Liposome composition and liposome-containing pharmaceutical composition

Abstract

A liposome composition which comprises (A) liposomes containing dihydrosphingomyelin and a diacylglycerol-polyethylene glycol, (B) a drug and (C) a sulfate and / or sucrose octasulfate, and in which (A) encapsulates (B), wherein: the diacylglycerol-polyethylene glycol is distearoylglycerol-polyethylene glycol; and (B) is 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide represented by formula (I) or pharmaceutically acceptable salt thereof.

Description

Liposome compositions and pharmaceutical compositions containing liposomes

[0001] The present invention relates to liposome compositions and pharmaceutical compositions comprising liposomes.

[0002] Liposomes are minute closed vesicles with an inner layer surrounded by one or more lipid bilayers, and can generally hold water-soluble substances in the inner phase and lipid-soluble substances in the lipid bilayer. Taking advantage of these properties of liposomes, formulations using liposome-related technology have been investigated, in which drugs are encapsulated in liposomes and delivered to target tissues. For example, a liposome composition containing diacylphosphatidylethanolamine, dihydrosphingomyelin, and cholesterols modified with a hydrophilic polymer has been proposed (Patent Document 1).

[0003] On the other hand, receptor tyrosine kinase inhibitors are known as molecularly targeted drugs that exhibit antitumor activity by inhibiting tyrosine kinases in cell membranes and cells. Among these, lenvatinib (compound name: 4-(3-chloro-4-(cyclopropylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide) is a tyrosine kinase inhibitor that targets receptors such as vascular endothelial growth factor receptors (VEGFR) 1-3 and fibroblast growth factor receptors (FGFR) 1-4 (Patent Document 2), and Patent Document 3 discloses a formulation using technology related to liposomes containing lenvatinib as a drug. Furthermore, Patent Document 1 above discloses a liposome composition containing sunitinib, a tyrosine kinase inhibitor, as a drug.

[0004] International Publication No. 2018 / 181963, U.S. Patent No. 7,253,286, Chinese Patent Application Publication No. 112603890

[0005] An object of the present invention is to provide a novel liposome composition which contains a tyrosine kinase inhibitor as a drug and has excellent retention in blood.

[0006] The present inventors have discovered that a liposome composition containing liposomes composed of specific lipids and containing (4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (hereinafter also referred to as "compound (I)") represented by the following formula (I) or a pharmaceutically acceptable salt thereof as a drug having tyrosine kinase inhibitory activity has unexpectedly long retention in blood, and have completed the present invention.

[0007] That is, the present disclosure provides the following [1] to

[19] : [1] A liposome composition comprising: (A) a liposome containing dihydrosphingomyelin and diacylglycerol-polyethylene glycol, (B) a drug, and (C) a sulfate and / or sucrose octasulfate, wherein (A) encapsulates (B), wherein the diacylglycerol-polyethylene glycol is distearoylglycerol-polyethylene glycol, and (B) is 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide represented by formula (I) or a pharmaceutically acceptable salt thereof. [2] The liposome composition according to [1], wherein (C) is a sulfate. [3] The liposome composition according to [1] or [2], wherein the dihydrosphingomyelin is a compound represented by formula (II-1), (II-2), (II-3), or (II-4), or a mixture thereof. [4] The liposome composition according to [1] or [2], wherein the dihydrosphingomyelin is a compound represented by formula (II-3). [5] The liposome composition according to any one of [1] to [4], further comprising (D) an organic acid. [6] The liposome composition according to [5], wherein (D) is at least one selected from the group consisting of succinic acid, citric acid, maleic acid, and salicylic acid. [7] The liposome composition according to any one of [1] to [6], wherein (A) further comprises a cholesterol. [8] The liposome composition according to any one of [1] to [7], further comprising (E) a basic compound. [9] The liposome composition according to any one of [1] to [8], wherein (C) is ammonium sulfate.

[10] The liposome composition according to any one of [5] to [9], wherein (D) is succinic acid.

[11] The liposome composition according to any one of [8] to

[10] , wherein (E) is sodium hydroxide or ammonia.

[12] The liposome composition according to any one of [1] to

[11] , wherein (A) further encapsulates (C).

[13] A pharmaceutical composition comprising the liposome composition according to any one of [1] to

[12] .

[14] An angiogenesis inhibitor comprising the liposome composition according to any one of [1] to

[12] .

[15] An antitumor agent comprising the liposome composition according to any one of [1] to

[12] .

[16] A method for preventing or treating tumors in a subject, comprising administering to the subject the liposome composition according to any one of [1] to

[12] .

[17] Use of the liposome composition according to any one of [1] to

[12] for producing a medicament for preventing or treating tumors.

[18] The liposome composition according to any one of [1] to

[12] for use in the prevention or treatment of tumors.

[19] A method for producing the liposome composition according to any one of [1] to

[12] , comprising the steps of: providing a dispersion of liposomes; and mixing the dispersion of liposomes with 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0008] According to the present invention, a novel liposome composition containing a tyrosine kinase inhibitor as a drug and having excellent retention in blood can be provided.

[0009] 1 is a graph showing the time course of drug blood concentration when the liposome compositions of Examples 2, 5, and 8, and an aqueous solution of the methanesulfonate of Compound (I) were intravenously administered to mice. 2 is a graph showing the results of an anti-PEG IgM antibody production evaluation test.

[0010] As used herein, the term "liposome" refers to a minute closed vesicle having an internal phase surrounded by a lipid bilayer. Examples of liposomes include small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), multilamellar vesicles (MLVs) having multiple concentric membranes, and multivesicular vesicles (MVVs) having multiple irregular membranes.

[0011] As used herein, the term "internal phase of a liposome" refers to the region surrounded by the lipid bilayer of a liposome, and when this region contains water, it is synonymous with "internal aqueous phase" and "internal aqueous phase of a liposome." The term "external phase of a liposome" refers to the region not surrounded by the lipid bilayer of a liposome (i.e., the region other than the internal phase and the lipid bilayer) when the liposome is dispersed in a liquid.

[0012] The liposome composition according to this embodiment contains (A) a liposome comprising dihydrosphingomyelin and diacylglycerol-polyethylene glycol (hereinafter also referred to as "component (A)"), (B) a drug (hereinafter also referred to as "component (B)"), and (C) a sulfate and / or sucrose octasulfate (hereinafter also referred to as "component (C)").

[0013] In the liposome composition according to this embodiment, the internal phase of the liposome encapsulates a drug (component (B)). In this specification, "encapsulating" refers to a form in which the liposome membrane itself encapsulates the drug, a form in which the drug is enclosed in a closed space formed by the liposome membrane, or a combination of these forms.

[0014] (A) Liposome The liposome composition according to this embodiment contains (A) a liposome containing dihydrosphingomyelin and diacylglycerol-polyethylene glycol.

[0015] Dihydrosphingomyelin is a phospholipid having two carbon chains in the molecule, and is useful as a lipid for further increasing the blood retention of the liposome composition encapsulating a drug used in the present invention. Specific examples of such lipids include compounds represented by the following formulas (II-1), (II-2), (II-3), and (II-4), or mixtures thereof.

[0016] The dihydrosphingomyelin may be, for example, a dihydrosphingomyelin obtained by reducing naturally occurring sphingomyelin using a common method, or a dihydrosphingomyelin obtained by chemical synthesis (synthetic dihydrosphingomyelin). The synthetic dihydrosphingomyelin preferably has a longer carbon chain, and exhibits excellent blood retention due to its high purity. Here, while dihydrosphingomyelin derived from naturally occurring substances such as chicken eggs is generally often the compound represented by formula (II-1) above, chemical synthesis allows the compounds represented by formulas (II-1) to (II-4) to be obtained with high purity.

[0017] The content of dihydrosphingomyelin in all lipid components constituting the liposome is not particularly limited, but may be 40 to 99.9 mol %, 45 to 96 mol %, or 48 to 64 mol %.

[0018] Diacylglycerol-polyethylene glycol is a compound in which the hydroxyl groups of diacylglycerol are modified with polyethylene glycol. The molecular weight of the polyethylene glycol in diacylglycerol-polyethylene glycol is not particularly limited, but is preferably 500 to 10,000 daltons, and more preferably 1,000 to 5,000 daltons. Examples of diacylglycerol-polyethylene glycol include dimyristoylglycerol-polyethylene glycol (DMG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), and distearoylglycerol-polyethylene glycol (DSG-PEG). One embodiment of diacylglycerol-polyethylene glycol is distearoylglycerol-polyethylene glycol (e.g., DSG-PEG2000).

[0019] The content of diacylglycerol-polyethylene glycol in all lipid components constituting the liposome is not particularly limited, and may be, for example, 0.1 to 15 mol %, 0.2 to 10 mol %, or 0.3 to 6 mol %.

[0020] In this embodiment, the component (A) may further contain cholesterols, which can further enhance the membrane stability of the liposome.

[0021] Examples of cholesterols include cholesterol, 3-cholestanone, and acyl cholesterol.

[0022] In this embodiment, when component (A) contains cholesterols, the content of cholesterols in all lipid components constituting the liposome is not particularly limited, and may be, for example, 0.1 to 50 mol %, 10 to 50 mol %, or 30 to 48 mol %.

[0023] In this embodiment, when component (A) contains a cholesterol, the content ratio of cholesterol relative to dihydrosphingomyelin in component (A) is not particularly limited, but may be 15 to 60 parts by mass, 20 to 50 parts by mass, or 21 to 35 parts by mass, from the viewpoint of increasing the encapsulation rate of the drug (component (B)).

[0024] The content of component (A) in the liposome composition is not particularly limited, but is preferably 0.001 to 50% by mass, more preferably 0.01 to 40% by mass, and even more preferably 0.01 to 30% by mass, based on the total mass of the liposome composition.

[0025] The average particle size of the liposomes is not particularly limited, but may be 20 to 500 nm, 50 to 300 nm, or 70 to 200 nm, from the viewpoints of feasibility of production and accumulation at tumor sites. The average particle size of the liposomes can be measured by dynamic light scattering.

[0026] (B) Drug The liposome composition according to this embodiment contains compound (I) or a pharmaceutically acceptable salt thereof as (B) drug.

[0027] Compound (I) (CAS No. 417719-46-1) is a known compound.

[0028] Compound (I) contained in the liposome composition according to this embodiment may be in the form of a free form or a pharmaceutically acceptable salt, such as a salt with an inorganic acid, a salt with an organic acid, a salt with an inorganic base, a salt with an organic base, or a salt with an acidic or basic amino acid.

[0029] Examples of salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Examples of salts with organic acids include salts with acetic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, p-toluenesulfonic acid, etc.

[0030] Examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, ammonium salts, etc. Examples of salts with organic bases include salts with diethylamine, diethanolamine, meglumine, N,N-dibenzylethylenediamine, etc.

[0031] Examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Examples of salts with basic amino acids include salts with arginine, lysine, ornithine, etc.

[0032] Compound (I) or a pharmaceutically acceptable salt thereof used in preparing the liposome composition according to this embodiment can be produced, for example, by the method described in Example 583 of Patent Document 1.

[0033] The pharmaceutically acceptable salt of compound (I) used in preparing the liposome composition according to this embodiment is not particularly limited as long as it is one of the above-mentioned salts, and examples thereof include salts of organic acids such as maleic acid.

[0034] The content of component (B) in the liposome composition according to this embodiment is not particularly limited, but may be 0.001 to 100 mg / mL, 0.05 to 80 mg / mL, 0.01 to 50 mg / mL, or 0.1 to 30 mg / mL. The content of component (B) in the liposome composition according to this embodiment is also not particularly limited, but may be 0.01 to 50 mass%, 0.1 to 40 mass%, 0.5 to 30 mass%, or 1 to 20 mass%, based on the total mass of the liposome composition.

[0035] The drug / total lipid weight ratio (D / L ratio) in the liposome composition according to this embodiment may be, for example, greater than 0.1. The D / L ratio in the liposome composition according to this embodiment may be, for example, 1.0 or less. The D / L ratio can be calculated from the cholesterol concentration in the liposome composition using the following formula: The total lipid concentration can be calculated based on the above formula, and the drug concentration in the liposome composition can be divided by the calculated total lipid concentration. Here, if the D / L ratio is a high value such as more than 0.1, a required amount of drug can be administered with a smaller amount of lipid, which may further reduce the burden on the body, such as lipid-derived toxicity.

[0036] The liposome composition of the present invention contains (C) sulfate and / or sucrose octasulfate. When the internal aqueous phase of the liposome contains component (C), the encapsulation rate and blood retention of the drug (component (B)) can be increased.

[0037] The content of component (C) in the internal aqueous phase of the liposome is not particularly limited, but may be, for example, 10 to 1000 mM.

[0038] Examples of sulfates include ammonium sulfate, ammonium hydrogen sulfate, and triethylamine sulfate.

[0039] The content of sulfate in the internal aqueous phase of the liposome is not particularly limited, but may be, for example, 50 to 1000 mM, 100 to 500 mM, or 150 to 400 mM.

[0040] Examples of sucrose octasulfate include sucrose octasulfate triethylamine (SOS-TEA) and sucrose octasulfate ammonium.

[0041] The content of sucrose octasulfate in the internal aqueous phase of the liposome is not particularly limited, but is, for example, 10 to 200 mM, 20 to 150 mM, or 30 to 100 mM.

[0042] One embodiment of component (C) is a sulfate, and another embodiment of component (C) is ammonium sulfate.

[0043] (D) Organic Acid The liposome composition according to this embodiment may further contain an organic acid (D) (hereinafter also referred to as "component (D)"). When the internal aqueous phase of the liposome further contains component (D), it is expected to have the effect of suppressing the degradation of the components constituting the liposome. Examples of organic acids include those having buffering capacity, such as succinic acid, citric acid, maleic acid, and salicylic acid. One embodiment of the organic acid is succinic acid.

[0044] The content of component (D) in the internal phase of the liposome is not particularly limited, but may be, for example, 0.1 to 300 mM, 5 to 200 mM, or 10 to 100 mM.

[0045] (E) Basic Compound The liposome composition according to this embodiment may further contain a basic compound (hereinafter also referred to as "component (E)"). Examples of basic compounds include alkali metal hydroxides (sodium hydroxide, potassium hydroxide, etc.) and amines (ammonia, alkylamine, etc.). One embodiment of the basic compound is sodium hydroxide. Another embodiment of the basic compound is ammonia.

[0046] pH of the Internal Aqueous Phase The pH of the internal aqueous phase in the liposome composition according to this embodiment is not particularly limited, and may be, for example, 1 to 6.

[0047] Method for Producing Liposome Composition The method for producing the liposome composition according to this embodiment is not particularly limited, but the composition can be produced, for example, by the steps of preparing a lipid mixture, preparing an aqueous phase, forming liposome particles, sizing the particles, replacing the aqueous phase liquid outside the liposomes, encapsulating a drug in the liposome particles, and removing the drug from the external aqueous phase.

[0048] <Preparation of Lipid Mixture> In preparing the lipid mixture, the components constituting the liposomes (dihydrosphingomyelin, diacylglycerol-polyethylene glycol, and, if necessary, cholesterols) are mixed with an organic solvent, and the mixture is heated to dissolve the components, thereby producing an oil phase. The organic solvent used in the oil phase is not particularly limited, and for example, a water-soluble organic solvent that is arbitrarily miscible with water can be used. Alternatively, an organic solvent that is not necessarily water-soluble can be used, and the components are mixed and dissolved, followed by removal of the organic solvent, which can serve as an alternative to the above preparation.

[0049] Examples of water-soluble organic solvents include alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and t-butanol; glycols such as glycerin, ethylene glycol, and propylene glycol; and polyalkylene glycols such as polyethylene glycol. Among these, alcohols are preferred. The alcohol is preferably at least one selected from ethanol, methanol, 2-propanol, and t-butanol, more preferably at least one selected from ethanol, 2-propanol, and t-butanol, and even more preferably ethanol.

[0050] <Preparation of Aqueous Phase> The aqueous phase may be water (distilled water, water for injection, etc.), physiological saline, various buffer solutions, or aqueous solutions of sugars (sucrose, etc.), or mixtures thereof (aqueous solvents). In the present invention, when a drug is encapsulated in liposome particles by the remote loading method described below, an aqueous solution containing component (C) may also be used as the aqueous phase.

[0051] The buffer solution is not limited to organic or inorganic solutions, but is preferably one that has a buffering effect at a hydrogen ion concentration close to that of body fluids, such as phosphate buffer, Tris buffer, citrate buffer, acetate buffer, histidine buffer, and Good's buffer. The internal phase of the liposome may be an aqueous solution in which liposomes are dispersed during liposome production, or may be newly added water, physiological saline, various buffers, or aqueous solutions of sugars, or mixtures thereof. Water that does not contain impurities (dust, chemicals, etc.) can be used as the external or internal aqueous phase.

[0052] Physiological saline refers to an inorganic salt solution adjusted to be isotonic with the human body and may further have a buffer function. Examples of physiological saline include saline containing 0.9 w / v% (mass / volume percent) sodium chloride, PBS, and Tris-buffered saline.

[0053] In this specification, the aqueous phase includes both the external aqueous phase and the internal aqueous phase. In this specification, the external aqueous phase refers to the aqueous solution in which liposomes are dispersed. For example, in the case of an injection, the solution occupying the outside of the liposomes in the liposome dispersion stored in a vial or pre-filled syringe is the external aqueous phase. Similarly, in the case of a liquid dispersed at the time of administration using the attached dispersion liquid or other dissolving liquid, the solution occupying the outside of the liposomes in the liposome dispersion is the external aqueous phase. In this specification, the internal aqueous phase refers to the aqueous phase in the closed vesicles separated by the lipid bilayer membrane of the liposome.

[0054] <Liposome Particle Formation> Liposomes are formed by mixing the lipid mixture with an aqueous phase. The liquid temperature during liposome particle formation can be adjusted as appropriate.

[0055] <Particle size regulation> The particle size of the obtained liposomes can be made uniform by extrusion treatment, etc. Extrusion treatment means that the liposomes are passed through a filter having fine pores, whereby physical shear force is applied to the liposomes, thereby causing them to become fine particles.

[0056] <Replacement of the liposome external aqueous phase liquid> In this embodiment, when a drug is encapsulated in liposome particles by the remote loading method described below, the liposome external aqueous phase liquid may be replaced. Examples of the replacement liquid that can be used include, but are not limited to, a 0.05 to 5% by mass aqueous sodium chloride solution, a 0.05 to 20% by mass aqueous glucose solution, and a 0.05 to 40% by mass aqueous sucrose solution. By using the replacement liquid described above to dialysis, ultracentrifugation, or ultrafiltration of the liposome solution, the aqueous solution containing component (C) present in the external aqueous phase can be removed, and liposomes whose external aqueous phase has been replaced with the replacement liquid can be obtained.

[0057] <Encapsulating a drug into liposome particles by remote loading method> In this embodiment, a drug can be encapsulated into liposome particles by the remote loading method. As used herein, the remote loading method refers to a method in which liposomes containing no drug are produced by the method described above, and the drug is introduced into the liposomes by adding the drug to the liposome external phase. In the remote loading method, component (C) (e.g., ammonium sulfate) can be used.

[0058] In the remote loading method, a drug added to the external aqueous phase migrates into the liposome and is incorporated into the liposome. The driving force used for this is a solubility gradient, an ion gradient, a pH gradient, or the like. For example, a method can be used in which a drug is introduced into the liposome interior using an ion gradient formed across the liposome membrane. The temperature at which a drug is encapsulated into liposome particles by the remote loading method is not particularly limited, but can be, for example, 20 to 90°C.

[0059] <Removal of Drug from External Aqueous Phase and Adjustment of Drug Concentration> The drug-encapsulated liposome solution may be subjected to dialysis, ultracentrifugation, filtration, etc. to remove the drug not contained in the liposomes. In addition, the drug concentration can be adjusted to the desired level by ultracentrifugation or ultrafiltration using a sucrose / histidine buffer or the like of a predetermined concentration as a replacement solution.

[0060] <Sterile filtration and sterile filling> The liposome composition obtained above may be subjected to sterile filtration and / or sterile filling. Sterile filtration can be performed using a filter with a pore size that can be sterilized (e.g., a 0.2 μm filtration sterilization filter). Known methods for sterile filling can be used.

[0061] The liposome composition according to this embodiment has the effect of providing excellent blood retention. Blood retention can be evaluated, for example, using the blood drug concentration 24 hours after administration of the liposome composition to an animal (such as a mouse) as an index. As confirmed in the Examples described below, the liposome composition according to this embodiment exhibits a high blood drug concentration of more than 800 ng / mL (administered at a drug dose of 2 mg / kg) after 24 hours in mice, demonstrating excellent blood retention.

[0062] The liposome composition according to this embodiment can be used as a pharmaceutical composition. The liposome composition according to this embodiment can also be used as an angiogenesis inhibitor or an antitumor agent. When the liposome composition according to this embodiment is used as an antitumor agent, examples of cancer types to which it can be applied include pancreatic cancer, gastric cancer, colon cancer, breast cancer, prostate cancer, lung cancer, kidney cancer, brain tumor, blood cancer, ovarian cancer, head and neck cancer, thyroid cancer, esophageal cancer, liver cancer, biliary tract cancer, pancreatic and digestive tract neuroendocrine tumors, malignant melanoma, endometrial cancer, sarcoma, and urothelial cancer.

[0063] The pharmaceutical composition according to this embodiment can be manufactured according to known methods, such as the method described in the General Provisions for Preparations of the Japanese Pharmacopoeia, Seventeenth Edition. The pharmaceutical composition according to this embodiment can be administered to a patient appropriately according to its dosage form.

[0064] The dose of the pharmaceutical composition according to this embodiment varies significantly depending on the type of target disease, the age, sex, weight, severity of symptoms, etc. of the patient, but the usual dose per adult is about 0.1 to 300 mg, calculated as the free form of compound (1).

[0065] The present invention will be described in more detail below with reference to examples. However, the present invention can be embodied in various forms and should not be construed as being limited to the examples set forth herein.

[0066] Proton nuclear magnetic resonance spectra were measured using a JEOL 500 (JMTC-500 / 54 / JJ, ECZ500RS) or a Bruker Ultrashield Plus 600 (BZH102 / 600 / 70F, D355 / 54-6026, PA BBO 600S3 BBF-HD-05 Z SP). Chemical shifts for proton nuclear magnetic resonance spectra are reported in δ units (ppm) relative to tetramethylsilane, and coupling constants are reported in hertz (Hz). Splitting patterns are abbreviated as follows: s: singlet, d: doublet, t: triplet, dd: double doublet, m: multiplet, brs: broad singlet, brd: broad doublet, and brt: broad triplet.

[0067] 1. Measurement of inhibitory effect on cell proliferation stimulated by vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) The inhibitory effect of compound (I) on cell proliferation stimulated by VEGF or bFGF was evaluated by the HUVEC 2D growth assay according to the following procedure. Normal human umbilical vein endothelial cells (HUVEC) were isolated according to a reported method (New Biochemistry Experiment Course "Cell Culture Techniques", pp. 197-202). The cells were cultured in 5% CO ２ The cells were cultured overnight in a collagen I-coated flask (IWAKI 4143-010) in EGM-2 medium (LONZA Inc. CC-3162) containing 10% fetal bovine serum (FBS: Sigma 172012) in an incubator (37°C). The next day, the culture supernatant was discarded and replaced with EBM-2 medium containing 2% FBS, followed by overnight culture. The next day, 1.5 x 10 cells were added to each well of a 96-well plate (Corning 3904) in Human Endothelial Serum Free Medium (SFM: Gibco 11111-044) containing 2% FBS and penicillin / streptomycin (Fujifilm Wako Pure Chemical Industries, Ltd.). ４ 100 μL of HUVEC cell suspension prepared at 100 μL / mL was added to each well, and the mixture was incubated in 5% CO２ The cells were cultured overnight in an incubator (37°C). The next day, 50 μL each of the test substance diluted with 2% FBS-SFM and VEGF (R&D systems 293-VE) or bFGF (gibco 13256-029) prepared in SFM medium containing 2% FBS to a final concentration of 20 ng / mL was added, and the cells were incubated under 5% CO ２ The cells were cultured in an incubator (37°C) for 3 days. 100 μL of the culture supernatant was aspirated and discarded from each well, and 100 μL of CellTiter-Glo® 2.0 Assay (Promega G9243) was added to each well, mixed, and incubated at room temperature for 20 minutes. ＴＭ Luminescence in each well was measured using a luminescence analyzer (PerkinElmer). The measured value when VEGF or bFGF was added without the addition of the test substance was set to 100%, and the measured value when the test substance, VEGF, or bFGF was not added was set to 0%, and the cell viability in the presence of the test substance was calculated. From this cell viability, the concentration of the test substance required to inhibit 50% of HUVEC proliferation in the presence of VEGF or bFGF (IC ５０ The IC values ​​of Compound (I) on cell proliferation stimulated by VEGF and bFGF were calculated. ５０ The values ​​were 1.75 nM and 343 nM, respectively.

[0068] 2. Preparation of Liposome Composition (1) Preparation of Lipid Mixture The total lipid weight listed in the liposome column of Table 1 was mixed with an equal weight of ethanol and dissolved at 80°C to obtain a lipid mixture. In Table 1, DSPC represents distearoylphosphatidylcholine, Egg-SM represents hen's egg sphingomyelin, Egg-DHSM represents hen's egg dihydrosphingomyelin, TS-DHSM represents synthetic dihydrosphingomyelin, DSG-PEG represents distearoylglycerol-polyethylene glycol, and Chol represents cholesterol. The DSPC used was LIPOID PC 18:0 / 18:0 (manufactured by LIPOID), the Egg-SM used was LIPOID E SM (manufactured by LIPOID), the Egg-DHSM was produced by hydrogenating Egg-SM as a starting material, and the DSG-PEG used was SUNBRIGHT GS-020 (manufactured by NOF Corp.). The TS-DHSM can be synthesized according to the method described below. The proton nuclear magnetic resonance spectrum of the synthesized TS-DHSM is as follows: 1 H-NMR Spectrum (600 MHz, CD3OD) δ(ppm): 0.92(6H, t, J=7.1Hz), 1.17-1.45(56H, m), 1.53-1.64(3H, m), 1.67(1H, brd, J=6.4Hz), 2.19-2.29(2H, m), 3.24(9H, s), 3.59-3.69(3H, m), 3.86-3.93(1H, m), 3.93-4.06(1H, m), 4.10-4.18(1H, m), 4.23-4.33(2H, m).

[0069] (2) Preparation of Aqueous Phase (2a) Preparation of Aqueous Phase 1 (Intraliposomal Aqueous Phase) In Table 1, the aqueous phase 1 of Examples 1, 2, 10, 11, and 12, and Comparative Examples 1 to 5 was obtained by adding an aqueous ammonia solution to an aqueous solution of sulfuric acid or methanesulfonic acid to adjust the pH to 4, and then adjusting the volume with water to the concentrations listed in Table 1. The aqueous phase 1 of Example 3 was obtained by mixing the aqueous phase 1 prepared in Examples 1 and 2, and Comparative Examples 1 to 4 with the aqueous phase 1 prepared in Comparative Example 5 in a 1:1 ratio. Examples 4 and 5 were obtained by desalting an aqueous solution of sucrose sodium octasulfate (manufactured by Amadis Chemical) through an ion exchange resin, adjusting the pH to 4 with triethylamine, and adjusting the volume with water to the concentrations listed in Table 1. Example 6 was obtained by dissolving ammonium sulfate and succinic acid in water, and then adjusting the volume with water to the concentrations listed in Table 1. In Examples 7 to 9 and Comparative Example 6, ammonium sulfate and succinic acid were dissolved in water, the pH was adjusted to 4 by adding an aqueous solution of a base listed in Table 1, and the volume was then adjusted with water to the concentrations listed. As an example, aqueous phase 1 in Examples 7 and 8 was obtained by weighing 9.25 g of ammonium sulfate and 1.18 g of succinic acid, adding water (160 mL) to dissolve the mixture, adding 5.8 mL of 1N aqueous sodium hydroxide to adjust the pH to 4, and then adjusting the volume to 200 mL with water. (2b) Preparation of aqueous phase 2 (external aqueous phase during drug encapsulation) L-histidine (9.3 g) and sodium chloride (18 g) were dissolved in water (1900 mL), the pH was adjusted to 6 with aqueous hydrochloric acid, and the volume was then adjusted to 2000 mL to obtain aqueous phase 2. (2c) Preparation of aqueous phase 3 (external aqueous phase after drug encapsulation) L-histidine (1.55 g) and sodium chloride (9 g) were dissolved in water (900 mL), the pH was adjusted to 7.5 with aqueous hydrochloric acid, and the volume was adjusted to 1000 mL to obtain aqueous phase 3.

[0070] (3) Preparation of liposomes Aqueous phase 1, which had been preheated to 80°C, was added to the lipid mixture and mixed by inversion. Using an extruder (manufactured by LIPEX) while heating at 80°C, the mixture was passed through a series of 100 nm pore size filters (manufactured by Whatman) to size the mixture. The resulting size-sized mixture was diluted with saline and then ultracentrifuged at approximately 300,000 g. The supernatant was removed, the mixture was suspended in saline, and then ultracentrifuged again at approximately 300,000 g. The supernatant was removed again, and the mixture was suspended in saline. The mixture was passed through a 0.45 μm pore size PES (polyethersulfone) filter (manufactured by Whatman) to obtain liposomes.

[0071] (4) Preparation of Liposomal Composition (Formulation) A drug solution was prepared by dissolving the drug (Compound (I) maleate or lenvatinib methanesulfonate) in a 5% glucose / HCl solution to a concentration of 0.75 mg / mL as the free form. The liposomes and drug solution were sequentially added to aqueous phase 2, which had been preheated to 60°C, and mixed at 60°C for 60 minutes. The drug concentration in aqueous phase 2 after mixing was 0.2 mg / mL, and the drug solution and liposomes were adjusted so that the drug to total lipid weight ratio was 0.17 to 0.2. The mixture was cooled to 4°C and then passed through a 0.45 μm pore PES filter (Whatman). The filtrate was diluted with saline and ultracentrifuged at approximately 300,000 g, after which the supernatant was removed. The mixture was resuspended in aqueous phase 3 and passed through a 0.22 μm pore PES filter (PALL) to obtain a liposome composition. The average particle size of the liposome composition was measured by dynamic light scattering using a particle size measuring device (Zetasizer NanoZS (Malvern)).

[0072] 3. Calculation of Drug / Total Lipid Weight Ratio (D / L Ratio) in Liposome Composition The cholesterol concentration and drug concentration in the liposome composition prepared above were measured using HPLC under the following conditions. (3-1) Cholesterol concentration Detection: UV 215 nm Column: ODS (C18) Mobile phase: methanol / tetrahydrofuran / 0.17 M ammonium acetate = 94 / 5 / 1 (v / v / v) (3-2) Drug concentration Detection: UV 252 nm Column: ODS (C18) Mobile phase A: water / acetonitrile / TFA = 990 / 10 / 1 Mobile phase B: water / acetonitrile / TFA = 100 / 900 / 1 Concentration of Mobile phase B: 20% Next, the D / L ratio of each liposome composition was calculated according to the method described in paragraph 0037 above. The calculation results are shown in Table 1.

[0073] 4. Measurement of Blood Drug Concentration (1) The liposome composition prepared above was administered intravenously to ICR male mice (dose: 2 mg / kg of drug, 4 mice per group). 24 hours after administration, whole blood was collected from the inferior vena cava under isoflurane anesthesia. The blood was centrifuged at approximately 1200 g for 10 minutes, and plasma was collected. The drug concentration of the collected plasma was quantified using LC-MS / MS. The blood drug concentrations 24 hours after administration are shown in Table 1.

[0074] When compound (I) was used as the drug, the liposome compositions of Examples 1 and 2, which contained hen's egg dihydrosphingomyelin or synthetic dihydrosphingomyelin in the liposomes, showed high blood drug concentrations and excellent blood retention, compared to the liposome compositions of Comparative Examples 1 and 2, which contained distearoylphosphatidylcholine or hen's egg sphingomyelin in the liposomes. On the other hand, the liposome compositions of Comparative Examples 3 and 4, in which the drug in Examples 1 and 2 was replaced with lenvatinib, showed significantly lower blood drug concentrations than the liposome compositions of Examples 1 and 2.

[0075]

[0076] 5. Measurement of Blood Drug Concentration (2) The liposome compositions of Examples 2, 5, and 8, and aqueous solutions of methanesulfonate of Compound (I) were administered to mice in the same manner as in 4. Measurement of Blood Drug Concentration (1) above, and blood samples were taken 15 minutes, 4 hours, and 24 hours after administration to measure the time course of the blood drug concentration. The results are shown in Figure 1.

[0077] The liposome compositions of Examples 2, 5, and 8 showed good blood drug concentration profiles even at 15 minutes, 4 hours, and 24 hours after administration, whereas the blood drug concentration of the aqueous solution of methanesulfonate of compound (I) decreased immediately after administration and dropped significantly after 24 hours. This demonstrates that the blood retention of compound (I) administered as an aqueous solution of methanesulfonate is significantly improved by administering the liposome composition of this embodiment.

[0078] 6. Anti-PEG IgM Antibody Production Evaluation Test It is known that repeated administration of polyethylene glycol-modified liposomes to a subject produces anti-PEG antibodies, resulting in a decrease in the blood retention of the drug after the second administration, known as the accelerated blood clearance (ABC) phenomenon. Here, by comparing the amount of anti-PEG antibodies produced, which is thought to be the cause of the ABC phenomenon, it is possible to evaluate the inhibitory effect on its occurrence. Thus, a liposome composition in which compound (I) is encapsulated in liposomes of this embodiment and liposomes not encapsulating compound (I) were administered to mice, and the amount of anti-PEG antibodies produced in each mouse was evaluated by the following method.

[0079] The liposome composition encapsulating compound (I), prepared in the same manner as in Example 8, and liposomes before encapsulating compound (I) were administered intravenously to male BALB / c mice via the tail at a dose of 4 mg / kg of DHSM per mouse (1 mg / kg of the drug for the liposome composition encapsulating compound (I)). The DHSM dose was measured using the same method as in (3-1) Measurement of cholesterol concentration in

[0073] . Ten days after administration, whole blood was collected from the inferior vena cava under isoflurane anesthesia, and serum was collected from the mice administered with the liposome composition encapsulating compound (I) and the mice administered with liposomes not encapsulating compound (I). The collected mouse serum was diluted 100-fold, and the amount of anti-PEG IgM antibody produced was evaluated by measuring the absorbance at 490 nm using a spectrophotometer (SpectraMax M2 (Molecular Devices)) according to the method reported in cited paper A (cited paper A: International Journal of Pharmaceutics. 15 (2012) 436: 636-643). The results are shown in Figure 2.

[0080] The serum collected from mice administered with the liposome composition encapsulating compound (I) had a significantly lower concentration of anti-PEG IgM antibodies than the serum collected from liposomes not encapsulating compound (I). This confirmed the effect of suppressing the occurrence of the ABC phenomenon by combining the liposome of this embodiment with compound (I). That is, it is expected that the combination of the liposome composition of this embodiment with compound (I) or a pharmaceutically acceptable salt thereof will suppress the occurrence of the ABC phenomenon and will exhibit high blood retention even after the second administration.

[0081] The maleate and methanesulfonate salts of compound (I) can be produced, for example, by the methods described below. However, these are illustrative and are not intended to limit the scope of the invention.

[0082] In the production examples, commercially available compounds were used where appropriate. The abbreviations used have the following meanings: CD ３OD: deuterated methanol DMSO-d ６ : Deuterated dimethyl sulfoxide DMA: N,N-dimethylacetamide

[0083] [Production Example 1] 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide maleate

[0084] 4-(3-Chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (5.70 g, 13.7 mmol) was added to DMA (23 mL) solvent and dissolved by heating and stirring at 80°C. Then, a solution of maleic acid (1.91 g, 16.5 mmol) in DMA (5.5 mL) was added and stirred for 5 minutes. 2-Butanone (350 mL) was added dropwise to the reaction solution over 1 hour, and the mixture was stirred at 40°C for 17 hours. The reaction solution was cooled to room temperature and stirred for 6 hours, after which the precipitated solid was collected by filtration. The resulting solid was further washed with 2-butanone and then subjected to ventilation drying and drying under reduced pressure at 70°C to obtain the title compound (6.40 g). 1 H-NMR Spectrum (500 MHz, DMSO-d6) δ(ppm): 1.08(3H, t, J=7.2Hz), 3.10-3.18(2H, m), 4.04(3H, s), 6.23(2H, s), 6.60(1H, d, J=5.5Hz), 7.00(1H, t, J=5.5Hz), 7.25(1H, dd, J=9.0, 2.8Hz), 7.50(1H, d, J=2.8Hz), 7.53(1H, s), 7.76(1H, brs), 7.87(1H, brs), 8.08(1H, s), 8.28(1H, d, J=9.6Hz), 8.67(1H, s), 8.72(1H, d, J=4.8Hz).

[0085] [Production Example 2] 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide methanesulfonate

[0086] To a suspension of 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinolinecarboxamide (1.00 g, 2.41 mmol) in methanol (20 mL) was added methanesulfonic acid (188 μL, 2.89 mmol) at 70° C. and stirred for 15 minutes. The reaction solution was cooled to room temperature and stirred for 16 hours, and then the precipitated solid was collected by filtration. The obtained solid was further washed with methanol and diethyl ether and dried under reduced pressure to obtain the title compound (1.06 g). 1 H-NMR Spectrum (500 MHz, DMSO-d6) δ(ppm): 1.09(3H, t, J=7.1Hz), 2.34(3H, s), 3.08-3.22(2H, m), 4.09(3H, s), 6.95(1H, d, J=6.3Hz), 7.06(1H, brt, J=4.9Hz), 7.34(1H, dd, J=9.0, 2.7Hz), 7.61-7.66(2H, m), 7.91(1H, brs), 7.97(1H, brs), 8.16(1H, s), 8.36(1H, d, J=9.3Hz), 8.73(1H, s), 8.97(1H, d, J=6.3Hz).

Claims

1. (A) Liposomes containing dihydrosphingomyelin and diacylglycerol-polyethylene glycol (B) Drugs, and (C) Containing sulfates and / or sucrose octasulfates, A liposome composition wherein (A) contains (B), The aforementioned diacylglycerol-polyethylene glycol is distearoylglycerol-polyethylene glycol, The above (B) is 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinoline carboxamide represented by formula (I) or a pharmaceutically acceptable salt thereof. A liposome composition. 【Chemistry 1】

2. The liposome composition according to claim 1, wherein (C) is a sulfate.

3. The liposome composition according to claim 1, wherein the dihydrosphingomyelin is a compound represented by formula (II-1), (II-2), (II-3), or (II-4), or a mixture thereof. 【Chemistry 2】

4. The liposome composition according to claim 1, wherein the dihydrosphingomyelin is a compound represented by formula (II-3). 【Transformation 3】

5. (D) The liposome composition according to claim 1, further comprising an organic acid.

6. The liposome composition according to claim 5, wherein (D) is at least one selected from the group consisting of succinic acid, citric acid, maleic acid, and salicylic acid.

7. The liposome composition according to claim 1, wherein (A) further contains cholesterols.

8. (E) The liposome composition according to claim 1, further comprising a basic compound.

9. The liposome composition according to claim 1, wherein (C) is ammonium sulfate.

10. The liposome composition according to claim 5, wherein (D) is succinic acid.

11. The liposome composition according to claim 8, wherein (E) is sodium hydroxide or ammonia.

12. A pharmaceutical composition comprising the liposome composition according to any one of claims 1 to 11.

13. An angiogenesis inhibitor comprising the liposome composition according to any one of claims 1 to 11.

14. An antitumor agent comprising the liposome composition according to any one of claims 1 to 11.

15. A method for producing a liposome composition according to any one of claims 1 to 11, A step of providing a liposome dispersion, The steps include mixing the liposome dispersion with 4-(3-chloro-4-(ethylaminocarbonyl)aminophenoxy)-7-methoxy-6-quinoline carboxamide represented by formula (I) or a pharmaceutically acceptable salt thereof, Methods that include... 【Chemistry 4】

Images