Drug delivery controllable liposomes

Abstract

The present invention relates to a liposome capable of controlling drug delivery and, more specifically, to a liposome containing, as membrane-forming lipids, a lamella forming lipid and an amphiphilic lipid having an isoprenoid-type fat chain.

Description

[Technical Field]

[0001] This invention relates to liposomes capable of controlling drug delivery. [Background technology]

[0002] Liposomes, which are closed vesicles with a lipid bilayer membrane, can encapsulate both hydrophilic and hydrophobic drugs, and generally exhibit excellent biocompatibility and biodegradability. Therefore, their use in drug delivery systems (DDS) as a drug delivery tool has attracted considerable attention.

[0003] Liposomes are known to accumulate in tumor tissue due to the Enhanced Permeation and Retention EPR (EPR) effect. In tumor tissue, gaps are formed between vascular endothelial cells, and vascular permeability is significantly increased compared to normal tissue. Therefore, macromolecules that do not permeate normal blood vessel walls leak out of the blood vessels and accumulate in tumor tissue, which is the EPR effect. Thus, liposomes have a drug delivery function to tumor tissue. However, the accumulation of typical liposomes in tumor tissue due to the EPR effect alone is not very high.

[0004] To achieve effective drug delivery systems (DDS), precise control of drug delivery and release is desired. Patent Document 1 discloses a pH-responsive liposome that releases a target substance in a basic environment, comprising a cationic amphiphilic molecule and at least one of anionic amphiphilic molecules and zwionic amphiphilic molecules as constituent lipids. Patent Document 2 discloses a temperature-sensitive liposome composed of a liposome membrane constituent lipid, a polymer compound having a heat-responsive portion and a hydrophobic portion, and PEG. However, conventional liposomes still have considerable room for improvement in terms of drug delivery amount and other aspects.

[0005] Non-lamellar liquid crystals (NLLCs) have been reported to have advantages such as a high drug content rate, ease of preparation, and high stability in macromolecular pharmaceuticals compared to conventional DDS carriers. However, non-lamellar liquid crystal-forming lipids have been reported to have hemolytic effects, and systemic administration has been considered difficult.

[0006] Patent Document 3 reports a topical skin preparation containing a low-viscosity non-lamellar liquid crystal-forming lipid that retains and sustains-release a drug within the non-lamellar liquid crystal. However, Patent Document 3 does not describe a method for more highly controlling drug delivery for such a formulation.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide liposomes capable of controlling drug delivery. Alternatively, an object of the present invention is to provide liposomes exhibiting an antitumor effect.

Means for Solving the Problems

[0009] As a result of intensive studies to solve the above problems, the present inventors have found that liposomes capable of effectively delivering drugs into cells and cell nuclei, liposomes capable of controlling drug delivery due to temperature responsiveness, and liposomes exhibiting an antitumor effect can be produced using amphiphilic lipids having isoprenoid-type fatty chains, and have thus completed the present invention.

[0010] That is, the present invention includes the following aspects.

[0011] [1] Liposomes containing a lamella-forming lipid and an amphiphilic lipid having an isoprenoid-type fatty chain as membrane-constituting lipids.

[0012] <000,096>[2] The liposome according to [1] above, wherein the amphiphilic lipid having an isoprenoid-type fatty chain is an amphiphilic compound represented by the following general formula (I). [Chemical formula] (In the formula, X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2, m represents 1 or 2, TIFF0007829229000002.tif58 represents a single bond or a double bond, and R represents a hydrophilic group having one or more hydroxyl groups)

[0013] [3] The liposome according to [2] above, wherein R in the above formula represents a hydrophilic group obtained by removing one hydroxyl group from glycerol, sorbitan, or propylene glycol.

[0014] [4] The liposome according to any one of [1] to [3] above, wherein the amphiphilic lipid having an isoprenoid-type fatty chain is mono O-(5,9,13-trimethyltetradeca-4-enoyl) glycerol, mono O-(5,9,13,17-tetramethyloctadecanoyl) glycerol, mono O-(5,9,13,17-tetramethyloctadeca-4-enoyl) sorbitan, or mono O-(5,9,13,17-tetramethyloctadeca-4-enoyl) propylene glycol.

[0015] [5] The liposome according to any one of [1] to [4] above, wherein the lamella-forming lipid contains at least one selected from the group consisting of phospholipids, steroids, and cationic lipids.

[0016] [6] The liposome according to any one of [1] to [5] above, wherein the lamella-forming lipid contains a phospholipid modified with a water-soluble polymer.

[0017] [7] A liposome according to any one of [1] to [6] above, wherein the lamellar-forming lipid comprises phosphatidylcholine, cholesterol, and PEGylated phosphatidylethanolamine.

[0018] [8] A liposome according to any one of [1] to [6] above, wherein the lamellar-forming lipid comprises phosphatidylethanolamine and 1,2-dialkylcarbonyloxy-3-mono, di, or trialkylammonium propane.

[0019] [9] A liposome according to any one of [1] to [8] above, comprising amphiphilic lipids having isoprenoid-type fatty acid chains in an amount of 5 to 40 mol% in mole fraction with respect to the total amount of membrane constituent lipids.

[0020]

[10] A temperature-responsive liposome as described in any of [1] to [9] above.

[0021]

[11] A liposome according to any of [1] to

[10] above, further comprising a drug.

[0022]

[12] The liposome described in

[11] above, wherein the drug is a nucleic acid.

[0023]

[13] The liposome described in

[12] above, wherein nucleic acid and liposome form a complex.

[0024]

[14] A drug delivery formulation for intracellular use, comprising any of the liposomes described in

[11] to

[13] above.

[0025]

[15] A formulation for delivering nucleic acids into the nucleus of a cell, comprising the liposome described in

[12] or

[13] above.

[0026]

[16] The preparation according to

[14] or

[15] above, wherein the cells are tumor cells.

[0027]

[17] A pharmaceutical preparation comprising any of the liposomes described in [1] to

[13] above.

[0028]

[18] The pharmaceutical preparation described in

[17] above, which is an antitumor agent.

[0029] This specification includes the disclosures of Japanese Patent Application No. 2020-135330, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0030] According to the present invention, it is possible to provide liposomes that allow for controllable drug delivery and are preferably temperature-responsive, as well as liposomes that exhibit antitumor effects. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1 shows a photograph of particulate formulation No. 4 taken with cryo-TEM at a magnification of 15,000x. The bars represent 100nm. [Figure 2] Figure 2 is a graph showing the change in fluorescence intensity ratio F / F0 of particulate formulations No. 6-12 with respect to temperature, which is used as an index for evaluating membrane fluidity. [Figure 3] Figure 3 is a photograph showing the temperature responsiveness (temperature sensitivity) of particulate formulations No. 1 to 4. [Figure 4] Figure 4 is a graph showing the interaction (hemolytic effect) between microparticle formulations No. 1-5 and biological membranes. A: 37°C, B: 45°C. [Figure 5] Figure 5 shows photographs of fluorescence observation results over time in tumor-bearing mice after intravenous administration of microparticle formulation No. 13 or 14. A: Microparticle formulation No. 13, B: Microparticle formulation No. 14. [Figure 6] Figure 6 is a graph showing the time course of fluorescence intensity at tumor sites after intravenous administration of particulate formulation No. 13 or 14. [Figure 7] Figure 7 is a photograph showing fluorescence images of organs extracted from mice administered with microparticle formulation No. 13 or 14. [Figure 8]Figure 8 shows a photograph of particulate formulation No. 27 taken with cryo-TEM at a magnification of 15,000x. The bars represent 100nm. [Figure 9] Figure 9 is a graph showing the intracellular uptake efficiency of fluorescently labeled molecules in groups treated with microparticle formulations No. 31-33 and in the untreated (untreated) group. A: Relationship between the number of cells that detected fluorescence and their fluorescence intensity, B: Relative value of the average fluorescence intensity of the groups treated with microparticle formulations No. 31-33 compared to the average fluorescence intensity of the untreated group (average fluorescence intensity ratio). [Figure 10] Figure 10 is a photograph of an electrophoretic image showing the nucleic acid complex state in nucleic acid-microparticle complexes derived from microparticle formulation No. 15 or 20. [Figure 11] Figure 11 is a graph showing the ratio of the brightness intensities of the nucleic acid (pcDNA3-EGFP) band released from the nucleic acid-microparticle complex. A: Microparticle formulation No. 15, A: Microparticle formulation No. 20. [Figure 12] Figure 12 shows fluorescence images of cells treated with each formulation. A: Microparticle formulation No. 15 (LCMGE), B: Microparticle formulation No. 16 (DOPE / DOTAP), C: Microparticle formulation No. 20 (MGE / DOPE / DOTAP (LNPMGE)), D: Microparticle formulation No. 23 (MGE / DOTAP), E: pcDNA3-EGFP alone, F: LFN2000-nucleic acid mixture. Fluorescence: GFP, Bar: 100 μm. Magnification: 10x. [Figure 13] Figure 13 shows the dot plots obtained from FACS analysis. A: pcDNA3-EGFP alone, B: microparticle formulation No. 16, C: microparticle formulation No. 17, D: microparticle formulation No. 18, E: microparticle formulation No. 19, F: microparticle formulation No. 20. [Figure 14] Figure 14 shows the dot plots obtained from FACS analysis. A: Particulate formulation No. 21, B: Particulate formulation No. 22, C: Particulate formulation No. 23, D: Particulate formulation No. 24, E: Particulate formulation No. 25. [Figure 15]Figure 15 shows the dot plots obtained from FACS analysis. A: Particulate formulation No. 26, B: Particulate formulation No. 27, C: Particulate formulation No. 28, D: Particulate formulation No. 29, E: Particulate formulation No. 30. [Figure 16] Figure 16 shows photographs of time-series fluorescence observations of tumor-bearing mice after intravenous administration of formulations No. 37-40. A: Microparticle formulation No. 37, B: Microparticle formulation No. 38, C: Formulation No. 39, D: Formulation No. 40. Arrows indicate tumors. [Figure 17] Figure 17 shows the time course of luminescence intensity, an indicator of tumor cell volume, in tumor-bearing mice with and without heating treatment after administration of the drug. A: Tumor on the left side of the back, with heating. B: Tumor on the right side of the back, without heating. [Modes for carrying out the invention]

[0032] The present invention will be described in detail below.

[0033] This invention relates to liposomes containing lamellar-forming lipids and amphiphilic lipids having isoprenoid-type fatty acid chains as membrane constituent lipids.

[0034] In this invention, liposomes refer to closed vesicles having a lipid bilayer. The lipid bilayer of the liposomes of this invention is mainly composed of lipids, but may also contain components other than lipids. The liposomes of this invention may have a single lamellar structure or a multi-lamellar structure having two or more lipid bilayers. In this invention, liposomes may also be referred to as microparticles.

[0035] The liposomes of the present invention are mainly composed of a lipid bilayer containing a lamellar-forming lipid and an amphiphilic lipid having an isoprenoid-type fatty acid chain (in one embodiment, a non-lamellar liquid crystal-forming lipid having an isoprenoid-type fatty acid chain). The lamellar-forming lipid as the membrane-constituting lipid of the liposome is sometimes also called the liposome-constituting lipid. The lamellar-forming lipid used as the membrane-constituting lipid in the present invention is not particularly limited, but it is preferable to include at least one selected from the group consisting of phospholipids, steroids, and cationic lipids. In one embodiment, the lamellar-forming lipid used as the membrane-constituting lipid in the present invention may include one selected from the group consisting of phospholipids, steroids, and cationic lipids, or consist of one of them. In another embodiment, the lamellar-forming lipid used as the membrane-constituting lipid in the present invention may include a phospholipid and a steroid, or consist of a phospholipid and a steroid. In another embodiment, the lamellar-forming lipid used as the membrane-constituting lipid in the present invention may include a phospholipid and a cationic lipid, or consist of a phospholipid and a cationic lipid. In another embodiment, the lamellar-forming lipid used as a membrane-constituting lipid in the present invention may include a steroid and a cationic lipid, or consist of a steroid and a cationic lipid. In another embodiment, the lamellar-forming lipid used as a membrane-constituting lipid in the present invention may include a phospholipid, a steroid and a cationic lipid, or consist of a phospholipid, a steroid and a cationic lipid. The liposome of the present invention may contain one or more (for example, two, three, four, five, or six or more) lamellar-forming lipids as membrane-constituting lipids.

[0036] The lamellar-forming lipid used in the liposomes of the present invention may contain one or more phospholipids. Examples of phospholipids include, but are not limited to, one or more phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerin, phosphatidic acid, and sphingomyelin. Examples of phosphatidylcholine include, but are not limited to, dimyristoylphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), soy phosphatidylcholine (SPC; also known as soy lecithin), hydrogenated soy phosphatidylcholine (HSPC; also known as hydrogenated soy lecithin), and egg yolk phosphatidylcholine (EPC; also known as egg yolk lecithin). Examples of phosphatidylethanolamines include, but are not limited to, dioleylphosphatidylethanolamine (DOPE) and distearoylphosphatidylethanolamine (DSPE). Examples of phosphatidylglycerin include, but are not limited to, dioleylphosphatidylglycerin sodium (DOPG-Na). In the present invention, phospholipids in the form of halides such as chlorides and bromides, alkali metal salts or alkaline earth metal salts, sulfates, nitrates, and other salts can also be used, and these are included in the range of the individual corresponding phospholipids listed above. The liposomes of the present invention may contain phospholipids in the form of their salts. The salts of phospholipids may be pharmaceutically acceptable salts. Liposomes containing phospholipids in the form of their salts are also included in the range of liposomes according to the present invention.

[0037] The phospholipid used in the present invention may be a phospholipid modified with a water-soluble polymer. The water-soluble polymer is not limited to the following, but may be, for example, polyethylene glycol (PEG), polyethylene glycol (PEG) derivatives, polyvinylpyrrolidone, polylactic acid, polyglycolic acid, etc. In one preferred embodiment, the phospholipid modified with the water-soluble polymer is a PEGylated phospholipid. A PEGylated phospholipid refers to a phospholipid to which polyethylene glycol (PEG) or a derivative thereof is attached (bonded). The polyethylene glycol (PEG) derivative may be PEG having any functional group and / or multiple arms. The polyethylene glycol (PEG) derivative may be polyethylene glycol having a functional group at the terminal, for example, alkoxyethylene glycol (monoalkoxy polyethylene glycol). Examples of alkoxyethylene glycols include methoxypolyethylene glycol (MPEG) and ethoxypolyethylene glycol. Polyethylene glycol may have any molecular weight, for example, polyethylene glycol with a molecular weight of 100,000 or less, 200 to 800,000, 300 to 15,000, or 500 to 5,000 (e.g., 2,000). The phospholipid modified with the water-soluble polymer may be any of the above-mentioned phospholipids. The phospholipid modified with the water-soluble polymer may be, for example, a PEGylated phosphatidylethanolamine, including PEGylated distearoylphosphatidylethanolamine, and a preferred example is DSPE-PEG2000. The phospholipid used in the present invention may be synthetic or naturally derived. In one embodiment, the phospholipid used in the present invention may be, for example, phosphatidylcholine such as dimyristoylphosphatidylcholine (DMPC), soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, or egg yolk phosphatidylcholine, or phosphatidylethanolamine such as distearoylphosphatidylethanolamine. Generally, liposomes using phospholipids modified with water-soluble polymers, i.e., liposomes modified with water-soluble polymers and whose surface is hydrated, exhibit high blood retention, but they do not interact well with cells and are not easily taken up by cells.However, the liposomes of the present invention, by using amphiphilic lipids having isoprenoid-type fatty acid chains together with lamellar-forming lipids as membrane components, promote cell interaction even when modified with water-soluble polymers.

[0038] The lamellar-forming lipid used in the liposomes of the present invention may contain one or more steroids. Examples of steroids include, but are not limited to, any steroid such as sterols, bile acids, and steroid hormones, with sterols being preferred. Examples of sterols include, but are not limited to, cholesterol, lanosterol, and ergosterol.

[0039] The lamellar-forming lipid used in the liposomes of the present invention may contain one or more cationic lipids. Examples of cationic lipids include, but are not limited to, 1,2-dialkylcarbonyloxy-3-mono, di, or trialkylammonium propane (1,2-dialkylcarbonyloxy-3-alkylammonium propane, 1,2-dialkylcarbonyloxy-3-dialkylammonium propane, or 1,2-dialkylcarbonyloxy-3-trialkylammonium propane), for example, 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP) (e.g., chloride), 1,2-dioleoyloxy-3-dimethylammonium propane or 1,2-dioleyloxy-3-dimethylaminopropane (DODAP), 1,2-dimyristoyloxy-3-trimethylammonium propane (DMTAP) (e.g., chloride), etc. In the present invention, cationic lipids in the form of halides such as chlorides and bromides, alkali metal salts or alkaline earth metal salts, sulfates, nitrates, and other salts may also be used, and these are included in the range of the individual corresponding cationic lipids listed above. The liposomes of the present invention may contain cationic lipids in the form of their salts. The salts of cationic lipids may be pharmaceutically acceptable salts. Liposomes containing cationic lipids in the form of their salts are also included in the range of liposomes according to the present invention. The liposomes of the present invention more preferably contain cationic lipids when they contain nucleic acids as drugs as described later, but may contain cationic lipids in other cases.

[0040] The lamellar-forming lipid used in the liposomes of the present invention preferably has a phase transition temperature of more than 20°C, and may be, for example, 21°C to 42°C, 23°C to 42°C, or 23°C to 40°C.

[0041] In one embodiment, the lamellar-forming lipid used in the liposomes of the present invention may include phosphatidylcholine (e.g., dimyristoylphosphatidylcholine), cholesterol, and PEGylated phosphatidylethanolamine (e.g., PEGylated distearoylphosphatidylethanolamine).

[0042] In one embodiment, the lamellar-forming lipid used in the liposomes of the present invention may include phosphatidylethanolamine (e.g., dioleylphosphatidylethanolamine) and 1,2-dialkylcarbonyloxy-3-mono, di, or trialkylammonium propane (e.g., 1,2-dioleoyloxy-3-trimethylammoniumpropane; 1,2-dioleoyloxy-3-trimethylammoniumpropane chloride, etc.).

[0043] The liposomes of the present invention include, in addition to lamellar-forming lipids, amphiphilic lipids having isoprenoid-type fatty acid chains. In one embodiment, the amphiphilic lipid having isoprenoid-type fatty acid chains in the present invention may be a lipid (non-lamellar liquid crystal-forming lipid) that has the ability to form a non-lamellar liquid crystal on its own in the presence of water without requiring other lipids.

[0044] In a preferred embodiment, the amphiphilic lipid having an isoprenoid-type fatty acid chain used in the liposome of the present invention is an amphiphilic compound represented by the following general formula (I).

[0045] [ka]

[0046] In general formula (I), X and Y each represent a hydrogen atom or together represent an oxygen atom. In general formula (I), n represents an integer from 0 to 2 (preferably 1 or 2), and m represents 1 or 2. In the amphiphilic compound represented by general formula (I), the combination of n and m may be any of the following: n=0, m=1; n=0, m=2; n=1, m=1; n=1, m=2; n=2, m=1; or n=2, m=2.

[0047] In the formula: TIFF0007829229000004.tif57 represents a single bond or a double bond.

[0048] In general formula (I), R represents a hydrophilic group having one or more hydroxyl groups (one or more hydroxyl groups), and is not limited to the following, but examples include a hydrophilic group obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, glycol (e.g., propylene glycol), and isosorbide. Note that a hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid may be a group from which the OH (hydroxyl group) contained in the carboxyl group of glyceric acid has been removed.

[0049] In relation to the present invention, glycol means a compound consisting of a chain or cyclic carbon, oxygen, and hydrogen atom in which two hydroxyl groups are bonded to two different carbon atoms. Regarding amphiphilic lipids having isoprenoid-type fatty acid chains used in the present invention, preferred examples of glycols include, but are not limited to, propylene glycol, ethylene glycol, butylene glycol, isoprene glycol (also known as 3-methyl-1,3-butanediol), diethylene glycol, and isosorbide.

[0050] In this invention, the notation in general formula (I) is: TIFF0007829229000005.tif610 means that the amphiphilic compound is either the E (cis) or Z (trans) geometric isomer, or a mixture thereof.

[0051] In general formula (I), the above amphiphilic compound having a hydrophilic group from which one hydroxyl group (OH) has been removed from any one of the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, mannitol, xylitol, sorbitan, and isosorbide is a non-lamellar liquid crystal forming lipid.

[0052] In one preferred embodiment of the liposome of the present invention, m=1 in general formula (I). In a further preferred embodiment of the liposome of the present invention, m=1 in general formula (I), and R is a hydrophilic group obtained by removing one hydroxyl group (OH) from glycerol.

[0053] Examples of amphiphilic compounds represented by general formula (I) include the amphiphilic compounds represented by general formula (II) below.

[0054] [ka]

[0055] In general formula (II), X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2 (0, 1, or 2), and m represents 1 or 2.

[0056] In general formula (II), R represents a hydrophilic group having one or more hydroxyl groups (one or more hydroxyl groups), and is not limited to the following, but represents a hydrophilic group obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, glycol (e.g., propylene glycol), and isosorbide. The hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid may be a group from which the OH (hydroxyl group) contained in the carboxyl group of glyceric acid has been removed.

[0057] Another example of an amphiphilic compound represented by general formula (I) is the amphiphilic compound represented by general formula (III) below.

[0058] [ka]

[0059] In general formula (III), X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2 (preferably 1 or 2), and m represents 1 or 2.

[0060] In general formula (III), R represents a hydrophilic group having one or more hydroxyl groups (one or more hydroxyl groups), and is not limited to the following, but examples include hydrophilic groups obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, glycol (e.g., propylene glycol), and isosorbide. A hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid may be a group from which the OH (hydroxyl group) contained in the carboxyl group of glyceric acid has been removed.

[0061] Another example of an amphiphilic compound represented by general formula (I) is the amphiphilic compound represented by general formula (IV) below.

[0062] [ka]

[0063] In general formula (IV), X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2 (preferably 1 or 2), and m represents 1 or 2.

[0064] In general formula (IV), R represents a hydrophilic group having one or more hydroxyl groups (one or more hydroxyl groups), and is not limited to the following, but examples include a hydrophilic group obtained by removing one hydroxyl group (OH) from any one selected from the group consisting of glycerol, erythritol, pentaerythritol, diglycerol, glyceric acid, triglycerol, xylose, sorbitol, ascorbic acid, glucose, galactose, mannose, dipentaerythritol, maltose, mannitol, xylitol, sorbitan, glycol (e.g., propylene glycol), and isosorbide. The hydrophilic group obtained by removing one hydroxyl group (OH) from glyceric acid may be a group from which the OH (hydroxyl group) contained in the carboxyl group of glyceric acid has been removed.

[0065] Examples of amphiphilic compounds represented by general formula (I) include glycerol-based, sorbitan-based, or propylene glycol-based compounds: Mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol, Mono-O-(5,9,13-trimethyltetradecanoyl)glycerol, Mono-O-(5,9,13-trimethyltetradeca-4,8,12-trienoyl)glycerol, Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol, Mono-O-(5,9,13,17-tetramethyloctadecanoyl)glycerol, Mono-O-(5,9,13,17-tetramethyloctadeca-4,8,12,16-tetraenoyl)glycerol, Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan, and Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)propylene glycol These are some examples, but are not limited to them.

[0066] As the amphiphilic lipid according to the present invention, salts of amphiphilic compounds represented by general formula (I), such as halides such as chloride and bromide, alkali metal salts or alkaline earth metal salts, sulfates, nitrates, etc., can also be used. These salts fall within the range of the individual corresponding amphiphilic compounds listed above. The liposomes of the present invention may contain the amphiphilic compound represented by general formula (I) in the form of its salt. The salt of the amphiphilic compound represented by general formula (I) may be a pharmaceutically acceptable salt. Liposomes containing the amphiphilic compound represented by general formula (I) in the form of its salt are also included within the range of liposomes according to the present invention.

[0067] The amphiphilic compound represented by general formula (I) used in the present invention can be synthesized by referring to the examples described below, or by following the synthesis method described in International Publication WO2014 / 178256 or International Publication WO2020 / 050423 (Patent Document 3). Alternatively, the amphiphilic compound represented by general formula (III) can be synthesized, for example, by following the synthesis method described in International Publication WO2011 / 078383. Furthermore, the amphiphilic compound represented by general formula (IV) can be synthesized, for example, by following the synthesis method described in International Publication WO2006 / 043705.

[0068] In one embodiment, the liposomes of the present invention may contain amphiphilic lipids having isoprenoid-type fatty acid chains in an amount such that the mole fraction (mol%) relative to the total amount of membrane constituent lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains) is 50 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, or 20 mol% or less. The liposomes of the present invention may contain amphiphilic lipids having isoprenoid-type fatty acid chains (total amount if two or more such amphiphilic lipids are used) in amounts such that, in mole fraction relative to the total amount of membrane constituent lipids, they are, for example, 5 mol% to 50 mol%, 5 mol% to 40 mol%, 10 mol% to 50 mol%, 20 mol% to 50 mol%, 10 mol% to 40 mol%, 5 mol% to 25 mol%, 5 mol% to 30 mol%, 5 mol% to 35 mol%, 10 mol% to 35 mol%, 20 mol% to 35 mol%, 20 mol% to 30 mol%, 10 mol% to 25 mol%, or 20 mol% to 25 mol%.

[0069] In one embodiment, the liposomes of the present invention may contain phospholipids as lamellar-forming lipids. The liposomes of the present invention may contain phospholipids (total amount if two or more types of phospholipids are used) in a mole fraction (mol%) relative to the total amount of membrane constituent lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains), for example, 5 mol% to 65 mol%, 20 mol% to 65 mol%, 25 mol% to 50 mol%, 25 mol% to 40 mol%, 30 mol% to 40 mol%, 10 mol% to 35 mol%, or 20 mol% to 35 mol%.

[0070] In one embodiment, the liposomes of the present invention may contain a steroid as a lamellar-forming lipid. The liposomes of the present invention may contain a steroid (total amount if two or more types of steroids are used) in an amount such that, in terms of mole fraction (mol%) relative to the total amount of membrane constituent lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains), it is, for example, 5 mol% to 70 mol%, 20 mol% to 50 mol%, 30 mol% to 50 mol%, or 30 mol% to 40 mol%.

[0071] In one embodiment, the liposomes of the present invention may contain cationic lipids (total amount if two or more cationic lipids are used), for example, 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), in an amount such that the mole fraction (mol%) relative to the total amount of membrane constituent lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains) is, for example, 40 mol% to 70 mol%, 50 mol% to 70 mol%, 55 mol% to 65 mol%, or 45 mol% to 55 mol%.

[0072] In one embodiment, the liposomes of the present invention may contain amphiphilic lipids having isoprenoid-type fatty acid chains (e.g., C17MGE) and phospholipids (e.g., DOPE) in a ratio of molar fraction (mol%) to the total amount of membrane constituent lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains), preferably 1:2 to 2:1, more preferably 1:1.5 to 1.5:1, and even more preferably 1:1.1 to 1.1:1.

[0073] In a preferred embodiment, the liposomes of the present invention may contain cationic lipids as lamellar-forming lipids. The liposomes of the present invention may contain cationic lipids, for example, 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP), in a mole fraction (mol%) of 45 mol% to 55 mol% (e.g., 50 mol%) relative to the total amount of membrane-constituting lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains), amphiphilic lipids having isoprenoid-type fatty acid chains (e.g., C17MGE), in a mole fraction (mol%) of 20 mol% to 25 mol% relative to the total amount of membrane-constituting lipids, and phospholipids (e.g., DOPE), in a mole fraction (mol%) of 20 mol% to 35 mol%.

[0074] In one embodiment, the liposomes of the present invention may contain amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol, and lamellar-forming lipids such as dimyristoylphosphatidylcholine (DMPC), cholesterol, and PEGylated distearoylphosphatidylethanolamine. Such liposomes of the present invention may contain, in mole fraction (mol%) of amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol, 25-55 mol% of DMPC, 30-50 mol% of cholesterol, and 4-10 mol% of PEGylated distearoylphosphatidylethanolamine (e.g., DSPE-PEG2000) relative to the total amount of membrane-constituting lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains). Such liposomes of the present invention may also contain, in mole fraction (mol%) of the total membrane lipid content, amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol in amounts of 5 mol% to 25 mol% or 20 mol% to 35 mol%, DMPC in amounts of 35 mol% to 55 mol%, cholesterol in amounts of 30 mol% to 50 mol%, and PEGylated distearoylphosphatidylethanolamine (e.g., DSPE-PEG2000) in amounts of 4 mol% to 10 mol%.

[0075] In one embodiment, the liposomes of the present invention may contain amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol, and lamellar-forming lipids, such as dioleylphosphatidylethanolamine (DOPE) and 1,2-dioleoyloxy-3-trimethylammoniumpropane chloride (DOTAP). Such liposomes of the present invention may contain, in mole fraction (mol%) of amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol, 15 mol% to 35 mol% or 20 mol% to 40 mol%, DOPE, and 40 mol% to 70 mol% of DOTAP, relative to the total amount of membrane-constituting lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains). Such liposomes of the present invention may also contain, in mole fraction (mol%) relative to the total amount of membrane constituent lipids, amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol in amounts of 5 mol% to 30 mol% or 10 mol% to 20 mol%, DOPE in amounts of 10 mol% to 35 mol%, and DOTAP in amounts of 55 mol% to 65 mol%.

[0076] In one embodiment, the liposomes of the present invention may contain amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol and 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP). Such liposomes of the present invention may contain, in mole fraction (mol%) of amphiphilic lipids having isoprenoid-type fatty acid chains, such as mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol and 50-60 mol% of DOTAP, relative to the total amount of membrane constituent lipids (moles of lamellar-forming lipids + moles of amphiphilic lipids having isoprenoid-type fatty acid chains). Such liposomes of the present invention may not contain phospholipids or steroids.

[0077] The liposomes of the present invention may or may not contain other substances in addition to lamellar-forming lipids and amphiphilic lipids having isoprenoid-type fatty acid chains.

[0078] The liposomes of the present invention may or may not contain a surfactant, but it is not necessary to include one. Examples of surfactants include nonionic surfactants such as block copolymers of hydrophilic ethylene oxide and hydrophobic propylene oxide (polyoxyethylene polyoxypropylene glycol), polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, and polyoxyethylene hydrogenated castor oil. As nonionic surfactants, those with a molecular weight of 1000 or more (more preferably 5000 or more) are more preferred. Examples of block copolymers of ethylene oxide and propylene oxide include polyoxyethylene (200) polyoxypropylene (70) glycol, polyoxyethylene (196) polyoxypropylene (67) glycol, polyoxyethylene (160) polyoxypropylene (30) glycol, and polyoxyethylene (120) polyoxypropylene (40) glycol. These block copolymers of ethylene oxide and propylene oxide are pluronic (R) , Poloxam (R) Unilube (R) Pronon (R) These are commercially available under various names. Particularly preferred examples of nonionic surfactants include polyoxyethylene (200) polyoxypropylene (70) glycol and polyoxyethylene (196) polyoxypropylene (67) glycol (also known as Pluronic Acid). (R) F127; Unilube 70DP-950B, Poloxamer (R) Examples include 407). Another example of a surfactant is P80 (polyoxyethylene sorbitan monooleate (20E.O.)).

[0079] The liposomes of the present invention typically have an internal aqueous phase containing an aqueous solvent. Examples of such aqueous solvents include, but are not limited to, phosphate-buffered saline (PBS), citrate buffer, citrate-phosphate buffer, acetate buffer, Tris-HCl buffer, hypotonic phosphate buffer, and water. The aqueous solvent may also contain other substances, such as drugs, as described later.

[0080] The liposomes of the present invention preferably do not contain non-lamellar liquid crystals. In one embodiment, the liposomes of the present invention may contain non-lamellar liquid crystal-forming lipids as membrane constituent lipids, but may not contain non-lamellar liquid crystals. The liposomes of the present invention are not non-lamellar liquid crystal microparticles.

[0081] The liposomes of the present invention can be prepared by conventional liposome manufacturing methods. Specifically, the liposomes of the present invention can be prepared by dissolving membrane-constituting lipids, such as lamellar-forming lipids and amphiphilic lipids having isoprenoid-type fatty acid chains, in an organic solvent (preferably ethanol, chloroform, etc.), mixing them uniformly, removing the organic solvent (such as alcohol) by treatment such as vacuum concentration, adding an aqueous solvent suitable for liposome preparation, such as a buffer solution (e.g., phosphate-buffered saline) or water, and then dispersing them using an ultrasonic homogenizer, high-pressure homogenizer, etc. (e.g., sonication). The sonication may be performed, for example, by applying an amplitude of 10-30% (20%) for 20-60 seconds (e.g., 20-40 seconds, preferably 30 seconds) 1-5 times (e.g., 2 times). It is preferable to perform the dispersion step, such as dispersion by sonication, within a range that does not cause the temperature to rise too high.

[0082] The liposomes of the present invention are preferably temperature-responsive and can exert a cell membrane destabilizing effect within a predetermined temperature range. In preferred embodiments, the liposomes of the present invention have little effect on the cell membrane at normal body temperature (approximately 35-38°C), but destabilize the cell membrane at higher temperatures, for example, 39-60°C or 44-48°C. In the present invention, cell membrane destabilization means interacting with the cell membrane, increasing the membrane fluidity of the cell membrane, thereby improving the efficiency of substance delivery into the cell and promoting cell lysis. The temperature-responsive liposomes of the present invention can be advantageously used for substance delivery into cells and induction of cell lysis based on their cell membrane destabilizing effect. The temperature-responsive liposomes of the present invention can also be advantageously used to suppress tumor growth by utilizing heating.

[0083] The liposomes of the present invention typically have an average particle diameter of 50 nm to 500 nm, preferably 80 nm to 400 nm, more preferably 80 nm to 300 nm, and even more preferably 100 nm to 300 nm.

[0084] The liposomes of the present invention typically have a PdI of 0.05 to 0.4, preferably 0.05 to 0.35, or 0.1 to 0.35, more preferably 0.05 to 0.3, or 0.1 to 0.3, for example, 0.15 to 0.25.

[0085] The liposomes of the present invention preferably have the property of accumulating at tumor sites due to the EPR effect (Enhanced Permeation and Retention Effect) when administered systemically. The liposomes of the present invention also accumulate in the liver and spleen when administered systemically. The liposomes of the present invention do not cause hemolysis at normal body temperature and can be safely administered systemically.

[0086] The liposomes of the present invention preferably further contain a drug. In the present invention, a drug is any substance (active ingredient) that is contained in or immobilized on a liposome, preferably retained inside or within the membrane of the liposome, for delivery into a cell. The drug may be an organic compound or an inorganic compound. The drug may be a water-soluble drug or a lipid-soluble (lipophilic, water-insoluble, or sparingly water-soluble) drug. Typically, water-soluble drugs are retained in the internal aqueous phase inside the liposome, and lipid-soluble drugs are embedded and retained within the membrane of the liposome. The drug may, but is not limited to, a physiologically active substance. The drug may, but is not limited to, a protein, peptide, amino acid, nucleic acid, etc. The drug may, but is not limited to, a therapeutic or preventive effect.

[0087] The drug may be a fluorescent substance such as a fluorescent protein, a dye, or a labeling substance such as a radioisotope. The liposomes of the present invention, which contain a labeling substance as the drug, can also be used as labeling agents (imaging agents) for labeling cells by delivering the labeling substance to cells.

[0088] The drug may be any nucleic acid, such as DNA, RNA, a DNA-RNA hybrid, or DNA or RNA containing artificial bases or modified nucleic acids. The nucleic acid may contain any gene. The nucleic acid may be an expression vector or expression cassette containing the transgene under the control of an expression promoter. The expression vector may be a plasmid vector, a viral vector, etc. The transgene may be any gene, such as DNA or RNA. The transgene may be a gene that produces an antitumor effect, such as a tumor suppressor gene, a cell growth regulator gene, or an apoptosis-inducing gene, or it may be a toxin protein gene. The nucleic acid may also be siRNA, shRNA, dsRNA, etc. RNA interference-inducing nucleic acids such as siRNA and shRNA may be designed for genes whose expression should be suppressed. The liposomes of the present invention can also facilitate the delivery of such nucleic acids into the nucleus of a cell. The nucleic acid may be for the treatment or prevention of any disease. In one embodiment, it is preferable that the liposomes of the present invention bind to the nucleic acid, forming a complex. The binding of the liposomes and nucleic acids is not particularly limited, but may be by electrostatic interaction, for example. The nucleic acid-liposome complex according to the present invention is particularly useful for the delivery of nucleic acids to cells. In a preferred embodiment, the nucleic acid-liposome complex according to the present invention includes, as membrane constituent lipids, an amphiphilic lipid having the above-mentioned isoprenoid-type fatty acid chain and a lamellar-forming lipid. The lamellar-forming lipid used in the nucleic acid-liposome complex includes at least one selected from the group consisting of phospholipids, steroids, and cationic lipids, but preferably includes a cationic lipid, and more preferably includes both a cationic lipid and a phospholipid. As described above, the cationic lipid is particularly preferred, but 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) is particularly preferred. As described above, the phospholipid is also particularly preferred, but phosphatidylethanolamine such as dioleylphosphatidylethanolamine (DOPE) is particularly preferred.

[0089] In one embodiment, the drug may be an antitumor agent. In another embodiment, the drug may be a treatment for liver disease (e.g., hepatitis) or spleen disease. The antitumor agent is not particularly limited, but examples include doxorubicin hydrochloride (DXR).

[0090] The liposomes of the present invention can be advantageously used for intracellular drug delivery. The present invention also provides intracellular drug delivery formulations comprising the liposomes of the present invention. The cells to which the drug is delivered may be any cells, but for example, in the case of systemic administration, they may be tumor cells. The cells to which the drug is delivered may also be hepatocytes or spleen cells.

[0091] The liposomes of the present invention, containing the above-mentioned nucleic acids, can be advantageously used for nucleic acid delivery into cells, particularly into the nucleus of cells. The present invention also provides a formulation for nucleic acid delivery into the nucleus of cells, comprising the liposomes of the present invention containing the above-mentioned nucleic acids. The cells delivering the nucleic acids may be any cells, but are preferably tumor cells. The cells delivering the nucleic acids may also be hepatocytes or spleen cells.

[0092] The present invention provides a drug delivery system for drugs, such as nucleic acids like genes.

[0093] The drug delivery formulations for intracellular use and nucleic acid delivery formulations for intracellular nuclei of the present invention may further contain pharmaceutically acceptable additives (e.g., carriers, excipients, buffers, pH adjusters, preservatives, colorants, flavorings, propellants, etc.).

[0094] The drug delivery formulation for cells and the nucleic acid delivery formulation for the nucleus of cells according to the present invention may be a reagent for in vitro testing (in vitro nucleic acid delivery reagent) or a pharmaceutical formulation.

[0095] The present invention provides a pharmaceutical formulation containing the liposomes of the present invention. In the present invention, "pharmaceutical formulation" can refer to a pharmaceutical composition. The pharmaceutical formulation of the present invention may contain pharmaceutically acceptable additives (e.g., carriers, excipients, buffers, pH adjusters, preservatives, colorants, flavorings, propellants, etc.) as long as the liposome form is maintained. Furthermore, the liposomes of the present invention can exhibit antitumor effects even without containing a drug (e.g., an antitumor agent). Therefore, a pharmaceutical formulation containing the liposomes of the present invention can be an antitumor formulation, regardless of whether or not it contains a drug (e.g., an antitumor agent).

[0096] The pharmaceutical formulation according to the present invention may be in any dosage form, but is preferably a liquid, capsule, spray, aerosol, injection, suppository, or depot formulation. The pharmaceutical formulation according to the present invention may be for the treatment or prevention of any disease, for example, for the treatment or prevention of various diseases such as neoplasms like cancer and benign tumors, skin diseases, and liver disorders.

[0097] The present invention also provides a method for gradually delivering a drug into the body, which includes applying the above-mentioned formulation according to the present invention to the body (particularly to the biological tissues within the body) of a subject (e.g., a patient). Here, the application into the body is preferably carried out by parenteral administration (e.g., systemic administration such as intravenous or intra-arterial administration, intraperitoneal, intramuscular, transdermal, subcutaneous, or intradermal administration, etc.), but the route of administration is not limited thereto. Parenteral administration can be carried out by systemic or local administration, for example, but systemic administration is more preferred. The subject to which the above-mentioned formulation according to the present invention is administered may be any animal, including mammals, birds, etc. (e.g., an animal having the above-mentioned disease), and may be, for example, a human, a chimpanzee, a gorilla, an orangutan, a primate, a dog, a cat, a rabbit, a ferret, a panda, a cow, a horse, a sheep, a goat, a pig, a mouse, a rat, etc. The subject is preferably a subject that requires administration of the liposome of the present invention or a pharmaceutical formulation containing it.

[0098] The liposomes of the present invention are preferably temperature-responsive, and more preferably, exhibit improved membrane fluidity and action on cell membranes under predetermined temperature conditions higher than body temperature. Therefore, by warming the target site under predetermined temperature conditions after administration, drug release from the liposomes can be promoted specifically at that site, and cell membrane destabilization can be induced. In other words, the liposomes of the present invention can promote cell death when used in combination with warming treatment. Since the liposomes of the present invention exhibit high tumor accumulation, combining them with warming treatment can yield a higher antitumor effect. The warming treatment can be performed at a temperature higher than body temperature (approximately 35-38°C), for example, 39-60°C, or for example, 44-48°C. The warming treatment can be performed using any means that can intensively (preferably specifically) warm the affected area, for example, by ultrasonic treatment or near-infrared irradiation (wavelength 650-2,500 nm, for example, wavelength 700-1,000 nm) that can warm up to the target temperature. It should be noted that heating is not necessarily required in this invention, and drug delivery using the liposomes of this invention is possible even without heating. In particular, for nucleic acid delivery, it is entirely possible to administer the liposomes of this invention without heating. The temperature-responsive liposomes of this invention can be used in pharmaceutical formulations that provide antitumor effects, i.e., in tumor (cancer) therapeutic agents. The dosage of a pharmaceutical formulation containing the liposomes of this invention can be appropriately determined by those skilled in the art based on the drug content in the liposomes.

[0099] The present invention also provides a method for suppressing tumor growth or a method for treating a tumor, comprising administering the liposomes of the present invention or a pharmaceutical formulation containing them to a subject (e.g., a patient). The route of administration of the liposomes of the present invention or formulations such as drug delivery formulations, nucleic acid delivery formulations, and pharmaceutical formulations containing them is not particularly limited, but parenteral administration (e.g., systemic administration such as intravenous or intra-arterial administration, intraperitoneal, intramuscular, transdermal, subcutaneous, or intradermal administration) is preferred, and parenteral administration may be by systemic or local administration, for example. The subject to whom the liposomes of the present invention or the formulations containing them are administered is preferably a subject that requires administration of the liposomes of the present invention or the formulations containing them, and more preferably a subject that has a tumor or is suspected of having a tumor. Alternatively, the subject may be a subject to whom drug delivery to the liver or spleen is desired, such as a subject with a disease of the liver or spleen. The target may be any animal, including mammals and birds (for example, animals with the above-mentioned diseases), such as humans, chimpanzees, gorillas, orangutans and other primates, dogs, cats, rabbits, ferrets, pandas, cows, horses, sheep, goats, pigs, mice, rats, etc. The dosage of the liposomes or formulations of the present invention can be appropriately determined by those skilled in the art, but for example, in the case of humans, it may be an amount equivalent to 0.0001 mg to 100 g or 1 mg to 50 g of liposomes, but is not limited thereto.

[0100] In the present invention, "tumor" encompasses malignant or benign neoplastic diseases. Target tumors (cancers) for which the liposomes of the present invention provide drug delivery or antitumor effects include, but are not limited to, breast cancer, liver cancer, spleen cancer, kidney cancer, pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, lung cancer, head and neck cancer, brain tumor, biliary tract cancer, bladder cancer, uterine cancer (endometrial cancer, cervical cancer, etc.), ovarian cancer, fallopian tube cancer, prostate cancer, leukemia, malignant lymphoma, multiple myeloma, and the like. Target organs for drug delivery by the liposomes of the present invention include, for example, the liver and spleen, and target diseases include diseases of the liver or spleen (e.g., hepatitis). [Examples]

[0101] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0102] [Example 1] Synthesis of amphiphilic lipids having isoprenoid-type fatty acid chains (1) Synthesis of mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol

[0103] [ka]

[0104] To a solution of 0.65 g (7.1 mmol) of glycerol and 0.59 g (4.3 mmol) of potassium carbonate in dry N,N-dimethylformamide (3.5 mL), 1.0 g (3.5 mmol) of methyl 5,9,13-trimethyltetradeca-4-enoate (methyl tetrahydrofarnesyl acetate) was slowly added dropwise at 80°C. After stirring at 100°C for 18 hours, 1 M hydrochloric acid was added to the reaction mixture, and it was extracted with ether. The extract was sequentially washed with saturated sodium bicarbonate solution and saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (mobile phase: ethyl acetate / hexane mixture) to obtain the title compound as a colorless, transparent liquid. Regarding the obtained compound, 1 The results of the H-NMR and viscosity measurements are as follows.

[0105] 1 H-NMR spectrum (300MHz, CDCl3, TMS) δ:0.80-0.90(m,9H),1.00-1.70(m,15H),1.97(td,J=7.8,17.0Hz,2H),2.13(t,J=6. 1Hz,1H,OH),2.25-2.45(m,4H),2.55(d,J=5.2Hz,1H,OH),3.50-4.00(m,3H),4.10-4.25(m,2H),5.08(t,J=6.7Hz,1H) Viscosity: 0.48Pa·s (shear rate 92 1 / s) The synthesized mono O-(5,9,13-trimethyltetradeca-4-enoyl) glycerol is also referred to as C17MGE.

[0106] (2) Synthesis of mono O-(5,9,13,17-tetramethyloctadeca-4-enoyl) glycerol

[0107] [Chemical formula]

[0108] Under a reduced pressure of 60 - 70 mmHg and a nitrogen stream, 28.2 g (80.0 mmol) of methyl 5,9,13,17-tetramethyloctadeca-4-enoate was gradually added dropwise to a dried N,N-dimethylformamide (48 mL) solution of 23.5 g (255 mmol) of glycerol and 0.55 g (4.0 mmol) of potassium carbonate at 80°C, and the mixture was stirred at the same temperature for 3 hours. The resulting reaction solution was diluted with an ethyl acetate / hexane mixed solvent (at a ratio of 1:1, 200 mL), washed with water, saturated aqueous sodium hydrogen carbonate, and saturated brine (twice), and then dried over magnesium sulfate. The residue obtained by filtration and concentration was purified by silica gel column chromatography (hexane / ethyl acetate = 100:0 - 30:70), and 13.3 g (yield 40%) of the title compound was obtained as a slightly yellow transparent liquid. Regarding the obtained compound, 1 The results of 1H-NMR measurement are as follows.

[0109] 1 1H-NMR spectrum (300 MHz, CDCl3, TMS) δ: 0.80 - 0.95 (m, 12H), 1.00 - 1.70 (m, 22H), 1.85 - 2.15 (m, 2H), 2.15 - 2.55 (m, 4H), 3.53 - 3.78 (m, 3H), 3.80 - 4.00 (m, 1H), 4.10 - 4.25 (m, 2H), 5.08 (dd, J = 6.9 Hz, J = 6.9 Hz, 1H) The synthesized mono O-(5,9,13,17-tetramethyloctadeca-4-enoyl) glycerol is also referred to as C22MGE.

[0110] [Example 2] Preparation of a microparticle formulation Microparticle formulations were prepared using the thin-film hydration method, a known method for preparing liposomes, according to the compositions shown in Table 1 below.

[0111] Specifically, first, as lipids, we used C17MGE (synthesized in Example 1), an amphiphilic lipid having an isoprenoid-type fatty acid chain, and dimyristoylphosphatidylcholine (DMPC; COATSOME), a type of phosphatidylcholine. (R) MC-4040, NOF Corporation), DSPE-PEG2000 (SUNBRIGHT (R) A mixed solution was prepared by uniformly mixing 10 mM ethanol solutions of DSPE-020CN (NOF Co., Ltd.) and cholesterol (Wako Special Grade, FUJIFILM Wako Pure Chemical Corporation), respectively. DSPE is distearoylphosphatidylethanolamine, and PEG is polyethylene glycol. The resulting mixed solution was concentrated under reduced pressure to completely remove the ethanol. Then, pH 7.4 phosphate-buffered saline (PBS(-)) was added to achieve a final lipid concentration of 1 mM, and the solution was allowed to stand at room temperature for 10 minutes. The solution was then ultrasonically treated at room temperature for several minutes using an ultrasonic cleaner (5510, BRANSON) to prepare a crude dispersion. Furthermore, this crude dispersion was ultrasonically treated twice for 30 seconds at 20% amplitude using an ultrasonic homogenizer (Sonics Vibra-Cell VCX-750, Sonics & Materials, Inc.) to prepare slightly turbid particulate formulations No. 1-5. These microparticle formulations were each prepared in volumes of 1 to 5 mL.

[0112] [Example 3] Evaluation of physical properties of particulate formulation The particle size distribution and zeta potential of particulate formulations No. 1-5 prepared in Example 2 were measured by dynamic light scattering using a zetasizing device, Nano-ZS (Malvern). Each emulsion was prepared by diluting it 1000-fold with PBS. Table 1 shows the average particle size (nm) (Z-Average), PdI (polydispersion index), and zeta potential (mV) obtained as the average values ​​of 5-6 measurements for each sample.

[0113] [Table 1]

[0114] The average particle size of the obtained particulate formulations was in the range of 112–168 nm, and they possessed appropriate PdI and zeta potentials. All of these particulate formulations were stable throughout the experimental process, with no visible aggregates.

[0115] Furthermore, structural analysis of particulate formulations No. 1-5 was performed using small-angle X-ray scattering (SAXS) with the NANO Viewer nanoscale X-ray structure evaluation system (Rigaku). Each particulate formulation No. 1-5 was introduced into a capillary under atmospheric pressure, and measurements were performed in a reduced-pressure system (the sample itself was under atmospheric pressure). As a result, no peaks characteristic of non-lamellar liquid crystals were observed in any of the particulate formulations. Therefore, it was shown that particulate formulations No. 1-5 do not form non-lamellar liquid crystals.

[0116] The microparticle morphology of microparticle formulation No. 4 was observed using a cryogenic transmission electron microscope (cryo-TEM) (JEM-3100FEF, JEOL Ltd.). Specifically, microparticle formulation No. 4 was first diluted with PBS to a total lipid concentration of 0.5 mM. 1 μL of this diluted solution was dropped onto a hydrophilized Cu microgrid (product number: 1643, 200 mesh, JEOL Ltd.) and blotted. Using a cryogenic sample preparation system (EM-CPC, Leica), the resulting grid was instantaneously frozen with liquefied ethane and then observed under liquid nitrogen temperature using cryo-TEM in bright-field mode. The defocus was 5-10 μm, adjusted as appropriate depending on the observation magnification. Figure 1 shows the image taken at a magnification of 15,000x. As shown in Figure 1, microparticles with liposome-like membrane structures (single lamellars) with a diameter of approximately 100 nm were observed.

[0117] Furthermore, microparticle formulation No. 5 showed a tendency towards decreased stability when observed similarly using cryo-TEM.

[0118] [Example 4] Changes in the physical properties of fine particles due to temperature changes (film fluidity) Ethanol solutions of each lipid were mixed according to the component ratios of the microparticle formulations No. 1 to 5 shown in Table 1. Furthermore, an ethanol solution of 1,6-diphenyl-1,3,5-hexatriene (DPH) (0.025 mM) was added as a fluorescent dye at a concentration of 0.01 mol% per 1,6-diphenyl-1,3,5-hexatriene (DPH) relative to the total lipid amount (C17MGE+DMPC+Cho+DSPE-PEG2000). The procedure was carried out in the same manner as in Example 2 so that the final total concentration of the lipids (C17MGE+DMPC+Cho+DSPE-PEG2000) and DPH was 1 mM, thereby preparing DPH-containing microparticle formulations No. 6 to 10 corresponding to microparticle formulations No. 1 to 5. Furthermore, as controls, by replacing DMPC in microparticle formulation No. 6 with dipalmitoylphosphatidylcholine (DPPC; COATSOME MC-6060, NOF Corporation) or dioleilphosphatidylcholine (DOPC; COATSOME MC-8181, NOF Corporation), C17MGE-free DPH-containing microparticle formulations No. 11 and 12 were prepared, respectively, using DPPC or DOPC as the phospholipid.

[0119] The membrane fluidity of the microparticles with respect to temperature changes was evaluated by adding 5 μL of a 1 M 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) ethanol solution as a quencher to 1 mL each of microparticle formulations No. 6 to 12 and observing the change in fluorescence intensity when the temperature was raised from 25°C to 60°C. DPH, which is localized in the lipid bilayer of the microparticles, exhibits fluorescence of a constant intensity, but when the lipid bilayer loosens and chemically reacts with TEMPO present outside, the fluorescence of DPH disappears. Using this principle (fluorescence quenching method), a lower fluorescence intensity ratio [F (fluorescence intensity in the presence of quencher) / F0 (fluorescence intensity in the absence of quencher)] indicates higher membrane fluidity.

[0120] For this fluorescence intensity measurement, a fluorescence spectrophotometer (RF-6000, Shimadzu Corporation, Japan) was used, and the measurement sample was observed at an excitation wavelength of 353 nm and an emission wavelength of 430 nm while the temperature was increased from 25°C to 60°C.

[0121] Figure 2 shows the change in fluorescence intensity ratio F / F0 of particulate formulations No. 6 to 12 with respect to temperature, with temperature (°C) on the horizontal axis and fluorescence intensity ratio F / F0 on the vertical axis.

[0122] As shown in Figure 2, in particulate formulation No. 11, which is based on DPPC with a phase transition temperature of 42°C and without C17MGE, the decrease in fluorescence intensity ratio began at 35°C and the disappearance of fluorescence intensity was completed by 50°C, at which point the decrease in fluorescence intensity stopped. In addition, in particulate formulation No. 12, which is based on DOPC with a phase transition temperature of 20°C and without C17MGE, the decrease in fluorescence intensity ratio almost stopped at 25°C, the start of measurement, and remained almost constant thereafter up to 60°C. On the other hand, in particulate formulation No. 6, which is based on DMPC with a phase transition temperature of 23°C and without C17MGE, the decrease in fluorescence intensity ratio continued slowly from 25°C to 60°C.

[0123] In DMPC-based microparticle formulations No. 7-10 containing C17MGE in a predetermined proportion, the decrease in fluorescence intensity ratio progressed slowly and continuously from 25°C to 60°C, similar to DMPC-based microparticle formulation No. 6 without C17MGE. However, the higher the proportion of C17MGE added, the lower the fluorescence intensity ratio tended to be at each temperature.

[0124] From these results, it was revealed that in DPH-containing microparticle formulations No. 6-12, there is a close correlation between the temperature at which membrane fluidity changes and the phase transition temperature of phosphatidylcholine incorporated into the microparticle formulation. Furthermore, it was found that the higher the proportion of C17MGE added, the higher the membrane fluidity at each temperature. This demonstrates that the addition of amphiphilic lipids with isoprenoid-type fatty acid chains can increase the membrane fluidity of microparticles in response to temperature.

[0125] [Example 5] Evaluation of the interaction between microparticle formulation and cell membrane in response to temperature changes To visualize the interaction between the microparticle formulation of the present invention and the cell membrane, an agglutination test was performed using red blood cells.

[0126] First, a red blood cell solution was prepared as follows: As an anticoagulant, trisodium citrate dihydrate (22 mg / mL), citrate monohydrate (8 mg / mL), and glucose (22 mg / mL) were dissolved in purified water to prepare a citrate-dextrose solution (ACD solution). Blood was collected from the left ventricle of a WBN / ILA-Ht hairless rat (male, 8 weeks old) using a syringe coated with heparin solution (1000 units / mL). To the obtained blood, 1 / 5 volume of ACD solution was quickly added and mixed by inversion. Then, the mixture was centrifuged at 3000 rpm for 20 minutes at 4°C using a hybrid high-speed refrigerated centrifuge (Model 6200, Kubota Shoji Co., Ltd.), followed by centrifugation at 3000 rpm for 10 minutes at room temperature using a benchtop centrifuge (Model 5200, Kubota Shoji Co., Ltd.). After that, the supernatant and the white precipitate (leukocytes, platelets) on top of the red blood cell fraction were removed as much as possible. To the obtained red blood cell fraction, three times the volume of PBS(-) was added, mixed by inversion, and centrifuged at 4,000 rpm for 10 minutes at room temperature, and the supernatant was removed. This procedure was repeated three times to obtain a washed red blood cell fraction. PBS(-) was added to this red blood cell fraction until it reached 2%, and a red blood cell solution was prepared. In this specification, PBS that does not contain calcium and magnesium is referred to as PBS(-).

[0127] Sixty microtubes (1.5 mL capacity, Eppendorf) containing 50 μL each of the above red blood cell solution were prepared. These microtubes were divided into five groups of 12 each. To each group, 100 μL of either the microparticle formulations No. 1-4 prepared in Example 2, or PBS(-) as a control, was added and mixed by pipetting. The five solutions (12 of each) obtained by mixing microparticle formulations No. 1-4 or PBS(-) with the red blood cell solution were heated for 15 minutes at 12 different temperatures (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48°C) using an aluminum block constant temperature bath (Dry Thermo Unit DTU-1B, Taitec Co., Ltd.). The entire volume of the solution in each microtube was transferred to each well of a 96-well round-bottom plate and allowed to stand for 120 minutes. After that, the state of the red blood cells in each well was visually observed. Furthermore, if red blood cells do not interact with other components, they will aggregate and settle in the center of the bottom of the well. However, if they do interact with other components, the colloid formed by the association of those components with the red blood cells will be dispersed and observed throughout the well.

[0128] Figure 3 shows images of the solution after heating at the 12 temperatures mentioned above, captured with a digital camera. As can be seen in Figure 3, when using microparticle formulations No. 2 and 3, which have a lower mole fraction of C17MGE, the state in which red blood cells interact with the microparticles and the colloid is dispersed throughout the well was observed at 47°C or higher, whereas when using microparticle formulation No. 4, which has a higher mole fraction of C17MGE, it was observed from a lower temperature of 44°C. On the other hand, when using microparticle formulation No. 1, which does not contain C17MGE, the state in which red blood cells interact with the microparticle formulation and the colloid is dispersed throughout the well was not observed from 37°C to at least 48°C, similar to the control using PBS(-).

[0129] These results indicate that amphiphilic lipids containing isoprenoid-type fatty acid chains incorporated into the microparticle formulation induce interaction (association) between the cell membrane and the microparticles.

[0130] [Example 6] Evaluation of the cell membrane destabilizing effect of microparticle formulations in response to temperature changes To visualize the destabilizing effect of the microparticle formulation of the present invention on cell membranes, a hemolysis test was performed using red blood cells.

[0131] First, a red blood cell test solution with appropriate absorbance was prepared as follows. An equal volume of PBS(-) was added to the washed red blood cell fraction obtained in the same manner as in Example 5 to obtain a red blood cell suspension. 250 μL of the red blood cell suspension was lysed with 4 times the volume (1 mL) of hypotonic buffer (10-fold dilution of PBS(-)) and centrifuged at 3,000 rpm for 5 minutes. The absorbance of the obtained supernatant was measured at 540 nm (the absorption wavelength of hemoglobin), and diluted with PBS(-) to obtain an absorbance of 2.0 to 2.5. The red blood cell test solution was obtained by diluting the red blood cell suspension with PBS(-) at the same dilution ratio to obtain an absorbance of 2.0 to 2.5.

[0132] Forty-five microtubes (1.5 mL capacity, Eppendorf) containing 40 μL of the above red blood cell test solution were prepared. These microtubes were divided into five groups of nine tubes each. To each group, four times the amount (160 μL) of one of the microparticle formulations No. 1 to 5 prepared in Example 2 was added to each microtube, and the mixture was then mixed by pipetting. Five different solutions (nine of each) obtained by mixing one of the microparticle formulations No. 1 to 5 with the red blood cell test solution were then heated in an aluminum block constant temperature bath (Dry Thermo Unit DTU-1B, Taitec Co., Ltd.) at 37°C or 45°C for 15 minutes, 30 minutes, 45 minutes, or 60 minutes. One sample solution from each group was kept unheated as a "heating time of 0 minutes" sample solution.

[0133] Next, each solution was centrifuged (3000 rpm, 5 minutes), and the supernatant was transferred to each well of a 96-well plate. Then, it was scanned using a microplate reader (Spectra Max). (R)The absorbance at 540 nm was measured using M2e (MOLECULAR DEVICES), and the hemolysis rate (%) of red blood cells in each well was determined. The hemolysis rate (%) was calculated by dividing the absorbance obtained for each solution by the absorbance obtained using hypotonic buffer (10-fold dilution of PBS(-)) instead of each solution.

[0134] Figure 4 shows graphs illustrating the change in hemolysis rates of five different solutions containing one of the microparticle formulations No. 1-5 and a red blood cell test solution, at 37°C (Figure 4A) and 45°C (Figure 4B), with respect to heating time. The horizontal axis represents heating time [minutes], and the vertical axis represents hemolysis rate [%]. The hemolysis rate values ​​are the mean ± standard deviation of three measurements.

[0135] At a heating temperature of 37°C, there was little difference in hemolysis rates between microparticle formulation No. 1 (which did not contain C17MGE) and microparticle formulations No. 2-4 (which contained C17MGE). Microparticle formulation No. 5 (which contained C17MGE) showed a tendency for the hemolysis rate to increase with heating time at 37°C. On the other hand, at a heating temperature of 45°C, there was little difference in hemolysis rates between microparticle formulation No. 1 (which did not contain C17MGE) and microparticle formulations No. 2 and 3 (which contained C17MGE at a lower mole fraction), similar to the case at 37°C. However, microparticle formulations No. 4 and 5 (which contained C17MGE at a higher mole fraction) showed an increase in hemolysis rates with heating time. This indicates that the incorporation of amphiphilic lipids with isoprenoid-type fatty acid chains into the microparticle formulations results in cell membrane disruption under heating conditions.

[0136] The results from Examples 4-6 showed that microparticle formulations containing amphiphilic lipids with isoprenoid-type fatty acid chains, such as C17MGE, could increase the membrane fluidity of the formulation itself by heating, and also promoted the interaction between the microparticles and the cell membrane, particularly inducing cell membrane destabilization. Microparticle formulations that did not contain amphiphilic lipids with isoprenoid-type fatty acid chains, such as C17MGE, showed increased membrane fluidity of the formulation itself, but did not show interaction with the cell membrane and did not induce cell membrane destabilization.

[0137] [Example 7] Pharmacokinetics of microparticle formulations after in vivo administration The pharmacokinetics of the microparticle formulations were evaluated by systemic administration to mice using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), a hydrophobic near-infrared fluorescent dye, in microparticle formulations No. 1 and 4, which contained C17MGE.

[0138] First, ethanol solutions of each lipid were mixed according to the component ratios of microparticle formulations No. 1 and 4 shown in Table 1. Then, an ethanol solution of DiR equivalent to 0.5 mol% of the total lipid amount (C17MGE+DMPC+Cho+DSPE-PEG2000) was added. The procedure was carried out in the same manner as in Example 2 so that the final total concentration of the lipids (C17MGE+DMPC+Cho+DSPE-PEG2000) and DiR was 1.5 mM. This prepared DiR-containing microparticle formulations No. 13 and 14, corresponding to microparticle formulations No. 1 and 4, respectively. Note that microparticle formulations No. 13 and 14 do not contain non-lamellar liquid crystals.

[0139] Next, BALB / c mice (female, 5 weeks old) were inoculated with 4T1-Luc cells, a mouse mammary cancer cell line that stably expresses luciferase (obtained from the JCRB Cell Bank (Japan) under JCRB number JCRB1447, 1.2 x 10⁶ cells). 6 Tumor-bearing mice, 12 days after subcutaneous transplantation of tumor cells, were subjected to general anesthesia using isoflurane. Then, 100 μL of microparticle formulation No. 13 or 14 was intravenously administered via the tail using a syringe (Terumo syringe 1 mL) equipped with a 27G needle. At 0.5, 1, 3, 6, 18, 24, and 45 hours after administration, the entire mouse under general anesthesia was observed using a fluorescence imaging device (IVIS Spectram) to track the time-dependent changes in fluorescence intensity at the tumor site. 45 hours after administration, the mice were euthanized, and each organ (heart, liver, spleen, kidney, lung, pancreas, tumor) was removed from the abdomen and observed using a fluorescence imaging device. For this observation, a filter set with an excitation wavelength of 745 nm and an fluorescence wavelength of 800 nm was used. Living Image was used for data analysis. (R) (PerkinElmer Corporation) was used.

[0140] Figure 5 shows fluorescence imaging images of the entire mouse at various time points after administration of microparticle formulation No. 13 or 14. Figure 6 shows the results of quantifying the fluorescence intensity of tumor sites using the ROI (Region of Interests) function based on the fluorescence imaging images in Figure 5. In Figure 6, the horizontal axis is time [h], and the vertical axis is total radiative efficiency [p / s] / [μW / cm]. 2 This shows (n=2~3). Figure 7 shows fluorescence imaging images of organs extracted from the abdomen of mice administered with microparticle formulation No. 13 or 14 (45 hours after administration).

[0141] As a result, it was revealed that microparticle formulation No. 14, which contains C17MGE, exhibited similar organ accumulation behavior to microparticle formulation No. 13, which is a typical liposomal formulation that does not contain C17MGE. Specifically, both microparticle formulations No. 13 and 14 administered to mice showed a time-dependent increase in accumulation at tumor sites, peaking at approximately 24 hours, but continuing to show high accumulation at tumor sites even after 45 hours. Furthermore, they also showed high accumulation in the liver and slight accumulation in the spleen. Notably, neither microparticle formulation showed accumulation in other organs such as the lungs, which could cause side effects. In addition, no external abnormalities were observed in the mice throughout this evaluation test.

[0142] Therefore, it was demonstrated that a microparticle formulation containing liposomes with C17MGE (without non-lamellar liquid crystals) can be safely administered to the body and accumulate at tumor sites. The accumulation of the microparticle formulation of the present invention at tumor sites is thought to be due to the EPR effect (Enhanced permeation and retention effect). Furthermore, as shown in the above examples, the microparticle formulation of the present invention does not interact with cells at body temperature (around 37°C), but interacts with cells and destabilizes the cell membrane when heated to 44-48°C. Therefore, it was considered that the microparticle formulation of the present invention can deliver drugs to tumor cells through temperature stimulation.

[0143] [Example 8] Preparation of a microparticle formulation Particulate formulations No. 15 containing non-lamellar liquid crystals and particulate formulations No. 16-30 without non-lamellar liquid crystals were prepared by adding cationic lipids to enable the complexation of negatively charged nucleic acids. In the case of particulate formulation No. 15, the proportion of C17MGE was increased to form non-lamellar liquid crystals, and a surfactant (Pluronic Acid) was added to disperse it. (R) F127) was added. In the case of microparticle formulations No. 16-30, the proportion of cationic (positively charged) lipids, which also contribute to liposome formation, was increased, and the surfactant (Pluronic (R) F127) was not included in the formula.

[0144] Specifically, in the preparation of particulate formulation No. 15 containing a non-lamellar liquid crystal dispersion, the following are used according to the weight fraction [wt%] shown in Table 2 below: C17MGE having an isoprenoid-type fatty acid chain, dioleylphosphatidylethanolamine (DOPE; COATSOME ME-8181, NOF Corporation), a type of phosphatidylethanolamine, 1,2-dioleoyloxy-3-trimethylammoniumpropane chloride (DOTAP; COATSOME CL-8181TA, NOF Corporation), a cationic lipid, and Pluronic (R) F127 (Unilube) (R) 70DP-950B (NOF, or Aldrich P2443) was added to a total of 50 mg in 5-20 mL round-bottom flasks, and 0.5 mL of chloroform was added to dissolve it uniformly. The mole fractions [mol%] of C17MGE, DOPE, and DOTAP, which correspond to lipids, were 91.3 mol%:4.2 mol%:4.5 mol%.

[0145] On the other hand, in the preparation of particulate formulations No. 16 to 30 that do not contain non-lamellar liquid crystals, C17MGE, DOPE, and DOTAP, each prepared as a 10 mM ethanol solution, were uniformly mixed in 5 to 20 mL round-bottom flasks according to the mole fractions [mol%] shown in Table 3 below, so that the total weight after ethanol removal was 50 mg. The ratio of each component in the lipids in the particulate formulations was set to 50 mol% or 60 mol% of DOTAP within an appropriate mole fraction range, and the C17MGE:DOPE ratio was set to 0:100 to 100:0.

[0146] After completely removing chloroform or ethanol from each obtained solution by vacuum concentration, pH 7.4 phosphate-buffered saline (PBS) was added to achieve a final combined lipid concentration of 2 mM, and the mixture was allowed to stand at room temperature for 10 minutes. The mixture was then treated with an ultrasonic cleaner (5510, BRANSON) at room temperature for several minutes to obtain a crude dispersion. Furthermore, this crude dispersion was ultrasonically treated twice for 30 seconds at 20% amplitude using an ultrasonic homogenizer (Sonics Vibra-Cell VCX-750, Sonics & Materials, Inc.) to prepare turbid or slightly turbid particulate formulations No. 15-30. Each of these particulate formulations was prepared in volumes of 0.5-1 mL.

[0147] Furthermore, using a similar procedure, a microparticle formulation (GMO microparticle formulation) was prepared according to the same composition and preparation method as microparticle formulation No. 20, except that glyceryl monooleate (GMO, Rikemar XO-100, NOF Corporation) was used instead of C17MGE. However, aggregates precipitated and separated into layers in this GMO microparticle formulation within at least half a day after preparation. Therefore, it was shown that GMO is not suitable as a lipid to replace C17MGE in the preparation of the microparticle formulation of the present invention.

[0148] [Example 9] Evaluation of physical properties of particulate formulations The particle size distribution and zeta potential of particulate formulations No. 15-30 prepared in Example 8 were measured in the same manner as in Example 3. For each measurement sample, the average particle size (nm) (Z-Average), PdI (polydispersion index), and zeta potential (mV) obtained as the average of three measurements are shown in Tables 2 and 3.

[0149] [Table 2]

[0150] [Table 3]

[0151] In particulate formulations No. 16-30 that do not contain non-lamellar liquid crystals, regardless of whether the mole fraction of DOTAP in lipids was 50 mol% or 60 mol%, the average particle size tended to increase as the proportion of C17MGE in the C17MGE:DOPE ratio increased, and the PdI also increased, leading to a tendency for the actual formulation to become unstable. Formulation No. 30, with a mole fraction of DOTAP in lipids of 60 mol% and C17MGE:DOPE = 100:0, showed a lower average particle size and greater stability, contrary to the above trend.

[0152] Furthermore, structural analysis of particulate formulations No. 15 to 30 was performed using small-angle X-ray scattering (SAXS), similar to Example 3. At least three scattering peaks were observed in the scattering intensity distribution obtained from particulate formulation No. 15. The ratio of the peaks showed a ratio of 1:√3:2, characteristic of inverse hexagonal liquid crystals, indicating that this formulation is a liquid crystal emulsion (hexosome) in which inverse hexagonal liquid crystal particles are dispersed in the aqueous phase. The scattering vector value of the peak located at the smallest angle was 1.42 nm⁻¹. On the other hand, no peaks characteristic of non-lamellar liquid crystals were observed in any of the particulate formulations No. 16 to 30.

[0153] Microparticle formulation No. 27 was observed using cryo-TEM, similar to Example 3. Figure 8 shows the image of microparticle formulation No. 27 at a magnification of 15,000x. Microparticles with liposome-like membrane structures (multilamellae) of 100 nm or more were observed.

[0154] [Example 10] Verification of the effect of intracellular introduction by fluorescent dyes The effect of fluorescent dyes on intracellular drug delivery was tested using HaCaT cells (300493-SF, Cellline Services Co., Ltd.), a human epidermal keratinocyte cell line.

[0155] Specifically, first, ethanol solutions of each lipid were mixed in the component ratios shown in Table 3 for particulate formulations No. 16, 20, and 23. Then, 0.5 mol% of rhodamine-PE (810150, Avanti Polar Lipid Co.) was added as a fluorescently labeled molecule relative to the total amount of lipids (C17MGE + DOPE + DOTAP). The procedure was carried out in the same manner as in Example 8 so that the final total concentration of rhodamine-PE and the lipids (C17MGE + DOPE + DOTAP) was 2 mM, thereby preparing fluorescently labeled particulate formulations No. 31 to 33, corresponding to particulate formulations No. 16, 20, and 23, respectively.

[0156] Next, pre-packaged HaCaT cells (4x10) in a 24-well plate. 4 Three of the four wells containing cells (per well) were treated by adding 10 μL each of one of the microparticle formulations No. 31-33. The four wells, including one untreated well (without microparticle formulation), were incubated at 37°C for 3 hours (under a 5% CO2 atmosphere). The intracellular uptake efficiency of the fluorescently labeled molecule was then measured using a flow cytometer (CytoFLEX, Beckman Coulter). Data for 10,000 cell counts was obtained for each measurement.

[0157] Figure 9A is a graph showing the amount of fluorescently labeled molecules taken up intracellularly in groups treated with microparticle formulations No. 31-33 and untreated groups, with the horizontal axis representing the intracellular fluorescence intensity [au] due to the fluorescently labeled molecule and the vertical axis representing the number of cells. Figure 9B shows the ratio (intracellular uptake efficiency) of the amount of fluorescently labeled molecules taken up intracellularly in groups treated with microparticle formulations No. 31-33 to the amount taken up intracellularly in the untreated group, calculated from Figure 9A. Intracellular uptake efficiency was calculated as the average fluorescence intensity ratio = fluorescence intensity of the formulation / fluorescence intensity of the untreated group.

[0158] As a result, the groups treated with microparticle formulations No. 31-33 all showed approximately twice the intracellular uptake efficiency of fluorescently labeled molecules compared to the untreated group. Therefore, it was demonstrated that microparticle formulations No. 31-33, like the microparticle formulations prepared in Example 2, can efficiently deliver drugs into cells.

[0159] [Example 11] Preparation of nucleic acid-microparticle complex and evaluation of its complex state Nucleic acid-microparticle complexes were prepared by mixing microparticle formulations No. 15-30, prepared in Example 8, with pcDNA3-EGFP (provided by Addgene, USA under MTA), a plasmid expression vector (pDNA) containing the EGFP gene, at an N / P ratio of 0.5, 1, or 2. The N / P ratio indicates the proportion of nucleic acid introduced into the microparticle formulation and is expressed as the amount (mol) of cationic lipid (in this case, DOTAP) in the microparticle formulation divided by the amount (mol) of nucleic acid. In other words, a larger N / P ratio indicates a higher proportion of cationic lipid to nucleic acid.

[0160] The nucleic acid complex state of nucleic acid-microparticle complexes derived from microparticle formulation No. 15 and No. 20, which were mixed with nucleic acids at an N / P ratio of 0.5, 1, or 2, was evaluated by electrophoresis using a horizontal electrophoresis apparatus (Mupid-exU, Mupid Inc.). Specifically, pcDNA3-EGFP alone and each nucleic acid-microparticle complex were loaded in amounts equivalent to 0.8 μg of nucleic acid, and electrophoresis was performed at 100 mV for 30 minutes using a 0.7% agarose gel. After staining with ethidium bromide, electrophoretic images were obtained. Furthermore, the brightness intensity of the bands for pcDNA3-EGFP alone and nucleic acids released from each nucleic acid-microparticle complex was determined using the image processing software ImageJ.

[0161] Figure 10 shows the electrophoretic image of the nucleic acid-microparticle complex. Figure 11 shows the ratio of the brightness intensity of the band of nucleic acid released from the nucleic acid-microparticle complex (pcDNA3-EGFP) to the brightness intensity of the band of pcDNA3-EGFP alone. A smaller brightness intensity ratio indicates a stronger complex state between the nucleic acid and microparticles.

[0162] As shown in Figures 10 and 11, both nucleic acid-microparticle complexes derived from microparticle formulations No. 15(A) and No. 20(B) showed a tendency for the amount of nucleic acid retained in the complex state to increase with increasing N / P ratio. The nucleic acid-microparticle complex derived from microparticle formulation No. 15 retained more than half of the nucleic acid in the complex state, suggesting a strong interaction between microparticles and nucleic acid. On the other hand, the nucleic acid-microparticle complex derived from microparticle formulation No. 20 showed a greater amount of free nucleic acid compared to the nucleic acid-microparticle complex derived from microparticle formulation No. 15, indicating a weaker interaction between microparticles and nucleic acid than that of the nucleic acid-microparticle complex derived from microparticle formulation No. 15.

[0163] Nucleic acid-microparticle complexes derived from microparticle formulations No. 15 and 20, which contain C17MGE, were retained as complexes after electrophoresis, although the strength of the interaction between the nucleic acid and microparticles varied. This indicates that all of them possess bioavailability under certain environmental conditions.

[0164] [Example 12] Evaluation of the effect of introducing nuclear nucleic acids by gene expression The effect of introducing nucleic acids into the nucleus via gene expression was evaluated using HaCaT cells (300493-SF, Cellline Services Co., Ltd.), a human epidermal keratinocyte cell line.

[0165] This evaluation included nucleic acid-microparticle complexes (containing 0.8 μg of pcDNA3-EGFP) derived from microparticle formulations No. 15-30, prepared in Example 11 with an N / P ratio of 2, pcDNA3-EGFP alone (0.8 μg), and the commercially available nucleic acid delivery reagent, Lipofectamine. (R) A mixture of 2000 (11668027, Invitrogen) and pcDNA3-EGFP alone (0.8 μg of pcDNA3-EGFP alone and Lipofectamine) (R) A 4 μL mixture (hereinafter referred to as LFN2000-nucleic acid mixture) was used.

[0166] Each well in a 24-well plate (4x10) contains pre-filled HaCaT cells. 4Nucleic acid-microparticle complexes derived from microparticle formulations No. 15-30, pcDNA3-EGFP alone, or an LFN2000-nucleic acid mixture were added to cells (per well), incubated at 37°C for 40 hours, and the expression status of EGFP, a type of green fluorescent protein (GFP), was observed using a fluorescence microscope (BZ-X710, Keyence Corporation, equipped with a Plan Fluorite 4×PH lens).

[0167] Figure 12 shows fluorescence images of cells treated with each formulation. Cells treated with pcDNA3-EGFP alone, the negative control, did not express GFP, while cells treated with the LFN2000-nucleic acid mixture, the positive control, strongly expressed GFP. Almost no GFP expression was observed in the nucleic acid-microparticle complex derived from microparticle formulation No. 15, which contains non-lamellar liquid crystals. Furthermore, the nucleic acid-microparticle complexes derived from microparticle formulations No. 16 and 23, which do not contain either C17MGE or DOPE and do not contain non-lamellar liquid crystals, showed lower GFP expression intensity than the LFN2000-nucleic acid mixture. In contrast, the nucleic acid-microparticle complex derived from microparticle formulation No. 20, which contains both C17MGE and DOPE but does not contain non-lamellar liquid crystals, showed GFP expression intensity equivalent to that of the LFN2000-nucleic acid mixture.

[0168] Furthermore, for each nucleic acid-microparticle complex derived from microparticle formulations No. 16-30, and specifically for pcDNA3-EGFP alone, fluorescence-activated cell sorting (FACS) analysis was performed using a flow cytometer (CytoFLEX, Beckman Coulter) to quantitatively capture the GFP expression status in treated cells. In this analysis, a laser wavelength of 488 nm was used, and the GFP expression status of HaCaT cells in each well treated as described above was examined using the data analysis software CytoExpert.

[0169] Figures 13-15 show dot plots obtained from HaCaT cells treated with nucleic acid-microparticle complexes derived from each microparticle formulation or pcDNA3-EGFP alone, with the x-axis representing FITC fluorescence intensity [au] (fluorescence intensity filtered at a wavelength capable of detecting GFP fluorescence) and the y-axis representing FSC (forward scatter light) intensity [au]. FSC intensity is proportional to the cell diameter and represents cell size. Dots with FITC fluorescence intensity above a certain intensity (threshold) represent cells expressing GFP, while dots below a certain intensity (threshold) represent cells that do not express GFP. The threshold was set at 4000 for pcDNA3-EGFP alone and 5000 for those derived from microparticle formulations No. 16-30. Dots that deviated significantly from the cell-specific population were excluded from the GFP expression count as dead cells or other cells in poor condition.

[0170] FACS analysis revealed that the GFP expression rate (GFP-expressing cell percentage) in HaCaT cells treated with pcDNA3-EGFP alone was 0.04%, while the GFP-expressing cell percentage improved in HaCaT cells treated with nucleic acid-microparticle complexes derived from microparticle formulations No. 16-30 (Table 4).

[0171] [Table 4]

[0172] In nucleic acid-microparticle complexes derived from microparticle formulations No. 16-23 containing DOTAP with a lipid mole fraction of 50 mol%, the nucleic acid-microparticle complex derived from microparticle formulation No. 20 with a C17MGE:DOPE ratio of 50:50 showed the highest percentage of GFP-expressing cells (17.2%), a significant improvement compared to cDNA3-EGFP alone. On the other hand, the nucleic acid-microparticle complex derived from microparticle formulation No. 16 with a C17MGE:DOPE ratio of 0:100 showed a low percentage of GFP-expressing cells (7.9%).

[0173] Furthermore, among nucleic acid-microparticle complexes derived from microparticle formulations No. 24-30 containing DOTAP with a lipid mole fraction of 60 mol%, the nucleic acid-microparticle complex derived from microparticle formulation No. 27 with C17MGE:DOPE=50:50 and the nucleic acid-microparticle complex derived from microparticle formulation No. 30 with C17MGE:DOPE=100:0, which had a small average particle size and stability, showed high GFP-expressing cell percentages of 18.8% and 22.6%, respectively. On the other hand, the nucleic acid-microparticle complex derived from microparticle formulation No. 24, which had a small average particle size and stability but C17MGE:DOPE=0:100, showed a low GFP-expressing cell percentage (9.0%).

[0174] Therefore, it was shown that C17MGE is essential for inducing GFP expression in cells, regardless of whether the mole fraction of DOTAP in lipids is 50 mol% or 60 mol%, and that a small average particle size and high formulation stability are also important.

[0175] The results from Examples 11 and 12 showed that microparticle formulations containing C17MGE and not containing non-lamellar liquid crystals (e.g., Nos. 19-22, 26, 27, and 30 in particular) are useful for inducing gene expression. Nucleic acid-microparticle complexes derived from other microparticle formulations had little to no effect on introducing nucleic acids into the nucleus (gene expression effect).

[0176] The results above demonstrate that microparticle formulations containing amphiphilic lipids with isoprenoid-type fatty acid chains, such as C17MGE, and not containing non-lamellar liquid crystals, can introduce complexed nucleic acids into cells (especially the cell nucleus) and are useful for gene delivery and gene expression. Therefore, microparticle formulations containing amphiphilic lipids with isoprenoid-type fatty acid chains, such as C17MGE, and not containing non-lamellar liquid crystals can be used in nucleic acid delivery systems and are useful, for example, in the manufacture of pharmaceutical formulations for gene therapy.

[0177] [Example 13] Synthesis of amphiphilic lipids having isoprenoid-type fatty acid chains (1) Synthesis of mono-O-(5,9,13,17-tetramethyloctadecanoyl)glycerol

[0178] [ka]

[0179] Under a nitrogen atmosphere, 2.5 g of 5% palladium-carbon was added to a solution of 20.6 g (50.0 mmol) of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)glycerol in ethyl acetate (62 mL). After replacing the nitrogen in the system with hydrogen, the mixture was stirred at room temperature under atmospheric pressure and hydrogen atmosphere for 42 hours. After replacing the hydrogen in the system with nitrogen, the 5% palladium-carbon was filtered off. The filtrate was purified by silica gel column chromatography (ethyl acetate) to obtain 20.2 g (98% yield) of the title compound as a colorless, transparent liquid. Regarding the obtained compound, 1 The results of the H-NMR measurement are as follows:

[0180] 1 H-NMR spectrum (300MHz, CDCl3, TMS) δ:0.7-0.9(m,15H),0.95-1.75(m,26H),2.13(t,J=6.0Hz,OH),2.34(t,J=7.7Hz,2 H),2.56(d,J=5.1Hz,OH),3.55-3.75(m,2H),3.94(m,1H),4.15(dd,J=6.0,11.7Hz,1H),4.20(dd,J=4.7,11.7Hz,1H)

[0181] Mono-O-(5,9,13,17-tetramethyloctadecanoyl)glycerol is also known as saturated C22MGE.

[0182] (2) Synthesis of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan

[0183] [ka]

[0184] 70.5 g (200 mmol) of methyl 5,9,13,17-tetramethyloctadeca-4-enoate and 54.7 g (300 mmol, sorbitan M-90, Sanko Chemical Industry Co., Ltd., containing 90% solids and 10% water, solids content: sorbitan 79-84%, isosorbide 15-18%) were added to a reaction vessel at room temperature, and the mixture was stirred at 120°C and 8 kPa for 1 hour. The pressure was released with nitrogen, and 2.2 g (40 mmol) of sodium methoxide and 0.04 g of sodium phosphinate monohydrate were added, followed by stirring at 160°C and 8 kPa for 1 hour. The pressure was released with nitrogen, and 1.1 g (20 mmol) of sodium methoxide was added, followed by further stirring at 160°C and 8 kPa for 1 hour. The pressure was released using nitrogen, and 1.1 g (20 mmol) of sodium methoxide was added again. The mixture was then stirred at 160°C and 8 kPa for 1.5 hours. After cooling to 60°C, 200 mL of ethyl acetate and 200 mL of 0.5 M hydrochloric acid were added while stirring. 600 mL of ethyl acetate was added to the resulting reaction mixture and extracted. The extract was sequentially washed with saturated sodium bicarbonate solution and saturated saline solution, dried over magnesium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (mobile phase: ethyl acetate / hexane mixture) to obtain 36.1 g (yield 37%) of the compound listed in the title as a light brown transparent liquid. The obtained fraction contained mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan and mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)isosorbide in a ratio of approximately 8:2 (by weight) (calculated from the TIC area value obtained by GC-MS measurement in ion mode EI+). The obtained fraction also contained a small amount of diester derived from sorbitan (estimated by GC-MS measurement and TLC analysis). Regarding the obtained fraction, 1 The results of the 1H-NMR measurement are as follows:

[0185] 1H-NMR spectrum (300MHz, CDCl3, TMS) δ:0.7-0.9(m,12H),0.9-1.8(m,22H),1.85- 2.0(m,2H),2.0-2.5(m,4H),3.5-4.9(m,6.4H),5.04(m,1H),5.0-5.2(m,0.6H)

[0186] Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan is also known as C22SOE.

[0187] The obtained fraction was used as mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)sorbitan fraction (C22 sorbitan ester fraction) in the examples described later.

[0188] (2) Synthesis of mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)propylene glycol

[0189] [ka]

[0190] Under reduced pressure of 60-70 mmHg and a nitrogen stream, 20.6 g (271 mmol) of propylene glycol and 0.211 g (1.53 mmol) of potassium carbonate were dissolved in 60 mL of dry N,N-dimethylformamide. 30.0 g (85.0 mmol) of methyl 5,9,13,17-tetramethyloctadeca-4-enoate was gradually added dropwise at 85°C, and the mixture was stirred at the same temperature for 3 hours. The resulting reaction solution was diluted with ethyl acetate / hexane mixed solvent (1:1,120 mL), washed with water, saturated sodium bicarbonate solution, and saturated brine (twice), and then dried over magnesium sulfate. After filtration and concentration, the resulting residue was purified by silica gel column chromatography (mobile phase: hexane / ethyl acetate = 100:0-70:30) to obtain 21.6 g (yield 63%) of the title compound as a pale yellow transparent liquid. Regarding the obtained compound, 1 The results of the H-NMR measurement are as follows:

[0191] 1 H-NMR spectrum (300MHz, CDCl3, TMS) δ:0.80-0.95(m,12H),0.95-1.70(m,25H),1.96(td,J=7.5,18Hz,2H),2.1(b rs,OH),2.25-2.42(m,4H),3.55-3.70(m,0.7H),3.85-4.15(m,1.95H),4.98(m,0.35H),5.08(t,J=6.8Hz,1H)

[0192] Mono-O-(5,9,13,17-tetramethyloctadeca-4-enoyl)propylene glycol is also known as C22PGE.

[0193] [Example 14] Preparation and physical property evaluation of particulate formulation Except for replacing C17MGE in the composition of microparticle formulation No. 4 (Table 1) with saturated C22MGE, C22SOE, or C22PGE (all amphiphilic lipids having isoprenoid-type fatty acid chains), microparticle formulations No. 34 to 36 shown in Table 5 below were prepared according to the preparation method for microparticle formulation No. 4 described in Example 2.

[0194] Furthermore, the particle size distribution and zeta potential of particulate formulations No. 34-36 were measured using dynamic light scattering with a zetasizing device, Nano-ZS (Malvern), in the same manner as in Example 3. For each measurement sample, the average particle size (nm) (Z-Average), PdI (polydispersion index), and zeta potential (mV) obtained as the average of 5-6 measurements are shown in Table 5.

[0195] [Table 5]

[0196] [Example 15] Evaluation of the effect of heating on the cytotoxicity of microparticle formulations The effect of heating on the cytotoxicity of microparticle formulations No. 4 and 34-36 was evaluated by measuring the activity of lactate dehydrogenase (LDH) released from cells into the culture medium under heating or non-heating conditions in the presence of these microparticle formulations. The Cytotoxicity LDH Assay Kit-WST (Dojin Chemical Research Institute) was used to measure LDH activity.

[0197] Specifically, 4T1-Luc cells (JCRB1447, luciferase-expressing breast cancer cell line) were seeded into 10 35mm dishes (1.5 x 10 5 Cells were placed in a dish and incubated overnight at 37°C. Then, 100 μL each of microparticle formulations No. 4 and 34-36 was added to two dishes per formulation. Of the two dishes containing each microparticle formulation, one dish was irradiated with near-infrared light (wavelength 980 nm, output 1.5 W; laser FC-W-980-1.5W (Changchun New Industries Optoelectronics Tech. Co. Ltd.)) for 20 minutes to create a "heated" sample, while the other dish was incubated at 37°C for another 20 minutes without the irradiation to create an "unheated" sample. Note that irradiating with near-infrared light for at least 20 minutes raised the temperature of the liquid culture medium in the dish to over 45°C.

[0198] Next, 100 μL of the supernatant from each dish was transferred to separate wells of a 96-well plate. For the color reaction, 100 μL of the Working solution prepared according to the instructions for use of the above kit was added to each well and incubated at room temperature for 30 minutes. After stopping the color reaction by adding 50 μL of the Stop solution included with the above kit to each well, the plates were scanned using a microplate reader (Spectra Max). (R)Absorbance at 490 nm was measured using M2e (MOLECULAR DEVICES). As controls, LDH activity was measured in the same manner as above for 4T1-Luc cells without the addition of the microparticle formulation and without the above-mentioned near-infrared heating (no microparticle formulation, no heating), and for 4T1-Luc cells with Lysis buffer added instead of the microparticle formulation and without the above-mentioned near-infrared heating (showing total LDH activity in the cells).

[0199] The percentage of cell damage [%] was calculated using the following formula. Cytotoxicity rate [%] = [(Absorbance of sample with or without microparticle formulation added, heated or unheated) - (Absorbance of sample without microparticle formulation added, unheated)] / [(Absorbance of sample with Lysis buffer added, unheated) - (Absorbance of sample without microparticle formulation added, unheated)] x 100

[0200] The increase in cytotoxicity due to heating was calculated using the following formula. Increase in cytotoxicity due to heating [pt] = (Percentage of cytotoxicity in heated samples with microparticle formulation added [%]) - (Percentage of cytotoxicity in unheated samples with microparticle formulation added [%])

[0201] The results are shown in Table 6. [Table 6]

[0202] As shown in the results above, similar to the results of Example 6, the rate of cytotoxicity in microparticle formulation No. 4 containing C17MGE increased significantly upon heating. Furthermore, an increase in the rate of cytotoxicity upon heating was also observed in microparticle formulations No. 34 to 36 containing saturated C22MGE, C22SOE, or C22PGE. This demonstrates that the microparticle formulation of the present invention can produce an antitumor effect using only liposomes, without the need to encapsulate an antitumor agent.

[0203] [Example 16] Antitumor effect of in vivo administration of microparticle formulation The antitumor effect of a microparticle formulation containing C17MGE, an amphiphilic lipid with isoprenoid-type fatty acid chains, encapsulated with doxorubicin hydrochloride (DXR) as an antitumor agent, on tumor-bearing mice was evaluated.

[0204] Specifically, particulate formulations No. 37 and 38 (Table 6), containing or not containing C17MGE, with DXR encapsulated, were prepared as follows. First, ethanol solutions of each lipid were mixed according to the same component ratio as particulate formulation No. 1 or 4 in Table 1, and then the ethanol was removed by vacuum concentration to prepare a thin film. Next, a 250 mM ammonium sulfate aqueous solution was added so that the final concentration of the lipids (total amount) was 1 mM, and the mixture was allowed to stand at room temperature for 10 minutes. The obtained sample solutions were subjected to sonication in the same manner as in Example 2 to prepare slightly turbid particulate formulations. Next, each microparticle formulation was centrifuged at 80,000 rpm for 30 minutes at 4°C (CS120GX, Hitachi Koki), and after removing the separated aqueous solution, it was redispersed with a 10% sucrose aqueous solution to achieve a total lipid concentration of 10 mM. Furthermore, 0.7 times the volume of the resulting redispersed solution was added to a 10% sucrose aqueous solution containing DXR (2 mg / mL), and the mixture was incubated at 60°C for 1 hour. The redispersed solution with added DXR was again centrifuged at 80,000 rpm for 30 minutes at room temperature, and after removing the separated aqueous solution ("Aqueous Solution A"), it was redispersed with PBS(-) to achieve a final lipid concentration of 10 mM, thereby preparing microparticle formulations No. 37 and 38. The particle size distribution and zeta potential of microparticle formulations No. 37 and 38 were measured by dynamic light scattering using a zetasizing nano-ZS (Malvern) in the same manner as in Example 3. Table 7 shows the DXR concentration (mg / mL), average particle size (nm) (Z-Average), PdI (polydispersion index), and zeta potential (mV) for particulate formulations No. 37 and 38.

[0205] As controls, a PBS(-) solution with a DXR concentration of 0.5 mg / mL was prepared as formulation No. 39, and a simple PBS(-) solution without DXR was prepared as formulation No. 40.

[0206] The DXR concentration (mg / mL) in particulate formulations No. 37 and 38 was calculated by quantifying the amount of DXR in the aqueous solution ("Aqueous Solution A") separated and removed as described above using HPLC (High-Performance Liquid Chromatography), and subtracting that quantified value from the amount of DXR added. The amount of DXR in the separated and removed aqueous solution ("Aqueous Solution A") was quantified based on a calibration curve using the ratio of the peak area of ​​DXR to the peak area of ​​the internal standard substance, after adding 1 / 20 volume of a mobile phase solution of butyl parahydroxybenzoate (0.1 mg / mL) as an internal standard substance. The analytical conditions were as follows.

[0207] • Column: Inertsil (R) ODS-3: Inner diameter 4.6 mm, length 25 cm, particle size 5 μm (GL Sciences Co., Ltd.) Mobile phase: 0.3% sodium lauryl sulfate / 0.14% phosphoric acid aqueous solution:acetonitrile = 1:1 Flow rate: 1.0 mL / min, Column temperature: 30°C, Injection volume: 10 μL • Detector: UV absorbance spectrophotometer (254 nm)

[0208] [Table 7]

[0209] BALB / c mice (female, 6 weeks old) were given 4T1-Luc cells (JCRB1447, 1x10) as tumor cells. 6 Tumor-bearing mice were prepared by subcutaneously transplanting cells into two locations on the left and right sides of the back, and then waiting 12 days. These tumor-bearing mice were administered preparations No. 37-40, and the tumor transplantation sites were heated, and the antitumor effect was evaluated (n=3).

[0210] Specifically, the luminescence intensity of the tumor transplant site was measured on the first day of the experiment (day 0), the formulation was administered on days 1, 4, 7, and 10 of the experiment, the tumor transplant site was heated on days 2, 5, 8, and 11, and the luminescence intensity of the tumor transplant site was measured again on days 3, 6, 9, and 12. In addition, the tumor volume of the tumor transplant site, the weight of the mice, and the appearance of the tumors were observed throughout the experiment.

[0211] The drug was administered intravenously into the tail (once daily) using a syringe (Terumo syringe 1 mL) equipped with a 29 gauge (G) needle, after general anesthesia had been administered with isoflurane to tumor-bearing mice.

[0212] The tumor volume at the transplant site is measured by measuring the short and long diameters of the tumor with calipers, and then calculating the tumor volume (mm³) at two locations on the left and right sides of the back of each mouse according to the following formula. 3 ) was calculated. Tumor volume (mm 3 )=[(minor axis) 2 x (major axis)) / 2

[0213] The tumor transplantation sites were heated to approximately 45°C by irradiating the tumor transplantation site on the left side of the back of each mouse, which was under general anesthesia, with near-infrared light (wavelength 980 nm, output 1.5 W) for 30 seconds, using the same method as in Example 15. The tumor transplantation sites on the right side of the back of each mouse were not heated.

[0214] To evaluate the antitumor effect, the luminescence intensity of the tumor transplantation site is measured using VivoGlo. TM 100 μL of 30 mg / mL saline solution of luciferin (P1043, Promega) was administered intraperitoneally to mice. Sixteen minutes later, the luminescence intensity (Total Flux [photons / sec]) at two tumor transplantation sites on the left and right sides of the back was measured using a fluorescence imaging system (IVIS Spectrum). The measurement conditions were open filter (all wavelength range), binning=medium, and f / stop=1. The luminescence intensity measured by this method indicates the activity level of luciferase expressed by cells, and therefore indicates the amount of viable cells.

[0215] Figure 16 shows the luminescence imaging image of the entire mouse on day 6 of the study, administered with formulations No. 37-40. Figure 17 shows a graph illustrating the results of measuring the luminescence intensity over time at two tumor transplantation sites on the left and right sides of the back.

[0216] Table 8 shows the increase in luminescence intensity at two tumor transplant sites on the left and right sides of the back from day 1 (day 0) to day 12. The percentage of suppression of the increase due to heating shown in Table 8 was calculated as follows: Rate of suppression of increase due to heating (%) = [(Increase without heating) - (Increase with heating)] / (Increase without heating) x 100

[0217] [Table 8]

[0218] As shown in Table 8, microparticle formulation No. 38, containing DXR and C17MGE, significantly suppressed tumor growth in both tumor transplantation sites on the left and right sides of the back, and showed the greatest suppression of tumor growth, particularly in the heated tumor transplantation site on the left side of the back. Formulation No. 39, a PBS solution containing DXR alone, also suppressed tumor growth, but the level of suppression was lower than that of microparticle formulation No. 38. Microparticle formulation No. 37, containing DXR but without C17MGE, did not sufficiently suppress tumor growth. As shown in the rate of suppression by heating, the effect of heating on tumor growth suppression was significantly greater in microparticle formulation No. 38.

[0219] Based on the tumor volume measurement results, when microparticle formulation No. 38 was administered, the increase in tumor volume at the tumor transplant site from the first day (day 0) to the 12th day of the study (mm²) 3 The tumor transplantation site was 110 mm on the left side of the back (with heating). 3 The tumor transplant site on the right side of the back (without heating) measured 323 mm. 3 The study showed that heating significantly suppressed the increase in tumor volume. While formulation No. 39 strongly attacks normal cells with DXR, formulation No. 38 has a significant advantage in that its DXR is retained within liposomes and has a property that makes it easily accumulate in tumor cells, thus offering greater safety for normal cells.

[0220] Furthermore, no abnormalities in the mice's body weight or external appearance were observed during this evaluation test.

[0221] The results above demonstrate that microparticle formulations containing antitumor agents such as DXR and amphiphilic lipids with isoprenoid-type fatty acid chains such as C17MGE not only accumulate in tumor cells when administered in vivo (Example 7), but also show an effect of suppressing tumor cell proliferation, and that this antitumor effect can be more significantly enhanced by combining it with heating.

[0222] All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

1. A liposome comprising lamellar-forming lipids and amphiphilic lipids having isoprenoid-type fatty acid chains as membrane constituent lipids, Amphiphilic lipids having isoprenoid-type fatty acid chains are given by the following general formula (I): 【Chemistry 1】 (In the formula, X and Y each represent a hydrogen atom or together represent an oxygen atom, n represents an integer from 0 to 2, and m represents 1 or 2.) 【change】 (where represents a single or double bond, and R represents a hydrophilic group from which one hydroxyl group has been removed, selected from the group consisting of glycerol, sorbitan, and glycol.) It is an amphiphilic compound represented by The lamellar-forming lipid includes at least one selected from the group consisting of phospholipids, steroids, and cationic lipids. Liposomes.

2. The liposome according to claim 1, wherein R in the formula represents a hydrophilic group obtained by removing one hydroxyl group from glycerol, sorbitan, or propylene glycol.

3. The liposome according to claim 1 or 2, wherein the amphiphilic lipid having an isoprenoid-type fatty acid chain is mono-O-(5,9,13-trimethyltetradeca-4-enoyl)glycerol, mono-O-(5,9,13,17-tetramethyloctadecanoyl)glycerol, mono-O-(5,9,13,17-tetramethyloctadecanoyl)sorbitan, or mono-O-(5,9,13,17-tetramethyloctadecanoyl)propylene glycol.

4. A liposome according to any one of claims 1 to 3, wherein the lamellar-forming lipid comprises a phospholipid modified with a water-soluble polymer.

5. The liposome according to any one of claims 1 to 4, wherein the lamellar-forming lipid comprises phosphatidylcholine, cholesterol, and PEG-modified phosphatidylethanolamine.

6. The liposome according to any one of claims 1 to 4, wherein the lamellar-forming lipid comprises phosphatidylethanolamine and 1,2-dialkylcarbonyloxy-3-mono, di, or trialkylammonium propane.

7. A liposome according to any one of claims 1 to 6, comprising amphiphilic lipids having isoprenoid-type fatty acid chains in an amount of 5 to 40 mol% in mole fraction relative to the total amount of membrane constituent lipids.

8. A liposome according to any one of claims 1 to 7, which is temperature-responsive.

9. A liposome according to any one of claims 1 to 8, further comprising a drug.

10. The liposome according to claim 9, wherein the drug is a nucleic acid.

11. The liposome according to claim 10, wherein nucleic acid and liposomes form a complex.

12. A drug delivery formulation for intracellular use, comprising liposomes according to any one of claims 9 to 11.

13. A formulation for delivering nucleic acids into the nucleus of a cell, comprising the liposomes described in claim 10 or 11.

14. The formulation according to claim 12 or 13, wherein the cells are tumor cells.

15. A pharmaceutical formulation comprising liposomes according to any one of claims 1 to 11.

16. The pharmaceutical preparation according to claim 15, which is an antitumor preparation.

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