WO2015166988A1 - リポソームの製造方法 - Google Patents
リポソームの製造方法 Download PDFInfo
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- WO2015166988A1 WO2015166988A1 PCT/JP2015/062985 JP2015062985W WO2015166988A1 WO 2015166988 A1 WO2015166988 A1 WO 2015166988A1 JP 2015062985 W JP2015062985 W JP 2015062985W WO 2015166988 A1 WO2015166988 A1 WO 2015166988A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/14—Liposomes; Vesicles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1277—Processes for preparing; Proliposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
- B01J13/06—Making microcapsules or microballoons by phase separation
- B01J13/12—Making microcapsules or microballoons by phase separation removing solvent from the wall-forming material solution
- B01J13/125—Making microcapsules or microballoons by phase separation removing solvent from the wall-forming material solution by evaporation of the solvent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/70—Nanostructure
- Y10S977/773—Nanoparticle, i.e. structure having three dimensions of 100 nm or less
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/902—Specified use of nanostructure
- Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
- Y10S977/906—Drug delivery
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/902—Specified use of nanostructure
- Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
- Y10S977/906—Drug delivery
- Y10S977/907—Liposome
Definitions
- the present invention relates to a method for producing liposomes.
- the present invention relates to a method for producing liposomes that can be suitably used for pharmaceutical applications.
- Liposomes are closed vesicles formed of lipid bilayers using lipids, and have an aqueous phase (inner water phase) in the space of the closed vesicles. Liposomes usually exist in a dispersed state in an aqueous solution outside the closed vesicles (outer aqueous phase). Liposomes take advantage of features such as barrier ability, compound retention ability, biocompatibility, freedom of particle size setting, easy degradability, surface modification, etc., for various applications such as immunosensors, artificial erythrocytes, and carriers for drug delivery systems. Has been studied. In carrier applications, liposomes can contain a wide variety of substances, including water-soluble compounds, fat-soluble small molecules, and polymers.
- the particle diameter should be about 200 nm or less from the viewpoint of permeation through a biological membrane.
- storage stability is preferably imparted from various viewpoints such as aggregation, sedimentation, and drug leakage.
- a drug such as a solution containing a liposome containing a drug
- high safety is required for the intravenous injection.
- Chlorinated solvents such as chloroform or additives such as dispersion aids not approved for use are not preferred.
- suitability for aseptic filtration is also required.
- an emulsification method may be mentioned as an industrial scale production method for liposomes.
- Patent Document 1 a solution in which at least one lipid is dissolved in at least one organic solvent at a temperature of about 35 ° C. to 55 ° C. is added to the aqueous phase, and the resulting mixture is stirred and dispersed.
- a method for producing an aqueous dispersion of lamellar lipid vesicles comprising the step of evaporating the solvent with a portion of the solvent water.
- the organic solvent is immiscible with water and in this method the amount of each of the two phases is adjusted to obtain an oil-in-water (O / W) dispersion.
- Patent Document 2 describes a method of preparing submicron-sized liposomes by adding an amphipathic lipid dissolved in ethanol to an aqueous phase while stirring with a magnetic stirrer.
- drug active ingredients or drug precursors, biological reagents or cosmetics can be added to the aqueous or organic phase to make liposomes.
- amphipathic lipids are limited to those that dissolve in ethanol, and the only applicable phospholipids are lecithins. Therefore, it is difficult to prepare liposomes in which various phospholipids are mixed at an arbitrary ratio.
- this method since the particle diameter increases when the solvent is removed, a treatment using a membrane filter having constant pores is necessary to make the particle diameter uniform.
- a phospholipid is dissolved in an organic solvent that can be mixed with an aqueous component such as alcohol, and after mixing and dispersing with a sufficient amount of the aqueous component to form a single phase, the solvent is evaporated to form a lipid thin film.
- an aqueous component such as alcohol
- a method of forming liposomes by forming and stirring a mixture obtained by adding an aqueous component to the lipid film is described. Although it is said that stable liposomes suitable for injection can be prepared by this method, there is no description of the particle size and dispersion stability of the produced liposomes.
- Patent Document 4 describes a method of forming fine particles by a two-stage emulsification step and a solvent removal step using a mixed solvent containing at least two kinds of organic solvents.
- a and two solvents of solvent group B consisting of a chlorinated solvent such as ester, alcohol, ether and chloroform are used in combination.
- an unfavorable dispersant as an injection such as pluronic for miniaturization. When these dispersants are not used, the particle size is about 135 nm, which is not sufficient for forming fine particles of 100 nm or less.
- storage stability required for pharmaceutical products is no description regarding storage stability required for pharmaceutical products.
- a surfactant such as a dispersant (in the present invention, the dispersant also includes a dispersion aid) is useful for atomization in the emulsification step in order to effectively lower the interfacial tension.
- the dispersant may exist at at least one of the hydrophilic and hydrophobic interfaces, and may promote leakage due to diffusion of the encapsulated drug.
- only a few materials have been approved for use as intravenous injections from the viewpoint of safety.
- the extrusion treatment is a method of extruding a mixture containing the liposome from a fine hole of the filter using an extruder to obtain a liposome. According to this method, a fine liposome with small size variation can be obtained. However, clogging of the extruder filter, deformation of the liposome, etc. may occur. It is not preferable for production.
- an object of the present invention is to provide a method for producing liposomes having safety and stability. Moreover, this invention makes it a subject to provide the manufacturing method of the liposome which has safety
- the inventors have Mixing an oil phase in which at least one lipid is dissolved in an organic solvent and an aqueous phase and stirring the aqueous solution containing the lipid, and evaporating the organic solvent from the aqueous solution containing the liposomes obtained in the stirring step
- a method for producing a liposome comprising a step, comprising:
- the ester organic solvent is at least one selected from ethyl acetate, methyl acetate, isopropyl acetate, t-butyl acetate, and methyl propionate.
- the ester organic solvent is ethyl acetate.
- the water-soluble organic solvent is an alcohol.
- the alcohol is at least one selected from ethanol, methanol, 2-propanol, and t-butanol.
- the mass ratio of the water-soluble organic solvent to the ester organic solvent is 90:10 to 30:70.
- the stirring step shearing at a peripheral speed of 20 m / sec or more is given to the aqueous solution containing lipid.
- the organic solvent is evaporated by heating.
- the concentration of the organic solvent contained in the aqueous solution containing the liposome is set to 15% by mass or less within 30 minutes after the start of the step of evaporating the organic solvent.
- an unfavorable organic solvent such as chloroform, methylene chloride, hexane, or cyclohexane, or an additive such as a dispersion aid that is not approved for intravenous use in pharmaceutical applications is used.
- a liposome having an average particle size of 200 nm or less, a uniform particle size distribution, and excellent storage stability, which is particularly suitable for pharmaceutical use, can be obtained.
- the production suitability (for example, aseptic filtration suitability) is good, the average particle size is 200 nm or less, the particle size distribution shows a uniform distribution, and is preserved. Liposomes with excellent stability can be obtained.
- the average particle size and particle size distribution suitable for pharmaceutical use can be efficiently produced on an industrial scale.
- production suitability eg, homogenization and refinement of liposomes without sizing treatment, aseptic filtration suitability) Etc.
- storage stability required for the preparation liposomes can be efficiently produced on an industrial scale.
- FIG. 1 is a plot of filtration volume (g) and time (min).
- FIG. 2 is a plot of liposome particle size (nm) and ethyl acetate ratio (%).
- process is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
- a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
- % means mass percentage.
- the amount of each component in the composition means the total amount of the plurality of substances present in the composition unless there is a specific notice when there are a plurality of substances corresponding to each component in the composition. To do.
- the present invention Mixing an oil phase in which at least one lipid is dissolved in an organic solvent and an aqueous phase and stirring an aqueous solution containing the lipid; and A method for producing a liposome, comprising a step of evaporating an organic solvent from an aqueous solution containing the liposome obtained in the stirring step,
- the organic solvent is a mixed solvent of a water-soluble organic solvent and an ester-based organic solvent, It is a manufacturing method of a liposome.
- a liposome is a closed vesicle formed of a lipid bilayer membrane using lipid, and has an aqueous phase (inner aqueous phase) in the space of the closed vesicle.
- the inner water phase includes water and the like.
- Liposomes usually exist in a dispersed state in an aqueous solution outside the closed vesicles (outer aqueous phase). Liposomes are single lamellae (also called single-layer lamellae or unilamellar, and double-layer membranes have a single structure), but they are multilayer lamellae (also called multi-lamellar, which have a large number of double-layer membranes in the shape of onions. In the present invention, from the viewpoint of safety and stability in pharmaceutical use, it is a single-lamellar liposome. Is preferred.
- the form of the liposome is not particularly limited as long as it can encapsulate drugs and the like.
- Encapsulation means that a drug or the like takes a form of encapsulation, encapsulation, adhesion, etc. with respect to the liposome.
- a form in which a drug or the like is enclosed in a closed space formed by a film a form in which the drug is encapsulated, a form in which a drug is attached to the surface of a liposome (for example, at least the outer membrane surface and the inner membrane surface of the lipid bilayer A form existing on one surface), etc., and a combination thereof may be used.
- the size (average particle size) of the liposome is not particularly limited, but is 2 to 200 nm, preferably 5 to 150 nm, more preferably 5 to 120 nm, and further preferably 5 to 100 nm.
- the “average particle diameter” means an average value of the diameters of liposomes measured by a light scattering method. Liposomes are preferably in the form of spheres or similar.
- the component (membrane component) constituting the lipid bilayer of the liposome is selected from lipids. Any lipid that can be dissolved in a mixed solvent of a water-soluble organic solvent and an ester-based organic solvent can be used. Specific examples of lipids include phospholipids, lipids other than phospholipids, cholesterols, and derivatives thereof. These components may be composed of a single type or multiple types of components.
- Phospholipids include natural or synthetic phospholipids such as phosphatidylcholine (lecithin), phosphatylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, or those hydrogenated (for example, And hydrogenated soybean phosphatidylcholine (HSPC)).
- hydrogenated phospholipids such as hydrogenated soybean phosphatidylcholine, sphingomyelin and the like are preferable, and hydrogenated soybean phosphatidylcholine is more preferable.
- the “phospholipid” includes phospholipid derivatives obtained by modifying phospholipids.
- lipids other than phospholipids include lipids that do not contain phosphate, and are not particularly limited, but include glycerolipids that do not have a phosphate moiety in the molecule, sphingolipids that do not have a phosphate moiety in the molecule, and the like. It is done.
- lipid other than phospholipid includes derivatives of lipids other than phospholipids obtained by modifying lipids other than phospholipids.
- a lipid other than phospholipid contains a basic functional group
- the lipid is called a cationized lipid.
- the cationized lipid can modify the liposome membrane, and can improve the adhesion to the cell that is the target site.
- cholesterols examples include cholesterol.
- the curvature of the lipid membrane increases. Since the distortion of the membrane arranged in the liposome is also increased, the water-soluble drug is more likely to leak. However, as a means for suppressing leakage, it is effective to add cholesterol or the like in order to fill the membrane distortion caused by lipids.
- the liposome may be added with a hydrophilic polymer or the like for the purpose of improving blood retention, fatty acid, diacetyl phosphate or the like as a membrane structure stabilizer, ⁇ -tocopherol or the like as an antioxidant. Good.
- additives such as dispersion aids that are not approved for use in intravenous injection in pharmaceutical applications, such as surfactants.
- the liposome of the present invention can also contain phospholipids, lipids other than phospholipids, and cholesterols modified with hydrophilic polymers as phospholipids, lipids other than phospholipids, cholesterols, and derivatives thereof.
- the hydrophilic polymer is not particularly limited, and examples thereof include polyethylene glycols, polyglycerols, polypropylene glycols, polyvinyl alcohol, styrene-maleic anhydride alternating copolymers, polyvinyl pyrrolidone, and synthetic polyamino acids.
- the hydrophilic polymers can be used alone or in combination of two or more.
- polyethylene glycols, polyglycerols, and polypropylene glycols are preferable from the viewpoint of blood retention of the preparation, and polyethylene glycol (PEG), polyglycerol (PG), and polypropylene glycol (PPG) are more preferable.
- Glycol (PEG) is the most versatile and has the effect of improving blood retention, and is preferred.
- the molecular weight of PEG is not particularly limited.
- the molecular weight of PEG is 500 to 10,000 daltons, preferably 1,000 to 7,000 daltons, more preferably 2,000 to 5,000 daltons.
- lipid modified with PEG PEG-modified lipid
- PEG-modified lipids include 1,2-distearoyl-3-phosphatidylethanolamine-PEG2000 (manufactured by NOF Corporation), 1,2-distearoyl-3-phosphatidylethanolamine-PEG5000 (manufactured by NOF Corporation), and distearoyl.
- examples include 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol such as glycerol-PEG2000 (manufactured by NOF Corporation).
- These PEG-modified lipids may be added so as to contain 0.3 to 50% by mass, preferably 0.5 to 30% by mass, more preferably 1 to 20% by mass with respect to the total lipid amount.
- a combination of hydrogenated soybean phosphatidylcholine (the main lipid contained in the liposome), 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol (a lipid used in combination with the main lipid), and a lipid of cholesterol preferable.
- the liposome of the present invention can contain at least one of a water-soluble drug or a fat-soluble drug as a drug.
- the form retained in the inner aqueous phase of the liposome is advantageous, but the lipid bilayer membrane is thin and soft, so that the drug may easily leak.
- a liposome having a particle size of about 200 nm or less and having safety and stability can be produced. Therefore, in the present invention, the drug that can be encapsulated in the liposome is preferably a water-soluble drug.
- the active ingredient drug that can be encapsulated in the liposome is not particularly limited. Specifically, enzymes, proteins, peptides, nucleic acids, low-molecular compounds, saccharides (oligosaccharides and polysaccharides), high-molecular compounds, etc. Examples thereof include substances having activity or pharmacological activity.
- the drug is preferably a low molecular compound, more preferably a water-soluble low molecular compound, from the viewpoint of stability.
- the drug can be made into a water-soluble drug or a fat-soluble drug by appropriately adding an additive or the like to the water-soluble drug or the fat-soluble drug.
- low molecular weight drugs include anthracyclines such as doxorubicin, daunorubicin, and epirubicin, cisplatins such as cisplatin and oxaliplatin, taxanes such as paclitaxel and docetaxel, vinca alkaloids such as vincristine and vinblastine, Examples include bleomycins such as bleomycin, sirolimus anticancer agents such as sirolimus, and antimetabolites such as methotrexate, fluorouracil, gemcitabine, cytarabine, and pemetrexed. Among these, water-soluble drugs such as doxorubicin, gemcitabine, and pemetrexed are preferable.
- Method for producing liposome In the method for producing the liposome of the present invention, It is obtained by mixing an oil phase in which at least one lipid is dissolved in an organic solvent and an aqueous phase and stirring an aqueous solution containing lipid (hereinafter sometimes referred to as a stirring step), and a stirring step. And a step of evaporating the organic solvent from the aqueous solution containing the liposome (hereinafter sometimes referred to as an evaporation step).
- the method for producing liposomes may include other steps in addition to the stirring step and the evaporation step as necessary.
- Oil phase As the organic solvent used as the oil phase, a mixed solvent of a water-soluble organic solvent and an ester organic solvent is used. In the present invention, it is preferable that substantially no organic solvent such as chloroform, methylene chloride, hexane, or cyclohexane is used as the organic solvent, and it is more preferable that these organic solvents are not used.
- the water-soluble organic solvent is not particularly limited, but is preferably an organic solvent having a property of being arbitrarily mixed with water.
- 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 polyethylene glycol. And polyalkylene glycols. Among these, alcohols are preferable.
- the alcohol is preferably at least one selected from ethanol, methanol, isopropanol, and t-butanol, more preferably at least one selected from ethanol, isopropanol, and t-butanol, and preferably ethanol. Further preferred.
- the ester organic solvent is not particularly limited, but is preferably an ester obtained from a reaction between an organic acid and an alcohol.
- the ester-based organic solvent is preferably at least one selected from ethyl acetate, methyl acetate, isopropyl acetate, t-butyl acetate, and methyl propionate.
- Ethyl acetate, isopropyl acetate, methyl propionate Is more preferable, and ethyl acetate is more preferable.
- the mixing ratio of the water-soluble organic solvent and the ester organic solvent is not particularly limited, but is in a mass ratio of 90:10 to 30:70, preferably 80:20 to 40:60, more preferably 80:20 to 70:30. If it is.
- the mixed solvent of the water-soluble organic solvent and the ester organic solvent may further contain an aqueous solvent described below such as water or a buffer solution.
- the aqueous solvent may be added, for example, in the range of 1 to 30% by mass.
- the pH of the mixed solvent is not particularly limited, but is preferably in the range of about 3 to 10, and more preferably about 4 to 9.
- the ester organic solvent may contain physiologically active substances such as various drugs soluble in these solvents.
- the mixing ratio of ethanol and ethyl acetate is not particularly limited, but is preferably 80:20 to 70:30 in terms of mass ratio.
- the concentration of the lipid is not particularly limited and can be appropriately adjusted. However, as a solution using a mixed solution of a water-soluble organic solvent and an ester organic solvent as a solvent, 40 g / L to 250 g / L, preferably 40 g. / L to 200 g / L.
- the aqueous phase means an outer aqueous phase and an inner aqueous phase.
- the outer aqueous phase in the present invention means an aqueous solution in which liposomes are dispersed.
- a solution occupying the outside of the liposome in a dispersion of liposomes stored in a vial or prefilled syringe package is the outer aqueous phase.
- the solution occupying the outside of the liposome in the dispersion of liposome is the outer aqueous phase of the attached dispersion or other solution dispersed at the time of administration at the time of administration.
- the inner aqueous phase in the present invention means an aqueous phase in a closed vesicle separated by a lipid bilayer membrane.
- aqueous solutions in which liposomes are dispersed include water (distilled water, water for injection, etc.), physiological saline, various buffer solutions, aqueous solutions of sugars, and mixtures (aqueous solvents) thereof.
- the buffer is not limited to organic or inorganic, but a buffer having a buffering action near the hydrogen ion concentration close to the body fluid is preferably used.
- Phosphate buffer, Tris buffer, citric acid Examples include a buffer solution, an acetate buffer solution, and a good buffer.
- the pH of the aqueous phase is not particularly limited, but may be 5 to 9, preferably 7 to 8.
- the internal aqueous phase of the liposome may be an aqueous solution in which the liposome is dispersed when the liposome is produced, or water, physiological saline, various buffers, aqueous solutions of saccharides, and mixtures thereof newly added. There may be. It is preferable that the water used as the outer aqueous phase or the inner aqueous phase does not contain impurities (dust, chemical substances, etc.).
- the physiological saline means an inorganic salt solution adjusted to be isotonic with the human body, and may further have a buffer function.
- Examples of the physiological saline include saline containing 0.9 w / v sodium chloride, phosphate buffered saline (hereinafter also referred to as PBS), Tris buffered saline, and the like.
- an oil phase in which at least one kind of lipid is dissolved in an organic solvent is mixed with an aqueous phase, and the aqueous solution containing the lipid is stirred.
- an emulsion in which the oil phase and the aqueous phase are emulsified in the O / W type is prepared.
- liposomes are formed by removing part or all of the organic solvent derived from the oil phase by the evaporation step described below. Alternatively, part or all of the organic solvent in the oil phase evaporates in the course of stirring and emulsification to form liposomes.
- an ultrasonic wave or a mechanical shear force is used for particle refinement, and an extruder process or a microfluidizer process through a filter having a fixed pore diameter is used for uniform particle diameter. It can be carried out. However, in the production method of the present invention, since liposomes can be made fine, it is preferable not to perform sizing treatment.
- the average particle size of the prepared liposome can be controlled by arbitrarily selecting the stirring speed and time. From the viewpoint of obtaining liposomes having safety and stability, it is preferable to apply shear to the aqueous solution containing lipid at a peripheral speed of 20 m / sec or more. Although it does not limit as shear, Specifically, it is preferable to give the shear of peripheral speed 20m / sec or more and 35m / sec or less, and it is more preferable to give the shear of peripheral speed 23m / sec or more and 30m / sec or less.
- the organic solvent is evaporated from the aqueous solution containing the liposomes obtained in the stirring step.
- the evaporation step is a step in which part or all of the organic solvent derived from the oil phase is forcibly removed as an evaporation step, and a portion or all of the organic solvent in the oil phase is a process of stirring and emulsification. Including at least one of the steps of spontaneous evaporation.
- the method for evaporating the organic solvent in the evaporation step is not particularly limited.
- the step of evaporating the organic solvent by heating, the step of continuing to stand still or gently stirring after emulsification, and the step of performing vacuum deaeration Do at least one.
- the concentration of the organic solvent contained in the aqueous solution containing the liposome is preferably 15% by mass or less within 30 minutes after the start of the step of evaporating the organic solvent.
- the liquid temperature at the time of carrying out the production method of the present invention can be adjusted as appropriate, but the liquid temperature at the time of mixing the oil phase and the aqueous phase is preferably not less than the phase transition temperature of the lipid used.
- the liquid temperature at the time of mixing the oil phase and the aqueous phase is preferably not less than the phase transition temperature of the lipid used.
- the liquid temperature at the time of mixing the oil phase and the aqueous phase is preferably not less than the phase transition temperature of the lipid used.
- the liquid temperature at the time of mixing the oil phase and the aqueous phase is preferably not less than the phase transition temperature of the lipid used.
- the liquid temperature at the time of mixing the oil phase and the aqueous phase is preferably not less than the phase transition temperature of the lipid used.
- the liquid temperature at the time of mixing the oil phase and the aqueous phase is preferably not less than the phase transition temperature of the lipid used.
- it is preferably 35 ° C. or more and 70 °
- the aqueous solution containing liposomes prepared through the stirring step and evaporation step is centrifuged, ultrafiltered, dialyzed, gel filtered, frozen to remove components not contained in the liposomes or to adjust the concentration and osmotic pressure. You may post-process by methods, such as drying.
- aseptic filtration In order to use the aqueous solution containing liposomes obtained by the method for producing liposomes of the present invention as a pharmaceutical product, aseptic filtration is preferably performed.
- a filtration method an unnecessary thing can be removed from the aqueous solution containing a liposome using a hollow fiber membrane, a reverse osmosis membrane, a membrane filter, etc.
- the liposome on the filter sterilization filter may be adsorbed or aggregated.
- liposomes having a specific average particle size and a uniform particle size distribution are obtained, there is an unexpected effect that there is little influence such as pressure loss when filtration is performed.
- the aseptic filtration step and the aseptic filling step described later are preferably performed at a temperature lower than the phase transition temperature of the lipid constituting the liposome.
- the lipid phase transition temperature is around 50 ° C., it is preferably about 0 to 40 ° C., and more specifically, it is preferably produced at about 5 to 30 ° C.
- the aqueous solution containing liposomes obtained after aseptic filtration is preferably aseptically filled for medical use.
- a known method can be applied for aseptic filling.
- a liposome preparation suitable for medical use can be prepared by filling the container aseptically.
- aqueous solvent, an additive and the like can be appropriately added to an aqueous solution containing liposomes obtained by the present invention to form a liposome preparation.
- the liposome preparation may contain at least one of a pharmaceutically acceptable stabilizer, an antioxidant, and a pH adjuster in relation to the administration route.
- the stabilizer is not particularly limited, and examples thereof include saccharides such as glycerol, mannitol, sorbitol, lactose, or sucrose.
- antioxidants examples include, but are not limited to, ascorbic acid, uric acid, tocopherol homologs (for example, four isomers of vitamin E, tocopherol ⁇ , ⁇ , ⁇ , and ⁇ ) cysteine, EDTA, and the like.
- the stabilizer and the antioxidant can be used alone or in combination of two or more.
- pH adjuster examples include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate and the like.
- the liposome preparation of the present invention comprises a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin , Methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, PBS, Sodium chloride, saccharide, biodegradable polymer, serum-free medium, additive acceptable as a pharmaceutical additive It may contain.
- a pharmaceutically acceptable organic solvent collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate,
- the liposome preparation preferably contains ammonium sulfate, L-histidine, purified sucrose, sodium hydroxide, hydrochloric acid, or the like.
- the container for filling the liposome preparation is not particularly limited, but is preferably a material having low oxygen permeability in order to prevent oxidation and the like.
- plastic container glass container; aluminum foil, aluminum vapor deposition film, aluminum oxide vapor deposition film, silicon oxide vapor deposition film, polyvinyl alcohol, ethylene vinyl alcohol copolymer, polyethylene terephthalate, polyethylene naphthalate, polyvinylidene chloride, etc. gas barrier layer
- the film can be shielded from light by adopting a back using a colored glass; an aluminum foil, an aluminum vapor-deposited film, or the like, if necessary.
- the gas in the container space and the chemical solution it is preferable to replace the gas in the container space and the chemical solution with an inert gas such as nitrogen in order to prevent oxidation due to oxygen present in the space in the container.
- an inert gas such as nitrogen
- the injection solution may be bubbled with nitrogen and the container is filled in a nitrogen atmosphere.
- the administration method of the liposome preparation is preferably parenteral administration.
- intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection can be selected.
- administration method of the liposome preparation include administration by syringe and infusion.
- the dosage of the drug contained in the liposome preparation is usually selected in the range of 0.01 mg to 100 mg per kg body weight per day.
- the liposome preparation of the present invention is not limited to these doses.
- Method for measuring the particle size of liposomes In the method for producing liposomes of the present invention, fine and homogeneous liposomes can be prepared.
- An example of a method for measuring the particle size of the liposome is a dynamic light scattering method.
- the dynamic light scattering method is generally known to obtain a particle size with high accuracy in a solution having a dilute particle concentration. That is, when the particle concentration of the solution is high, the influence of multiple scattering (a phenomenon in which once scattered light hits other particles and is dispersed again) may not be ignored. However, diluting the aqueous solution containing the liposome obtained in the present invention may not correctly obtain the value of the particle size in the actual solution. In addition, in the industrialization of pharmaceutical preparations, the presence of slight coarse particles may have a significant effect on filterability during removal of foreign substances and aseptic filtration. However, it is difficult to detect such coarse particles when diluted. There are many cases.
- a dynamic light scattering measurement device using a Michelson interferometer can be used for the measurement of the liposome particle size according to the present invention.
- a dynamic light scattering measurement device using a Michelson interferometer can be used for the measurement of the liposome particle size.
- a low-coherence dynamic light scattering measurement device using a Mach-Zehnder interferometer that can be measured well.
- the influence of multiple scattering can be suppressed, and the particle size and particle size distribution can be accurately measured without diluting the solution containing liposomes.
- it can be suitably used with very high sensitivity.
- a measuring method it can measure with reference to patent 5325679.
- the method for producing liposomes of the present invention can obtain liposomes having an average particle size of 200 nm or less and excellent storage stability. Liposomes obtained by the method for producing liposomes of the present invention can be applied to pharmaceuticals, cosmetics, foods and the like, and are particularly useful for pharmaceutical use.
- the mixing ratio in the solvent composition means a volume ratio.
- DSPE-PEG N- (carbonyl-methoxypolyethylene glycol 2000) -1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt
- Example 3 a) Preparation of oil phase Hydrogenated soybean phosphatidylcholine, cholesterol and DSPE-PEG were mixed in a molar ratio of 76/19/5, and then an organic solvent (ethanol / ethyl acetate 75/25) was added and 70 ° C was added. Was heated to dissolve the lipid to obtain an oil phase.
- a sample for measurement was prepared by diluting a liposome solution 13 times with pure water.
- the particle diameter of the prepared sample was measured using a dynamic light scattering (DLS) apparatus (FPAR-1000AS, manufactured by Otsuka Electronics Co., Ltd.). Cumulant average particle size was adopted as a representative value of particle size.
- the results are shown in Table 1.
- the particle size (nm) in Table 1 indicates the particle size measured by a dynamic light scattering measurement device using a Michelson interferometer.
- FIG. 1 shows the results of evaluating the filterability of a sample solution containing liposomes prepared by changing the ethanol / ethyl acetate ratio.
- eta / ethyl acetate means ethanol / ethyl acetate
- the numerical value means a ratio (volume ratio) to ethanol / ethyl acetate.
- LC-DLS low coherence dynamic light scattering
- the LC-DLS measurement was carried out as follows using a measuring apparatus consisting of a Mach-Zender interferometer. Apparatus: Light source SLD, wavelength 835 ⁇ 22 nm, AD conversion speed Max 1 ⁇ sec / ch, immersion measurement using fiber probe (measurement conditions are temperature 25 ° C., modulation frequency 10 kHz, sampling time 20 ⁇ sec interval per integration 65536 points, integration 300 Times).
- the measured scattered light fluctuation signal was Fourier-transformed, and only the modulation signal was extracted from the obtained power spectrum to obtain an autocorrelation function.
- the obtained autocorrelation function was converted into a particle size distribution by the histogram method.
- the sample to be measured was measured as a stock solution without any treatment such as dilution. In particular, the amount of coarse material on the order of microns was measured. The results are shown in Table 1.
- the coarse particle ratio in Table 1 indicates the abundance ratio of coarse substances of 500 nm or more.
- sample solution About 50 ⁇ L of sample (liposome preparation solution) was weighed with Microman, and about 950 ⁇ L of methanol weighed with Microman was added thereto. At this time, it was shaken for about 1 minute, and it was visually confirmed that the solution became transparent.
- sample liposome preparation solution
- 100 ⁇ L of the solution of (1) above was weighed with a Microman, and about 950 ⁇ L of water for injection weighed with a micropipette was added. The solution was shaken for about 1 minute, then sonicated for about 1 minute, and further shaken for about 10 seconds.
- sample solution Preparation of sample solution (1) About 50 ⁇ L of sample (liposome preparation solution) was weighed with Microman, and about 450 ⁇ L of PBS weighed with a micropipette was added thereto and mixed by inversion well to obtain a uniform solution. (2) The solution of (1) above is weighed with about 100 ⁇ L micropipette, dropped onto a previously washed ultrafiltration membrane, and centrifuged. Centrifugation was performed at 7400 ⁇ g for 30 minutes at 5 ° C. (3) A solution obtained by shaking the filtrate obtained in the operation (2) for 10 seconds was used as a sample solution. b) Measurement 3. Measurement was performed in the same manner as in c).
- Preservability The sample solution was stored at 5 ° C. for 18 months. 3. above. And 4. Using the total amount of gemcitabine in the preparation measured in step 1 and the amount of gemcitabine present in the external aqueous phase, the storage leakage was calculated according to the following formula.
- Preservative leakage (%) 100 ⁇ (Outer aqueous phase drug concentration after 18 months storage ⁇ Outer aqueous phase drug concentration immediately after preparation of preparation) / Inner aqueous phase drug concentration was measured in the same manner. The results are shown in Table 1.
- Examples 4-1 to 4-7) Drug-encapsulated liposomes were prepared in the same manner as in Example 3 except that the mixing ratio of the organic solvent was adjusted as shown in Table 2.
- the particle size of the liposomes obtained in Examples 4-1 to 4-7 was measured by the method described below.
- a sample for measurement was prepared by diluting a liposome solution 100 times with a solution obtained by diluting 1 ⁇ PBS (Gibco, Life Technology) 10 times with pure water.
- the particle diameter of the prepared sample was measured using a dynamic light scattering (DLS) apparatus (FPAR-1000AS, manufactured by Otsuka Electronics Co., Ltd.). Cumulant average particle size was adopted as a representative value of particle size.
- the results are shown in Table 2.
- the particle sizes of the liposome samples of Examples 4-1 to 4-5 having the same solvent composition hardly change, and the production method of the present invention can produce liposomes very stably. It turns out that it is a method.
- the particle size tends to increase, but liposome particles having a particle size of 200 nm or less can be prepared. It can be seen that the production method of the present invention is a versatile invention capable of producing liposome particles having a particle size of 200 nm or less in a wide range of solvent ratios.
- the particle size of the liposomes obtained in Examples 5-1 to 5-5 was measured by the method described below.
- the sample for measurement was prepared by diluting the liposome solution 13 times with pure water.
- the particle diameter of the prepared sample was measured using a dynamic light scattering (DLS) apparatus (FPAR-1000AS, manufactured by Otsuka Electronics Co., Ltd.). Cumulant average particle size was adopted as a representative value of particle size.
- DLS dynamic light scattering
- liposome particles having a particle size of 200 nm or less can be produced.
- ethyl acetate is preferably in the range of 20% to 30%. If the amount of ethyl acetate is small, foaming during emulsification increases and loss of emulsification shear force to the lipid membrane occurs. If the amount of ethyl acetate is large, the oil droplets of ethyl acetate are combined to increase the particle size.
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Abstract
Description
リポソームは、バリア能、化合物保持能、生体適合性、粒径設定の自由度、易分解性、表面修飾性等の特徴を生かして、免疫センサー、人工赤血球、薬物送達システムのキャリヤーなど多様な応用が検討されてきた。キャリヤーの用途において、リポソームは、水溶性化合物、脂溶性低分子、高分子と幅広い物質を内包することができる。
しかし、両親媒性脂質はエタノールに溶解するものに限定されており、適用できるリン脂質はレシチン類のみである。従って、各種リン脂質を任意の割合で混合したリポソームを作製することは困難である。また、この方法では脱溶剤すると粒子径が大きくなってしまうため、粒子径を揃えるためには一定細孔を有したメンブレンフィルターなどを用いた処理が必要になる。
少なくとも1種の脂質が有機溶媒に溶解している油相と水相とを混合して脂質を含む水溶液を攪拌する工程、及び
攪拌する工程で得られたリポソームを含む水溶液から有機溶媒を蒸発させる工程を含むリポソームの製造方法であって、
有機溶媒が水溶性有機溶媒及びエステル系有機溶媒の混合溶媒である、リポソームの製造方法を用いることによって、注射剤として安全性に懸念がある有機溶剤又は添加剤を使用することなく、薬剤を内包して高い保存安定性を有する微細、かつ、均質なリポソームを調製できることを見出し、本発明を完成するに至った。
好ましくは、エステル系有機溶媒が酢酸エチル、酢酸メチル、酢酸イソプロピル、酢酸t-ブチル、及びプロピオン酸メチルから選ばれる少なくとも1種である。
好ましくは、エステル系有機溶媒が酢酸エチルである。
好ましくは、水溶性有機溶媒がアルコール類である。
好ましくは、アルコールがエタノール、メタノール、2-プロパノール、及びt-ブタノールから選ばれる少なくとも1種である。
好ましくは、水溶性有機溶媒とエステル系有機溶媒との質量比が、90:10~30:70である。
好ましくは、攪拌する工程において、脂質を含む水溶液に周速20m/sec以上のせん断を与える。
好ましくは、有機溶媒を蒸発させる工程において、有機溶媒を加熱することにより蒸発させる。
また本明細書において「~」を用いて示された数値範囲は、「~」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。
本発明において、特にことわらない限り、%は、質量百分率を意味する。
少なくとも1種の脂質が有機溶媒に溶解している油相と水相とを混合して脂質を含む水溶液を攪拌する工程及び、
攪拌する工程で得られたリポソームを含む水溶液から有機溶媒を蒸発させる工程を含むリポソームの製造方法であって、
有機溶媒が水溶性有機溶媒及びエステル系有機溶媒の混合溶媒である、
リポソームの製造方法である。
リポソームとは、脂質を用いた脂質二重膜で形成される閉鎖小胞体であり、その閉鎖小胞の空間内に水相(内水相)を有する。内水相には、水等が含まれる。リポソームは通常、閉鎖小胞外の水溶液(外水相)に分散した状態で存在する。リポソームはシングルラメラ(単層ラメラ又はユニラメラとも呼ばれ、二重層膜が一重の構造である。)であっても、多層ラメラ(マルチラメラとも呼ばれ、タマネギ状の形状の多数の二重層膜の構造である。個々の層は水様の層で仕切られている。)であってもよいが、本発明では、医薬用途での安全性及び安定性の観点から、シングルラメラのリポソームであることが好ましい。
リポソームは球状またはそれに近い形態をとることが好ましい。
本発明のリポソームは、薬剤として水溶性薬剤又は脂溶性薬剤の少なくとも一つを含むことができる。
本発明のリポソームの製造方法では、
少なくとも1種の脂質が有機溶媒に溶解している油相と水相とを混合して脂質を含む水溶液を攪拌する工程(以下、攪拌工程と呼ぶことがある)、及び
攪拌する工程で得られたリポソームを含む水溶液から有機溶媒を蒸発させる工程(以下、蒸発工程と呼ぶことがある)を含む。リポソームの製造方法は、必要に応じて、攪拌工程及び蒸発工程の他に、他の工程を含んでよい。
油相として用いられる有機溶媒として、水溶性有機溶媒及びエステル系有機溶媒の混合溶媒を用いる。本発明では、有機溶媒として、クロロホルム、塩化メチレン、ヘキサン、又はシクロヘキサンといった有機溶剤を実質的に用いないことが好ましく、これらの有機溶剤を用いないことがより好ましい。
水相とは、外水相及び内水相を意味する。
本発明における外水相とは、リポソームを分散する水溶液を意味する。たとえば注射剤の場合においては、バイアル瓶やプレフィルドシリンジ包装されて保管されたリポソームの分散液のリポソームの外側を占める溶液が外水相となる。また、添付された分散用液やその他溶解液により投与時に用時分散した液についても同様に、リポソームの分散液のリポソームの外側を占める溶液が外水相となる。
本発明における内水相とは、脂質二重膜を隔てた閉鎖小胞内の水相を意味する。
リポソームを製造する際に、リポソームを分散する水溶液(外水相)としては、水(蒸留水、注射用水等)、生理食塩水、各種緩衝液、糖類の水溶液及びこれらの混合物(水性溶媒)が好ましく用いられる。緩衝液としては、有機系、無機系に限定されることはないが、体液に近い水素イオン濃度付近に緩衝作用を有する緩衝液が好適に用いられ、リン酸緩衝液、トリス緩衝液、クエン酸緩衝液、酢酸緩衝液、グッドバッファーなどがあげられる。水相のpHは特に限定されないが、5~9、好ましくは7~8であればよい。例えば、リン酸緩衝液(例えば、pH=7.4)を用いることが好ましい。リポソームの内水相は、リポソームを製造する際に、リポソームを分散する水溶液であってもよいし、新たに添加される、水、生理食塩水、各種緩衝液、糖類の水溶液及びこれらの混合物をあってもよい。外水相または内水相として用いる水は、不純物(埃、化学物質等)を含まないことが好ましい。
生理食塩水とは、人体と等張になるように調整された無機塩溶液を意味し、さらに緩衝機能を持っていてもよい。生理食塩水としては、塩化ナトリウムを0.9w/v%含有する食塩水、リン酸緩衝生理食塩水(以下、PBSともいう)及びトリス緩衝生理食塩水などが挙げられる。
攪拌工程では、少なくとも1種の脂質が有機溶媒に溶解している油相と水相とを混合して脂質を含む水溶液を攪拌する。脂質が有機溶媒に溶解している油相及び水相を混合し撹拌することで、油相及び水相がO/W型に乳化した乳化液が調製される。混合後、油相由来の有機溶媒の一部又は全部を後述する蒸発工程によって除去することにより、リポソームが形成される。又は、油相中の有機溶媒の一部又は全部が撹拌・乳化の過程で蒸発して、リポソームが形成される。
蒸発工程では、攪拌する工程で得られたリポソームを含む水溶液から有機溶媒を蒸発させる。本発明において、蒸発工程とは、油相由来の有機溶媒の一部又は全部を蒸発工程として強制的に除去する工程、及び油相中の有機溶媒の一部又は全部が撹拌・乳化の過程で自然に蒸発する工程の少なくとも一つを含む。
本発明のリポソームの製造方法によって得られた、リポソームを含む水溶液を医薬品とするために、無菌ろ過を行うことが好ましい。ろ過の方法としては、中空糸膜、逆浸透膜、メンブレンフィルター等を用いて、リポソームを含む水溶液から不要な物を除去することができる。本発明では、特に限定されないが、滅菌できる孔径をもつフィルター(好ましくは0.2μmのろ過滅菌フィルター等)によってろ過することが好ましい。通常、ろ過工程において、ろ過滅菌フィルターへのリポソームが吸着又は凝集が発生することがある。しかし、本発明では、特定の平均粒子径及び均一な粒子径分布を有するリポソームが得られるため、ろ過を行う時に圧損などの影響が少ないという予想外の効果を有する。
無菌ろ過の後に得られたリポソームを含む水溶液は、医療用途として無菌充填することが好ましい。無菌充填の方法は公知のものが適用できる。容器に無菌的に充填することで医療用として好適なリポソーム製剤が調製できる。
本発明のリポソームの製造方法では、微細かつ均質なリポソームを作成することができる。リポソームの粒径の測定方法として、動的光散乱法を挙げることができる。
溶剤組成における混合比は容量比を意味する。例えば、「エタノール/酢酸エチル=90/10」は、容量比で90%エタノール/10%酢酸エチルを意味する。
a)油相の調製
水素添加大豆ホスファチジルコリン、コレステロール及びN-(カルボニル-メトキシポリエチレングリコール2000)-1,2-ジステアロイル-sn-グリセロ-3-ホスホエタノールアミンナトリウム塩(以下、DSPE-PEGともいう)を57/38/5のモル比となるように混合し、次いで有機溶媒(エタノール/酢酸エチル=75/25)を加えて70℃に加温して脂質を溶解し油相とした。
177mMの硫酸アンモニウム水溶液を調製し水相とした。
b)で調製した水相を70℃に加温し、水相/油相=8/3の容積比になるようにa)で調製した油相を添加した後、周速30m/s、角速度19000rpmにて30分間攪拌した。
c)で作製した乳化液を脂質の相転位温度以上に加温しながら窒素を送気することで有機溶媒を除去し、さらに窒素送気を停止して、脂質の相転位温度以上で3時間加温してリポソームを得た。
調製に用いる有機溶媒の組成を(エタノール/酢酸エチル=75/25)から(エタノール/酢酸エチル=90/10)に変更した以外、実施例1と同様にしてリポソームを得た。
a)油相の調製
水素添加大豆ホスファチジルコリン、コレステロール及びDSPE-PEGを76/19/5のモル比となるように混合し、次いで有機溶媒(エタノール/酢酸エチル=75/25)を加えて70℃に加温して脂質を溶解し油相とした。
4mMリン酸緩衝液(pH7.86)を調製し水相とした。
水相を70℃に加温し、水相/油相=8/3の容積比となるように油相を添加した後、周速20m/s、13000rpmにて30分攪拌した。その後、相転位温度以上に加温しながら窒素を送気することで有機溶剤を除去し、さらにタンジェンシャルフローフィルトレーションによりリポソーム液の脂質濃度を120~150mMの範囲で濃縮しながら、リポソームの外水相を0.09%塩化ナトリウム水溶液に置換することで有機溶剤を完全に除去し、薬剤未内包リポソームを得た。
1)PBSの調製
塩化ナトリウム81.63g、リン酸水素二ナトリウム十二水和物29.01g、リン酸二水素ナトリウム二水和物2.29gを注射用水948gで溶解し、PBSとした。
2)ゲムシタビン内包リポソーム液の調製
ゲムシタビン塩酸塩7.68g、PBS31.99g、日局注射用水44.83g、8M水酸化ナトリウム1.60mLを混合し、薬物溶液とした。つづいて、4つのバイアルにそれぞれ薬物溶液17.64mL、薬物未内包リポソーム18.00mL、8M水酸化ナトリウム0.36mLを混合した。混合後、約70℃で30分加温した後に室温まで冷却した。その後、薬剤リポソーム混液を孔径0.2μmの除菌用フィルターでろ過し、更にタンジェンシャルフローフィルトレーションにより薬剤リポソーム混液を9.4%スクロース/10mMヒスチジン水溶液で透析精製することで未内包のゲムシタビンを除去し、次にリポソームを孔径0.2μmの滅菌フィルターでろ過することで無菌のゲムシタビン内包リポソーム液を得た。
油相の調製に用いる有機溶媒の組成を(エタノール/酢酸エチル=75/25)から(エタノール/酢酸エチル=100/0)に変更した以外、実施例1と同様にしてリポソームを得た。
油相の調製に用いる有機溶媒の組成を(エタノール/酢酸エチル=75/25)から(エタノール/酢酸エチル=0/100)に変更した以外は実施例1と同様にして実施したが、脂質を完全に溶解することができない(脂質の溶解性が乏しいために乳化できない)ために、リポソームを調製することができなかった。
測定用の試料は、リポソーム液を純水で13倍に希釈して調製した。調製した試料の粒径は動的光散乱(DLS)装置(FPAR-1000AS、大塚電子株式会社製)を用いて測定した。キュムラント平均粒子径を粒径の代表値として採用した。
結果を表1に示す。表1における粒径(nm)は、マイケルソン型干渉計による動的光散乱測定装置による測定での粒径を示す。
リポソーム液の総脂質濃度が25~30mMになるように調整した後、25℃に保温した。エクストルーダー(Thermobarrel extruder、Lipex社製)に0.2μmフィルター(Sartobran P、Sartorius stedim社製)を設置し、注射用水を0.05MPaで通液することでフィルターを洗浄し、次に25℃に保温したリポソームを0.3MPaで加圧ろ過した。ろ液は種々の時間間隔で回収し、ろ液重量を測定した。1mLのろ過液をろ過するのに要する時間からろ過性を測定した。結果を表1及び図1に示す。表1のフィルターろ過性について、A:ろ過可能、B:ろ過可能であるが目詰まりを起こす、C:ろ過不可能(1mLろ過できず)をそれぞれ意味する。図1は、エタノール/酢酸エチルの比を変えて調製したリポソームを含む試料溶液に対するろ過性を評価した結果を示す。図1において、エタ/酢エチは、エタノール/酢酸エチルを意味して、数値はエタノール/酢酸エチルに対する比率(容量比)を意味する。また、ろ過性の程度が、粗大粒子の存在量と関連するか確認するため、粗大粒子の検出力が高い、低コヒーレンス動的光散乱(LC-DLS)測定を実施した。LC-DLS測定はマッハツエンダー型干渉計からなる測定装置を用い、以下のように実施した。
装置:光源SLD、波長835±22nm、AD変換速度Max 1μsec/ch、ファイバープローブによる液浸測定(測定条件は温度25℃、変調周波数10kHz、1回積算あたりのサンプリング時間20μsec間隔65536点、積算300回)。
解析法は測定した散乱光揺らぎの信号をフーリエ変換し、得られたパワースペクトルから変調信号のみを抜き出して自己相関関数を求めた。求められた自己相関関数をヒストグラム法により粒度分布に換算した。
測定する試料は希釈などの処理をすることなく、原液のまま測定した。特に、ミクロンオーダーの粗大物量を測定した。
結果を表1に示す。表1における粗大粒子比率は、500nm以上の粗大物の存在比率を示す。
調製したリポソーム液をメタノールに溶かしフィルターろ過した試料溶液、及びゲムシタビン塩酸塩を希釈調製した検量線標準溶液を用いて、液体クロマトグラフィー/紫外可視吸光度検出法により測定した。
ゲムシタビン塩酸塩約10mgを量り、約10mLの1×PBS(Gibco、Life Technology社製)に溶かした。この液に1×PBS(Gibco、Life Technology社製)を加えて、ゲムシタビン塩酸塩濃度0.1、1.0、5.0、又は10.0μg/mLの溶液を調製し、検量線標準液とした。
(1)試料(リポソーム製剤溶液)約50μLをマイクロマンで量りとり、これにマイクロマンで量りとった約950μLのメタノールを加えた。このとき、約1分間振とうし、溶液が透明になることを目視で確認した。
(2)上記(1)の溶液100μLをマイクロマンで量りとり、マイクロピペットで量りとった約950μLの注射用水を加えた。この液を約1分間振とうした後、約1分間超音波処理をし、さらに約10秒間振とうした。
(3)上記(2)の溶液をDISMICフィルター(穴径0.45μm)でろ過した溶液を試料溶液とした。
液体クロマトグラフィー/紫外可視吸光度検出法により以下の条件で測定した。
測定波長:272nm、カラム:Waters AtlantisT3φ4.6×150mm、5μm
カラム温度:40℃付近の一定温度
移動相A、Bはいずれも水/メタノール/トリフルオロ酢酸混液で、移動相の送液は移動相A及びBの混合比を変えて濃度勾配を制御した。
流量:毎分1.0mL、注入量:10μL、オートサンプラー温度:25℃付近の一定温度で測定を行った。
試料(リポソーム製剤)の希釈溶液から限外ろ過によりリポソームを取り除いた試料溶液、及びゲムシタビン塩酸塩を希釈調製した検量線標準溶液を用いて、液体クロマトグラフィー/紫外可視吸光度検出法により測定した。
(1)試料(リポソーム製剤溶液)約50μLをマイクロマンで量りとり、これにマイクロピペットで量りとった約450μLのPBSを加え、十分に転倒混和し均一な溶液とした。
(2)上記(1)の溶液を約100μLマイクロピペットで量り、あらかじめ洗浄した限外ろ過膜に滴下し遠心分離する。遠心分離は7400×g、30分間、5℃の条件で行った。
(3)上記(2)の操作のろ液を10秒間振とうした溶液を試料溶液とした。
b)測定
3.c)と同様に測定した。
試料溶液を5℃で18ケ月保存した。
上記3.及び4.で測定した製剤中の全ゲムシタビン量及び外水相中に存在するゲムシタビン量を用いて、保存性漏出を次の式により算出した。
式:保存性漏出(%)=100×(18ケ月保存後外水相薬剤濃度-製剤調製直後の外水相薬剤濃度)/内水相薬剤濃度
また、粒径を1.と同様に測定した。
結果を表1に示す。
有機溶媒の混合比率を表2のように調整した以外は、実施例3と同様に薬剤内包リポソームを調製した。
測定用の試料は、1×PBS(Gibco、Life Technology社製)を純水で10倍に希釈した液で、リポソーム液を100倍に希釈して調製した。調製した試料の粒径は動的光散乱(DLS)装置(FPAR-1000AS、大塚電子株式会社製)を用い測定した。キュムラント平均粒子径を粒径の代表値として採用した。
結果を表2に示す。
a)油相の調製
水素添加大豆ホスファチジルコリン、コレステロール及びDSPE-PEGを57/38/5のモル比となるように混合し、次いで有機溶媒を加えて70℃に加温して脂質を溶解し油相とした。有機溶媒の比率について、実施例5-1;(エタノール/酢酸エチル=88/12)、実施例5-1;(エタノール/酢酸エチル=79/21)、実施例5-3;(エタノール/酢酸エチル=75/25)、実施例5-4;(エタノール/酢酸エチル=70/30)、実施例5-5;(エタノール/酢酸エチル=63/37)に調製した。
187mMの硫酸アンモニウム水溶液を調製し水相とした。
b)で調製した水相を70℃に加温し、水相/油相=8/3の容積比になるようにa)で調製した油相を添加した後、周速30m/s、角速度19000rpmで30分間攪拌し、リポソーム液を得た。
測定用の試料は、リポソーム液を純水により13倍に希釈して調整した。調製した試料の粒径は動的光散乱(DLS)装置(FPAR-1000AS、大塚電子株式会社製)を用い測定した。キュムラント平均粒子径を粒径の代表値として採用した。
結果を表3及び図2に示す。
Claims (8)
- 少なくとも1種の脂質が有機溶媒に溶解している油相と水相とを混合して脂質を含む水溶液を攪拌する工程、及び
攪拌する工程で得られたリポソームを含む水溶液から有機溶媒を蒸発させる工程を含むリポソームの製造方法であって、
有機溶媒が水溶性有機溶媒及びエステル系有機溶媒の混合溶媒であるリポソームの製造方法。 - エステル系有機溶媒が酢酸エチル、酢酸メチル、酢酸イソプロピル、酢酸t-ブチル、及びプロピオン酸メチルから選ばれる少なくとも1種である請求項1に記載の製造方法。
- エステル系有機溶媒が酢酸エチルである請求項1に記載の製造方法。
- 水溶性有機溶媒がアルコール類である請求項1から3のいずれか一項に記載の製造方法。
- アルコールがエタノール、メタノール、2-プロパノール、及びt-ブタノールから選ばれる少なくとも1種である請求項4に記載の製造方法。
- 水溶性有機溶媒とエステル系有機溶媒との質量比が、90:10~30:70である請求項1から5のいずれか一項に記載の製造方法。
- 攪拌する工程において、脂質を含む水溶液に周速20m/sec以上のせん断を与える請求項1から6のいずれか一項に記載の製造方法。
- 有機溶媒を蒸発させる工程において、有機溶媒を加熱することにより蒸発させる請求項1から7のいずれか一項に記載の製造方法。
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WO2021201267A1 (ja) | 2020-04-03 | 2021-10-07 | 富士フイルム株式会社 | 抗腫瘍剤 |
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