Emulsion and method for producing the same

Abstract

The present invention relates to an emulsion and a method for producing the same. [Subject] Provide a novel emulsion containing nimodipine. [Solution] An emulsion comprising nimodipine, phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer, tocopherol and water. [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. Except for the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups.]

Description

Technical Field

[0001] The present invention relates to an emulsion and a method for producing the same, and more particularly to an emulsion containing lipid nanoparticles containing nimodipine and a method for producing the same. Background Art

[0002] Nimodipine is known as a calcium channel blocker. In addition, it is known that nimodipine can easily penetrate the blood-brain barrier and improve the blood supply to the brain. Therefore, nimodipine is widely used clinically to improve blood circulation in the recovery period of acute cerebrovascular injury, cerebral vasospasm after subarachnoid hemorrhage, ischemic nerve damage caused by it, hypertension, migraine, etc.

[0003] As a commercially available nimodipine preparation, for example, nimodipine (Bayer) is known. In addition, as an emulsified composition of nimodipine, for example, a nimodipine injection composition is disclosed in Patent Document 1, characterized in that it contains 0.02-0.23 mass concentration% of nimodipine, 2-30 mass concentration% of injection oil, 0.8-3 mass concentration% of emulsifier, 0-0.1 mass concentration% of complexing agent, 0-0.3 mass concentration% of stabilizer and 1-3 mass concentration% of osmotic pressure regulator. In addition, for example, a nimodipine submicroemulsion injection containing amino acids is disclosed in Patent Document 2.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application No. 2021-535932

[0007] Patent Document 2: Chinese Patent Application Publication No. 105796494 Summary of the invention

[0008] Problem that the invention aims to solve

[0009] In addition to using 25% high concentration ethanol to dissolve nimodipine, nimotong also uses an organic solvent other than ethanol (PEG400). In addition, nimotong needs to be mixed with other diluents when used, but in order to inhibit the precipitation of nimodipine, it is mixed with a diluent such as physiological saline before administration. When ethanol is diluted to dissolve nimodipine, the precipitation of nimodipine crystals begins slowly. Therefore, a complicated intravenous infusion set consisting of a three-way stopcock and an injection pump is used, and the drip speed needs to be very slow, which is a great burden on the medical site. Moreover, during the administration of nimodipine injection (generally more than 5 hours), patients must endure the vascular pain caused by the ethanol used in nimodipine injection, which aggravates the pain.

[0010] If micelles or emulsions are used as drug carriers for nimodipine, it is expected that the stability of nimodipine and the compliance of patients during administration can be greatly improved. Several emulsified compositions of nimodipine are known, such as those disclosed in Patent Documents 1 and 2, but it cannot be said that sufficient options are provided.

[0011] Therefore, an object of the present invention is to provide a novel emulsion containing nimodipine. Another object of the present invention is to provide a method for producing the emulsion.

[0012] Solutions for solving problems

[0013] The present invention relates to, for example, the following aspects. [1]

[0015] An emulsion comprising nimodipine, phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer, tocopherol and water.

[0016]

[0017] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.] [2]

[0019] The emulsion according to [1], wherein the phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin. [3]

[0021] The emulsion according to [1] or [2], wherein the neutral fat is at least one selected from the group consisting of soybean oil, sesame oil, olive oil, rapeseed oil, corn oil and medium-chain fatty acid triglycerides. [4]

[0023] The emulsion according to any one of [1] to [3], wherein R6 is a linear alkyl group having 8 to 18 carbon atoms. [5]

[0025] The emulsion according to any one of [1] to [4], wherein the buffer is at least one selected from the group consisting of citric acid and salts thereof, phosphoric acid and salts thereof, and tris(hydroxymethyl)aminomethane and salts thereof. [6]

[0027] The emulsion according to any one of [1] to [5], wherein the acid dissociation constant pK of the buffer is a In the range of 7 to 9. [7]

[0029] The emulsion according to any one of [1] to [6], wherein the pH is 5 or more and 9 or less. [8]

[0031] The emulsion according to any one of [1] to [7], wherein the content of nimodipine is 0.02% by mass or more and 0.2% by mass or less based on the total amount of the emulsion,

[0032] The content of the phospholipid is 1% by mass or more and 3% by mass or less based on the total amount of the emulsion.

[0033] The content of the neutral fat is 2% by mass or more and 10% by mass or less based on the total amount of the emulsion.

[0034] The content of the hyaluronic acid derivative is 0.1% by mass or more and 1% by mass or less based on the total amount of the emulsion.

[0035] The content of the buffer is 0.01% by mass or more and 0.2% by mass or less in terms of anhydrous content based on the total amount of the emulsion.

[0036] The tocopherol content is 0.01% by mass or more and 0.2% by mass or less based on the total amount of the emulsion. [9]

[0038] The emulsion according to any one of [1] to [8], which is an injection.

[10]

[0040] The emulsion according to any one of [1] to [9], which is used for administration without dilution.

[11]

[0042] A method for producing an emulsion, comprising:

[0043] a) heating and dissolving nimodipine in an organic solvent;

[0044] b) adding phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer and tocopherol to an aqueous solvent, followed by a step of coarse emulsification; and

[0045] c) a step of adding dropwise the solution obtained in step a) to the crude emulsion obtained in step b) to disperse the mixture, and further subjecting the dispersed liquid to a high-pressure emulsification treatment.

[0046]

[0047] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.]

[12]

[0049] A method for producing an emulsion, comprising:

[0050] A) the step of heating and dissolving nimodipine in an organic solvent;

[0051] B) adding phospholipids, a buffer and a hyaluronic acid derivative represented by the general formula (1) into an aqueous solvent for dispersion;

[0052] C) a step of mixing the solution obtained in step A) with a neutral lipid and tocopherol; and

[0053] D) a step of adding dropwise the solution obtained in step C) to the dispersion obtained in step B) to perform rough emulsification, and further subjecting the rough emulsified liquid to a high-pressure emulsification treatment.

[0054]

[0055] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.]

[0056] Effects of the Invention

[0057] According to the present invention, a novel emulsion containing nimodipine can be provided. According to the present invention, a method for producing the emulsion can also be provided. DETAILED DESCRIPTION

[0058] Hereinafter, the mode for implementing the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0059] <Features of the Invention>

[0060] (Lotion)

[0061] The present invention is characterized in providing an emulsion comprising nimodipine, phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer, tocopherol and water.

[0062]

[0063] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.]

[0064] (Method for producing emulsion)

[0065] The present invention is characterized in that it provides a method for producing an emulsion (first production method), which comprises: a) a step of heating and dissolving nimodipine in an organic solvent; b) a step of heating and dissolving phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer and tocopherol in an aqueous solvent, followed by a step of crude emulsification; and c) a step of dripping the solution obtained in step a) into the crude emulsion obtained in step b) and dispersing the mixture, and further subjecting the dispersed liquid to a high-pressure emulsification treatment.

[0066]

[0067] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.]

[0068] In addition, the present invention is characterized in providing a method for producing an emulsion (second production method), which includes: A) a step of heating and dissolving nimodipine in an organic solvent; B) a step of adding a phospholipid, a buffer and a hyaluronic acid derivative represented by the general formula (1) to an aqueous solvent for dispersion; C) a step of mixing the solution obtained in step A) with a neutral lipid and tocopherol; and D) a step of dropping the solution obtained in step C) into the dispersion obtained in step B) to perform coarse emulsification, and further performing a high-pressure emulsification treatment on the coarse emulsified liquid.

[0069]

[0070] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. n represents an integer of 1 to 2500. However, the case where R1, R2, R3, R4 and R5 are all hydroxyl groups is excluded.]

[0071] <Lotion>

[0072] The emulsion of this embodiment contains nimodipine, phospholipids, neutral lipids, a hyaluronic acid derivative represented by the general formula (1), a buffer, tocopherol and water.

[0073] The emulsion of the present embodiment is an oil-in-water (O / W) emulsion in which lipid nanoparticles containing nimodipine are dispersed in an aqueous phase. In the lipid nanoparticles, nimodipine is encapsulated in a shell consisting of at least phospholipids and hyaluronic acid derivatives. The shell consisting of at least phospholipids and hyaluronic acid derivatives has a structure in which hyaluronic acid derivatives are coated on a lipid membrane layer formed by phospholipids (and shell constituents other than hyaluronic acid derivatives such as lipids other than phospholipids as required). The hyaluronic acid derivatives do not form a covalent bond with the constituents of the lipid membrane layer, but are adsorbed on the lipid membrane layer at the portion (particularly the portion of R6) of the group shown in -O-CH2-CH(OH)-CH2-OR6.

[0074] (Nimodipine)

[0075] Nimodipine is a compound also known as 3-(2-methoxyethyl)-5-propane-2-yl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. The nimodipine used in the emulsion of this embodiment may be an R-isomer, an S-isomer, or a mixture of an R-isomer and an S-isomer (e.g., a racemic mixture containing an R-isomer and an S-isomer in a ratio of 1:1).

[0076] The content of nimodipine in the emulsion of the present embodiment can be, for example, 0.001% by mass to 1% by mass, 0.005% by mass to 0.8% by mass, 0.01% by mass to 0.5% by mass, 0.015% by mass to 0.3% by mass, or 0.02% by mass to 0.2% by mass, based on the total amount of the emulsion.

[0077] The content of nimodipine in the emulsion of the present embodiment may be 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, or 95% by mass or more, based on the content before storage, for example, after storage at 40° C. for 1 month.

[0078] (Phospholipids)

[0079] The emulsion of the present embodiment contains phospholipids as constituents of lipid nanoparticles. As phospholipids, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingosine phospholipids, and sphingomyelin and the like can be cited. One phospholipid can be used alone, or two or more can be used in combination.

[0080] As the phospholipid, phospholipids derived from animal or plant materials such as egg yolk, soybean, or rapeseed can be used without particular limitation.

[0081] The content of phospholipids in the emulsion of the present embodiment only needs to be an amount sufficient to form the above-mentioned lipid membrane layer. For example, based on the total amount of the emulsion, it can be 0.5% by mass to 10% by mass, 0.6% by mass to 8% by mass, 0.7% by mass to 6% by mass, 0.8% by mass to 5% by mass, 0.9% by mass to 4% by mass, or 1% by mass to 3% by mass.

[0082] (Neutral fat)

[0083] The emulsion of the present embodiment contains neutral lipids. Neutral lipids are used, for example, to assist the dissolution of nimodipine. As neutral lipids, vegetable oils such as soybean oil, sesame oil, rapeseed oil, safflower oil, olive oil, castor oil, corn oil, cottonseed oil, rice oil, sunflower oil, grape seed oil and wheat germ oil, medium-chain fatty acid triglycerides (MCT), and long-chain fatty acid triglycerides (LCT) can be cited. Neutral lipids can be used alone or in combination of two or more.

[0084] The content of neutral fat in the emulsion of this embodiment can be, for example, from 1 mass % to 20 mass %, from 1.2 mass % to 18 mass %, from 1.4 mass % to 16 mass %, from 1.6 mass % to 14 mass %, from 1.8 mass % to 12 mass %, or from 2 mass % to 10 mass %, based on the total amount of the emulsion.

[0085] (Hyaluronic acid derivative)

[0086] The emulsion of the present embodiment contains a hyaluronic acid derivative as a constituent component of lipid nanoparticles.

[0087] The hyaluronic acid derivative according to the present embodiment is represented by the following general formula (1).

[0088]

[0089] The hyaluronic acid derivative of the present embodiment has the following structure: a part or all of the hydroxyl groups bonded to the carbon atoms at positions 4 and 6 of N-acetylglucosamine constituting hyaluronic acid, the hydroxyl groups bonded to the carbon atoms at positions 2 and 3 of glucuronic acid constituting hyaluronic acid, and the carboxyl group bonded to the carbon atom at position 5 are substituted by the group represented by -O-CH2-CH(OH)-CH2-OR6. It should be noted that, in the group represented by -O-CH2-CH(OH)-CH2-OR6, the oxygen atom recorded on the far left is bonded to the carbon atom constituting hyaluronic acid.

[0090] In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6. R6 represents a linear or branched alkyl or alkenyl group. It should be noted that, when there are multiple R1s (when n is 2 or more), the R1s contained in different repeating units may be the same or different. Similarly, when there are multiple R2s (when n is 2 or more), the R2s contained in different repeating units may be the same or different. When there are multiple R3s (when n is 2 or more), the R3s contained in different repeating units may be the same or different. When there are multiple R4s (when n is 2 or more), the R4s contained in different repeating units may be the same or different. When there are multiple R5s (when n is 2 or more), the R5s contained in different repeating units may be the same or different.

[0091] Examples of the straight-chain or branched alkyl group represented by R6 include a straight-chain or branched alkyl group having 8 to 22 carbon atoms, a straight-chain or branched alkyl group having 8 to 21 carbon atoms, a straight-chain or branched alkyl group having 8 to 20 carbon atoms, a straight-chain or branched alkyl group having 8 to 19 carbon atoms, and a straight-chain or branched alkyl group having 8 to 18 carbon atoms.

[0092] More specific examples of the linear or branched alkyl group represented by R6 include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 2-methylbutyl, 1-methylbutyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, n-heptyl, n-octyl, isooctyl, 2-ethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl (myristyl), n-hexadecyl (palmityl), n-octadecyl (stearyl), and n-eicosyl.

[0093] Examples of the straight-chain or branched alkenyl group represented by R6 include a straight-chain or branched alkenyl group having 8 to 22 carbon atoms, a straight-chain or branched alkenyl group having 9 to 21 carbon atoms, a straight-chain or branched alkenyl group having 10 to 20 carbon atoms, a straight-chain or branched alkenyl group having 11 to 19 carbon atoms, and a straight-chain or branched alkenyl group having 12 to 18 carbon atoms.

[0094] More specific examples of the straight-chain or branched alkenyl group represented by R6 include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tetradecenyl and oleyl.

[0095] As the linear or branched alkyl or alkenyl group represented by R6, a linear or branched alkyl group is preferred, and a linear alkyl group is more preferred, from the viewpoint of safety to a living body.

[0096] In the general formula (1), n ​​represents an integer of 1 to 2500. For example, n may be 2 to 2500, 4 to 2450, 6 to 2400, 8 to 2350, 10 to 2300, 12 to 2250, 14 to 2200, 16 to 2150, 17 to 2100, 18 to 2050, or 19 to 2000.

[0097] In the general formula (1), when all of R1, R2, R3, R4 and R5 are hydroxyl groups, hyaluronic acid is obtained, and therefore the general formula (1) excludes the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups.

[0098] The modification rate of the hyaluronic acid derivative of the present embodiment can be, for example, 0.1% or more and 50% or less. In this specification, the modification rate of the hyaluronic acid derivative is a value representing the number of groups shown in -O-CH2-CH(OH)-CH2-OR6 contained in each constituent unit of hyaluronic acid as a percentage. Specifically, it is a value calculated by the following formula.

[0099] Modification rate of hyaluronic acid derivative = (number of groups represented by -O-CH2-CH(OH)-CH2-OR6) / number of constituent units of hyaluronic acid × 100 (%)

[0100] Here, one structural unit of hyaluronic acid refers to one structural unit composed of a disaccharide of glucuronic acid and N-acetylglucosamine. The modification rate of the hyaluronic acid derivative can be determined, for example, by 1 H-NMR spectrum analysis. 1In the H-NMR spectrum, a peak representing the proton of the methyl group (-CH3) of -NHC(=O)CH3 (N-acetyl) bonded to N-acetylglucosamine contained in the constituent unit of hyaluronic acid is observed near 2.0 ppm. Similarly, a peak representing the proton of the methylene group (-CH2-) contained in the alkyl or alkenyl group represented by R6 in the general formula (1) is observed near 1.3 ppm. The modification rate can be calculated from the integral value of the peak near 2.0 ppm and the integral value of the peak near 1.3 ppm.

[0101] From the perspective of improving emulsion stability, the modification rate of the hyaluronic acid derivative of this embodiment can be, for example, 0.5% to 45%, 1% to 40%, 2% to 30%, 3% to 20%, or 4% to 16%.

[0102] The hyaluronic acid derivative of the present embodiment can be produced, for example, by reacting hyaluronic acid or its salt with a compound represented by the following general formula (2) (hereinafter also referred to as "Compound 1"). It should be noted that, in order to improve reactivity, the raw material hyaluronic acid or its salt can also be substituted with an alkylammonium salt before reacting with Compound 1.

[0103]

[0104] [R6 in the general formula (2) has the same meaning as R6 in the general formula (1).]

[0105] The raw material hyaluronic acid or its salt can be extracted from biological tissues such as animals (e.g., cockscombs, umbilical cords, skin, joint fluid), and can be obtained by culturing microorganisms, animal cells or plant cells (e.g., fermentation methods using bacteria of the genus Streptococcus), or by chemical or enzymatic synthesis. The salt of hyaluronic acid is not particularly limited, and is preferably a food or pharmaceutically acceptable salt, and can include, for example, sodium salts, potassium salts, calcium salts, zinc salts, magnesium salts, ammonium salts, alkylammonium salts, etc.

[0106] The average molecular weight of the raw material hyaluronic acid or its salt can be, for example, 411 to 1,020,000. From the perspective of improving the adsorption ease, emulsification power and emulsification stability of lipid nanoparticles on the shell, it can be 1,000 to 1,000,000, 2,000 to 800,000, 4,000 to 700,000, 5,000 to 600,000, 6,000 to 500,000, 7,000 to 400,000, or 8,000 to 300,000.

[0107] In this specification, the average molecular weight of hyaluronic acid or a salt thereof is a value measured by the following method.

[0108] About 0.05g of hyaluronic acid and / or its salt (this product) is accurately weighed, dissolved in a sodium chloride solution with a concentration of 0.2mol / L, and a solution of 100mL is accurately prepared. 8mL, 12mL and 16mL of the solution are accurately weighed, and a sodium chloride solution with a concentration of 0.2mol / L is added respectively to accurately prepare a solution of 20mL, and the above 100mL solution and 20mL solution are used as sample solutions. For these sample solutions and a sodium chloride solution with a concentration of 0.2mol / L, the viscosity (formula (A)) is measured at 30.0±0.1°C by the viscosity determination method (method 1 capillary viscometer method) of the general test method of the Japanese Pharmacopoeia (15th Amendment), and the reduced viscosity (formula (B)) at each concentration is calculated. A curve graph is drawn with the reduced viscosity as the vertical axis and the concentration (g / 100mL) of the dry product converted relative to this product as the horizontal axis, and the intrinsic viscosity is obtained from the intersection of the straight line connecting each point and the vertical axis. The intrinsic viscosity obtained here is substituted into the formula (formula (C)) of Laurent to calculate the average molecular weight (M) (reference: TC Laurent, M. Ryan, A. Pietruszkiewicz,: BBA, 42, 476-485 (1960)).

[0109] (Formula A): Specific viscosity = {Number of seconds required for sample solution to flow down) / (Number of seconds required for 0.2 mol / L sodium chloride solution to flow down)} - 1

[0110] (Formula B): Reduced viscosity (dL / g) = specific viscosity / (concentration of the dry product converted to g / 100 mL))

[0111] (Formula C): Intrinsic viscosity (dL / g) = 3.6 × 10 -4 M 0.78

[0112] The hyaluronic acid derivatives may be used alone or in combination of two or more.

[0113] The content of the hyaluronic acid derivative in the emulsion of the present embodiment can be an amount sufficient to cover the above-mentioned lipid membrane layer and to improve the emulsifying power and emulsification stability. For example, based on the total amount of the emulsion, it can be 0.01% by mass to 10% by mass, 0.03% by mass to 8% by mass, 0.05% by mass to 6% by mass, 0.07% by mass to 4% by mass, 0.08% by mass to 3% by mass, 0.09% by mass to 2% by mass, or 0.1% by mass to 1% by mass.

[0114] From the perspective of improving emulsification power and emulsion stability, the content ratio of the hyaluronic acid derivative to the phospholipid in the emulsion of this embodiment (content of hyaluronic acid derivative (mass %): content of phospholipid (mass %)) can be, for example, in the range of 55:1 to 1:25, or in the range of 50:1 to 1:20, or in the range of 45:1 to 1:15.

[0115] (Buffer)

[0116] The emulsion of the present embodiment includes a buffer. The buffer is used to inhibit the decomposition of nimodipine and improve storage stability. As the buffer, for example, at least one selected from the group consisting of organic acids and salts thereof having a carboxyl group such as citric acid and its salts, phosphoric acid and its salts, and trishydroxymethylaminomethane and its salts can be cited. As a buffer, since the effect of inhibiting the decomposition of nimodipine and improving storage stability is more significant, it is preferred to use a buffer having an acid dissociation constant pKa in the range of 7 to 9.

[0117] The content of the buffer in the emulsion of the present embodiment can be an amount sufficient to improve the stability of nimodipine. For example, based on the total amount of the emulsion, the content of the buffer can be 0.002% by mass to 1% by mass, 0.004% by mass to 0.8% by mass, 0.006% by mass to 0.6% by mass, 0.008% by mass to 0.4% by mass, or 0.01% by mass to 0.2% by mass in terms of anhydrous content.

[0118] (Tocopherol)

[0119] The emulsion of the present embodiment includes tocopherol. Tocopherol is used to inhibit the decomposition of nimodipine and improve storage stability. As tocopherol, for example, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol can be cited. There are multiple optical isomers in these tocopherols, but the tocopherol used in the emulsion of the present embodiment can be any optical isomer, preferably a racemic body (such as dl-α-tocopherol). In the emulsion of the present embodiment, tocopherol can be used alone or in combination of two or more.

[0120] The content of tocopherol in the emulsion of the present embodiment only needs to be an amount sufficient to improve the stability of nimodipine. For example, based on the total amount of the emulsion, it can be 0.002% by mass to 1% by mass, 0.004% by mass to 0.8% by mass, 0.006% by mass to 0.6% by mass, 0.008% by mass to 0.4% by mass, or 0.01% by mass to 0.2% by mass.

[0121] (water)

[0122] The emulsion of the present embodiment contains water. As water, for example, distilled water, normal water, purified water, sterilized purified water, water for injection, and distilled water for injection, as defined in the 18th revised Japanese Pharmacopoeia, can be cited. Water constitutes the external phase (aqueous phase) of the lipid nanoparticles containing nimodipine. The external phase includes, for example, an aqueous solvent (e.g., water, buffer) used when forming an aqueous phase, an organic solvent used when forming an oil phase, etc.

[0123] The content of water in the emulsion of the present embodiment may be the remainder of the above-mentioned components, and specifically, for example, may be more than 0% by mass and less than 100% by mass based on the total amount of the emulsion.

[0124] <Other ingredients>

[0125] The emulsion of the present embodiment may further contain other components acceptable as pharmaceuticals within a range that does not inhibit the effects of the present invention. Examples of other components include solvents or solubilizers, emulsifying aids, osmotic pressure regulators, and pH regulators.

[0126] (Solvent or solubilizer)

[0127] The solvent or solubilizing agent is used, for example, to assist in the solubilization of nimodipine. As the solvent or solubilizing agent, for example, an organic solvent or a surfactant can be used.

[0128] Examples of the organic solvent include ethanol, dimethylformamide (DMF), propylene glycol, polyethylene glycol, acetone, dimethylformamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone, and tetrahydrofuran.

[0129] Examples of the surfactant include polyoxyethylene hydrogenated castor oil, polysorbate, glycerol fatty acid ester, and sorbitan fatty acid ester.

[0130] (Emulsifying agent)

[0131] Emulsifying aids are used to improve the emulsification stability of emulsions.

[0132] Examples of the emulsifying aid include surfactants such as polyoxyethylene hydrogenated castor oil, polysorbate, glycerol fatty acid esters, and sorbitan fatty acid esters; higher alcohols such as behenyl alcohol and stearyl alcohol; and higher fatty acids such as oleic acid and sodium oleate or their salts.

[0133] (Osmotic pressure regulator)

[0134] The osmotic pressure regulator is used to adjust the osmotic pressure ratio of the emulsion to a desired value.

[0135] Examples of the osmotic pressure regulator include sugars such as sorbitol, xylitol, mannitol, glucose, trehalose, maltose, sucrose, raffinose, lactose, and dextran; salts such as sodium chloride and potassium chloride; and concentrated glycerin.

[0136] (pH adjuster)

[0137] Examples of the pH adjuster include strong acids such as hydrochloric acid and strong bases such as sodium hydroxide.

[0138] The emulsion of this embodiment uses nimodipine, phospholipids, neutral lipids, hyaluronic acid derivatives represented by general formula (1), buffers, tocopherols and water in combination, so that nimodipine has excellent storage stability and excellent emulsification stability. Therefore, the emulsion of this embodiment may substantially not contain organic solvents (such as ethanol), synthetic surfactants and amino acids, etc., or the content thereof may be lower than that in the past.

[0139] The content of the organic solvent in the emulsion of the present embodiment can be, for example, less than 5% by mass, less than 4% by mass, less than 3% by mass, or less than 2.5% by mass based on the total amount of the emulsion. In addition, the content of ethanol in the emulsion of the present embodiment can be, for example, less than 5% by mass, less than 4% by mass, less than 3% by mass, or less than 2.5% by mass based on the total amount of the emulsion. The content of the synthetic surfactant in the emulsion of the present embodiment can be, for example, less than 5% by mass, less than 4% by mass, less than 3% by mass, less than 2% by mass, less than 1% by mass, or less than 0% by mass based on the total amount of the emulsion. The content of the amino acid in the emulsion of the present embodiment can be, for example, less than 5% by mass, less than 4% by mass, less than 3% by mass, less than 2% by mass, less than 1% by mass, or less than 0% by mass based on the total amount of the emulsion.

[0140] The particle size of the lipid nanoparticles contained in the emulsion of the present embodiment is preferably more than 0nm and less than 400nm. Thus, it becomes an emulsion suitable for use as an injection. In this specification, "particle size" refers to the average particle size measured by dynamic light scattering. The particle size can be measured by a particle size measuring device (e.g., Zetasizer Nano, Malvern Panalytical Co., Ltd.) using a dynamic light scattering method. From the perspective of excellent emulsification and being suitable for use as an injection, the particle size of the lipid nanoparticles contained in the emulsion of the present embodiment can exceed 0nm and be less than 350nm, exceed 10nm and be less than 300nm, more than 15nm and less than 250nm, more than 20nm and less than 200nm. In addition, the average particle size of the lipid nanoparticles contained in the emulsion of the present embodiment just after preparation and the change of the average particle size after being stored at 40°C for 1 month can be within 80nm, and can also be within 70nm, within 60nm, within 50nm, within 40nm, and within 35nm. It should be noted that the average particle size of the lipid nanoparticles contained in the emulsion of the present embodiment after storage is within the above range.

[0141] The emulsion of the present embodiment has excellent storage stability of nimodipine, and the ratio of the nimodipine content immediately after preparation to the nimodipine content after storage at 40°C for one month (after storage at 40°C for one month / immediately after preparation) can be within the range of 95% to 105%.

[0142] The pH of the emulsion of the present embodiment is not particularly limited, and for example, may be 3 or more and 11 or less. From the perspective of inhibiting the decomposition of nimodipine and improving storage stability, the pH of the emulsion of the present embodiment is preferably 4 or more and 10 or less, more preferably 5 or more and 9 or less, further preferably 6 or more and 9 or less, and even more preferably 7 or more and 9 or less.

[0143] The emulsion of the present embodiment can be used as a pharmaceutical composition, for example. In addition, since the emulsion of the present embodiment contains nimodipine, it can improve blood circulation in the recovery period of acute cerebrovascular injury, cerebral vasospasm after subarachnoid hemorrhage, ischemic nerve damage caused thereby, hypertension, migraine, etc., and can therefore be used as a therapeutic composition for cerebral hemorrhagic diseases such as hypertension, stroke, migraine, subarachnoid hemorrhage, etc.

[0144] The emulsion of the present embodiment can be used as, for example, an injection, a fat emulsion, or the like.

[0145] The subject to which the emulsion of the present embodiment is administered may be, for example, a mammal such as a human.

[0146] The dosage, timing, and period of administration of the emulsion of the present embodiment can be appropriately set according to the target disease, the attributes (sex, age, etc.) of the administration subject, and the like.

[0147] The emulsion of this embodiment can also be administered without dilution.

[0148] <Method for producing emulsion>

[0149] The emulsion of the present embodiment can be obtained by a production method (first production method) comprising the following steps: a) a step of heating and dissolving nimodipine in an organic solvent; b) a step of adding phospholipids, neutral lipids, a hyaluronic acid derivative represented by the general formula (1), a buffer and tocopherol to an aqueous solvent, followed by a step of crude emulsification; and c) a step of dropping the solution obtained in step a) into the crude emulsion obtained in step b) and dispersing the mixture, and further subjecting the dispersed liquid to a high-pressure emulsification treatment.

[0150] The emulsion of the present embodiment can also be obtained by a production method (second production method) comprising the following steps: A) a step of heating and dissolving nimodipine in an organic solvent; B) a step of adding a phospholipid, a buffer and a hyaluronic acid derivative represented by the general formula (1) to an aqueous solvent for dispersion; C) a step of mixing the solution obtained in step A) with a neutral lipid and tocopherol; and D) a step of dropping the solution obtained in step C) into the dispersion obtained in step B) to perform coarse emulsification, and further performing a high-pressure emulsification treatment on the coarse emulsified liquid.

[0151] (First Manufacturing Method)

[0152] (Step a)

[0153] In step a), nimodipine is heated and dissolved in an organic solvent. The organic solvent used can be any solvent that can dissolve nimodipine, and examples thereof include ethanol, dimethylformamide (DMF), propylene glycol, acetone, dimethylformamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone, tetrahydrofuran, and the like.

[0154] The temperature during heating and dissolving in step a) can be set, for example, in the range of 40 to 80° C. The temperature during heating and dissolving is preferably in the range of 50 to 70° C., and in the range of 55 to 65° C.

[0155] In step a), components other than nimodipine may be dissolved in an organic solvent as needed, but from the perspective of inhibiting the decomposition of nimodipine, phospholipids, neutral lipids and hyaluronic acid derivatives are preferably not dissolved in an organic solvent together. In other words, the solution obtained in step a) preferably does not contain phospholipids, neutral lipids and hyaluronic acid derivatives.

[0156] In step b), phospholipids, neutral lipids, hyaluronic acid derivatives represented by general formula (1), buffers and tocopherol are added to an aqueous solvent, followed by crude emulsification. Examples of aqueous solvents include distilled water, normal water, purified water, sterile purified water, water for injection and distilled water for injection as defined in the 18th revised Japanese Pharmacopoeia. The buffer added in step b) may be added in the form of a buffer solution.

[0157] In step b), the phospholipids, neutral lipids, the hyaluronic acid derivative represented by the general formula (1), the buffer and tocopherol may be added to the aqueous solvent and dissolved, for example, by stirring, etc. Alternatively, the solvent may be heated to 40 to 80° C. for dissolution as required.

[0158] In step b), the hyaluronic acid derivative represented by the general formula (1) may be first added to an aqueous solvent, heated to dissolve, and then phospholipids, neutral lipids, buffering agents and tocopherol may be added while stirring to form a coarse emulsification.

[0159] In the first manufacturing method, the order of performing step a) and step b) is arbitrary. For example, step a) may be performed first, or step b) may be performed first, or step a) and step b) may be performed simultaneously.

[0160] (Step c))

[0161] In step c), the solution obtained in step a) is added dropwise to the crude emulsion obtained in step b) and dispersed, and the dispersed liquid is further subjected to high-pressure emulsification. By implementing step c), the emulsion of the present embodiment is obtained. The dispersion before the high-pressure emulsification treatment can be implemented, for example, by slowly adding the solution obtained in step a) to the crude emulsion obtained in step b) at a certain speed, and then heating and stirring at 50 to 70°C at 5,000 to 15,000 rpm for 1 to 30 minutes. The particle size of the lipid nanoparticles contained in the emulsion obtained in the dispersion before the high-pressure emulsification treatment is generally in the range of 1 nm to 500 nm.

[0162] High-pressure emulsification treatment can be implemented, for example, using a high-pressure emulsifier. High-pressure emulsification treatment can be implemented, for example, by passing liquid 3 to 30 times at a pressure of 50 to 200 MPa under a temperature condition of 50 to 70°C. As a high-pressure emulsifier, a box-type high-pressure homogenizer such as a microfluidizer (manufactured by Microfluidics), Nanomizer (manufactured by YOSHIDA KIKAI CO., LTD.), STARBURST (manufactured by SUGINO MACHINE LIMITED CO., LTD.), a Gaulin-type homogenizer (manufactured by APV), a Rannie-type homogenizer (manufactured by Rannie), a high-pressure homogenizer (manufactured by Niro Soavi), a homogenizer (manufactured by Sanwa Machinery Co., Ltd.), a high-pressure homogenizer (manufactured by IZUMI FOOD MACHINERY), and a homogenizing valve-type high-pressure homogenizer (manufactured by ICA) can be cited. The particle size of the lipid nanoparticles contained in the emulsion obtained in the high-pressure emulsification treatment is usually in the range of 1 nm to 250 nm.

[0163] (Other steps)

[0164] The first production method may further include a step of sterilizing the emulsion in addition to the above steps, if necessary.

[0165] In the sterilization step, the emulsion obtained in step c) is sterilized. The sterilization of the emulsion can be carried out according to conventional methods. Specifically, for example, the emulsion can be sterilized by passing it through a membrane filter with a pore size of 0.2 to 0.5 μm (for example, a nylon syringe filter). In addition, for example, the emulsion can be sterilized by heating and pressurizing the emulsion (for example, 121° C., 20 minutes) using a moist heat sterilizer.

[0166] (Second Manufacturing Method)

[0167] (Step A)

[0168] In step A), nimodipine is dissolved in an organic solvent by heating. Step A) can be carried out in the same manner as step a) of the first production method.

[0169] (Step B)

[0170] In step B), phospholipids, a buffer and a hyaluronic acid derivative represented by the general formula (1) are added to an aqueous solvent for dispersion. Examples of aqueous solvents include distilled water, normal water, purified water, sterile purified water, water for injection and distilled water for injection as defined in the 18th revised Japanese Pharmacopoeia. The buffer added in step B) may be added in the form of a buffer solution.

[0171] In step B), the phospholipid, the buffer and the hyaluronic acid derivative represented by the general formula (1) may be added to an aqueous solvent and then dispersed by, for example, stirring.

[0172] In step C), the solution obtained in step A) is mixed with neutral lipid and tocopherol. In step C), neutral lipid and tocopherol may be added to the solution obtained in step A) and then mixed, for example, by stirring.

[0173] In the second manufacturing method, the order of performing step A) and step B) is arbitrary. For example, step A) may be performed first, step B) may be performed first, or step A) and step B) may be performed simultaneously. If step C) is performed for a long time, it may promote the decomposition of nimodipine, so it is preferably performed for as short a time as possible. That is, it is preferred to perform step C) after performing step A) and step B).

[0174] In step D), the solution obtained in step C) is added dropwise to the dispersion obtained in step B) and coarse emulsification is performed, and the coarse emulsified liquid is further subjected to high-pressure emulsification treatment. By implementing step D), the emulsion of the present embodiment is obtained. The coarse emulsification before the high-pressure emulsification treatment can be performed, for example, by slowly adding the solution obtained in step C) to the dispersion obtained in step B) at a certain speed, and then heating and stirring at 50 to 70°C at 5,000 to 15,000 rpm for 1 to 30 minutes. The particle size of the lipid nanoparticles contained in the emulsion obtained in the coarse emulsification before the high-pressure emulsification treatment is generally in the range of 1 nm to 500 nm.

[0175] The high-pressure emulsification treatment can be carried out in the same manner as step c) of the first production method.

[0176] (Other steps)

[0177] The second production method may further include a step of sterilizing the emulsion in addition to the above steps, if necessary. The sterilization step may be carried out in the same manner as described in the first production method.

[0178] Example

[0179] Hereinafter, the present invention will be described in more detail based on Examples etc. However, the present invention is not limited to the following Examples.

[0180] The correspondence between the components and abbreviations used in the Examples is as follows.

[0181] NP: Nimodipine, manufactured by Tokyo Chemical Industry Co., Ltd.

[0182] PC: PC-98T (trade name), manufactured by Kewpie Corporation (egg yolk lecithin purified to a phosphatidylcholine content of 98% or more)

[0183] PL: PL-100M (trade name), Kewpie Corporation (egg yolk lecithin containing 80% phosphatidylcholine and 20% phosphatidylethanolamine)

[0184] MCT: COCONARD RK (trade name), manufactured by Kao Corporation (tricaprylin)

[0185] LCT: Japanese Pharmacopoeia soybean oil, manufactured by KANEDA Co., Ltd. (long-chain fatty acid triglyceride with the carbon number of fatty acids mainly being 18)

[0186] HA-C12: hyaluronic acid derivative prepared in Experimental Example 1

[0187] Tris: 2-amino-2-hydroxymethyl-1,3-propanediol 999 (trade name) (trishydroxymethylaminomethane), manufactured by Fujifilm Wako Co., Ltd.

[0188] VE: Vitamin E, dl-α-tocopherol special grade (trade name), manufactured by Kanto Chemical Co., Ltd. (dl-α-tocopherol)

[0189] [Test Example 1: Investigation of Factors that Decompose Nimodipine]

[0190] <Manufacturing of Hyaluronic Acid Derivatives>

[0191] A hyaluronic acid derivative was prepared by the method described in Example 1 of Japanese Patent No. 4845071. Specifically, 5.0 g of hyaluronic acid (average molecular weight 8000, manufactured by Kewpie Corporation) was dissolved in 500 mL of water in a 1L beaker, and a 40 w / v% tetrabutylammonium hydroxide aqueous solution was added while stirring to adjust the pH to 7.2. After pH adjustment, freeze drying was performed to obtain 10.2 g of tetrabutylammonium salt of hyaluronic acid. 1.0 g of tetrabutylammonium salt of hyaluronic acid, C 20, and 10.2 g of tetrabutylammonium salt of hyaluronic acid were added to a 30 mL sample bottle. 12～13 2.0 g of alkyl glycidyl ether (reaction reagent) (made by Yokkaichi Synthetic Co., Ltd.) and 10 mL of dimethylformamide (DMF) were reacted in a water bath at 80°C for 8 hours while stirring. After the reaction was completed, 10 mL of 12.5 w / v% sodium chloride aqueous solution was added and the pH was adjusted to 1.0 with 8 w / v% hydrochloric acid. Then, 50 mL of ethanol was slowly added while stirring to precipitate hyaluronic acid. Then, the pH was adjusted to 7.0 with 25 w / v% sodium hydroxide, the precipitate was recovered by filtration, and washed 3 times with 50 mL of 80 v / v% ethanol aqueous solution. The obtained precipitate was vacuum dried at 60°C to obtain 0.48 g of a hyaluronic acid derivative represented by the following general formula (1).

[0192]

[0193] [In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6.]

[0194] In the hyaluronic acid derivative produced in Test Example 1, R6 is an alkyl group having 12 or 13 carbon atoms, and n is an integer of 1-50.

[0195] <Preparation and stability evaluation of ethanol preparations containing nimodipine>

[0196] Nimodipine was dissolved in ethanol in a manner of 0.2 mass % to prepare a 0.2 mass % NP ethanol solution. After adding each component to a 10 mL vial in a manner of forming the composition shown in Table 1, the container was sealed and stirred to prepare an ethanol preparation containing nimodipine. The prepared ethanol preparation containing nimodipine was stored at 60°C, and nimodipine was quantitatively measured at an appropriate time. The results are shown in Table 1.

[0197] <Quantitative determination of nimodipine>

[0198] The quantitative determination of nimodipine was performed by high performance liquid chromatography (HPLC) under the following conditions.

[0199] Mobile phase: a mixture of water: methanol: acetonitrile (27:35:38)

[0200] Column: Octadecyl silylated silica gel column (inner diameter 4.6 mm, length 250 mm, particle size 5 μm)

[0201] Temperature: 30℃

[0202] Detection method: Ultraviolet (UV) detector (detection wavelength 237nm)

[0203] [Table 1]

[0204]

[0205] ND: Not Determined

[0206] As shown in Table 1, ethanol or water did not promote the decomposition of nimodipine. On the other hand, it was found that phospholipids (PL, PC), hyaluronic acid derivatives (HA-C12) or neutral lipids (MCT) promoted the decomposition of nimodipine. In particular, it was found that hyaluronic acid derivatives (HA-C12) significantly promoted the decomposition of nimodipine.

[0207] [Test Example 2: Search for a substance that inhibits the decomposition of nimodipine caused by phospholipids]

[0208] Searches were conducted for substances that can inhibit the degradation of nimodipine by the phospholipids identified in Test Example 1. The results of the searches are extracted and described.

[0209] <Preparation and stability evaluation of ethanol preparations containing nimodipine>

[0210] 0.2 mass % NP ethanol solution was prepared in the same manner as in Test Example 1. After citric acid and trisodium citrate (both anhydrous, manufactured by Fujifilm Wako Co., Ltd.) were dissolved in water in a manner of 0.5 mass %, they were mixed to pH 5.0 to prepare 0.5 mass % citric acid buffer (pH 5.0). After sodium dihydrogen phosphate (anhydrous, manufactured by Fujifilm Wako Co., Ltd.) and disodium hydrogen phosphate (anhydrous, manufactured by Kanto Chemical Co., Ltd.) were dissolved in water in a manner of 0.5 mass %, they were mixed to pH 7.0 to prepare 0.5 mass % phosphate buffer (pH 7.0). After Tris was dissolved in water in a manner of 0.5 mass %, it was adjusted to pH 9.0 with 1M hydrochloric acid to prepare 0.5 mass % Tris buffer (pH 9.0). VE was dissolved in ethanol in a manner of 1 mass %, and a 1 mass % VE ethanol solution was prepared.

[0211] After adding each component to a 10 mL vial in a manner to form the composition shown in Table 2, the container was sealed and stirred to prepare an ethanol preparation containing nimodipine. The prepared ethanol preparation containing nimodipine was stored at 60°C, and nimodipine was quantitatively determined at an appropriate time. The quantitative determination of nimodipine was performed using the same method as in Test Example 1. The results are shown in Table 2.

[0212] [Table 2]

[0213]

[0214] As shown in Table 2, it was found that the degradation of nimodipine by phospholipids was suppressed by the addition of a buffer or tocopherol.

[0215] [Test Example 3: Confirmation of the effect of inhibiting the decomposition of nimodipine by tocopherol content]

[0216] The relationship between the tocopherol content and the decomposition inhibitory effect of nimodipine was confirmed.

[0217] <Preparation and stability evaluation of ethanol preparations containing nimodipine>

[0218] A 0.2 mass % NP ethanol solution and a 1 mass % VE ethanol solution were prepared in the same manner as in Test Example 2.

[0219] After adding each component to a 10 mL vial in a manner to form the composition shown in Table 3, the container was sealed and stirred to prepare an ethanol preparation containing nimodipine. The prepared ethanol preparation containing nimodipine was stored at 60°C, and nimodipine was quantitatively determined at an appropriate time. The quantitative determination of nimodipine was performed using the same method as in Test Example 1. The results are shown in Table 3.

[0220] [Table 3]

[0221]

[0222] As shown in Table 3, it was found that the decomposition of nimodipine by phospholipids was suppressed by the addition of tocopherol, and that the inhibitory effect was observed even when the amount of tocopherol added was 0.01% by mass.

[0223] [Test Example 4: Evaluation of the influence of buffer on emulsification and evaluation of emulsification stabilization by hyaluronic acid derivatives]

[0224] Emulsions containing nimodipine-containing lipid nanoparticles were prepared and the effects of buffers and hyaluronic acid derivatives on emulsification were evaluated.

[0225] <Manufacturing of Hyaluronic Acid Derivatives>

[0226] The hyaluronic acid derivative (HA-C12) was produced by the same method as in Test Example 1.

[0227] <Production of an emulsion containing lipid nanoparticles containing nimodipine>

[0228] An emulsion containing lipid nanoparticles containing nimodipine was prepared by the following method.

[0229] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PC, MCT, LCT, concentrated glycerol and 0.5% by mass citric acid buffer (pH 5.0) were added to pure water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 5 times at 150 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics) to prepare an emulsion (O / W type emulsion) of lipid nanoparticles encapsulating NPs. Each component was used in a manner to form the composition shown in Table 4.

[0230] <Evaluation of Particle Size>

[0231] The particle size of the lipid nanoparticles contained in the emulsion of each test example was measured immediately after preparation and after storage at 60° C. for 1 week. The particle size was measured by dynamic light scattering using a particle size measuring device (Zetasizer Nano, manufactured by Malvern Panalytical Co., Ltd.). The results are shown in Table 4.

[0232] [Table 4]

[0233]

[0234] As shown in Table 4, it can be seen that by adding a buffer (citric acid buffer (pH 5.0)), the particle size of the lipid nanoparticles increased, and the emulsification stability of the emulsion was significantly reduced. On the other hand, it can be seen that by adding a hyaluronic acid derivative, the increase in the particle size of the lipid nanoparticles can be suppressed even in the presence of a buffer, and the emulsification stability is improved.

[0235] [Test Example 5: Preparation and evaluation of emulsion containing lipid nanoparticles containing nimodipine]

[0236] <Manufacturing of Hyaluronic Acid Derivatives>

[0237] The hyaluronic acid derivative was produced by the same method as in Test Example 1.

[0238] <Preparation and stability evaluation of emulsion containing lipid nanoparticles containing nimodipine>

[0239] An emulsion containing lipid nanoparticles containing nimodipine was prepared by the following method.

[0240] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, VE, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to pure water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 5 times at 150 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics) and then filled into a vial to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Example 5-1). Each component was used in a manner to form the composition shown in Table 5.

[0241] NP was mixed with PL, MCT and ethanol, heated to 60°C for dissolution, and a lipid ethanol solution was prepared. HA-C12 was added to pure water heated to 60°C, and stirred for 5 minutes to prepare an aqueous phase. While stirring the aqueous phase heated to 60°C, the lipid ethanol solution was added dropwise, and coarse emulsification was performed for 5 minutes to obtain a coarse emulsion. After the coarse emulsion was treated 5 times with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics) at 150MPa, the treated product was filled into a vial to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NP (Comparative Example 5-1). Each component was used in a manner to form the composition shown in Table 5.

[0242] Immediately after preparation and after storage at 60° C. for 2 weeks, nimodipine was quantitatively determined for each emulsion by the same method as in Test Example 1. The results are shown in Table 5.

[0243] [Table 5]

[0244]

[0245] As shown in Table 5, it can be seen that if nimodipine is heated and dissolved together with lipids, the decomposition of nimodipine is promoted, so the quantitative value immediately after preparation is reduced (93% for Example 5-1 and 74% for Comparative Example 5-1). In addition, it can be seen that the storage stability of nimodipine in the emulsion of Comparative Example 5-1, which does not contain a buffer and tocopherol, is low (the difference before and after storage of Example 5-1 is -2%, while that of Comparative Example 5-1 is -20%). In addition, it can be seen that the hyaluronic acid derivative does not contribute to the storage stability of nimodipine.

[0246] [Test Example 6: Preparation and evaluation of emulsion containing lipid nanoparticles containing nimodipine]

[0247] <Manufacturing of Hyaluronic Acid Derivatives>

[0248] The hyaluronic acid derivative was produced by the same method as in Test Example 1.

[0249] <Preparation and stability evaluation of emulsion containing lipid nanoparticles containing nimodipine>

[0250] An emulsion containing lipid nanoparticles containing nimodipine was prepared by the following method.

[0251] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, VE, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to pure water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 10 times at 120 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics), and the treated product was passed through a 0.2 μm membrane filter and filled into a vial to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Example 6-1). Each component was used in a manner to form the composition shown in Table 6.

[0252] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to pure water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 5 times at 150 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics) and then filled into a vial to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Comparative Example 6-1). Each component was used in a manner to form the composition shown in Table 6.

[0253] For each emulsion, nimodipine was quantitatively measured immediately after preparation and after storage at 40°C for 1 month by the same method as in Test Example 1. In addition, for each emulsion, particle size was evaluated immediately after preparation and after storage at 40°C for 1 month by the same method as in Test Example 4. The results are collectively shown in Table 6.

[0254] [Table 6]

[0255]

[0256] As shown in Table 6, it can be seen that the emulsion of Example 6-1 and the emulsion of Comparative Example 6-1 differ in whether or not tocopherol is contained, and the emulsion of Example 6-1 containing tocopherol has more excellent storage stability of nimodipine.

[0257] [Test Example 7: Preparation and evaluation of emulsion containing lipid nanoparticles containing nimodipine]

[0258] <Manufacturing of Hyaluronic Acid Derivatives>

[0259] The hyaluronic acid derivative was produced by the same method as in Test Example 1.

[0260] <Preparation and stability evaluation of emulsion containing lipid nanoparticles containing nimodipine>

[0261] An emulsion containing lipid nanoparticles containing nimodipine was prepared by the following method.

[0262] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, VE, sodium oleate, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to pure water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 10 times at 120 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics), and then the treated product was passed through a 0.2 μm membrane filter and filled into a vial. Next, a wet heat sterilizer was used for sterilization at 121°C for 20 minutes to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Examples 7-1 to 7-3). Each component was used in a manner to form the composition shown in Table 7.

[0263] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, VE, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to pure water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 10 times at 120 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics), and then the treated product was passed through a 0.45 μm membrane filter and filled into a vial. Next, a wet heat sterilizer was used to sterilize the mixture at 121°C for 20 minutes to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Example 7-4). Each component was used in a manner to form the composition shown in Table 7.

[0264] For each emulsion, nimodipine was quantitatively measured immediately after preparation and after storage at 60°C for 2 weeks or 4 weeks by the same method as in Test Example 1. In addition, for each emulsion, particle size was evaluated immediately after preparation and after storage at 60°C for 2 weeks or 4 weeks by the same method as in Test Example 4. The results are collectively shown in Table 7.

[0265] [Table 7]

[0266]

[0267] It is understood that the emulsions of Examples 7-1 to 7-4 satisfying the constitution of the present invention are all excellent in the storage stability and emulsification stability of nimodipine.

[0268] [Test Example 8: Preparation and evaluation of emulsion containing lipid nanoparticles containing nimodipine]

[0269] <Manufacturing of Hyaluronic Acid Derivatives>

[0270] The hyaluronic acid derivative was produced by the same method as in Test Example 1.

[0271] <Preparation and stability evaluation of emulsion containing lipid nanoparticles containing nimodipine>

[0272] An emulsion containing lipid nanoparticles containing nimodipine was prepared by the following method.

[0273] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, VE, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to water heated to 60°C and coarsely emulsified for 5 minutes to obtain a coarse emulsion (1). While stirring the coarse emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and coarsely emulsified for 5 minutes to obtain a coarse emulsion (2). The coarse emulsion (2) was treated 10 times at 120 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics), and then the treated product was passed through a 0.45 μm membrane filter and filled into a vial. Next, a wet heat sterilizer was used for sterilization at 121°C for 20 minutes to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Examples 8-1 to 8-2). Each component was used in a manner to form the composition shown in Table 8.

[0274] For each emulsion, nimodipine was quantitatively measured immediately after preparation and after storage at 40°C for 1 month by the same method as in Test Example 1. In addition, for each emulsion, particle size was evaluated immediately after preparation and after storage at 40°C for 1 month by the same method as in Test Example 4. The results are collectively shown in Table 8.

[0275] [Table 8]

[0276]

[0277] It is found that the emulsions of Examples 8-1 to 8-2 satisfying the constitution of the present invention are all excellent in the storage stability and emulsification stability of nimodipine.

[0278] [Test Example 9: Preparation and evaluation of emulsion containing lipid nanoparticles containing nimodipine]

[0279] <Manufacturing of Hyaluronic Acid Derivatives>

[0280] The hyaluronic acid derivative was produced by the same method as in Test Example 1.

[0281] <Preparation and stability evaluation of emulsion containing lipid nanoparticles containing nimodipine>

[0282] An emulsion containing lipid nanoparticles containing nimodipine was prepared by the following method.

[0283] NPs were mixed with ethanol and heated to 60°C for dissolution to prepare an NP ethanol solution. HA-C12, PL, MCT, LCT, VE, concentrated glycerol and 0.5% by mass Tris buffer (pH 9.0) were added to water heated to 60°C, and the mixture was roughly emulsified for 5 minutes to obtain a crude emulsion (1). While stirring the crude emulsion (1) heated to 60°C, the NP ethanol solution was added dropwise and roughly emulsified for 5 minutes to obtain a crude emulsion (2). The crude emulsion (2) was treated 10 times at 120 MPa with a high-pressure emulsifier (microfluidizer, manufactured by Microfluidics), and the treated product was passed through a 0.2 μm membrane filter and filled into a vial to prepare an emulsion (O / W type emulsion) containing lipid nanoparticles encapsulating NPs (Examples 9-1 to 9-2). Each component was used in a manner to form the composition shown in Table 9.

[0284] For each emulsion, nimodipine was quantitatively measured immediately after preparation and after storage at 60°C for 1 week by the same method as in Test Example 1. In addition, for each emulsion, particle size was evaluated immediately after preparation and after storage at 60°C for 1 week by the same method as in Test Example 4. The results are collectively shown in Table 9.

[0285] [Table 9]

[0286]

[0287] It is found that the emulsions of Examples 9-1 to 9-2 satisfying the constitution of the present invention are all excellent in the storage stability and emulsification stability of nimodipine.

Claims

1. An emulsion comprising nimodipine, phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer, tocopherol and water, The modification rate of the hyaluronic acid derivative is 4% to 16%, The modification rate is a value expressing the number of groups represented by -O-CH2-CH(OH)-CH2-OR6 contained in each structural unit of hyaluronic acid as a percentage. In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6; R6 represents a linear or branched alkyl or alkenyl group; n represents an integer of 1 to 2500; wherein, Except when all of R1, R2, R3, R4 and R5 are hydroxyl groups, The buffer is tris(hydroxymethyl)aminomethane and its salt, The content of nimodipine is 0.02 mass % or more and 0.2 mass % or less based on the total amount of the emulsion. The content of the phospholipid is 1% by mass or more and 3% by mass or less based on the total amount of the emulsion. The content of the neutral fat is 2% by mass or more and 10% by mass or less based on the total amount of the emulsion. The content of the hyaluronic acid derivative is 0.1% by mass or more and 1% by mass or less based on the total amount of the emulsion. The content of the buffer is 0.01% by mass or more and 0.2% by mass or less in terms of anhydrous content based on the total amount of the emulsion. The tocopherol content is 0.01% by mass or more and 0.2% by mass or less based on the total amount of the emulsion. The content of nimodipine in the emulsion after storage at a temperature equivalent to 40° C. for 1 month is 95% by mass or more based on the content before storage. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, The neutral fat is at least one selected from the group consisting of soybean oil, sesame oil, olive oil, rapeseed oil, corn oil and medium-chain fatty acid triglycerides.

2. The emulsion according to claim 1, wherein The R6 is a linear alkyl group having 8 to 18 carbon atoms.

3. The emulsion according to claim 1, wherein The acid dissociation constant pK of the buffer a In the range of 7 to 9. The emulsion according to claim 1 , which is an injection. The emulsion according to claim 1 , which is used in a manner of administration without dilution.

6. A method for producing an emulsion, comprising: a) heating and dissolving nimodipine in an organic solvent; b) adding phospholipids, neutral lipids, a hyaluronic acid derivative represented by the following general formula (1), a buffer and tocopherol to an aqueous solvent, followed by a step of coarse emulsification; and c) adding the solution obtained in step a) dropwise to the crude emulsion obtained in step b) to disperse the mixture, and further subjecting the dispersed liquid to high pressure emulsification treatment, The modification rate of the hyaluronic acid derivative is 4% to 16%, The modification rate is a value expressing the number of groups represented by -O-CH2-CH(OH)-CH2-OR6 contained in each structural unit of hyaluronic acid as a percentage. In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6; R6 represents a linear or branched alkyl or alkenyl group; n represents an integer from 1 to 2500; except for the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups, The buffer is tris(hydroxymethyl)aminomethane and its salt, The content of nimodipine is 0.02 mass % or more and 0.2 mass % or less based on the total amount of the emulsion. The content of the phospholipid is 1% by mass or more and 3% by mass or less based on the total amount of the emulsion. The content of the neutral fat is 2% by mass or more and 10% by mass or less based on the total amount of the emulsion. The content of the hyaluronic acid derivative is 0.1% by mass or more and 1% by mass or less based on the total amount of the emulsion. The content of the buffer is 0.01% by mass or more and 0.2% by mass or less in terms of anhydrous content based on the total amount of the emulsion. The tocopherol content is 0.01% by mass or more and 0.2% by mass or less based on the total amount of the emulsion. The content of nimodipine in the emulsion after storage at a temperature equivalent to 40° C. for 1 month is 95% by mass or more based on the content before storage. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, The neutral fat is at least one selected from the group consisting of soybean oil, sesame oil, olive oil, rapeseed oil, corn oil and medium-chain fatty acid triglycerides.

7. A method for producing an emulsion, comprising: A) the step of heating and dissolving nimodipine in an organic solvent; B) adding phospholipids, a buffer and a hyaluronic acid derivative represented by the general formula (1) into an aqueous solvent for dispersion; C) a step of mixing the solution obtained in step A) with a neutral lipid and tocopherol; as well as D) adding the solution obtained in step C) dropwise to the dispersion obtained in step B) to perform rough emulsification, and further subjecting the rough emulsified liquid to high pressure emulsification treatment, The modification rate of the hyaluronic acid derivative is 4% to 16%, The modification rate is a value expressing the number of groups represented by -O-CH2-CH(OH)-CH2-OR6 contained in each structural unit of hyaluronic acid as a percentage. In the general formula (1), R1, R2, R3, R4 and R5 each independently represent a hydroxyl group or a group represented by -O-CH2-CH(OH)-CH2-OR6; R6 represents a linear or branched alkyl or alkenyl group; n represents an integer from 1 to 2500; except for the case where all of R1, R2, R3, R4 and R5 are hydroxyl groups, The buffer is tris(hydroxymethyl)aminomethane and its salt, The content of nimodipine is 0.02 mass % or more and 0.2 mass % or less based on the total amount of the emulsion. The content of the phospholipid is 1% by mass or more and 3% by mass or less based on the total amount of the emulsion. The content of the neutral fat is 2% by mass or more and 10% by mass or less based on the total amount of the emulsion. The content of the hyaluronic acid derivative is 0.1% by mass or more and 1% by mass or less based on the total amount of the emulsion. The content of the buffer is 0.01% by mass or more and 0.2% by mass or less in terms of anhydrous content based on the total amount of the emulsion. The tocopherol content is 0.01% by mass or more and 0.2% by mass or less based on the total amount of the emulsion. The content of nimodipine in the emulsion after storage at a temperature equivalent to 40° C. for 1 month is 95% by mass or more based on the content before storage. The phospholipid is at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and sphingomyelin, The neutral fat is at least one selected from the group consisting of soybean oil, sesame oil, olive oil, rapeseed oil, corn oil and medium-chain fatty acid triglycerides.

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