WO2015166987A1 - リポソーム組成物及びその製造方法 - Google Patents
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- WO2015166987A1 WO2015166987A1 PCT/JP2015/062984 JP2015062984W WO2015166987A1 WO 2015166987 A1 WO2015166987 A1 WO 2015166987A1 JP 2015062984 W JP2015062984 W JP 2015062984W WO 2015166987 A1 WO2015166987 A1 WO 2015166987A1
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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
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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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
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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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
-
- 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
- A61K9/1278—Post-loading, e.g. by ion or pH gradient
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Definitions
- the present invention relates to a liposome composition and a method for producing the same.
- the present invention relates to a liposome composition that can be suitably used for pharmaceutical use and a method for producing the same.
- 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 size needs to be about 200 nm or less from the viewpoint of permeation through a biological membrane.
- the nano-sized fine particles are preferably provided with storage stability 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 that are not approved for use (hereinafter, the dispersant also includes a dispersion aid, etc.) are not preferred.
- suitability for aseptic filtration is also required.
- the liposome composition containing a drug needs to deliver the drug to the affected area and release the required amount of the drug.
- compatibility between the ease of drug release and storage stability of the liposome composition is a conflicting issue, and it is desired that both can be achieved.
- Patent Document 1 describes a production method in which liposomes are prepared by an emulsification method without going through a lipid film drying and solidifying step.
- a manufacturing method using a water-soluble emulsifier that does not break the liposome lipid membrane as an emulsifier, specifically, pluronic as a nonionic surfactant is disclosed.
- surfactants are expected to have a significant effect on the strength of liposome membranes and need improvement.
- Patent Document 2 describes that a drug that is 2.0 to 5.0 times hypertonic than the biological fluid osmotic pressure of a warm-blooded animal is contained in a liposome that is a small unilamellar vesicle having a phase transition temperature of 40 to 45 ° C.
- a liposome composition enclosing a liquid is disclosed. It is also disclosed that this liposome composition is a liposome composition obtained through a drying and solidifying step of a mixed lipid membrane.
- this liposome composition has the property of suppressing release at a low temperature and releasing it at a high temperature for thermotherapy.
- a liposome composition having a long-term storage stability necessary for practical use and having a drug release rate on the order of several tens of hours and a method for producing the same have not been sufficiently established. It is desired.
- JP 2013-022482 A Japanese Patent Laid-Open No. 06-009374
- the drug is hardly released out of the liposome due to strong interactions such as hydrophobic interaction and electrostatic interaction. Therefore, the structure of the liposome composition after production can be maintained, and long-term storage stability is easily ensured. In this case, since the interaction is too strong, it is difficult to release the drug to the affected area. Therefore, it is ideal that the drug is encapsulated in an internal aqueous phase of the liposome in a dissolved state. Furthermore, by making the liposome composition hypertonic, the release of the drug from the liposome composition is promoted and more suitable. Drug delivery can be realized. However, the hypertonic condition makes it easy for the drug to leak from the liposome composition, and it is difficult to ensure long-term storage stability.
- the average particle size of the liposome is a fine particle of 100 nm or less.
- the curvature (curvature) of the liposome membrane is increased, making it difficult to encapsulate the drug.
- the present invention A liposome composition having an inner aqueous phase obtained from lipid emulsified by dissolving in an organic solvent and an aqueous solution in which the liposome constituting the outer aqueous phase is dispersed, wherein the liposome is dissolved in water
- the liposome composition contains a drug and has an osmotic pressure in the inner aqueous phase of 2 to 8 times that of the outer aqueous phase.
- the liposome has a single lamellar structure.
- the average particle diameter of the liposome is 5 nm or more and 100 nm or less.
- the liposome comprises at least hydrogenated soy phosphatidylcholine, 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol, and cholesterol.
- the present invention is a pharmaceutical composition containing the liposome composition described above.
- the present invention An emulsification step in which a lipid is dissolved in an organic solvent to form liposomes without going through a drying and solidification step.
- the liposome obtained after the emulsification step is used in the next step without being subjected to the extrusion treatment.
- the drug loading step and the osmotic pressure adjustment step are performed simultaneously.
- the liposome composition of the present invention even when the osmotic pressure of the inner aqueous phase is not less than 2 times and not more than 8 times the osmotic pressure of the outer water phase, It is possible to provide a liposome composition that contains a sex drug and is excellent in storage stability.
- the osmotic pressure of the inner aqueous phase is not less than 2 times and not more than 8 times the osmotic pressure of the outer aqueous phase, and encapsulates the dissolved water-soluble drug.
- a liposome composition capable of achieving a release rate on the order of several tens of hours.
- the production suitability for example, aseptic filtration suitability
- the average particle size is 100 nm or less
- the particle size distribution shows a uniform distribution, and is preserved.
- a liposome composition having excellent stability can be obtained.
- the average particle size and particle size distribution, production suitability (for example, aseptic filtration suitability, etc.) suitable for pharmaceutical use, and preservation necessary as a preparation A liposome composition containing a water-soluble drug in a dissolved state that can achieve both stability can be efficiently produced on an industrial scale.
- FIG. 1 is a DSC thermogram of a liposome (Example 1) produced by an emulsification method.
- FIG. 2 is a DSC thermogram of a liposome (Comparative Example 1) produced by the Bangham method.
- FIG. 3 is a plot of the relationship between the amount of drug encapsulated in the liposome and the elapsed time.
- FIG. 4 is a plot of the relationship between the abundance rate (non-encapsulation rate) of the drug present in the outer aqueous phase and the elapsed time.
- FIG. 5 is a plot of the relationship between the volume average particle size and the elapsed time.
- FIG. 6 is a plot of the relationship between drug release rate and elapsed time.
- the term “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 relates to a liposome composition having an inner aqueous phase obtained from an emulsified lipid dissolved in an organic solvent, and an aqueous solution in which the liposome constituting the outer aqueous phase is dispersed, wherein the liposome is dissolved. It is a liposome composition that encapsulates a water-soluble drug in a state and has an osmotic pressure of the inner aqueous phase of 2 to 8 times that of the outer aqueous phase.
- 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 is a liposome capable of encapsulating a drug.
- “Encapsulation” means that the drug is in a form contained in the inner aqueous phase with respect to the liposome.
- a form in which a drug is enclosed in a closed space formed by a film, a form in which the drug is included in the film itself, and the like 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 preferably contains phospholipids, lipids other than phospholipids, cholesterols and derivatives thereof, and phospholipids, lipids other than phospholipids, and cholesterols are modified with hydrophilic polymers. .
- hydrophilic polymer examples include, but are not limited to, polyethylene glycols, polyglycerols, polypropylene glycols, polyvinyl alcohol, styrene-maleic anhydride alternating copolymers, polyvinyl pyrrolidone, and synthetic polyamino acids. Said hydrophilic polymer can be used individually or in combination of 2 types or more, respectively. Among these, 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.
- phase transition temperature or Tm of the measurement object can be measured.
- the phase transition temperature or Tm refers to the low steric disorder of the lipid (liquid-disordered phase) from the low-temperature gel phase (solid-ordered phase) characterized by regular lipids in the lipid bilayer of liposomes. ) Denotes the temperature of transition to a hot fluid phase.
- the homogeneity of the composition of the obtained liposome membrane can be known from the shape of the phase transition peak (endothermic peak derived from the destruction of the lipid structure) by DSC thermogram.
- the differential scanning calorimetry can be performed using, for example, a differential scanning calorimeter.
- the differential scanning calorimeter is not particularly limited, and for example, DSC 120 manufactured by Seiko Instruments Inc. (SII) can be used.
- the rate of temperature rise is not particularly limited with respect to the liposome sample, it can be performed at a temperature rise of 0.2 ° C./min to 20 ° C./min.
- the scanning temperature range is not limited, and may be a range in which a desired endothermic peak can be observed according to the phase transition temperature of the liposome. As an example, scanning from around 5 ° C. to around 100 ° C. may be performed.
- FIG. 1 is a DSC thermogram obtained for a liposome produced by the emulsification method of the present invention (Example 1) and a liposome produced by the Bangham method (Comparative Example 1).
- liposomes with high osmotic pressure in the inner aqueous phase it is known that part of the lipid membrane forms a finger-linked gel phase (L ⁇ I phase), and the finger-linked gel phase and lamellar gel (L ⁇ 'phase)
- An endothermic peak attributed to these two components is produced.
- two clear endothermic peaks were obtained, but in the liposome obtained in Comparative Example 1, the endothermic peak was not separated into two.
- Example 1 of the present invention two peaks are clearly shown, indicating that the composition of the liposome membrane is uniform.
- the liposome of the present invention can contain at least one water-soluble drug as a drug.
- a water-soluble drug a form in which it is retained in the inner aqueous phase of the liposome is advantageous.
- the lipid bilayer membrane is thin and soft, so that the drug may easily leak.
- liposomes having safety and stability can be produced even if the particle diameter is about 100 nm or less.
- the drug contained in the drug may be any water-soluble drug that can be encapsulated in liposomes.
- the water-soluble drug is preferably a low molecular compound from the viewpoint of stability.
- water-soluble 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, bleomycin, etc.
- antimetabolites such as sirolimus anticancer agents such as bleomycin and sirolimus, methotrexate, fluorouracil, gemcitabine and cytarabine.
- water-soluble drugs such as doxorubicin, gemcitabine, and pemetrexed are preferable.
- the water-soluble drug encapsulated in the liposome of the present invention exists in a dissolved state in the inner aqueous phase of the liposome.
- the dissolved state is considered to be encapsulated in the dissolved state when the amount of the drug filled with respect to the volume of the liposome is equal to or lower than the saturated solubility of the drug in the composition solution of the inner aqueous phase.
- water-soluble drug to be encapsulated in a dissolved state those having a solubility of 1 mg / ml or more in water are preferable, and those having a solubility of 10 mg / ml or more are more preferable.
- the method for producing the liposome of the present invention comprises: An emulsification step in which a lipid is dissolved in an organic solvent to form liposomes without going through a drying and solidification step.
- the production method of the liposome composition may include other steps such as an evaporation step of evaporating the organic solvent used in the emulsification step, as necessary.
- the emulsification step in which the liposome is formed by emulsifying the mixed lipid dissolved in the organic solvent without passing through the drying and solidification step is not limited as long as it is an emulsification step. This is a step of forming fine particles in an emulsification step including a solvent. If necessary, liposomes can be formed by evaporating (desolving) the organic solvent used in the emulsification step.
- an oil phase in which at least one kind of lipid is dissolved in an organic solvent is mixed with an aqueous phase, and an aqueous solution containing the lipid is stirred to emulsify.
- an emulsion in which the oil phase and the aqueous phase are emulsified in an 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.
- the stirring method ultrasonic waves or mechanical shearing force is used for particle refinement. Further, in order to make the particle diameter uniform, an extruder process or a microfluidizer process through a filter having a fixed pore diameter can be performed. If an extruder or the like is used, the secondary vesicle liposomes can be separated into single vesicle liposomes. In the present invention, it is preferable from the viewpoint of simplifying the production process that the liposome in a state in which no drug is loaded is used in the next step without being subjected to the extrusion 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.
- 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 at all.
- 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.
- alcohols are preferable.
- the alcohol is preferably at least one selected from ethanol, methanol, 2-propanol, and t-butanol, more preferably at least one selected from ethanol, 2-propanol, and t-butanol. More preferably.
- 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 evaporation step may be provided as necessary.
- the organic solvent is evaporated from the aqueous solution containing the liposomes obtained in the emulsification 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 emulsification process is free of components contained in the liposomes, or is subjected to centrifugal separation, ultrafiltration, dialysis, gel filtration, lyophilization, etc., in order to remove components or adjust the concentration or osmotic pressure. You may post-process by the method.
- the obtained liposome can be made uniform in particle size by using a dialysis method, a filtration method, an extrusion treatment or the like.
- a dialysis method a filtration method, an extrusion treatment or the like.
- a centrifugal separation method, a dialysis method, a gel filtration method, or the like can be used.
- the extrusion treatment means a step of applying physical shearing force and atomizing by passing the liposome through a filter having pores.
- the liposome dispersion liquid and the filter can be rapidly atomized by keeping the temperature at a temperature equal to or higher than the phase transition temperature of the membrane constituting the liposome.
- the drug is dissolved in an aqueous medium to be hydrated / swelled, and heated by a method such as heating above the phase transition temperature, sonication or extrusion.
- a method such as heating above the phase transition temperature, sonication or extrusion.
- the drug can be dissolved in the aqueous phase during lipid emulsification and encapsulated in the inner aqueous phase.
- the drug is easily released by making the internal aqueous phase of the liposome hypertonic (pressure difference from the external aqueous phase) by the osmotic pressure adjusting step.
- the release speed can be controlled by setting the osmotic pressure.
- Methods, such as a dialysis, can be employ
- the osmotic pressure can be adjusted.
- the osmotic pressure of the inner aqueous phase is 2 to 8 times the osmotic pressure of the outer aqueous phase with respect to the liposome having the inner aqueous phase obtained from the emulsified lipid.
- the osmotic pressure of the outer aqueous phase is increased by, for example, dialysis or the like by setting the inner aqueous phase and the outer aqueous phase of the liposome not encapsulating the drug to a high osmotic pressure.
- the drug contained in the inner aqueous phase may leak and the osmotic pressure of the inner aqueous phase may decrease.
- the inner aqueous phase is replaced with a solution having a high osmotic pressure, and then the removal of the outer aqueous phase drug and the lowering of the outer aqueous phase osmotic pressure are simultaneously performed by dialysis. It is possible to obtain a liposome composition capable of satisfying both ease of release and storage stability.
- the osmotic pressure of the inner aqueous phase is 2 to 8 times, preferably 2.5 to 6 times, more preferably 3 to 5 times the osmotic pressure of the outer aqueous phase. Is double. It is generally known that the lipid bilayer of a liposome exhibits a structure such as a bilayer structure or a finger-fitting structure by making it twice or more. When the osmotic pressure of the inner aqueous phase is more than twice that of the outer aqueous phase, the liposome begins to have a finger-fitting structure from a bilayer structure.
- conditions for various lipids may be set in order to obtain a suitable finger structure, but it is preferable to control by adjusting the cholesterol ratio. As a result, it is possible to obtain a liposome composition that can achieve both drug release and storage stability.
- the solutes of the outer aqueous phase and the inner aqueous phase are homogenized, and the osmotic pressure at that time is defined as the osmotic pressure of the inner aqueous phase of the completed liposome composition. it can.
- the heating operation is limited to the case where the solute of the inner aqueous phase is sufficiently retained, for example, to suppress the lipid phase transition or less.
- the osmotic pressure of the outer aqueous phase can be defined by the osmotic pressure of the dialysate used in the final dialysis step.
- the solute composition concentration of the outer aqueous phase and the solute composition concentration of the inner aqueous phase are quantified and the osmotic pressure of the composition solution is measured.
- the osmotic pressure of the inner aqueous phase and the outer aqueous phase can be obtained.
- Measurement of osmotic pressure may be performed according to the osmotic pressure measuring method described in the 16th revision Japanese Pharmacopoeia. Specifically, the osmolality can be obtained by measuring the degree of freezing point (freezing point) of water.
- the freezing point depression degree of water is defined by the solute molar concentration, and the osmolality can be obtained from the solute molar concentration.
- the outer aqueous phase in the present invention is an aqueous solution that occupies the outside of the liposome.
- 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 that occupies the outside of the liposome is the outer aqueous phase of the attached dispersion or other solution that is dispersed at the time of administration at the time of administration.
- the osmotic pressure of the external water phase has an important effect on the living body during administration.
- the osmotic pressure of the outer aqueous phase in the present invention is preferably 200 to 400 mOsmol / L, more preferably 250 to 350 mOsmol / L, and most preferably isotonic with the body fluid.
- aseptic filtration In order to obtain an aqueous solution containing liposomes obtained by the method for producing a liposome composition of the present invention as a pharmaceutical composition, 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 composition suitable for medical use can be prepared by filling the container aseptically.
- An aqueous solvent, an additive, and the like can be appropriately added to an aqueous solution containing liposomes obtained by the present invention to obtain a pharmaceutical composition containing the liposome composition.
- the pharmaceutical composition may comprise at least one of pharmaceutically acceptable tonicity agents, stabilizers, antioxidants, and pH adjusters in relation to the route of administration.
- the isotonic agent is not particularly limited, but for example, inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, glycerol, mannitol, sorbitol, etc.
- inorganic salts such as sodium chloride, potassium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, glycerol, mannitol, sorbitol, etc.
- examples include polyols, sugars such as glucose, fructose, lactose, or sucrose.
- 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 adjusters examples include sodium hydroxide, citric acid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate.
- the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, 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, saccharides, biodegradable polymers, serum-free medium, and pharmaceutical acceptable additives It may be.
- a pharmaceutically acceptable organic solvent collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium al
- the pharmaceutical composition preferably contains ammonium sulfate, L-histidine, purified sucrose, sodium hydroxide, hydrochloric acid and the like.
- the container filled with the pharmaceutical composition is not particularly limited, but is preferably a material having low oxygen permeability.
- gas barrier layer made of 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.
- a back using a colored glass, an aluminum foil, an aluminum vapor-deposited film, or the like can be used to shield the light.
- a container filled with a pharmaceutical composition 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 pharmaceutical composition is preferably parenteral administration.
- intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, and intrathecal injection can be selected.
- Specific administration methods of the liposome composition include administration by syringe and infusion.
- the dosage of the drug contained in the pharmaceutical composition is usually selected in the range of 0.01 mg to 100 mg per kg body weight per day.
- the liposome composition of the present invention is not limited to these doses.
- a liposome composition that can achieve both drug release and storage stability can be obtained.
- the liposome composition of the present invention can be applied to pharmaceuticals, cosmetics, foods and the like, and is 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
- the osmotic pressure of this liquid is 1039 mOsm / L, which becomes the osmotic pressure of the inner aqueous phase of the liposome composition to be completed.
- this solution was heated at 70 ° C. for 10 minutes, cooled to 40 ° C., and diluted with a 1018 mM sucrose / 37 mM histidine solution. After dilution, they were combined into a drug loading solution.
- gemcitabine hydrochloride concentration 0.71 mg / mL, particle diameter 75.7 nm, inner aqueous phase osmotic pressure 1039 mOsm / L, outer aqueous phase osmotic pressure 285 mOsm / L, and the osmotic pressure of the inner aqueous phase with respect to the outer aqueous phase are A 3.6 times drug-encapsulating liposome composition was obtained.
- the osmotic pressure obtained from the solute molar concentration of this liquid is 1039 mOsm / L, which becomes the inner aqueous phase osmotic pressure of the liposome composition liquid to be completed.
- PBS prepared in d) of Example 1 was diluted 2.7 times by weight, and the drug solution was diluted 3 times.
- gemcitabine concentration 1.02 mg / mL, particle size 89.6 nm, inner aqueous phase osmotic pressure 1039 mOsm / L, outer aqueous phase osmotic pressure 285 mOsm / L, and the osmotic pressure of the inner aqueous phase with respect to the outer aqueous phase is 3.
- a 6-fold drug-encapsulating liposome composition was obtained.
- FIG. 1 shows a DSC thermogram of a liposome (Comparative Example 1) produced by a liposome produced by an emulsification method (Example 1).
- FIG. 2 shows a DSC thermogram of a liposome produced by the Bangham method (Comparative Example 1). 20 ⁇ L of drug-encapsulated liposome solution is filled and sealed in an aluminum hermetic pan (manufactured by Tzero), and measured from DSC Q2000 (manufactured by TA Instrument) from 25 ° C. to 70 ° C. at a scanning speed of 0.5 ° C./min. did.
- Liposome Example 1 produced by the emulsification method of the present invention clearly shows two peaks, a peak indicating a finger-assembled gel phase and a peak indicating a lamellar gel phase, and it can be seen that the composition of the liposome membrane is uniform.
- Example 2 In the preparation of the oil phase, 49.9 g, 6.1 g, and 12.9 g of hydrogenated soybean phosphatidylcholine, cholesterol, and DSPE-PEG were used, respectively, and the water phase / oil phase mixture was rotated at a peripheral speed of 19 m with a rotary stirring emulsifier. / S was mixed at 12,000 rpm for 30 minutes, and 10 ⁇ PBS (manufactured by Nippon Gene) was used instead of PBS prepared in d) of Example 1 in the drug loading step, and diluted to 2.7 times weight.
- PBS manufactured by Nippon Gene
- the drug-encapsulated liposome of Example 2 was filled in a 2 mL glass vial and stored at 5 ° C., and a part of the sample was sampled at a certain time. Using this sample, the following various evaluations were performed to evaluate the stability of the liposome composition in the present invention.
- the drug encapsulated in the liposomes was extracted by diluting (volume) 50 ⁇ L of the sampled sample with methanol. Subsequently, the extract was diluted 10 times with water (volume), and the amount of drug contained in this solution was quantified by HPLC. FIG. It has been found that the drug in the liposome composition of the present invention is sufficiently stable over a long period of 12 months.
- Unencapsulated rate (%) (Drug concentration in filtrate ⁇ 10) ⁇ Drug concentration in formulation ⁇ 100
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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。
この場合、相互作用が強すぎるため、患部に薬物を放出させることが困難になる。そこで、薬物をリポソームの内水相に溶解状態で内包することが理想的であり、さらにリポソーム組成物を高張の条件にすることで、リポソーム組成物からの薬物の放出を促進し、より適したドラッグデリバリーが実現できる。ただし、高張の条件にすることで、リポソーム組成物から薬物が漏出し易くなり、長期の保管安定性を担保することが困難であった。
有機溶媒に溶解して乳化された脂質から得られる、内水相を有するリポソームと、外水相を構成するリポソームを分散する水溶液とを有するリポソーム組成物であって、リポソームが溶解状態の水溶性薬物を内包し、内水相の浸透圧が外水相の浸透圧に対して2倍以上8倍以下である
リポソーム組成物である。
好ましくは、リポソームがシングルラメラの構造を有する。
好ましくは、リポソームの平均粒子径が5nm以上100nm以下である。
好ましくは、リポソームが、水素添加大豆ホスファチジルコリン、1,2-ジステアロイル-3-ホスファチジルエタノールアミン-ポリエチレングリコール、及びコレステロールを少なくとも含む。
乾燥固化工程を経ずに、有機溶媒に溶解した脂質を乳化してリポソームを形成する乳化工程、
乳化工程で得られたリポソームに水溶性薬物を内包させる薬物ローディング工程、及び
リポソームの内水相の浸透圧を外水相の浸透圧に対して2倍以上8倍以下に調整する浸透圧調整工程
を有するリポソーム組成物の製造方法である。
好ましくは、乳化工程の後に得られるリポソームをエクストリュージョン処理せずに次の工程に用いる。
好ましくは、薬物ローディング工程及び浸透圧調整工程を同時に行う。
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。
本明細書において組成物中の各成分の量は、組成物中に各成分に該当する物質が複数存在する場合、特に断らない限り、組成物中に存在する当該複数の物質の合計量を意味する。
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。
これらの中でも、製剤の血中滞留性の観点から、ポリエチレングリコール類、ポリグリセリン類、ポリプロピレングリコール類が好ましく、ポリエチレングリコール(PEG)、ポリグリセリン(PG)、ポリプロピレングリコール(PPG)がより好ましく、ポリエチレングリコール(PEG)は最も汎用であり、血中滞留性を向上させる効果があり、好ましい。
示差走査熱量測定(differential scanning calorimetry:DSC)によれば、測定物がもつ相転移温度又はTmが測定できる。相転移温度又はTmとは、リポソームの脂質二重膜において規則的な脂質を特徴とする低温のゲル相(固体-規則相)から、脂質が高い立体不規則性を有する(液体-不規則相)高温の流体相に移行する温度を意味する。具体的には、DSCサーモグラムによる相転移ピーク(脂質構造の破壊に由来する吸熱ピーク)の形状により、得られたリポソームの膜の組成の均一さを知ることができる。
本発明のリポソームは、薬物として水溶性薬物の少なくとも一つを含むことができる。
水溶性薬物の場合、リポソームの内水相に保持する形態が有利となるが、脂質二分子膜は薄く、やわらかいために薬物が漏出しやすくなることがある。しかし、本発明のリポソームの製造方法によれば、粒子径を100nm程度以下にしても、安全性及び安定性を有するリポソームが製造できる。
本発明のリポソームに内包した水溶性薬物は、リポソームの内水相に溶解状態で存在している。ここで、溶解状態とは、リポソームの体積に対して充填した薬物の量が、その内水相の組成液での薬物の飽和溶解度以下の場合、溶解状態で内包されたものとみなす。また、飽和溶解度以上においても、Cryo-TEMで薬物結晶が観察されないこと、XRD測定で結晶格子に起因する回折パターンが観察されない場合は、脂質膜が作る物理化学的な環境による溶解促進や、一部薬物が脂質膜に取り込まれるなどして大部分が溶解していることを示し、溶解状態で内包されたものとみなす。また、リポソーム内部で固体物を形成させて、薬物を封入させるローディング方法により内包したものは、水溶性の高い薬物であっても、本発明でいう溶解状態ではない。
本発明のリポソームの製造方法は、
乾燥固化工程を経ずに、有機溶媒に溶解した脂質を乳化してリポソームを形成する乳化工程、
乳化工程で得られたリポソームに水溶性薬物を内包させる薬物ローディング工程、及び
リポソームの内水相の浸透圧を外水相の浸透圧に対して2倍以上8倍以下に調整するリモートローディング工程
を有するリポソーム組成物の製造方法である。リポソーム組成物の製造方法は、必要に応じて、乳化工程で用いた有機溶媒を蒸発させる蒸発工程等、他の工程を含んでよい。
乳化工程では、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して乳化する。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し、乳化することで、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また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。
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まったく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。
本発明では、必要に応じて蒸発工程を設けてもよい。蒸発工程では、乳化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また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または全部が撹拌・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。
エクストリュージョン処理とは、細孔を有するフィルターにリポソームを通過させることで、物理的なせん断力を施し、微粒化する工程を意味する。リポソームを通過させる際、リポソーム分散液及びフィルターを、リポソームを構成する膜の相転移温度以上の温度に保温することで、速やかに微粒化することができる。
本発明の薬物ローディング工程では、リポソームに水溶性薬物を封入させる場合、水和・膨潤させる水性媒体に薬物を溶解し、相転移温度以上での加熱、超音波処理又はエクストルージョン等の方法により薬物をリポソームの内水相に内包させることができる。また、脂質乳化時の水相に薬物を溶解させ内水相に内包させることもできる。
本発明では、浸透圧調整工程によって、リポソームの内水相を高張にすること(外水相との圧力差)によって、薬物が放出しやすくなる。浸透圧を設定することにより、リリース速度が制御できる。浸透圧調整工程として、特に限定されないが、薬物ローディング工程の後に透析などの方法が採用できる。これにより浸透圧を調整することができる。また、本発明では、薬物ローディング工程及び浸透圧調整工程(好ましくは内水相の浸透圧調整)を同時に行うことが、生産効率の観点から好ましい。
本発明のリポソーム組成物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組成物とするために、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)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組成物が調製できる。
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76/19/5のモル比となるようにそれぞれ16.6g、2.0g、4.3g取り、次いで有機溶媒(エタノール/酢酸エチル=3/1)405mlを加えて70℃に加温して脂質を溶解し油相とした。
6mM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、回転かき混ぜ式乳化機にて、周速20m/s、13000rpmにて30分間混合した。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と水とを蒸発させ、乳化前の容積に対して約1/10の体積になるまで濃縮し、薬物未内包リポソームを得た。このときの粒子径は66.9nmであった。
薬物ローディング
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。ゲムシタビン塩酸塩29.57g、PBS123.09g、日局注射用水172.59g、8M水酸化ナトリウム6.17mLを混合し、薬物溶液とした。つづいて、二つの容器にそれぞれ薬物溶液137.2mL、薬物未封入リポソーム140.0mL、8M水酸化ナトリウム2.8mLを混合した。この液の浸透圧は1039mOsm/Lであり、これが完成するリポソーム組成物の内水相の浸透圧となる。次にこの液を70℃で10分間加熱した後40℃まで冷却し、1018mMスクロース/37mMヒスチジン溶液で希釈した。希釈後、ひとつにまとめ、薬物ローディング液とした。
透析液として275mMスクロース/10mMヒスチジン水溶液を調製した。この液の溶質モル濃度より求めた浸透圧は285mOsm/Lであった。この透析液を用いて室温にて透析を行い、薬物ローディング液の外水相に存在する未封入のゲムシタビン塩酸塩と各溶質を除去し、透析液で外水相を置換した。以上の工程より、ゲムシタビン塩酸塩濃度0.71mg/mL、粒子径75.7nm、内水相浸透圧1039mOsm/L、外水相浸透圧285mOsm/L、内水相の外水相に対する浸透圧が3.6倍の薬物内包リポソーム組成物を得た。
a)脂質薄膜の作製
ナス型フラスコに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それぞれ1660mg、431mg、205mg取り、次いで有機溶媒(クロロホルム/メタノール=5/1)20mlを加えて溶解させた。つづいて、エバポレーターにて有機溶媒を除去し、ナスフラスコの内壁面に脂質薄膜を得た。
6mMリン酸緩衝液(pH7.86)を調製し水相とした。
脂質薄膜が形成されたナスフラスコに水相を20mL加え60℃にて脂質薄膜を水和した後、超音波洗浄機にナスフラスコを浸し脂質薄膜を分散することで、粗大な薬物未内包リポソーム液を得た。エクストルーダー(Mini Extruder、Avanti Polar Lipids社製)を用い、0.2μmフィルター及び0.05μmフィルターを順次通過させることで整粒し、薬物未内包リポソームを得た。得られた薬物未内包リポソームは、粒子径83.2nmであった。
薬物ローディング
ゲムシタビン塩酸塩1.1g、実施例1のd)で作製したPBS4.6g、日局注射用水6.5g、8M水酸化ナトリウム0.2mLを混合し、薬物溶液とした。つづいて、薬物溶液に薬物未封入リポソーム12.12g、8M水酸化ナトリウム0.2mLを混合した。次にこの液を70℃で10分間加熱した後40℃まで冷却した。この液の溶質モル濃度より求めた浸透圧は1039mOsm/Lであり、これが完成するリポソーム組成液の内水相浸透圧となる。つづいて、実施例1のd)で作製したPBSを2.7倍重量希釈し、薬物溶液を3倍希釈した。
透析液として275mMスクロース/10mMヒスチジン水溶液を調製した。この液の溶質モル濃度より求めた浸透圧は285mOsm/Lであった。この透析液を用いて室温にて透析を行い、薬物ローディング液の外水相に存在する未封入のゲムシタビン塩酸塩と各溶質を除去し、透析液で外水相を置換した。以上の工程より、ゲムシタビン濃度1.02mg/mL、粒子径89.6nm、内水相浸透圧1039mOsm/L、外水相浸透圧285mOsm/L、内水相の外水相に対する浸透圧が3.6倍の薬物内包リポソーム組成物を得た。
図1は、乳化法で製造されたリポソーム(実施例1)で製造されたリポソーム(比較例1)のDSCサーモグラムを示す。図2は、バンガム法で製造されたリポソーム(比較例1)のDSCサーモグラムを示す。20μLの薬剤内包リポソーム液をアルミニウムハーメチックパン(Tzero社製)に充填密封し、DSC Q2000(TA Instrument社製)にて、0.5℃/分の走査速度で25℃から70℃まで測定を実施した。本発明の乳化法で製造されたリポソーム実施例1は、指組みゲル相を示すピーク及びラメラゲル相を示すピークの2つのピークが明瞭に示され、リポソーム膜の組成が均一であることが分かる。
油相の調製において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をそれぞれ49.9g、6.1g、12.9g用いたこと、水相/油相混合液を回転かき混ぜ式乳化機にて周速19m/s、12000rpmにて30分間混合したこと、薬物ローディング工程において実施例1のd)で作製したPBSの代わりに10×PBS(ニッポンジーン社製)を使用し、2.7倍重量に希釈した実施例1のd)で作製したPBSの代わりに10×PBS(ニッポンジーン社製)を用いたこと以外は実施例1同様に作製し、ゲムシタビン濃度0.72mg/mL、粒子径84nm、薬物未内包率2.1%、内水相浸透圧1039mOsm/kg、外水相浸透圧285mOsm/kg、内水相の外水相に対する浸透圧が3.6倍の薬物内包リポソーム組成物を得た。
実施例2の薬物内包リポソームを2mLガラスバイアルに充填し5℃にて保管し、一定の時点で試料として一部サンプリングを実施した。この試料を用いて、下記各種評価を実施し、本発明におけるリポソーム組成物の安定性を評価した。
サンプリングした試料50μLをメタノールで20倍希釈(体積)することでリポソームに内包された薬物を抽出した。つづいて、抽出液を水で10倍希釈(体積)し、この液に含まれる薬物量をHPLCにて定量した結果を図3に示す。12ヶ月の長期間に渡り、本発明のリポソーム組成物における薬物が充分に安定であることが分かった。
サンプリングした試料50μLを水で10倍希釈(体積)し、限外ろ過フィルター(アミコンウルトラ-0.5 10kDa、ミリポア社製)を用い7400×g、30分間、4℃の条件で遠心ろ過を実施した。回収したろ液に含まれる薬物量をHPLCにて定量し、外水相に存在する薬剤の存在率(未内包率)を次の式により算出し、結果を図4に示す。
サンプリングした試料を1×PBS(Gibco、Life Technology社製)で33倍希釈(体積)し、FPAR-1000AS(大塚電子社製)を用い、動的光散乱法にて体積平均粒子径を測定した。結果を図5に示す。
サンプリングした試料50μLをマウス血漿で20倍希釈し、37℃で24時間インキュベートした。つづいて限外ろ過フィルター(アミコンウルトラ-0.5 10kDa、ミリポア社製)を用い7400×g、30分間、4℃の条件で遠心ろ過を実施した。回収したろ液に含まれる薬物量をHPLCにて定量し、血漿中に放出された薬物放出率を次の式により算出した。
式:薬物放出率(%)=(ろ液中の薬物濃度×20)÷製剤中薬物濃度×100
結果を図6に示す。
Claims (8)
- 有機溶媒に溶解して乳化された脂質から得られる、内水相を有するリポソームと、外水相を構成するリポソームを分散する水溶液とを有するリポソーム組成物であって、リポソームが溶解状態の水溶性薬物を内包し、内水相の浸透圧が外水相の浸透圧に対して2倍以上8倍以下であるリポソーム組成物。
- リポソームが、シングルラメラの構造を有する請求項1に記載のリポソーム組成物。
- リポソームの平均粒子径が、5nm以上100nm以下である請求項1又は2に記載のリポソーム組成物。
- リポソームが、水素添加大豆ホスファチジルコリン、1,2-ジステアロイル-3-ホスファチジルエタノールアミン-ポリエチレングリコール、及びコレステロールを少なくとも含む請求項1から3のいずれか一項に記載のリポソーム組成物。
- 請求項1から4のいずれか一項に記載のリポソーム組成物を含有する医薬組成物。
- 乾燥固化工程を経ずに、有機溶媒に溶解した脂質を乳化してリポソームを形成する乳化工程、
乳化工程で得られたリポソームに水溶性薬物を内包させる薬物ローディング工程、及び
リポソームの内水相の浸透圧を外水相の浸透圧に対して2倍以上8倍以下に調整する浸透圧調整工程
を有するリポソーム組成物の製造方法。 - 乳化工程の後に得られるリポソームをエクストリュージョン処理せずに次の工程に用いる請求項6に記載のリポソーム組成物の製造方法。
- 薬物ローディング工程及び浸透圧調整工程を同時に行う請求項6又は7に記載のリポソーム組成物の製造方法。
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WO2017078009A1 (ja) * | 2015-11-02 | 2017-05-11 | 富士フイルム株式会社 | リポソーム組成物およびその製造方法 |
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JPWO2015166987A1 (ja) | 2017-04-20 |
EP3138558B1 (en) | 2023-06-07 |
US20170042813A1 (en) | 2017-02-16 |
EP3138558A4 (en) | 2017-03-08 |
US10772834B2 (en) | 2020-09-15 |
US20170202774A1 (en) | 2017-07-20 |
EP3138558A1 (en) | 2017-03-08 |
JP6276847B2 (ja) | 2018-02-07 |
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