary dilution by mixing the secondary dilution solution with a fourth solution containing water (which may be defined similarly to any of the first, second, and third solutions) in a third mixing area. Here, the average particle size of the liposome population produced is controlled to the desired range described above (typically, an average particle size of 90-110 nm and a polydispersity (PdI) of the particle size distribution of 0.2 or less) by adjusting at least one condition selected from the group consisting of the concentration of the alcohol in the primary dilution solution, the concentration of the lipid, the predetermined time, and the temperature at the time of mixing. In one embodiment, the preparation of a solution containing a CD1d ligand (e.g., KRN7000), a lipid, and an alcohol may include the step of dissolving the CD1d ligand (e.g., KRN7000) and the lipid in the alcohol. Heating may be applied during the dissolution process.

[0066] The average particle size of the liposome population can be adjusted by adjusting at least one of the following: the alcohol concentration in the primary dilution solution, the lipid concentration, the predetermined delivery time, and the mixing temperature. By gradually adjusting the alcohol concentration, the average particle size of the liposome population can be finely tuned. Furthermore, by gradually adjusting the alcohol concentration and then the predetermined delivery time, a liposome population with a desired average particle size can be produced. In this case, a particle size distribution that is narrow enough to be acceptable for pharmaceutical use can be achieved. Moreover, by adjusting the mixing temperature, the average particle size of the liposome population can be further precisely tuned.

[0067] As the lipids contained in the first solution, the above-mentioned components can be used as components other than the CD1d ligand compound that forms the liposomes constituting the group of liposomes contained in the liposome formulation 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 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, Furthermore, 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 (by weight) of the CD1d ligand compound (e.g., KRN7000) in the first solution may be, for example, 1-21% (w / w), preferably 5-15% (w / w), more preferably 7-13% (w / w), and even more preferably 9-11% (w / w) (10±1% (w / w)) of the total weight of components other than the CD1d ligand compound (e.g., KRN7000) that form liposomes (i.e., lipids).

[0068] In one embodiment, the first solution contains, as a component other than alcohol, a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In another embodiment, the component other than alcohol in the first solution consists of a CD1d ligand compound (e.g., KRN7000), DOPC, DOPG-Na, DPPC, DPPG-Na, and Cho. In these embodiments, the relative content (weight) of each component is 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

[0069] The alcohol contained in the first solution includes a monohydric or dihydric alcohol containing 1 to 6 carbon atoms. Alternatively, the alcohol contained in the first solution includes a monohydric or dihydric alcohol. In another embodiment, the alcohol contained in the first solution includes a monohydric alcohol. In a particular embodiment, the alcohol contained in the first solution includes a monohydric alcohol containing 1 to 3 carbon atoms. In a specific embodiment, the alcohol contained in the first solution includes methanol, ethanol, or isopropyl alcohol, or a combination thereof. Preferably, the alcohol contained in the first solution is ethanol.

[0070] In one embodiment, the second solution and / or the third solution may contain the alcohol present in the first solution at a lower concentration than the first solution.

[0071] 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 described in WO 2019 / 088193.

[0072] Any solution used in the manufacturing method of the present invention, including the first solution, the second solution, and the third solution, may optionally contain additives such as osmotic pressure regulators, stabilizers, antioxidants, and pH adjusters. Examples of additives such as osmotic pressure regulators, stabilizers, antioxidants, and pH adjusters are as described above.

[0073] The second and third solutions are preferably buffered aqueous solutions. The second and third solutions preferably contain an osmotic pressure regulator and a pH adjuster. The osmotic pressure regulator is preferably sucrose. The pH adjuster is preferably L-histidine and its hydrochloride salt. The second and third solutions are preferably adjusted to be isotonic or nearly isotonic (e.g., 285 ± 50 mOsm / L) with human body fluid (plasma). When sucrose is used as the osmotic pressure regulator, 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 regulator, 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). In one embodiment, the second and third solutions are buffer aqueous solutions that are isotonic or nearly isotonic (e.g., 285±50 mOsm / L) with respect to human body fluids (plasma) containing 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 one embodiment, the second and third solutions are buffer aqueous solutions that are isotonic or nearly isotonic (e.g., 285±50 mOsm / L) with respect to human body fluids (plasma) containing 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 and third solutions 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, including the first, second, and third solutions, and any solution containing water used in the production method of the present invention is, for example, 5.3 to 7.3 (6.3 ± 1.0), preferably 5.8 to 6.8 (6.3 ± 0.5). By doing so, the pH of the suspension of the liposome population or the internal solution of the liposomes obtained in the final product 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).

[0074] When the alcohol concentration (by weight) in the primary dilution solution is adjusted to a certain value (also called the "flowability point"), the average particle size of the liposome population changes over time, while conversely, at alcohol concentrations below the flowability point, the particle size of the liposomes hardly changes. In one embodiment, the flowability point may vary depending on the composition of the liposomes, temperature, and pressure. In one embodiment, the flowability point may vary depending on the type of alcohol in the primary dilution solution. In one embodiment, the flowability point may vary depending on the composition of the liposomes and / or the presence or absence of a drug supported on the liposomes, provided the type of alcohol in the primary dilution solution is the same.

[0075] In one embodiment, in the step of delivering the primary dilution solution from the first mixing region to the second mixing region through a delivery pipe over a predetermined time, the alcohol concentration and temperature are adjusted so that the alcohol concentration in the primary dilution solution is above the fluidity transition point. For example, the delivery pipe is heated to 85±5°C. Furthermore, in the step of delivering the secondary dilution solution from the second mixing region to the third mixing region through a delivery pipe over a predetermined time, the alcohol concentration and temperature are adjusted so that the alcohol concentration in the secondary dilution solution is below or equal to the fluidity transition point. For example, the delivery pipe is set to 20±5°C. That is, the step of delivering the primary dilution solution from the first mixing region to the second mixing region through a delivery pipe over a predetermined time is performed at a temperature above the phase transition temperature of the lipids in the primary dilution solution, and the step of delivering the secondary dilution solution from the second mixing region to the third mixing region through a delivery pipe over a predetermined time is performed at a temperature below the phase transition temperature of the secondary dilution solution. By controlling the reaction conditions in this way, in the primary dilution solution, the liposomes or their membranes become unstable, and heating above the phase transition temperature increases the fluidity of the lipids, thus increasing the frequency of fusion when they come into contact with each other due to Brownian motion, etc. Therefore, the fusion of the generated liposomes proceeds uniformly over time, and the particle size increases while maintaining a constant particle size distribution. On the other hand, 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 so as not to change.

[0076] Stainless steel capillary tubes (SSCTs) can be used as the liquid delivery pipes in steps B and D, and the reaction mixture may be mixed using an in-line mixer.

[0077] In one embodiment, the average particle size of the liposome population can be controlled to the desired range described above (typically, an average particle size of 90-110 nm and a polydispersity (PdI) of the particle size distribution of 0.2 or less) by adjusting the predetermined time (holding time) for the primary dilution solution to reach the second mixing region from the first mixing region (if any, a predetermined time for the secondary dilution solution to reach the third mixing region from the second mixing region). In one embodiment, the predetermined time for the primary dilution solution to reach the second mixing region from the first mixing region (if any, the time for the secondary dilution solution to reach the third mixing region) is controlled by at least one of the length and velocity of the flow path between the mixing regions. In one embodiment, the time for the primary dilution solution to reach the second mixing region from the first mixing region is controlled by the velocity between the first and second mixing regions.

[0078] Furthermore, by controlling the temperature in each step of the manufacturing method of the present invention, the lipid concentration in the primary dilution solution, the pressure in each step, etc., the average particle size of the liposome population can be controlled to the above-mentioned desired range (typically, an average particle size of 90 to 110 nm and a polydispersity (PdI) of the particle size distribution of 0.2 or less).

[0079] In one embodiment, the manufacturing method of the present invention includes a step of adjusting the composition of the solution after preparing a group of liposomes. In one embodiment, the manufacturing method of the present invention includes a step of adjusting the liposome concentration after preparing a group of liposomes. The step of adjusting the composition of the solution and the step of adjusting the liposome concentration may be performed simultaneously or separately. For example, the steps of adjusting the composition of the solution and adjusting the liposome concentration can be performed simultaneously by using a hollow fiber membrane column, as disclosed in International Publication No. 2016 / 024510. Means for adjusting the liposome concentration and solution composition in the solution containing the prepared group of liposomes include, but are not limited to, ultrafiltration and dialysis. For example, this step adjusts the pH of the solution containing the prepared group of liposomes (liposome suspension) to 5.3 to 7.3 (6.3 ± 1.0), preferably 5.8 to 6.8 (6.3 ± 0.5).

[0080] In one embodiment, the surface of the liposomes constituting the group of liposomes contained in the liposome formulation of the present invention may be modified with a modifier. Examples of modifiers include, but are not limited to, polyethylene glycol (PEG), Ficol, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, polyvinylpyrrolidone, polyvinyl methyl ether, polyvinyl methyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethylcellulose, hydroxyethylcellulose, polyaspartamide, synthetic polyamino acids, and derivatives thereof. Modification of liposomes with PEG or PEG derivatives may make them more likely to remain in the bloodstream for a longer period. Furthermore, modifying liposomes with targeting molecules (e.g., antibodies) that have affinity for specific tissues may make it easier for liposomes to reach target tissues. In one embodiment, the surface of the liposomes constituting the group of liposomes contained in the liposome formulation of the present invention is not modified with a modifying agent.

[0081] In one embodiment, the liposome formulation of the present invention is stable and maintains an average particle size of 90-110 nm (preferably 92.9-101.0 nm, 93.1-101.0 nm, 93.5-101.0 nm, 94.6-101.0 nm, 95.7-101.0 nm, 95.8-101.0 nm, 95.9-101.0 nm) for at least one month (for example, 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 testing conditions).

[0082] 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 group of liposomes is suspended in an aqueous solvent, or as a lyophilized solid (i.e., a lyophilized formulation).

[0083] In one embodiment, the group of liposomes is included 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 group of liposomes described above suspended in an aqueous solvent. The liposome suspension formulation of the present invention is suitably used for administering a group of CD1d ligand-containing liposomes 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 alcohols include methanol, ethanol, isopropyl alcohol, or combinations thereof. The alcohol is preferably ethanol. The aqueous solvent is preferably water.

[0084] The aqueous solvent may, if necessary, contain pharmaceutically acceptable additives such as osmotic pressure regulators, stabilizers, antioxidants, and pH adjusters.

[0085] Osmotic pressure regulators are not particularly limited, but examples include 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, or sucrose.

[0086] Stabilizers are not particularly limited, but examples include sugars such as glycerol, mannitol, sorbitol, lactose, or sucrose, and sterols such as cholesterol.

[0087] While not particularly limited, antioxidants include, for example, ascorbic acid, uric acid, and tocopherol congeners (e.g., vitamin E). Note that tocopherol has four isomers, α, β, γ, and δ, and any of them can be used in this invention.

[0088] Examples of pH adjusting agents include any basic or acidic compound, such as sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, L-histidine, and their hydrochloride salts.

[0089] The aqueous solvent is preferably a buffered aqueous solution containing an osmotic pressure regulator and a pH adjuster. The osmotic pressure regulator is preferably sucrose or maltose, and more preferably sucrose. The pH adjuster is preferably L-histidine and its hydrochloride salt.

[0090] In addition to the additives mentioned above, other additives such as pain relievers and preservatives may be added to the aqueous solvent as needed.

[0091] Examples of pain relievers include glucose, benzyl alcohol, mepivacaine hydrochloride, xylocaine hydrochloride, procaine hydrochloride, and carbocaine hydrochloride.

[0092] Examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0093] When preparing a lyophilized formulation by freeze-drying the liposome suspension formulation of the present invention, it is preferable to add a cryoprotective agent to the aqueous solvent in order to suppress the aggregation or fusion of liposomes or the breakdown of the lipid membrane. Examples of cryoprotective agents 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.

[0094] Since sugars such as sucrose have diverse functions, including osmotic pressure regulators, stabilizers, and cryoprotectors, aqueous solvents preferably contain sugars such as sucrose.

[0095] It is preferable to adjust the aqueous solvent (i.e., liposome suspension) to be isotonic or nearly isotonic (e.g., 285 ± 100 mOsm / L) with human body fluid (plasma). When sucrose is used as an osmotic pressure adjusting agent, it is preferable that the sucrose concentration of the aqueous solvent be about 9.0 to 11.0% (10.0 ± 1.0)% (w / v).

[0096] The pH of the aqueous solvent (i.e., liposome suspension) is, for example, 5.3 to 7.3 (6.3 ± 1.0), and is preferably adjusted to 5.8 to 6.8 (6.3 ± 0.5). In one embodiment, the aqueous solvent is a buffer aqueous solution containing sucrose, L-histidine, and L-histidine hydrochloride, with a pH of 5.8 to 6.8 (6.3 ± 0.5) and isotonic or nearly isotonic (e.g., 285 ± 50 mOsm / L) to human body fluids (plasma), and the sucrose concentration is preferably 9.0 to 11.0 (10.0 ± 1.0)% (w / v). As an example, the theoretical osmotic pressure of a buffer aqueous solution containing L-histidine (15 mM), L-histidine hydrochloride (5 mM), and sucrose (10.0% (w / v)) is 311.6 mOsm / L.

[0097] The amount of CD1d ligand compound-containing liposomes contained in the liposome suspension formulation of the present invention is not particularly limited, but for example, the concentration of the CD1d ligand compound (e.g., KRN7000) is 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).

[0098] In one embodiment, the group of liposomes is included 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 product (referred to as the liposome lyophilized formulation of the present invention). The lyophilized product of the present invention can be obtained by subjecting the group of liposomes described above suspended in an aqueous solvent (i.e., the liposome suspension formulation of the present invention described above) to a lyophilization process.

[0099] For example, the liposome suspension of the present invention can be divided and filled into containers such as vials, and then frozen at a temperature of about -20 to -80°C to obtain a frozen composition. The liposome freeze-dried formulation of the present invention can then be obtained by sublimating the water in the frozen composition under reduced pressure conditions (e.g., 10 Pa or less). To suppress aggregation or fusion of liposomes or breakdown of the lipid membrane during freeze-drying, it is preferable to add the above-mentioned cryoprotective agent to the liposome suspension.

[0100] By dispersing the liposome lyophilized formulation of the present invention in an aqueous solvent (e.g., water), a liposome suspension formulation of the present invention that satisfies the above conditions can be reconstituted. The reconstituted liposome suspension formulation of the present invention is then administered.

[0101] In this specification, the average particle size and polydispersity of the particle size distribution of the liposome population contained in a lyophilized formulation means the average particle size and polydispersity of the particle size distribution of the liposome population contained in a suspension formulation obtained by dispersing and reconstituting the lyophilized formulation in an aqueous solvent (e.g., water).

[0102] The liposomal formulation of the present invention can be administered orally or parenterally, but its pharmacokinetics in vivo are particularly improved when administered by injection. Compared to conventional formulations, the peak blood concentration of the CD1d ligand compound (e.g., KRN7000) is increased and the blood half-life is extended, making it suitable for injection administration. The liposomal formulation of the present invention can be used, for example, for intravenous injection, intramuscular injection, intradermal injection, subcutaneous injection, or intra-organ injection, and is preferably used for intravenous injection.

[0103] When the liposome formulation of the present invention is used as an injectable formulation, it can be stored and used in a form filled in a container. The container is preferably a sealed container. Examples of sealed container forms include ampoules, vials, or bags. Examples of container materials include glass and plastic. When filling containers such as ampoules or vials with the liposome formulation of the present invention, the gas phase of the container space may be replaced with an inert gas. A preferred example of an inert gas is nitrogen. For example, each container may be filled with liposomes containing a single-injectable dose of a CD1d ligand compound (e.g., KRN7000). For example, when using KRN7000 as the CD1d ligand compound, the liposome formulation of the present invention may be filled into a 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.

[0104] The liposomal formulation of the present invention exhibits improved pharmacokinetics in vivo, resulting in increased peak blood concentrations and extended blood half-lives of CD1d ligand compounds (e.g., KRN7000) compared to conventional formulations. Furthermore, the liposomal formulation of the present invention has high IL-4 induction ability while exhibiting low IFN-γ induction ability, enabling effective Treg induction, thus offering excellent immunosuppressive or immune tolerance induction effects. Therefore, the liposomal formulation 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. Autoimmune diseases include, but are not limited to, systemic lupus erythematosus, scleroderma, polyarteritis, myasthenia gravis, multiple sclerosis, autoimmune thyroiditis, type 1 diabetes mellitus, rheumatoid arthritis, Sjögren's syndrome, ANCA-associated vasculitis, Takayasu's arteritis, Behçet's disease, adult-onset Still's disease, relapsing polychondritis, IgA vasculitis, polymyalgia rheumatica, antiphospholipid syndrome, ankylosing spondylitis, Kawasaki disease, Crohn's disease, ulcerative colitis, psoriasis vulgaris, bullous pemphigoid, primary biliary cirrhosis, primary sclerosing cholangitis, and idiopathic interstitial pneumonia.

[0105] By administering an effective amount of the liposomal formulation of the present invention to a subject in need (e.g., a human), it is possible to prevent the onset of GVHD, organ transplant rejection, autoimmune diseases, etc., in that subject, or to treat GVHD, organ transplant rejection, autoimmune diseases, etc. In the case of preventing or treating GVHD, subjects in need include individuals who have already received transplants of allogeneic tissues (bone marrow, blood, etc.) or cells (hematopoietic stem cells, etc.) and have not yet developed GVHD, but who are at high risk of developing GVHD in the future; individuals who have received transplants of allogeneic tissues (bone marrow, blood, etc.) or cells (hematopoietic stem cells, etc.) and have developed GVHD; and individuals who are scheduled to receive transplants of allogeneic tissues (bone marrow, blood, etc.) or cells (hematopoietic stem cells, etc.). When preventing or treating organ transplant rejection, those who need these include individuals who have already received allogeneic or heterogeneous organ or cell transplants and have not yet developed rejection but are at high risk of developing rejection in the future, individuals who have received allogeneic or heterogeneous tissue or cell transplants and have developed rejection, and individuals who are scheduled to receive allogeneic or heterogeneous tissue or cell transplants. The present invention provides a liposomal formulation for use in the prevention or treatment of GVHD, organ transplant rejection, autoimmune diseases, etc. In one embodiment, GVHD may be caused by allogeneic hematopoietic stem cell transplantation.

[0106] In this specification, "effective dose" means the amount that produces the desired effect (e.g., therapeutic effect) in a subject, for example, that in a subject administered this dose, the symptoms or condition of the disease are alleviated, reduced, or eliminated, or its progression is delayed or suppressed, compared to a subject who did not receive this dose. The effective dose can be determined by a physician as appropriate, depending on the subject's age, weight, sex, and the severity of their symptoms.

[0107] In this specification, “prevention” means, with respect to a disease or disorder (e.g., GVHD, organ transplant rejection), preventing such a condition from occurring before it occurs, reducing the risk of such a condition occurring, or mitigating or reducing such a condition.

[0108] All references cited herein, including publications and patent documents, are incorporated herein by reference to the same extent as they are individually and specifically referred to and their entire contents are specifically described.

[0109] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. [Examples]

[0110] [Test Example 1] Evaluation of the manufacturing and stability of the comparative formulation (hereinafter referred to as RGI-2001-001). The manufacturing process for RGI-2001-001 is described below. Step 1: RGI-2001-001 intermediate (KRN7000 / lyophilized lipid mixture) Step 1a) Preparation of 90% (by weight / by weight) tert-butanol solution Water for injection was placed in a beaker and heated to 55-60°C. Tert-butanol was dissolved on a hot plate while stirring. The tert-butanol was poured into a pyrogen-free glass container (Pyrex), and water for injection was added to a final concentration of 90% (by weight), and the mixture was stirred for 10 ± 5 minutes while heating to 55-60°C.

[0111] Step 1b) Dissolution of 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 A lipid mixture containing (molar ratio 15:15:15:15:40) was to which tert-butanol was added to a final concentration of 100 g / L, and the mixture was stirred at 45±5°C until clear.

[0112] Step 1c) Preparation of KRN7000 / cyclohexane solution Powder of KRN7000 ((2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecantriol) (manufactured by Reguimmune) 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.

[0113] Step 1d) Preparation of KRN7000 / lipid mixture solution A lipid mixture solution was added to a KRN7000 / cyclohexane solution and stirred at 47±1°C until clear to obtain a KRN7000 / lipid mixture solution.

[0114] Step 1e) Freeze drying A glass container containing the KRN7000 / lipid mixture solution was carefully rotated in a dry ice / acetone bath to ensure uniform freezing of the solution to the inner wall of the container. After freezing, the glass container was left standing in dry ice for a minimum of 1 hour (maximum of 24 hours). The glass container containing the frozen KRN7000 / lipid mixture was then placed in a freeze-dryer, and the following steps were carried out. • After the temperature sensor of the manufactured product reached -40°C or below, it was frozen for a minimum of 2 ± 0.5 hours. After freezing was complete, the temperature of the frozen product was maintained at -40°C or below. • The vacuum level was set to 250 microns or less. The shelf temperature was raised from below -40°C to -35±3°C over a period of 6±0.5 hours or more. The shelf temperature was maintained at -35±3℃ for 12±0.5 hours. The shelf temperature was raised from -35±3℃ to 25±3℃ over a period of 4±0.5 hours or more. The product's temperature sensor was maintained at 25±3°C for 12±0.5 hours until the solenoid-type bleed valve became inoperable. After the vacuum level reached its maximum, the product was maintained at 25±3℃ for a minimum of 83 hours. After freeze-drying was complete and the chamber pressure reached atmospheric pressure, the glass containers containing the product were immediately sealed with caps 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, further freeze-drying was performed at 25±3°C for 24 hours.

[0115] Step 2: Manufacturing of RGI-2001-001 active pharmaceutical ingredient Step 2a) Preparation of buffer solution for formulation Sucrose was added to sterile water for injection to a final concentration of 10%, L-histidine to a final concentration of 15 mM, and L-histidine hydrochloride hydrate to a final concentration of 5 mM. The mixture was then stirred for 15 minutes to adjust the final pH to 6.5 ± 0.2. The resulting buffer solution was passed through a sterile filter (pore size 0.2 μm) before the next step.

[0116] Step 2b) Hydration of RGI-2001-001 intermediate The frozen RGI-2001-001 intermediate (KRN7000 / lipid mixture) was allowed to return to room temperature and stand for 30 minutes. The amount of formulation buffer to be 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 sterile conditions and stirred at 30-45°C for approximately 60 minutes until completely hydrated.

[0117] Process 2c) High-pressure extrusion The hydrated RGI-2001-001 intermediate solution was passed five times through an extruder fitted with a polycarbonate membrane filter (pore size 0.2 μm) at a target pressure (not exceeding 600 psi) of 200-300 psi under sterile conditions. Next, the resulting filtrate was passed once through an extruder fitted with a polycarbonate membrane filter (pore size 0.1 μm). As a final step, the resulting filtrate was passed ten times through an extruder fitted with two polycarbonate membrane filters (pore size 0.1 μm). The final collected material was passed through a sterile filter (pore size 0.2 μm) and stored at 2-8°C as the RGI-2001-001 active pharmaceutical ingredient.

[0118] The results of the accelerated (25°C) test for RGI-2001-001 are shown in the table below.

[0119] [Table 1]

[0120] The average particle size of RGI-2001-001 was 124 nm immediately after manufacturing and remained around 120 nm during the subsequent 6-month accelerated testing period. Furthermore, the DOPG-Na content after 6 months exceeded the specified limit. These results suggest that the comparative formulation (RGI-2001-001) exhibits uniform and stable average particle size of around 120 nm.

[0121] [Test Example 2] Method of manufacturing and evaluation of the stability 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 buffer solution for formulation Sucrose, L-histidine, L-histidine hydrochloride hydrate, and sterile water for injection were added to a single-use sterile bag, and the mixture was completely dissolved at room temperature. After adding sterile water for injection to achieve a final sucrose concentration (weight / volume) of 10% and a final sucrose density of 1.038 g / mL, the mixture was passed through a sterile filter (pore size 0.2 μm) and collected in a sterile plastic container.

[0122] Step 2a) Dissolution of RGI-7000 and lipids KRN7000 (manufactured by RegiImmune) (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%) The substance was added to ethanol, dissolved at 85±5℃, and sonication was continued until the solution became clear.

[0123] Step 2b) Preparation of RGI-2001-003 active pharmaceutical ingredient solution An ethanol solution containing KRN7000 and lipids, along with a formulation buffer, was passed through a stainless steel capillary tube (SSCT) set to 85±5°C and mixed in an inline mixer to obtain the crude product of the RGI-2001-003 active pharmaceutical ingredient solution. The crude product was mixed with a formulation buffer in another inline mixer and then cooled by passing it through an SSCT set to 20±5°C. The liposome particle size of the RGI-2001-003 active pharmaceutical ingredient was finely adjusted to an average particle size of approximately 100 nm by changing the pump speed.

[0124] Step 2c) Concentration and dialysis filtration The RGI-2001-003 active pharmaceutical ingredient was concentrated to 60% using a polyethersulfone resin hollow fiber membrane module (excluding substances less than 500 kDa), and then dialyzed with 10 times the weight of the formulation buffer.

[0125] Step 2d) Adjustment of the concentration of the active pharmaceutical ingredient (API) RGI-2001-003 The weight of KRN7000 in the RGI-2001-003 active pharmaceutical ingredient was confirmed by high-performance liquid chromatography, and then the final concentration was adjusted to 5.0 ± 0.5 mg / mL by dilution with sterile water for injection and concentration by diafiltration. The average particle size was adjusted to approximately 100 nm by dynamic light scattering.

[0126] Step 2e) Sterilization of the RGI-2001-003 active pharmaceutical ingredient. The product was collected in a sterilized plastic bag after passing it through a sterile filter (0.2 μm). The RGI-2001-003 active pharmaceutical ingredient was stored in a refrigerated (5±3℃) under light-shielding conditions until vial filling.

[0127] The results of the accelerated (25°C) test for RGI-2001-003 are shown in the table below.

[0128] [Table 2]

[0129] RGI-2001-003 was manufactured according to the technology described in Patent Documents 2 and 3, with the average liposome particle size set to 100 nm. The KRN7000 content and lipid composition of RGI-2001-003 are identical to those of RGI-2001-001. The average particle size was approximately 96 nm immediately after manufacturing, very close to the set value, and remained around 100 nm until the end of the accelerated testing. No deviation from the specifications was observed. These results suggest that the RGI-2001-003 liposome formulation possesses the target average particle size (100 nm) and exhibits excellent long-term stability.

[0130] [Example 3] Analysis of pharmacokinetics in human clinical trials In a Phase 1 clinical trial, RGI-2001-001 was administered intravenously to six patients who underwent hematopoietic stem cell transplantation at a dose of 100 μg / kg as KRN7000 within 30 minutes post-transplantation. 2 mL of peripheral blood was collected pre-transplant and at 0.5, 1, 2, 4, 6, 8, 24, 48, 72, and 96 hours post-transplant, and plasma components were recovered. KRN7000 in each plasma sample was isolated and identified by 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 post-transplantation to seven patients who underwent hematopoietic stem cell transplantation. 2 mL of peripheral blood was collected before transplantation and at 0.5, 2, 4, 6, 8, 24, and 48 hours after transplantation. Plasma components were recovered, and KRN7000 in the plasma was isolated and identified by LC-MS / M. These patients received RGI-2001-003 intravenously at a dose of 100 μg / kg as KRN7000 for 5 to 6 consecutive weeks. On day 14, 2 mL of peripheral blood was again collected before administration and at 0.5 and 4 hours after administration. Plasma components were recovered, and KRN7000 in the plasma was isolated and identified by LC-MS / M. Furthermore, NKT response and activated Treg count (Ki-67+%) were monitored over time in patients who received RGI-2001-003.

[0131] As a result, the peak blood concentration (Cmax) and blood half-life (t1 / 2) were 187.6 ng / mL and 23.4 hours in the RGI-2001-001 administration group, compared to 881 ng / mL and 35.8 hours in the RGI-2001-003 administration group. This result indicates that the RGI-2001-003 administration group had a significantly higher peak blood concentration (Cmax) and a significantly longer blood half-life (t1 / 2) compared to the RGI-2001-001 administration group (Table 3). Furthermore, in terms of the temporal changes in pharmacokinetics, RGI-2001-003 showed a typical decay curve, while RGI-2001-001 showed an irregular trend (Figure 1). Plasma levels of KRN7000 were similar between the initial and subsequent administrations of RGI-2001-003, and patient exposure to KRN7000 was similar on day 0 and day 14 even after repeated weekly administrations of RGI-2001-003 (Table 4, Figure 1). RGI-2001-003 administration induced an increase in NKT cell response and regulatory T cell count (Table 5).

[0132] [Table 3]

[0133] [Table 4]

[0134] [Table 5]

[0135] [Test Example 4] Evaluation of drug efficacy in mice It has been reported that after intravenous administration, RGI-2001 is taken up by splenic marginal zone B cells, which then release KRN7000 intracellularly. KRN7000 then binds to the CD1d molecule and is presented on the cell surface, inducing IL-4 production in iNKT cells and inducing regulatory T cells (Treg) (Non-Patent Literature 1). On the other hand, when dendritic cells take up RGI-2001, it acts to induce IFN-γ production in iNKT cells.

[0136] C57BL / 6 mice (female, 8 weeks old; Claire) were administered 2 μg of RGI-2001-001 or RGI-2001-003 as a KRN7000 dose via the tail vein, and orbital blood samples were collected 2 and 24 hours later. Plasma concentrations of IFN-γ, IL-4, and IL-10 were measured using the Cytometric Bead Array method (BD Biosciences). As a result, IFN-γ was lower in the RGI-2001-003 group than in the RGI-2001-001 group, while IL-4 was higher in the RGI-2001-003 group than in the RGI-2001-001 group (Figure 2). On the other hand, IL-10 production was similar in both groups, or slightly higher in the RGI-2001-003 group than in the RGI-2001-001 group (Figure 3). The results suggest that RGI-2001-003, compared to RGI-2001-001, is more easily taken up by marginal zone B cells due to its average particle size being adjusted to around 100 nm. As a result, RGI-2001-003 more strongly induces IL-4 production in iNKT cells and more effectively induces Treg cells. On the other hand, in contrast to IL-4, the activity of RGI-2001-003 in inducing IFN-γ production in iNKT cells was suggested to be lower than that of RGI-2001-001.

[0137] [Test Example 5] Measurement of particle size The particle size and polydispersity of the liposome formulation produced in Test Example 2 were measured by dynamic light scattering. <Condition 1> The liposome formulation produced in Test Example 2 was stored in PBS (Ca 2+ After dilution 1000 times (without containing [unspecified substance]), particle size and polydispersity were analyzed by DLS (dynamic light scattering, backscattering) at a temperature of 25°C using a Malvern ZetaSizer Nano ZS. The results are shown in Table 6.

[0138] [Table 6]

[0139] <Condition 2> The liposome formulation produced in Test Example 2 was stored in PBS (Ca 2+ The sample was diluted 1000 times (without containing [unspecified substance]), and the particle size and polydispersity were analyzed by DLS (dynamic light scattering, 90°C scattering). The results are shown in Table 7.

[0140] [Table 7]

[0141] Based on the results of these tests, the average particle size of the RGI-2001-003 liposome formulation when Test Examples 3 and 4 were performed was estimated to be in the range of 92.9 to 101.0 nm.

[0142] [Test Example 7] In Study Example 3, seven patients who received RGI-2001-003 were followed up. Of the seven patients, one transiently developed grade II acute GVHD and one transiently 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 a mild case with lesions only on the skin, and the symptoms were transient. There was no recurrence of GVHD more than one year after hematopoietic stem cell transplantation. These results suggest that RGI-2001-003 has a superior effect in suppressing the development of GVHD.

[0143] [Table 8] [Industrial applicability]

[0144] The present invention provides a liposomal formulation containing KRN7000 that can remain in the bloodstream for a long period and maintain a high concentration of KRN7000 in the blood for an extended period. The liposomal formulation of the present invention is expected to have excellent preventive or therapeutic effects against GVHD.

[0145] This application is based on Japanese Patent Application No. 2020-201802 (filing date: December 4, 2020), the contents of which are fully incorporated herein.

Claims

1. A liposome formulation comprising a group of liposomes containing a CD1d ligand compound, wherein the average particle size of the group of liposomes is 90 to 110 nm, and the polydispersity of the particle size distribution is 0.2 or less.

2. A liposome formulation according to claim 1, wherein the average particle size is 92.9 to 101.0 nm.

3. A liposome formulation according to claim 1 or 2, wherein the polydispersity of the particle size distribution is 0.133 or less.

4. A liposome formulation according to any one of claims 1 to 3, wherein the number of liposomes with a particle diameter of less than 50 nm is 10% or less of the total population of liposomes.

5. A liposome formulation according to any one of claims 1 to 4, wherein the number of liposomes with a particle diameter greater than 450 nm is 10% or less of the total population of liposomes.

6. A liposome formulation according to any one of claims 1 to 5, wherein the average particle size is maintained at 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. A liposome formulation according to any one of claims 1 to 6, wherein the CD1d ligand compound is α-galactosylceramide.

8. The liposome formulation according to claim 7, wherein α-galactosylceramide is (2S,3S,4R)-1-o-(α-D-galactopyranosyl)-2-(N-hexecosanoylamino)-1,3,4-octadecanetriol.

9. A liposome formulation according to any one of claims 1 to 8, wherein the group of liposomes is included as a liposome suspension.

10. The liposome formulation according to claim 9, wherein the pH of the liposome suspension is 5.8 to 6.

8.

11. A liposome formulation according to any one of claims 1 to 8, wherein the group of liposomes is contained as a lyophilized product.

12. A liposome formulation according to any one of claims 1 to 11, for administration by injection.

13. A liposome formulation according to any one of claims 1 to 12, for the prevention or treatment of graft-versus-host disease.

14. The liposome formulation according to claim 13, wherein graft-versus-host disease is caused by allogeneic hematopoietic stem cell transplantation.

15. A liposome formulation according to any one of claims 1 to 12, for the prevention or treatment of organ transplant rejection.

16. The liposome formulation according to claim 15, wherein the organ transplant is the transplantation of allogeneic organs or cells.

17. The liposome formulation according to claim 15, wherein the organ transplant is the transplantation of a different organ or cells.

18. A method for reducing the risk of developing graft-versus-host disease in a subject who is at risk of developing graft-versus-host disease, comprising administering an effective amount of the liposome formulation described in any one of claims 1 to 12 to the subject.

19. The method according to claim 18, wherein the subject at risk of developing graft-versus-host disease is a subject who has received 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 an effective amount of the liposome formulation described in any one of claims 1 to 12 to the subject.

21. The method according to any one of claims 18 to 20, wherein 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 who is at risk of developing organ transplant rejection, comprising administering an effective amount of the liposome formulation described in any one of claims 1 to 12 to the subject.

23. The method according to claim 22, wherein the subject at risk of developing organ transplant rejection is a subject who has received an allogeneic or heterogeneous organ or cell transplant, or a subject who is scheduled to receive an allogeneic or heterogeneous organ or cell transplant.

24. A method for treating organ transplant rejection in a subject who has developed organ transplant rejection, comprising administering an effective amount of the liposome formulation described in any one of claims 1 to 12 to the subject.

25. The method according to any one of claims 22 to 24, wherein the organ transplant is the transplantation of allogeneic organs or cells.

26. The method according to any one of claims 22 to 24, wherein the organ transplant is the transplantation of a different organ or cells.

27. A liposome formulation according to any one of claims 1 to 12, for use in the prevention or treatment of graft-versus-host disease.

28. The liposome formulation according to claim 27, wherein graft-versus-host disease is caused by allogeneic hematopoietic stem cell transplantation.

29. A liposome formulation according to any one of claims 1 to 12, for use in the prevention or treatment of organ transplant rejection.

30. The liposome formulation according to claim 29, wherein the organ transplant is the transplantation of allogeneic organs or cells.

31. The liposome formulation according to claim 29, wherein the organ transplant is the transplantation of a different organ or cells.

32. Use of a liposome formulation according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for the prevention or treatment of graft-versus-host disease.

33. The use according to claim 32, wherein graft-versus-host disease is caused by allogeneic hematopoietic stem cell transplantation.

34. Use of a liposome formulation according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for the prevention or treatment of organ transplant rejection.

35. The use according to claim 34, wherein the organ transplant is the transplantation of allogeneic organs or cells.

36. The use according to claim 34, wherein the organ transplant is the transplantation of a different organ or cells.

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