WO2015166985A1 - リポソーム組成物及びその製造方法 - Google Patents
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- WO2015166985A1 WO2015166985A1 PCT/JP2015/062982 JP2015062982W WO2015166985A1 WO 2015166985 A1 WO2015166985 A1 WO 2015166985A1 JP 2015062982 W JP2015062982 W JP 2015062982W WO 2015166985 A1 WO2015166985 A1 WO 2015166985A1
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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
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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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
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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
- A61K9/1278—Post-loading, e.g. by ion or pH gradient
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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 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 such as dispersion aids not approved for use are not preferred.
- suitability for aseptic filtration is also required.
- Patent Document 1 describes a gemcitabine-encapsulated drug carrier that can suppress the release rate of gemcitabine and maintain the local concentration of gemcitabine for a long period of time.
- a suitable release rate is obtained by containing cholesterol as a membrane constituent component in a specific ratio.
- a carrier containing cholesterol at a ratio of 0 to less than 35 mol% A drug carrier comprising liposomes encapsulating gemcitabine is described.
- the drug release rate from the liposome decreases as the cholesterol ratio decreases.
- Patent Document 2 discloses an aqueous solution containing liposomes in an aqueous medium, wherein the liposomes have an aqueous internal space separated from the medium by a membrane containing cholesterol and phosphatidylcholine, and the internal space is supplemented with irinotecan and sucrose 8 sulfate.
- An aqueous solution containing is disclosed.
- this liposome composition is sufficiently allowed to have an osmotic pressure in the liposome as high as 727 mmol / kg.
- a polyanion such as sucrose 8-sulfate
- Example 64 describes that an aqueous solution having an osmotic pressure of 727 mmol / kg was prepared.
- 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 diameter 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 attack on cancer cells is greatly affected by the exposure time of the drug.
- a drug such as an antimetabolite that inhibits DNA synthesis attacks only some cells in the DNA synthesis phase, and therefore effective cell killing cannot be obtained if the exposure time is short.
- the body metabolism after administration is fast, sufficient exposure time in the tumor cannot be obtained, and the expected drug effect is often not obtained.
- QOL quality of life
- the present invention has a long-term storage stability that is practically necessary, and by making the inner aqueous phase hypertonic, the release of the drug can be appropriately adjusted, and the drug on the order of several tens of hours It is an object of the present invention to provide a liposome composition having a release rate of 5 and a method for producing the same.
- the inventors have long-term storage stability necessary for practical use, and by making the inner aqueous phase hypertonic, the drug release can be adjusted moderately, on the order of several tens of hours.
- the present inventors have found a liposome composition having a drug release rate and a method for producing the same, and have completed the present invention.
- a liposome composition comprising a liposome having an inner aqueous phase and an aqueous solution in which the liposome constituting the outer aqueous phase is dispersed;
- the liposome encapsulates the drug in a dissolved state, and the osmotic pressure of the inner aqueous phase is 2 to 8 times the osmotic pressure of the outer aqueous phase,
- the liposome composition has a drug release rate from liposomes of 10% / 24 hr to 70% / 24 hr at 37 ° C. in plasma.
- the drug is at least one of an anticancer agent or an antimetabolite.
- the lipid constituting the liposome includes at least hydrogenated soybean phosphatidylcholine, 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol, and cholesterol.
- the average particle diameter of the liposome is 5 nm or more and 100 nm or less.
- the release rate of the drug from the liposome is 10% / 24 hr or less at 37 ° C. in physiological saline containing no blood component.
- 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.
- a method for producing a liposome composition having an osmotic pressure adjusting step of adjusting to hypertonicity is a method for producing a liposome composition having an osmotic pressure adjusting step of adjusting to hypertonicity.
- the osmotic pressure adjusting step adjusts the osmotic pressure of the inner aqueous phase of the liposome to 2 to 8 times the osmotic pressure of the outer aqueous phase.
- 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 it has long-term storage stability necessary for practical use, and by making the inner aqueous phase hypertonic, it is possible to moderately adjust the drug release, and in the order of several tens of hours.
- a liposome composition having a drug release rate and a method for producing the same can be provided.
- FIG. 1 is a plot of the relationship between incubation time and external water phase rate.
- FIG. 2 is a plot of the relationship between incubation time and release rate.
- FIG. 3 is a plot of the relationship between the drug concentration of gemcitabine hydrochloride and Cell Viability.
- FIG. 4 is a plot of the relationship between time after administration and free gemcitabine concentration in plasma.
- FIG. 5 is a plot of the relationship between tumor volume and days after transplantation when Capan-1 cells were transplanted into mice to form subcutaneous tumors.
- FIG. 6 is a plot of the relationship between the body weight of mice and the number of days after transplantation when Capan-1 cells were transplanted into mice to form subcutaneous tumors.
- FIG. 7 is a plot of the relationship between the number of days after transplantation and the tumor volume when BxPC-3 cells were transplanted into mice to form subcutaneous tumors.
- FIG. 8 is a plot of the relationship between the number of days after transplantation and the body weight of mice when BxPC-3 cells were transplanted into mice to form subcutaneous tumors.
- FIG. 9 is a plot of the relationship between the number of days after transplantation and the survival rate when SUIT-2 cells were transplanted into mice to form tumors.
- FIG. 10 is a plot of the relationship between the elapsed time of the liposome composition of the present invention and the drug concentration of gemcitabine hydrochloride.
- FIG. 11 is a plot of the relationship between the elapsed time of the liposome composition of the present invention and the abundance (non-encapsulation rate) of gemcitabine hydrochloride.
- FIG. 12 is a plot of the relationship between the elapsed time of the liposome composition of the present invention and the particle size of the liposome.
- FIG. 13 is a plot of the relationship between the elapsed time of the liposome composition of the present invention and the drug release rate released into plasma.
- FIG. 14 is a plot of the relationship between time after administration of the liposome composition of the present invention and plasma drug concentration.
- 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.
- Encapsulation rate refers to the ratio (mass ratio or mol ratio) of the drug encapsulated in the liposome to the incorporated drug (preparation amount) when the encapsulated drug carrier is formed by mixing the liposome components and the drug. ).
- “Release” means that the drug encapsulated in the liposome passes through the lipid membrane constituting the liposome and exits from the liposome.
- “Release rate” refers to the ratio (weight ratio or mol ratio) between a drug that exits from a liposome encapsulating a liposome component and the drug and the drug encapsulated in the liposome.
- “Slow release (release) rate” means that a small amount of drug goes out of the liposome per unit time.
- “Retention in blood” means a subject (“subject” or “individual”) to which a liposome composition (or a pharmaceutical composition containing the liposome composition) is administered, preferably human (patient), mouse, monkey In mammals such as livestock, it means a property (state) in which a drug encapsulated in liposomes is present in blood.
- “Tumor” (used in the present invention in the same meaning as “cancer (cancer)”) is specifically esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, laryngeal cancer, lung cancer, prostate cancer, bladder Examples include solid tumors such as cancer, breast cancer, uterine cancer, ovarian cancer, and Kaposi's sarcoma, and humoral tumors such as leukemia.
- the site where a tumor develops is a tumor cell, tissue, organ or organ and the interior thereof.
- a liposome composition comprising a liposome having an inner aqueous phase and an aqueous solution in which the liposome constituting the outer aqueous phase is dispersed;
- the liposome encapsulates the drug in a dissolved state, and the osmotic pressure of the inner aqueous phase is 2 to 8 times the osmotic pressure of the outer aqueous phase,
- the release rate of the drug from the liposome is 10% / 24 hr or more and 70% / 24 hr or less at 37 ° C. in plasma, It is a liposome composition.
- 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.
- lipid any lipid that can be dissolved in a mixed solvent of a water-soluble organic solvent and an ester-based organic solvent can be arbitrarily 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.
- the liposome composition of the present invention preferably does not contain an anionic polymer (polyanion).
- polyanion since the release property can be controlled by the osmotic pressure of the inner aqueous phase, it has an advantage that it is excellent in versatility and the drug that can be used for the liposome is not limited.
- 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, and bleomycin. And antimetabolites such as methotrexate, fluorouracil, gemcitabine, cytarabine, and pemetrexed, and the like. Among these, water-soluble drugs such as doxorubicin, gemcitabine, and pemetrexed are preferable.
- 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
- 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.
- dissolution may be promoted by the physicochemical environment created by the lipid membrane, or partly It shows that most of the drug is dissolved, for example, by taking it into the lipid membrane, and is considered to be encapsulated in a dissolved state.
- a highly water-soluble drug that is encapsulated by a loading method in which a solid substance is formed inside the liposome and the drug is encapsulated is not in the dissolved state referred to in the present invention.
- 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 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.
- 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 mixture of a water-soluble organic solvent and an ester organic solvent as a solvent, 40 g / L to 250 g / L, preferably 100 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 higher 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 and swollen, and the drug is dissolved by a method such as heating above the phase transition temperature, ultrasonic treatment, or extrusion. It can be included in the internal aqueous phase of the liposome. Alternatively, 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 inner aqueous phase of the liposome hypertonic (pressure difference) 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 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, intraocular 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.
- Release rate means the amount of drug that exits the liposome per unit time.
- the release rate in plasma at 37 ° C. is preferably 10% by mass / 24 hr to 70% by mass / 24 hr, more preferably 20% by mass / 24 hr to 60% by mass / 24 hr, more preferably 20% by mass / 24 hr. More preferably, it is 50 mass% / 24 hr or less.
- the release speed depends on the temperature, it is preferable to measure under a constant temperature condition.
- the temperature is not particularly limited, but it is preferably measured within the range of body temperature (35 ° C. or more and 38 ° C. or less).
- the drug contained in the liposome is an anticancer drug
- the release rate is less than 10% by mass / 24 hr, sufficient exposure time in the body as an anticancer drug cannot be obtained, and the expected efficacy
- liposomes containing anticancer drugs remain in the body for an unnecessarily long time, and accumulate in tissues that are difficult to distribute, such as skin, resulting in unexpected toxicity. May develop.
- the dose is greater than 70% by mass / 24 hr, the amount of drug to be exposed per unit time increases, so that the maximum blood concentration increases, resulting in increased toxicity.
- the leaked drug is transferred to tissues other than the tumor site. It is not preferable because the retention in the blood decreases due to distribution or rapid metabolism.
- the drug contained in the liposome can be measured by a method such as liquid high-speed chromatograph or mass spectrum.
- the dose of the drug varies depending on the administration subject, target organ, symptom, administration method, and the like.
- Is preferably administered by intravenous injection at about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg per day.
- an amount converted according to body weight and surface area can be administered with respect to the above-mentioned dose per 60 kg body weight.
- a tumor in the present invention, a tumor can be transplanted into a model animal (preferably mouse or rat) in order to measure the tumor volume.
- a model animal preferably mouse or rat
- the liposome composition of the present invention is administered to a subject such as a mammal, the effect of inhibiting the growth of the tumor volume can be observed. Inhibition of tumor volume growth depends on the drug used, the combination of lipids and the like comprising the liposomes, and the effective dose.
- inhibiting the growth of the tumor volume is meant at least one of being able to inhibit tumor growth, achieve tumor stasis, and substantial or complete tumor regression.
- the tumor cell transplantation for example, 100 tumor cells is distributed so that the model animal is distributed to a treatment group and a control group and the tumor cells are established. After growing to ⁇ 1000mm 2 you can start.
- the dosage can be administered at 0.01-100 mg / kg based on body weight at the start of treatment.
- the model animal is a mouse
- the weight of each group of mice was measured every day until the minimum weight was reached. The body weight of each group was then measured until the end of the experiment. Tumors can be measured with calipers, etc. until the final sacrifice for sampling, until the tumor reaches 2000 mm 3 or until the animal dies
- Tumor volume in a mammalian subject can be measured using any art-recognized method. For example, using the caliper measurement, using the formula: (a ⁇ b 2 ) ⁇ 0.5 (where “a” is the maximum diameter and “b” is the length of the minor axis) Tumor volume can be assessed. In humans, the tumor volume can be measured by techniques such as diagnostic imaging such as computed tomography (CT) scanning and magnetic resonance imaging (MRI) scanning.
- CT computed tomography
- MRI magnetic resonance imaging
- the liposome composition of the present invention and the method for producing the same have long-term storage stability that is practically necessary, and by making the inner aqueous phase hypertonic, the drug release can be appropriately adjusted and is on the order of several tens of hours. Liposome compositions with controlled drug release 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.
- All phases mean an oil phase and an aqueous phase.
- the osmotic pressure at this time becomes the inner aqueous phase osmotic pressure of the drug-encapsulating liposome.
- the particle size was adjusted by sequentially passing through a 0.2 ⁇ m filter and a 0.05 ⁇ m filter using an extruder (Mini Extruder, manufactured by Avanti Polar Lipids) under heating at 70 to 80 ° C.
- volume average particle diameter was measured by a dynamic light scattering method using Nanotrac UPA-UT (manufactured by Nikkiso Co., Ltd.) after diluting the sample 1000 times with water. The results are shown in Table 3.
- the release rate in plasma can be arbitrarily controlled by adjusting the osmotic pressure of the inner aqueous phase relative to the outer aqueous phase.
- Example 3 a) Preparation of the oil phase 16.6 g, 2.0 g, 4.3 g of hydrogenated soybean phosphatidylcholine, cholesterol and DSPE-PEG to a molar ratio of 76/19/5, respectively, and then an organic solvent (ethanol / acetic acid) Ethyl 75/25) 405 ml was added and heated to 70 ° C. to dissolve the lipid to obtain an oil phase.
- an organic solvent ethanol / acetic acid
- a drug-encapsulated liposome composition having a gemcitabine concentration of 0.68 mg / mL, a particle diameter of 73 nm, and an inner aqueous phase / outer aqueous phase osmotic pressure ratio of 3.6 times was obtained.
- Example 3 Measurement of Release Rate and External Aqueous Phase Ratio in Plasma 50 ⁇ L of the liposome composition obtained in Example 3 was adjusted so that mouse plasma would be 0%, 1%, 3%, 10%, 33%, and 100%, respectively. It was diluted 20 times (volume) with a solution diluted with PBS (Gibco, Life Technology), incubated at 37 ° C. for 24 hours, and 100 ⁇ L was collected at 0, 1, 4, 9, and 24 hours. Subsequently, centrifugal filtration was performed at 7400 ⁇ g for 30 minutes at 4 ° C. using an ultrafiltration filter (Amicon Ultra-0.5 10 kDa, manufactured by Merck Millipore).
- PBS Gibco, Life Technology
- Example 4 was prepared in the same manner as in Example 3 except that a drug loading solution was prepared by the method described below. Preparation of drug loading solution Gemcitabine hydrochloride 1.03 g, 10 ⁇ PBS (Gibco, manufactured by Life Technology) 17.92 g, JP injection water 10.76 g, 1M sodium hydroxide 3.42 mL were mixed to obtain a drug solution. . Subsequently, 27.0 mL of the drug solution and 27.0 mL of drug-unencapsulated liposomes were mixed. After heating at 70 ° C. for 10 minutes, the mixture was allowed to cool at room temperature for 30 minutes to obtain a drug loading solution. The osmotic pressure of this liquid was 1014 mOsm / L.
- Comparative Example 4 was prepared in the same manner as in Example 4 except that the amount of JP injection water was increased and the osmotic pressure was adjusted to 239 mOsm / L for the reduced amount of 10 ⁇ PBS (Gibco, manufactured by Life Technology).
- Comparative Example 5 was prepared in the same manner as in Example 4 except that the amount of JP injection water was reduced and the osmotic pressure was adjusted to 1482 mOsm / L for an amount of 10 ⁇ PBS (Gibco, Life Technology) increased.
- the average particle size is obtained by diluting the sample 100 times by weight with 1 ⁇ PBS (Gibco, Life Technology), and using a dynamic light scattering method using FPAR-100AS (Otsuka Electronics). Was measured. The results are shown in Table 5.
- Capan-1 human pancreatic cancer cell line
- free drug (gemcitabine hydrochloride) solution and drugs encapsulated in the liposome compositions of Example 4 and Comparative Examples 4 and 5 were added at various concentrations, and then cultured until 144 hours later.
- the cell viability was calculated from the amount of luminescence obtained by Luminescent Cell Viability Assay after completion of the culture. The results are shown in FIG.
- the drug action concentration (IC50) that inhibits cancer cell growth by 50% was determined.
- the free drug was 7 nM
- the liposome composition of Example 4 was 6 nM
- Comparative Example 4 was 22 nM
- Comparative Example 5 was 5 nM. It can be seen from FIG. 3 that the effectiveness of the liposome composition of Comparative Example 4 having a low release rate is significantly reduced.
- the liposome compositions of Example 4 and Comparative Examples 4 and 5 were administered to 7-week-old male BALB / c mice as a drug amount by 1 mg / kg tail vein injection under satiety conditions. Blood was collected for 15 minutes, 4 hours, and 24 hours after administration, and centrifuged to quantify the concentration of free gemcitabine in plasma. The results are shown in FIG. In addition, the blood concentration half-life (hereinafter also referred to as T1 / 2 ) was determined.
- the liposome composition of Example 4 was 15 hr
- Comparative Example 4 was 13 hr
- Comparative Example 5 was 13 hr.
- Example 5 was prepared in the same manner as Example 3 except that the scale of the drug loading solution was different.
- water for injection was reduced by increasing the PBS (10 ⁇ ) prepared in d) 1) of Example 3 to adjust the osmotic pressure
- Comparative Example 6 was prepared in d) 1) of Example 3. It was prepared in the same manner as in Example 3 except that the amount of water for injection was reduced by further increasing PBS (10 ⁇ ). The release speed was measured in the same manner as in Example 4.
- the sample was diluted 33 times with 1 ⁇ PBS (Gibco, Life Technology), and the cumulant average particle size was measured by a dynamic light scattering method using FPAR-100AS (Otsuka Electronics). The results are shown in Table 6.
- Capan-1 subcutaneous transplantation tumor-bearing model mouse 1 ⁇ 10 7 Capan-1 cells, a human pancreatic cancer cell line, were transplanted subcutaneously into the flank of female nude mice to form subcutaneous tumors.
- free gemcitabine aqueous solution 240 mg / 10 mL / kg / week
- liposome compositions of Examples 5 and 6 and Comparative Example 6 4 mg / 10 mL / kg / week
- solvent reference (9.4% sucrose) was started.
- a free gemcitabine aqueous solution, a liposome composition and a solvent reference were administered via the tail vein, and one course was performed once a week for a total of three courses.
- the tumor volume 32 days after transplantation was measured with calipers, and the inhibitory effect of subcutaneous tumors was evaluated. The results are shown in FIG.
- body weight variation was measured as a measure of toxicity. The results are shown in FIG.
- Example 7 was prepared in the same manner as Example 3 except that the preparation of the drug loading solution described below was different.
- Preparation of drug loading solution Gemcitabine hydrochloride 1.02 g, 10 ⁇ PBS (Gibco, Life Technology) 17.82 g, JP injection water 10.76 g, 1M sodium hydroxide 3.42 mL were mixed to obtain a drug solution. .
- 27.00 mL of the drug solution, 27.00 mL of drug-unencapsulated liposomes, and 3.42 mL of 1M sodium hydroxide were placed in the vial and mixed. The mixture was heated at 70 ° C. for 10 minutes and then allowed to cool at 40 ° C. for 30 minutes.
- This solution was used as a drug loading solution.
- the osmotic pressure is 1084 mOsm / L, which becomes the inner aqueous phase osmotic pressure of the completed liposome composition.
- the release speed was measured in the same manner as in Example 4.
- the sample was diluted 100 times with 1 ⁇ PBS (Gibco, Life Technology), and the cumulant average particle size was measured by a dynamic light scattering method using FPAR-100AS (Otsuka Electronics). The results are shown in Table 7.
- BxPC-3 subcutaneous transplantation tumor-bearing model mouse This test confirmed the effect on free gemcitabine against cancer cells that are insensitive to in vivo.
- 5 ⁇ 10 6 BxPC-3 cells, a human pancreatic cancer cell line that is insensitive to free gemcitabine in vivo were transplanted subcutaneously into the flank of female nude mice to form subcutaneous tumors.
- administration of a free gemcitabine aqueous solution 240 mg / 10 mL / kg / week
- the liposome compositions of Examples 6 to 9 4 mg / 10 mL / kg / week
- tumor administration was significantly improved in the administration group of Example 7 in spite of equivalent weight loss indicating toxicity, and surprisingly high tumor suppressive effect was also obtained against the free gemcitabine low-sensitivity cell line. It turns out to have.
- Example 8 was prepared in the same manner as Example 3 except that the scale of the drug loading solution was different. The release speed was measured in the same manner as in Example 4. For the average particle size, the sample was diluted 33 times with 1 ⁇ PBS (Gibco, Life Technology), and the cumulant average particle size was measured by a dynamic light scattering method using FPAR-100AS (Otsuka Electronics). The results are shown in Table 8.
- Example 9 Example 3 d) Instead of PBS (10 ⁇ ) prepared in 1), 10 ⁇ PBS (Gibco, manufactured by Life Technology) was used, the internal aqueous phase osmotic pressure was different, and the scale of the drug loading solution was Except for differences, it was prepared in the same manner as in Example 3, gemcitabine concentration 0.71 mg / mL, particle diameter 84 nm, drug non-encapsulation rate 2.1%, inner aqueous phase osmotic pressure 940 mOsm / L, outer aqueous phase osmotic pressure 285 mOsm / L A drug-encapsulated liposome composition having an osmotic pressure of 3.3 times the inner aqueous phase relative to the outer aqueous phase was obtained.
- the drug-encapsulated liposome of Example 9 was filled in a 2 mL 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, and the stability of the liposome composition in the present invention was measured.
- 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. The results are shown in FIG. It has been found that the drug in the liposome composition of the present invention is sufficiently stable over a long period of 24 months.
- Unencapsulated rate (%) (Drug concentration in filtrate ⁇ 10) ⁇ Drug concentration in formulation ⁇ 100 The results are shown in FIG.
- Example 10 The same procedure as in Example 3 was performed except for the drug loading step.
- drug loading 1.9 g of pemetrexed disodium heptahydrate, 2.4 g of sodium chloride, and 24 g of water for injection were mixed and dissolved by heating at 45 ° C. to obtain a drug solution.
- 8.0 mL of the drug solution and 8.0 mL of the drug non-encapsulated liposome were mixed and heated at 70 ° C. for 10 minutes to obtain a drug loading solution. This solution was dialyzed to complete the liposome composition.
- the liposome composition of Example 10 was administered to 8-week-old male C3H mice as a drug amount by 6 mg / kg tail vein injection under non-fasting conditions. Blood was collected at 15 minutes, 1 hour, 4 hours, and 24 hours after administration, and the concentration of free pemetrexed in plasma was quantified to determine T1 / 2 . The results are shown in FIG. In the case of the aqueous drug solution, T 1/2 was 2 hours, whereas in the liposome composition of Example 10, 17 hours was sufficiently longer than the aqueous drug solution, and the DDS preparation had a sufficiently long residence in blood. It was confirmed to show sex.
- Table 9 shows the osmotic pressure ratio, release rate, in vitro test, and in vivo test related to the liposome composition of the present invention. From the results shown in Table 9, the liposome encapsulates the dissolved drug, the osmotic pressure of the inner aqueous phase is 2 to 8 times the osmotic pressure of the outer aqueous phase, and the drug release rate from the liposome However, it can be seen that the liposome composition that is 10% / 24 hr or more and 70% / 24 hr or less in plasma at 37 ° C. has good cancer cell growth inhibitory activity and has excellent blood retention.
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Abstract
Description
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。
また、曝露時間を充分に得るために、希薄濃度の抗がん剤を点滴によって長時間曝露させる投与方法もあるが、患者が点滴時間中拘束されるなどQOL(quality of life;生活の質)の観点において好ましくない。
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倍以下であり、
リポソームからの薬物のリリース速度が、血漿中37℃において10%/24hr以上70%/24hr以下であるリポソーム組成物である。
好ましくは、薬物が抗がん剤又は代謝拮抗剤の少なくとも一つである。
好ましくは、リポソームを構成する脂質が、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。
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、リポソームからの薬物のリリース速度が、血液成分を含まない生理食塩水中37℃において10%/24hr以下である。
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
内包されなかった薬物水溶液を低張の液で置換することで内水相の浸透圧を外水相の浸透圧に対して高張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、浸透圧調整工程が、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。
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。
「放出」とは、リポソームに封入された薬物が、リポソームを構成する脂質膜を通過して、リポソームの外部へ出ることを意味する。
「放出率」とは、リポソームの構成成分と薬物とを封入したリポソームから外部へ出る薬物と、リポソームに封入された薬物との比率(重量比またはmol比)をいう。
「リリース(放出)速度が遅い」とは、単位時間あたりにリポソームの外部へ出る薬物量が少ないことを意味する。
「血中滞留性」とは、リポソーム組成物(又はリポソーム組成物を含有する医薬組成物)を投与した対象(「被検体」又は「個体」であり、好ましくはヒト(患者)、マウス、サル、家畜などの哺乳動物)において、リポソームに封入された状態の薬物が血液中に存在する性質(状態)を意味する。
「腫瘍」(本発明では、「がん(癌)」と同義で用いる)とは、具体的には食道癌、胃癌、結腸癌、直腸癌、肝臓癌、喉頭癌、肺癌、前立腺癌、膀胱癌、乳癌、子宮癌、卵巣癌、カポジ肉腫などの固形腫瘍、また白血病などの液性腫瘍が挙げられる。腫瘍が発生する部位は、腫瘍の細胞、組織、器官または臓器及びそれらの内部などである。
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倍以下であり、
リポソームからの薬物のリリース速度が、血漿中37℃において10%/24hr以上70%/24hr以下である、
リポソーム組成物である。
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。
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。
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、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倍以下に調整する薬物ローディング工程
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(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。
リリース速度とは、単位時間あたりにリポソームの外部へ出る薬物量を意味する。本発明において、リリース速度は、血漿中37℃において、10質量%/24hr以上70質量%/24hr以下が好ましく、20質量%/24hr以上60質量%/24hr以下がより好ましくは20質量%/24hr以上50質量%/24hr以下がさらに好ましい。
また、薬物のリリース速度を測定する際、薬物の投与量は、投与対象、対象臓器、症状、投与方法などによっても異なるが、例えば、注射剤では、例えば、ヒト(患者;体重60kgとして)においては、一日につき約0.01~30mg程度、好ましくは約0.1~20mg程度、より好ましくは約0.1~10mg程度を静脈注射により投与するのが好ましい。他の動物の場合も、上記の体重60kg当たりの投与量に対して、体重及び表面積に応じて換算した量を投与することができる。
本発明では、腫瘍体積を測定するために、モデルとなる動物(好ましくはマウスもしくはラット)に対して腫瘍を移植できる。本発明のリポソーム組成物を哺乳動物等の対象に投与する場合、腫瘍の体積の増殖抑制という効果が観測できる。腫瘍の体積の増殖抑制は、使用される薬物、リポソームを構成する脂質等の組合せ、及び有効量に依存する。腫瘍体積の増殖抑制とは、腫瘍成長を抑制し得るか、腫瘍静止を達成し得るか、及び実質的若しくは完全な腫瘍退縮の少なくとも一つを意味する。
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。
全相とは、油相及び水相を意味する。
a)油相の調製
水素添加大豆ホスファチジルコリン、コレステロールを76/19のモル比となるようにそれぞれ1.79g、0.22g取り、次いで有機溶媒(エタノール/酢酸エチル=1/1)15mlを加えて70℃に加温して脂質を溶解し油相とした。
ゲムシタビン塩酸塩を注射用水と10×PBS(pH7.4)(ニッポンジーン社製)を用い、8mg/mLとなるように溶解した。その際、注射用水とPBSの比率を変更することで、下表の浸透圧になるよう調整した。
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、乳化機(エクセルオートホモジナイザーED-3、日本精機製作所製)にて、3000rpmにて10分間混合し、つぎに6000rpmにて10分間混合し、つぎに12000rpmにて10分間混合した。その後、水素添加大豆ホスファチジルコリン、コレステロール、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)とのモル比が76/19/5となるように0.41gのDSPE-PEGを水溶液として添加した。つづいて、70℃での加温を維持し攪拌を続けることで有機溶媒と水を蒸散させ、表2に示す浸透圧となった時点で加温と攪拌を止め、蒸散を停止した。このときの浸透圧が完成する薬物内包リポソームの内水相浸透圧となる。つづいて、70~80℃の加温下でエクストルーダー(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た。
透析液として水で10倍容量に希釈した10×PBS(pH7.4)(ニッポンジーン社製)を調製した。この液の浸透圧は307mOsm/Lであった。この浸透圧が完成する薬物内包リポソームの外水相浸透圧となる。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。
体積平均粒子径は試料を水で1000倍重量に希釈し、ナノトラックUPA-UT(日機装社製)を用いた動的光散乱法で測定した。結果を表3に示す。
実施例1、2及び比較例1~3のリポソーム組成物50μLをマウス血漿で20倍希釈(体積)し、37℃で24時間インキュベートし、0、1、4、9、24時間の時点で100μL採取した。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、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、リリース速度及び外水相率を次の式により算出した。
式:リリース速度(%/24hr)=(インキュベーション24時間後のろ液中の薬物量-インキュベーション前のろ液中の薬物量)×20÷リポソーム組成物の全相に含まれる薬量×100
式:外水相率(%)=(ろ液中の薬物濃度×10)÷製剤中薬物濃度×100
結果を表3及び図1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=75/25)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で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67.0nmであった。
薬物ローディング
1)PBS(10×)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(10×)とした。実施例3ではここで調製したPBSを用いた。
2)薬物ローディング液の調製
ゲムシタビン塩酸塩7.68g、PBS(10×)31.99g、日局注射用水44.83g、8N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、薬物未封入リポソーム18.00mL、8N水酸化ナトリウム0.36mLを混合した。70℃で10分間加熱した後40℃で30分間かけて放冷した。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浸透圧になる。つづいて、2.7倍希釈したPBS(10×)で希釈した。1つのバイアルにまとめて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0.68mg/mL、粒子径73nm、内水相/外水相浸透圧比3.6倍の薬物内包リポソーム組成物を得た。
実施例3で得られたリポソーム組成物50μLを、それぞれマウス血漿が0%、1%、3%、10%、33%、100%となるようにPBS(Gibco、Life Technology社製)で希釈した溶液で20倍希釈(体積)し、37℃で24時間インキュベートし、0、1、4、9、24時間の時点で100μL採取した。つづいて、限外ろ過フィルター(アミコンウルトラ-0.5 10kDa、メルクミリポア社製)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、リリース速度及び外水相率を次の式により算出した。
式:リリース速度(%/24hr)=(インキュベーション24時間後のろ液中の薬物量-インキュベーション前のろ液中の薬物量)×20÷リポソーム組成物全相に含まれる薬量×100
式:外水相率(%)=(各インキュベーション時点のろ液中の薬物量-インキュベーション前のろ液中の薬物量)×20÷リポソーム組成物全相に含まれる薬量×100
結果を表4及び図2に示す。
実施例4は下記に記載する方法で薬物ローディング液を調製した以外は実施例3同様に作製した。
薬物ローディング液の調製
ゲムシタビン塩酸塩1.03g、10×PBS(Gibco、Life Technology社製)17.92g、日局注射用水10.76g、1M水酸化ナトリウム3.42mLを混合し、薬物溶液とした。つづいて、薬物溶液27.0mL、薬物未封入リポソーム27.0mLを混合した。70℃で10分間加熱した後室温で30分間かけて放冷し、薬物ローディング液とした。この液の浸透圧は1014mOsm/Lであった。
比較例4は、10×PBS(Gibco、Life Technology社製)を減量した分量について日局注射用水を増量し、浸透圧を239mOsm/Lに調整した以外は実施例4と同様に作製した。
比較例5は、10×PBS(Gibco、Life Technology社製)を増量した分量について日局注射用水を減量し、浸透圧を1482mOsm/Lに調整した以外は実施例4と同様に作製した。
平均粒子径は試料を1×PBS(Gibco、Life Technology社製)で100倍重量に希釈し、FPAR-100AS(大塚電子)を用いた動的光散乱法でキュムラント平均粒子径を測定した。結果を表5に示す。
実施例4及び比較例4、5で得られたリポソーム組成物50μLをマウス血漿で20倍希釈(体積)し、37℃で24時間インキュベートし、限外ろ過フィルター(アミコンウルトラ-0.5 10kDa、メルクミリポア社製)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、リリース速度を次の式により算出した。
式:リリース速度(%/24hr)=(インキュベーション24時間後のろ液中の薬物量-インキュベーション前のろ液中の薬物量)×20÷リポソーム組成物の内水相に含まれる薬量×100
結果を表5に示す。
Capan-1(ヒト膵がん細胞株)を2×103Cells/mL/wellを96ウェルプレートに播種した。つづいて、遊離薬物(ゲムシタビン塩酸塩)溶液、実施例4及び比較例4、5のリポソーム組成物に内包された薬物を各濃度添加した後、144時間後まで培養した。培養終了後にLuminescent Cell Viability Assayにより得られた発光量から細胞生存率を算出した。結果を図3に示す。また、がん細胞増殖を50%阻害する薬物作用濃度(IC50)を求めた。遊離薬物が7nM、実施例4のリポソーム組成物が6nM、比較例4が22nM、比較例5が5nMを示した。図3からリリース速度が低い比較例4のリポソーム組成物における有効性が顕著に低下していることが分かる。
7週齢雄BALB/cマウスに飽食条件下で実施例4及び比較例4、5のリポソーム組成物を、薬物量として1mg/kg尾静脈注射により投与した。投与後15分間、4時間、24時間で採血し、遠心分離を施し血漿中の遊離ゲムシタビン濃度を定量した。結果を図4に示す。また、血中濃度半減期(以下、T1/2ともいう)を求めた。実施例4のリポソーム組成物が15hr、比較例4が13hr、比較例5が13hrを示した。マウス中における遊離ゲムシタビンのT1/2は0.7hrであり、いずれのリポソーム組成物も血中滞留性が大幅に向上しているが、リリース速度が90%/hrである比較例4は、比較的滞留性が乏しいことが分かった。このことは、薬物が速やかな代謝を受けるため、血中での漏出が多過ぎると濃度が低下することを示している。
実施例5は薬物ローディング液のスケールが異なる以外は実施例3と同様に作製した。実施例6は浸透圧調節のため実施例3のd)1)で調製したPBS(10×)を増量した分注射用水を減量し、比較例6は実施例3のd)1)で調製したPBS(10×)をさらに増量した分注射用水を減量した以外は実施例3と同様に作製した。
実施例4と同様にリリース速度を測定した。
平均粒子径は試料を1×PBS(Gibco、Life Technology社製)で33倍重量に希釈し、FPAR-100AS(大塚電子)を用いた動的光散乱法でキュムラント平均粒子径を測定した。
結果を表6に示す。
ヒト膵臓癌細胞株であるCapan-1細胞1×107個を雌性ヌードマウスの脇腹部皮下に移植し皮下腫瘍を形成させた。移植後11日目から遊離ゲムシタビン水溶液(240mg/10mL/kg/週)、実施例5、6及び比較例6のリポソーム組成物(4mg/10mL/kg/週)並びに溶媒参照(9.4%スクロース)を投与開始した。遊離ゲムシタビン水溶液、リポソーム組成物及び溶媒参照を尾静脈投与して、1週間1回を1クール、合計3クール実施した。移植後32日後の腫瘍体積をノギスで測定し、皮下腫瘍の抑制効果について評価した。結果を図5に示す。また、毒性を示す尺度として体重変動を測定した。結果を図6に示す。
実施例7は下記に記載する薬物ローディング液の調製が異なる以外は実施例3と同様に作製した。
薬物ローディング液の調製
ゲムシタビン塩酸塩1.02g、10×PBS(Gibco、Life Technology社製)17.82g、日局注射用水10.76g、1M水酸化ナトリウム3.42mLを混合し、薬物溶液とした。つづいて、バイアルに薬物溶液27.00mL、薬物未封入リポソーム27.00mL、1M水酸化ナトリウム3.42mLを入れ、混合した。70℃で10分間加熱した後40℃で30分間かけて放冷した。この液を薬物ローディング液とした。浸透圧は1084mOsm/Lであり、これが完成するリポソーム組成物の内水相浸透圧になる。
実施例4と同様にリリース速度を測定した。
平均粒子径は試料を1×PBS(Gibco、Life Technology社製)で100倍重量に希釈し、FPAR-100AS(大塚電子)を用いた動的光散乱法でキュムラント平均粒子径を測定した。
結果を表7に示す。
遊離ゲムシタビンに対してin vivoにおいて低感受性である癌細胞に対する効果を確認した試験である。
遊離ゲムシタビンに対してin vivoで低感受性となるヒト膵臓癌細胞株であるBxPC-3細胞5×106個を雌性ヌードマウスの脇腹部皮下に移植し皮下腫瘍を形成させた。移植後46日目から遊離ゲムシタビン水溶液(240mg/10mL/kg/週)及び実施例6~9のリポソーム組成物(4mg/10mL/kg/週)を投与開始した。遊離ゲムシタビン水溶液を腹腔内投与、リポソーム組成物と溶媒対照(9.4%スクロース)を尾静脈投与として、1週間1回を1クールとして、合計2クール実施した。移植後60日後の腫瘍体積をノギスで測定し、皮下腫瘍の抑制効果について評価した。結果を図7に示す。また、毒性を示す尺度として体重変動を測定した。結果を図8に示す。
実施例8は薬物ローディング液のスケールが異なる以外は実施例3同様に作製した。
実施例4と同様にリリース速度を測定した。
平均粒子径は試料を1×PBS(Gibco、Life Technology社製)で33倍重量に希釈し、FPAR-100AS(大塚電子)を用いた動的光散乱法でキュムラント平均粒子径を測定した。
結果を表8に示す。
ヒト膵臓癌細胞株であるSUIT-2細胞1×106個を雌性ヌードマウスの膵臓に移植し腫瘍を形成させた。癌細胞を移植しないが、同様の開腹手術を施した参照群をsham群とした。転移及び腹膜播種の認められる移植後7日目から遊離ゲムシタビン水溶液(240mg/10mL/kg)、実施例8のリポソーム組成物(4mg/10mL/kg)及び溶媒参照(9.4%スクロース)を尾静脈投与し、移植後91日までの生存曲線を求めた。結果を図9に示す。
実施例3のd)1)で調製したPBS(10×)の代わりに10×PBS(Gibco、Life Technology社製)を用いたこと、内水相浸透圧が異なること及び薬物ローディング液のスケールが異なること以外は実施例3同様に作製し、ゲムシタビン濃度0.71mg/mL、粒子径84nm、薬物未内包率2.1%、内水相浸透圧940mOsm/L、外水相浸透圧285mOsm/L、内水相の外水相に対する浸透圧が3.3倍の薬物内包リポソーム組成物を得た。
実施例9の薬物内包リポソームを2mLバイアルビンに充填し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測定した。
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10に示す。24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(ミリポア製アミコンウルトラ-0.5 10kDa)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た。
式:未内包率(%)=(ろ液中の薬物濃度×10)÷製剤中薬物濃度×100
結果を図11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、動的光散乱法測定を大塚電子社製FPAR-1000ASにて体積平均粒子径を測定した。結果を図12に示す。12ヶ月の長期間に渡り粒子径の変化がほとんどなく、本発明のリポソーム組成物の粒子は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。つづいて外ろ過フィルター(ミリポア製アミコンウルトラ-0.5 10kDa)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13に示す。
薬剤ローディング工程以外は、実施例3と同様に作製した。薬剤ローディングは、ペメトレキセド2ナトリウム7水和物1.9g、塩化ナトリウム2.4g、注射用水24gを混合し、45℃で加熱して溶解させ、薬物溶液とした。続いて、薬物溶液8.0mL、薬剤未封入リポソーム8.0mLを混合し、70℃で10分加熱し、薬剤ローディング液とした。この液を透析してリポソーム組成物を完成させた。
Claims (10)
- 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倍以下であり、
リポソームからの薬物のリリース速度が、血漿中37℃において10%/24hr以上70%/24hr以下であるリポソーム組成物。 - 薬物が、抗がん剤又は代謝拮抗剤の少なくとも一つである請求項1に記載のリポソーム組成物。
- リポソームを構成する脂質が、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又は2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から3のいずれか一項に記載のリポソーム組成物。
- リポソームからの薬物のリリース速度が、血液成分を含まない生理食塩水中37℃において10%/24hr以下である請求項1から4のいずれか一項に記載のリポソーム組成物
- 請求項1から5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、及び内包されなかった薬物水溶液を低張の液で置換することで内水相の浸透圧を外水相の浸透圧に対して高張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。 - 浸透圧調整工程が、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倍以下に調整する請求項7に記載の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7又は8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7~9のいずれか一項に記載のリポソーム組成物の製造方法。
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US11166914B2 (en) | 2015-11-02 | 2021-11-09 | Fujifilm Corporation | Tumor therapeutic agent and kit containing gemcitabine liposome composition |
US11166913B2 (en) | 2015-11-02 | 2021-11-09 | Fujifilm Corporation | Tumor therapeutic agent and kit containing gemcitabine liposome composition |
WO2019244978A1 (ja) | 2018-06-20 | 2019-12-26 | 富士フイルム株式会社 | ゲムシタビンを内包するリポソーム組成物および免疫チェックポイント阻害剤を含む組合せ医薬 |
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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JPWO2015166985A1 (ja) | 2017-04-20 |
US20170042810A1 (en) | 2017-02-16 |
US10646442B2 (en) | 2020-05-12 |
EP3138557A1 (en) | 2017-03-08 |
JP6263609B2 (ja) | 2018-01-17 |
EP3138557B1 (en) | 2023-06-07 |
EP3138557A4 (en) | 2017-03-08 |
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