WO2017097196A1 - 脂质体的制备方法 - Google Patents

脂质体的制备方法 Download PDF

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Publication number
WO2017097196A1
WO2017097196A1 PCT/CN2016/108840 CN2016108840W WO2017097196A1 WO 2017097196 A1 WO2017097196 A1 WO 2017097196A1 CN 2016108840 W CN2016108840 W CN 2016108840W WO 2017097196 A1 WO2017097196 A1 WO 2017097196A1
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Prior art keywords
phosphatidylcholine
liposome
water
group
aqueous phase
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English (en)
French (fr)
Inventor
张喜全
董平
张焕青
程艳菊
周浩
蒋波
刘飞
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Priority to EP16872387.2A priority Critical patent/EP3388055B1/en
Priority to JP2018529305A priority patent/JP6884783B2/ja
Priority to US15/782,025 priority patent/US20180361342A1/en
Priority to CN201680071216.4A priority patent/CN108289846B/zh
Publication of WO2017097196A1 publication Critical patent/WO2017097196A1/zh
Anticipated expiration legal-status Critical
Priority to US16/928,424 priority patent/US20200338519A1/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/08—Simple coacervation, i.e. addition of highly hydrophilic material
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
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    • 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
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • 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
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Disinfection or sterilisation of materials or objects, in general; Accessories therefor
    • A61L2/02—Disinfection or sterilisation of materials or objects, in general; Accessories therefor using physical processes
    • A61L2/022—Filtration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2103/00—Materials or objects being the target of disinfection or sterilisation
    • A61L2103/05—Living organisms or biological materials

Definitions

  • the present application relates to novel liposome preparation methods and liposomes prepared by such methods.
  • emulsions and micelle formulations have some disadvantages.
  • emulsions are thermodynamically unstable systems, which tend to accumulate, fuse, flocculate, oxidize, degrade, hydrolyze, etc. during storage, thereby affecting the quality of the emulsion and the efficacy of the drug.
  • micellar formulations typically utilize a surfactant to form micelles to solubilize the drug.
  • surfactants can cause toxic side effects during clinical use, trigger hypersensitivity reactions, and affect drug safety.
  • liposomes can alter the in vivo distribution of drugs, reduce the toxicity of drugs, alleviate allergic reactions and immune responses, and delay the release of drugs
  • liposome preparations obtained by existing methods are usually in a liquid state and are thermodynamically unstable.
  • the system has the problem of low stability, low encapsulation efficiency, easy leakage of the drug, bacterial growth and sedimentation during storage, difficulty in controlling the particle size, and wide particle size distribution. Even after reconstitution into a liquid liposome after freeze-drying, it is difficult to be reconstituted and the reconstituted liposome has a large particle size and a wide particle size distribution.
  • Mexiticol is insoluble or almost insoluble in water and aqueous media and is a poorly water-soluble drug.
  • the stability is poor and the micelle formulation is too toxic. Therefore, there is an urgent need to develop new formulations suitable for poorly water-soluble or water-insoluble drugs and methods for their preparation.
  • the application provides a method of preparing a liposome comprising:
  • the present application also provides another method of preparing liposomes comprising:
  • the lyoprotectant is added to the aqueous phase of step (1) or prior to the sterile filtration in step (3).
  • the present application provides a liposome obtained by the above preparation method, characterized in that the liposome is reconstituted by adding water or an aqueous solvent, and the reconstituted liposome has a particle diameter of 50 to 400 nm.
  • a reaction including a “catalyst” includes a catalyst, or two or more catalysts.
  • the term “or” is generally used in its meaning including “and/or” unless it is specifically defined otherwise.
  • the application provides a method of preparing a liposome comprising:
  • the lyoprotectant may be added in the aqueous phase of step (1) or prior to the freeze drying in step (3).
  • water for injection is added and dispensed prior to performing the freeze-drying in step (3).
  • step (3) prior to lyophilization in step (3), water for injection is added and sterilized for filtration and then dispensed.
  • the present application also provides another method of preparing liposomes comprising:
  • the lyoprotectant is added to the aqueous phase of step (1) or prior to the sterile filtration in step (3).
  • the phospholipid is selected from one or more of egg yolk phosphatidylcholine, hydrogenated egg phosphatidylcholine, soybean phosphatidylcholine or hydrogenated soybean phosphatidylcholine. More preferably, the phospholipid is selected from the group consisting of egg yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine a combination of bases. Still more preferably, the phospholipid is selected from the group consisting of a combination of egg yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine in a weight ratio of 3:1.
  • the organic solvent is selected from the group consisting of anhydrous ethanol, 95% ethanol, methanol, propanol, chloroform, dichloromethane, tert-butanol, n-butanol, acetone, methylpyrrolidone One or more of ethyl acetate, isopropyl ether or diethyl ether.
  • the organic solvent is selected from the group consisting of absolute ethanol, 95% ethanol or tert-butanol. More preferably, the organic solvent is selected from the group consisting of anhydrous ethanol.
  • the weight ratio of the substance to be encapsulated to the phospholipid is from 1:1 to 1:500.
  • the weight ratio of the substance to be encapsulated to the phospholipid is from 1:1 to 1:100. More preferably, the weight ratio of the substance to be encapsulated to the phospholipid is from 1:1 to 5:50. Still more preferably, the weight ratio of the substance to be encapsulated to the phospholipid is 1:20.
  • the weight ratio of the substance to be encapsulated to the organic solvent is from 1:1 to 1:100.
  • the weight ratio of the substance to be encapsulated to the organic solvent is from 1:9 to 1:50. More preferably, the weight ratio of the substance to be encapsulated to the organic solvent is 1:30.
  • the aqueous phase comprises or consists essentially of water, for example, deionized water, distilled water, purified water, water for injection, and the like, preferably water for injection.
  • the organic phase and the aqueous phase can be mixed under nitrogen.
  • the polycarbonate membrane has a pore size selected from the group consisting of 0.015, 0.03, 0.05, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 5.0, 8.0, 10.0 or 12.0 microns, preferably 0.1 microns or 0.2 microns.
  • a layer of polyester film can be added under the polycarbonate film.
  • the extrusion may be performed in any manner as long as a large particle size liposome can be passed through the membrane to become a small particle size liposome.
  • This step requires controlling the temperature of the feed liquid to be 25-80 ° C, preferably 55-65 ° C.
  • the lyoprotectant is selected from one or more of the group consisting of mannitol, glucose, galactose, sucrose, lactose, maltose, or trehalose.
  • the lyoprotectant is selected from one or more of sucrose, trehalose or mannitol. More preferably, the lyoprotectant is selected from the group consisting of sucrose or a combination of sucrose and mannitol. Still more preferably, the lyoprotectant is a combination of sucrose or a weight ratio of sucrose to mannitol of 2:1.
  • an antioxidant may also be added to the organic phase in step (1).
  • the antioxidant is selected from the group consisting of sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, ascorbyl palmitate, t-butyl p-hydroxyanisole (BHA), di-tert-butyl-p-cresol (BHT).
  • BHA t-butyl p-hydroxyanisole
  • BHT di-tert-butyl-p-cresol
  • the antioxidant is selected from the group consisting of sodium metabisulfite, t-butyl p-hydroxyanisole, Di-tert-butyl-p-cresol, vitamin E acetate. More preferably, the antioxidant is selected from the group consisting of di-tert-butyl-p-cresol or sodium metabisulfite.
  • the aqueous phase in step (1) may further comprise a metal ion chelating agent.
  • the metal ion chelating agent is selected from the group consisting of disodium edetate, sodium edetate, 1,2-diaminocyclohexanetetraacetic acid, diethylenetriaminepentaacetic acid, and N-(2-hydroxyethyl)-B. Trisodium diamine triacetate or N-bis(2-hydroxyethyl)glycine.
  • the metal ion chelating agent is selected from the group consisting of disodium edetate or sodium calcium edetate.
  • the pH adjusting agent may also be added prior to performing freeze drying in step (3) or after adding a lyoprotectant in step (3).
  • the pH adjusting agent is selected from the group consisting of hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, citric acid, tartaric acid, maleic acid, sodium hydroxide, sodium hydrogencarbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate or sodium citrate.
  • the pH adjusting agent is selected from the group consisting of hydrochloric acid or sodium hydroxide.
  • the pH is adjusted in the range of 2-10, preferably 4-7.
  • the liposome obtained by the preparation method of the present application is rapidly reconstituted after addition of water or an aqueous solvent, and the reconstituted liposome has a particle diameter of 50 to 400 nm, preferably 100 to 250 nm. .
  • the particle size dispersion coefficient is ⁇ 0.5, preferably, the particle size dispersion coefficient is ⁇ 0.23.
  • Figure 4 Distribution of fluorescent marker IR623 and IR623-labeled mexican liposome in mouse tumor sites and organs.
  • the body fluid; the obtained liposome solution is extruded through a 0.1 ⁇ m polycarbonate film to obtain a liposome liquid having a certain particle size and a certain particle size distribution; adding a formula amount of sucrose, and adding water for injection Dilute to 1000g; dispense into neutral borosilicate glass controlled injection bottle (Western bottle) and freeze-dry.
  • the encapsulation efficiency was >99% as measured by ultrafiltration.
  • Preparation process the formulated amount of tacrolimus, egg yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine are dissolved in the formula amount of absolute ethanol, and dissolved by heating at 60 ° C, as an organic phase; % of water for injection, heated to 60 ° C, as an aqueous phase; while shearing the aqueous phase, the organic phase was added to the aqueous phase to obtain a liposome feed; the resulting liposome solution was passed through a 0.1 ⁇ m polycarbonate The film was extruded 3 times; the amount of sucrose was added, and diluted with water for injection to 1000 g; it was dispensed into a neutral borosilicate glass controlled injection bottle (west bottle) and then freeze-dried. The encapsulation efficiency was >99% as measured by ultrafiltration.
  • Preparation process preparing a formula amount of alprazolam, egg yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine in a formula amount of absolute ethanol, heating and dissolving at 60 ° C, as an organic phase; taking a formula of 70 % of water for injection, heated to 60 ° C, as an aqueous phase; while shearing the aqueous phase, the organic phase was added to the aqueous phase to obtain a liposome feed; the resulting liposome solution was passed through a 0.1 ⁇ m polycarbonate The film was extruded 3 times; the amount of sucrose was added, and diluted with water for injection to 1000 g; it was dispensed into a neutral borosilicate glass controlled injection bottle (west bottle) and then freeze-dried. The encapsulation efficiency was >99% as measured by ultrafiltration.
  • a liposome solution is formed; the obtained liposome solution is extruded through a 0.2 ⁇ m polycarbonate film; a formula amount of sucrose is added, and diluted with water for injection to 100 g; It is obtained by lyophilization in a neutral borosilicate glass controlled injection bottle (west bottle).
  • Preparation process taking the formula amount of mexiticol, polyoxyethylene castor oil, glycerin and absolute ethanol, heating and dissolving in a 45 ° C water bath, dissolving and sterilizing, filtering, and dispensing.
  • Preparation process taking the formula amount of mexicot and tert-butanol, heating and dissolving, sterilizing and filtering, and lyophilizing after dispensing to obtain mexiticol powder.
  • Preparation process Take the formula amount of ELP, glycerin and absolute ethanol, mix and sterilize and filter, and dispense to obtain special solvent.
  • Preparation process formulating amount of mexiticol, egg yolk phosphatidylcholine and hydrogenated soybean phosphatidylcholine in a formula amount of absolute ethanol, heating and dissolving at 60 ° C, as an organic phase; taking a formula of 70 % of water for injection, heated to 60 ° C, as an aqueous phase; while shearing the aqueous phase, the organic phase was added to the aqueous phase to obtain a liposome feed; the resulting liposome solution was passed through a 0.1 ⁇ m polycarbonate The film was extruded 3 times; the amount of sucrose was added, and diluted with water for injection to 1000 g; it was dispensed into a neutral borosilicate glass controlled injection bottle (west bottle). As a result, the obtained sample showed a significant change in the trait after standing at 40 ° C for 15 days, that is, the white emulsion became a yellow emulsion.
  • Comparative Examples 3, 4, 5 and 7, 10 batches of the pharmaceutical composition containing mexitic can be prepared.
  • Each batch of samples was stored at 40 ° C for 15 days, and the traits and related substances in each sample were examined and compared with the results on day 0, and the results were as follows.
  • Example 2 one batch of the pharmaceutical composition containing mexeticol was prepared; according to the formulation and preparation process of Comparative Example 5, one batch of micelle preparation containing mexican was prepared.
  • the obtained two batches of the pharmaceutical preparations were subjected to an acute toxicity test of mice, an acute toxicity test of rats, and a toxicity test of rat administration for 4 weeks in mice and rats at the same dose.
  • the toxicity test results of the two pharmaceutical preparations were compared and the results were as follows:
  • Example 2 According to the formulation and preparation process of Example 2, one batch of the pharmaceutical composition containing mexeticol was prepared; according to the formulation and preparation process of Comparative Example 5, one batch of micelle preparation containing mexican was prepared. The resulting two batches of the drug were tested for long-term toxicity in rats. Rats were administered at doses of 60, 30 and 10 mg/kg, respectively. The test results of the two pharmaceutical preparations were compared and the results were as follows:
  • One batch of the pharmaceutical composition containing mexicilin was prepared in accordance with the formulation and preparation process of Example 2. Eighteen SD rats were divided into 3 groups, 6 in each group, 3 females and 3 males. After administration of 30 mg/kg of methacetal to the rats in each group, blood samples and tissues were anesthetized at 15 min, 2 h and 6 h after administration, and blood samples and tissues were treated to obtain plasma and Tissue homogenate samples were established by LC-MS/MS to determine mesitylene and its active metabolite SN-38 in plasma and tissue homogenates (chemical name: 20(s)-7-ethyl-10-hydroxyl Camptothecin), the results are shown in Figures 1 and 2.
  • Figure 1 shows that mexicokon is mainly distributed in the rectum, liver, lung, plasma, colon, kidney, ovary and heart.
  • Figure 2 shows that SN-38 is mainly distributed in the colon, rectum, liver, lung, plasma, ovary, In the jejunum, ileum, duodenum and kidneys.
  • the concentrations of ciscocon and SN-38 in the rectum were very high, while in the colon, although the concentration of mexeticol was lower than that of plasma, the concentration of the active metabolite SN-38 of mexican was the highest, indicating the application of the present application.
  • the pharmaceutical composition is concentrated in a particular organ or tissue. Both mexican and SN-38 have the lowest concentrations in the brain and testis.
  • Example 1 According to the formulation of Example 1 (additional addition of about 0.8% (w/w, based on the total amount of phospholipids in the formulation) of DSPE with fluorescent probe IR623 (added and dissolved in the organic phase) And a preparation process to prepare a batch of methiocan pharmaceutical composition (particle size about 100 nm) containing a fluorescent probe (IR623).
  • the mice injected with mexicokonium liposome with fluorescent probe with a particle diameter of 100 nm were all distributed with fluorescence signals at 0.5 h. The fluorescence signal began to concentrate at the tumor site from 4 h, and the fluorescence signal intensity of the tumor site at 8 h. The strongest, the fluorescent signal of the tumor site began to weaken after 8h, and there was still a fluorescent signal at the tumor site at 48h (see Figure 3 for the results).
  • mice injected with mexicokonium liposome with fluorescent probe with a diameter of 400 nm showed obvious fluorescence signal in the abdomen at 0.5 h, and the abdominal fluorescent signal increased at 4 h, and the fluorescent signal was concentrated in the abdomen. (The results are shown in Figure 3).
  • the tumor-bearing mice were dissected, and the organs in each body (tumor, liver, spleen, kidney and intestine) were observed in the living imager for the fluorescence distribution of each organ. From Figure 4, we can see that the fluorescence of the organs of mice injected with free fluorescent probes is very weak and almost completely metabolized. In the organs of mice injected with mexicokonium liposome having a fluorescent probe having a particle diameter of 100 nm, the tumor has strong fluorescence compared to other organs. Fluorescence probe is injected at a particle size of 400 nm. The fluorescence of the liver is the strongest in the organs of mice that are needle-derived with mexitronic liposomes.
  • the mexicokonium liposome with a fluorescent probe with a particle diameter of 100 nm has passive tumor targeting, while the mexican liposome with a fluorescent probe with a particle diameter of 400 nm is mainly Liver accumulation. Fluorescent probes are mainly excreted through the intestinal and renal metabolism.

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Abstract

一种脂质体的制备方法，其包括：（1）将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得到脂质体料液；（2）将步骤（1）所得脂质体料液经聚碳酸脂膜挤出；（3）冷冻干燥。

Description

脂质体的制备方法
相关申请的引用
本申请要求于2015年12月08日向中华人民共和国国家知识产权局提交的申请号为201510897554.0的中国发明专利申请的权益。
技术领域
本申请涉及新的脂质体制备方法及由此方法制备的脂质体。
背景技术
为了提高水溶性差或水不溶性药物的溶解度，乳剂、胶束和脂质体等制剂技术被用于此类药物的制剂开发中。但乳剂和胶束制剂存在一些缺点。例如，乳剂属于热力学不稳定系统，在储存过程中易出现聚集、融合、絮凝、氧化、降解、水解等现象，从而影响乳剂的质量和药物的疗效。再如，胶束制剂通常会利用表面活性剂来形成胶束以增溶药物。然而，表面活性剂在临床使用时会产生毒副作用，引发超敏反应，影响用药安全。虽然脂质体能够改变药物的体内分布、降低药物的毒性、减轻变态反应和免疫反应，并延缓药物的释放，但是，通过已有方法得到的脂质体制剂通常为液体状态，属于热力学不稳定系统，存在稳定性低的问题，而且包封率低、药物较易渗漏、在储存过程中会出现细菌滋生和沉降凝聚、并难以控制粒径且粒径分布宽。即使冷冻干燥后再复溶成液体脂质体时，难以被复溶且复溶后的脂质体的粒径很大且粒径分布很宽。
中国专利(专利号：ZL201110355747.5)公开了如式I所示的化合物(又称美西替康)，
Figure PCTCN2016108840-appb-000001
美西替康在水及水性介质中不溶或几乎不溶，属于水难溶性药物。该专利提及可以将该药物配制成乳液、微乳液或胶束制剂。然而，我们发现将该药物配制成乳液、微乳液或胶束制剂后，稳定性很差且胶束制剂的毒性太大。因此，亟需开发新的适用于水溶性差或水不溶性药物的制剂及其制备方法。
发明概述
一方面，本申请提供了脂质体的制备方法，其包括：
(1)将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得脂质体料液；
(2)将步骤(1)所得的脂质体料液经聚碳酸脂膜挤出；
(3)冷冻干燥。
另一方面，本申请还提供了脂质体的另一制备方法，其包括：
(1)将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得脂质体料液；
(2)将步骤(1)所得的脂质体料液经聚碳酸脂膜挤出；
(3)加入注射用水并除菌过滤，分装和冷冻干燥；
其中在步骤(1)的水相中或者在实施步骤(3)中的除菌过滤之前加入冻干保护剂。
再一方面，本申请提供了通过上述制备方法获得的脂质体，其特征在于，所述脂质体加水或水性溶剂后复溶，复溶后的脂质体粒径为50-400nm。
发明详述
在以下的说明中，包括某些具体的细节以对各个公开的实施方案提供全面的理解。然而，相关领域的技术人员会认识到，不采用一个或多个这些具体的细节，而采用其它方法、部件、材料等的情况下可实现实施方案。
除非本申请中另外要求，在整个说明书和其后的权利要求书中，词语“包括(comprise)”及其英文变体例如“包括(comprises)”和“包括(comprising)”应解释为开放式的、含括式的意义，即“包括但不限于”。
在整个本说明书中提到的“一实施方案”或“实施方案”或“在另一实施方案中”或“在某些实施方案中”意指在至少一实施方案中包括与该实施方案所述的相关的具体参考要素、结构或特征。因此，在整个说明书中不同位置出现的短语“在一实施方案中”或“在实施方案中”或“在另一实施方案中”或“在某些实施方案中”不必全部指同一实施方案。此外，具体要素、结构或特征可以任何适当的方式在一个或多个实施方案中结合。
应当理解，在本申请说明书和附加的权利要求书中用到的单数形式的冠词“一”(对应于英文“a”、“an”和“the”)包括复数的对象，除非文中另外明确地规定。因此，例如提到的包括“催化剂”的反应包括一种催化剂，或两种或多种催化剂。还应当理解，术语“或”通常以其包括“和/或”的含义而使用，除非文中另外明确地规定。
一方面，本申请提供了脂质体的制备方法，其包括：
(1)将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得脂质体料液；
(2)将步骤(1)所得的脂质体料液经聚碳酸脂膜挤出；
(3)冷冻干燥。
在本申请的一些实施方案中，可以在步骤(1)的水相中或者在实施步骤(3)中的冷冻干燥之前加入冻干保护剂。
在本申请的一些实施方案中，在实施步骤(3)中的冷冻干燥之前，加入注射用水并分装。
在本申请的一些实施方案中，在实施步骤(3)中的冷冻干燥之前，加入注射用水并除菌过滤，然后分装。
另一方面，本申请还提供了脂质体的另一制备方法，其包括：
(1)将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得脂质体料液；
(2)将步骤(1)所得的脂质体料液经聚碳酸脂膜挤出；
(3)加入注射用水并除菌过滤，分装和冷冻干燥；
其中在步骤(1)的水相中或者在实施步骤(3)中的除菌过滤之前加入冻干保护剂。
在本申请的一些实施方案中，所述待包封的物质可以是药物，也可以是其他物质。优选的，所述待包封的物质选自药物。更优选的，所述待包封的物质选自美西替康、多西他赛、紫杉醇、阿霉素、两性霉素B、他克莫司、伊立替康、前列地尔、利培酮、西地那非、利多卡因、芬太尼、布比卡因、地塞米松、曲前列环素、阿柏西普、非布索坦、长春瑞滨、头孢匹胺钠、异环磷酰胺、氨柔比星、夫西地酸钠、头孢美唑钠、还原型谷胱甘肽、依达拉奉、加替沙星、盐酸氟西汀、阿苯达唑、米托蒽醌、阿普唑仑、万古霉素、头孢克洛、头孢克肟、盐酸氢溴索或阿托伐他汀。还更优选的，所述待包封的物质选自美西替康、多西他赛、紫杉醇、他克莫司或阿普唑仑。
在本申请的一些实施方案中，所述磷脂选自蛋黄磷脂酰胆碱、氢化蛋黄磷脂酰胆碱、大豆磷脂酰胆碱、氢化大豆磷脂酰胆碱、双软脂酸磷脂酰胆碱、二癸酰基磷脂酰胆碱、二棕榈酰磷脂酰胆碱、磷酯酰丝氨酸、磷脂酰肌醇、磷脂酰乙醇胺、培化磷脂酰乙醇胺、磷脂酰甘油、磷脂酰胆碱、二鲸蜡磷酸酯、二肉豆蔻酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二月桂酰磷脂酰胆碱、二油酰磷脂酰胆碱、二芥酰基磷脂酰胆碱、1-肉豆蔻酰基-2-棕榈酰基磷脂酰胆碱、1-棕榈酰基-2-硬脂酰基磷脂酰胆碱、1-棕榈酰基-2-肉豆蔻酰基磷脂酰胆碱、1-硬脂酰基-2-肉豆蔻酰基磷脂酰胆碱、1-硬脂酰基-2-棕榈酰基磷脂酰胆碱、1-肉豆蔻酰基-2-油酰基磷脂酰胆碱、1-棕榈酰基-2-油酰基磷脂酰胆碱、1-硬脂酰基-2-油酰基磷脂酰胆碱、二肉豆蔻酰基磷脂酰乙醇胺、二棕榈酰基磷脂酰乙醇胺、二硬脂酰基磷脂酰乙醇胺、二油酰基磷脂酰乙醇胺、二芥酰基磷脂酰乙醇胺或1-棕榈酰基-2-油酰基磷脂酰乙醇胺中的一种或多种。优选的，所述磷脂选自蛋黄磷脂酰胆碱、氢化蛋黄磷脂酰胆碱、大豆磷脂酰胆碱或氢化大豆磷脂酰胆碱中的一种或多种。更优选的，所述磷脂选自蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆 碱的组合。还更优选的，所述磷脂选自其中蛋黄磷脂酰胆碱与氢化大豆磷脂酰胆碱的重量比为3:1的组合。
在本申请的一些实施方案中，所述有机溶剂选自无水乙醇、95％的乙醇、甲醇、丙醇、三氯甲烷、二氯甲烷、叔丁醇、正丁醇、丙酮、甲基吡咯烷酮、乙酸乙酯、异丙醚或乙醚中的一种或多种。优选的，所述有机溶剂选自无水乙醇、95％的乙醇或叔丁醇。更优选的，所述有机溶剂选自无水乙醇。
在本申请的一些实施方案中，待包封的物质与磷脂的重量比为1:1-1:500。优选的，待包封的物质与磷脂的重量比为1:1-1:100。更优选的，待包封的物质与磷脂的重量比为1:15-1:50。还更优选的，待包封的物质与磷脂的重量比为1:20。
在本申请的一些实施方案中，待包封的物质与有机溶剂的重量比为1:1-1:100。优选的，待包封的物质与有机溶剂的重量比为1:9-1:50。更优选的，待包封的物质与有机溶剂的重量比为1:30。
在本申请的一些实施方案中，所述水相包含水作为主要组分或基本上由水组成，例如，去离子水、蒸馏水、纯化水、注射用水等，优选注射用水。
在本申请的一些实施方案中，所述有机相与水相混合时的温度为25-80℃。优选的，有机相与水相混合时的温度为55-65℃。
在本申请的一些实施方案中，有机相与水相可在氮气保护下进行混合。
在本申请的一些实施方案中，所述聚碳酸酯膜(Polycarbonate)的孔径选自0.015、0.03、0.05、0.08、0.1、0.2、0.4、0.6、0.8、1.0、2.0、3.0、5.0、8.0、10.0或12.0微米，优选0.1微米或0.2微米。任选地，可在聚碳酸酯膜的下面再加一层聚酯膜(Polyester)。
在本申请的一些实施方案中，所述挤出可以是以任何方式实施的挤出，只要能使大粒径的脂质体通过所述膜后变成小粒径的脂质体即可，该步骤需要控制料液的温度为25-80℃，优选为55-65℃。
在本申请的一些实施方案中，所述冻干保护剂选自甘露醇、葡萄糖、半乳糖，蔗糖、乳糖、麦芽糖或海藻糖中的一种或多种。优选的，所述冻干保护剂选自蔗糖、海藻糖或甘露醇中的一种或多种。更优选的，所述冻干保护剂选自蔗糖或蔗糖与甘露醇的组合。还更优选的，所述冻干保护剂为蔗糖或蔗糖与甘露醇的重量比为2:1的组合。
任选地，在本申请的一些实施方案中，还可以向步骤(1)中的所述有机相中加入抗氧化剂。所述抗氧化剂选自亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、维生素C、抗坏血酸棕榈酸酯，叔丁基对羟基茴香醚(BHA)、二叔丁基对甲酚(BHT)、维生素E醋酸酯、半胱氨酸和蛋氨酸中的一种或多种。优选的，所述抗氧化剂选自焦亚硫酸钠、叔丁基对羟基茴香醚、 二叔丁基对甲酚、维生素E醋酸酯。更优选的，所述抗氧化剂选自二叔丁基对甲酚或焦亚硫酸钠。
任选地，在本申请的一些实施方案中，步骤(1)中的所述水相中还可以包含金属离子螯合剂。所述金属离子螯合剂选自依地酸二钠、依地酸钙钠、1,2-二氨基环己烷四乙酸、二乙三胺五乙酸、N-(2-羟乙基)-乙二胺三乙酸三钠或N-二(2-羟乙基)甘氨酸。优选的，所述金属离子螯合剂选自依地酸二钠或依地酸钙钠。
任选地，在本申请的一些实施方案中，还可以在步骤(3)中实施冷冻干燥之前或者在步骤(3)中加入冻干保护剂之后加入pH调节剂。所述pH调节剂选自盐酸、硫酸、乙酸、磷酸、枸橼酸、酒石酸、马来酸、氢氧化钠、碳酸氢钠、磷酸氢二钠、磷酸二氢钠或枸橼酸钠。优选的，所述pH调节剂选自盐酸或氢氧化钠。在本申请的一些实施方案中，pH的调节范围为2-10，优选为4-7。
在本申请的一些实施方案中，通过本申请的制备方法得到的脂质体在加入水或水性溶剂之后快速复溶，复溶后的脂质体的粒径为50-400nm，优选100-250nm。在本申请的一些实施方案中，粒径分散系数≤0.5，优选地，粒径分散系数≤0.23。
本申请的制备方法具有如下优点中的一个或多个：(1)制备工艺简单，只需溶解、保温、混合、挤出和冻干等步骤，特别适合工业化大规模生产；(2)冻干前的脂质体料液经0.22微米过滤除菌后，然后无菌灌装和冻干，在工业生产上容易实现且保证产品是无菌的；(3)冻干后的脂质体稳定性好，其粒径、含量、有关物质、包封率等关键的质量指标同第0月时相比，无明显变化；(4)冻干后的产品几乎无溶剂残留。
与通过本领域中的常规方法制得的脂质体相比，通过本申请的制备方法所得到的脂质体具有如下优点中的一个或多个：(1)包封率高(＞99％)，在贮存过程中药物无渗漏且包封率不会降低；(2)本申请的脂质体经聚碳酸脂膜多次挤出后粒径分布很窄，分散系数在0.18以下，很好地控制了脂质体的粒径及粒径分布；(3)与普通制剂(例如，胶束制剂或乳剂等)相比，本申请的脂质体经动物实验证实降低了药物制剂的毒性，在体内特定的器官和组织集中分布，具有靶向性，增强了药物制剂的药效；(4)本申请的脂质体为固体，与液体形式的脂质体相比，稳定性显著增加、具有更好的重现性，易被复溶且更易于储存和运输。
本申请的脂质体可以是普通脂质体、长循环脂质体、热敏脂质体、免疫脂质体，或其他具有特殊功效的脂质体。
本申请的脂质体可以通过任何适当的途径向患者或受试者给药，例如静脉内给药、动脉内给药、肌肉给药、腹膜内给药、皮下给药、关节内给药、鞘内给药、侧脑室内给药、鼻腔喷雾、肺部吸入、口服给药以及本领域技术人员已知的其他合适的给药途径。可以利用本申 请所得的脂质体治疗的组织病变包括但不限于膀胱、肝脏、肺部、肾脏、骨骼、软组织、肌肉、乳房等组织的病变。
附图说明
图1：向大鼠静脉注射30mg/kg美西替康脂质体后，美西替康在体内的组织分布图。
图2：向大鼠静脉注射30mg/kg美西替康脂质体后，活性代谢产物SN38在体内的组织分布图。
图3：荧光标记物IR623和经IR623标记的美西替康脂质体在小鼠体内的分布图。
图4：荧光标记物IR623和经IR623标记的美西替康脂质体在小鼠肿瘤部位和各脏器的分布图。
实施例
通过以下实施例示例性说明本申请的具体制备方法，但本申请的保护范围不局限于此，本领域技术人员在本申请披露的技术范围内，根据本申请的技术方案及其发明构思进行的等同替换或改变都应涵盖在本申请的保护范围之内。
实施例1
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例2
配方：
15kg配制量 配方量
美西替康 30g
蛋黄磷脂酰胆碱 450g
氢化大豆磷脂酰胆碱 150g
无水乙醇 900g
蔗糖 900g
注射用水 加至15kg
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.2μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至15kg；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例3
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配方量的蔗糖和配制量的70％的注射用水，在60℃下加热溶清后作为水相；边剪切或搅拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.4μm聚碳酸酯膜挤出3次；再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例4
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 40g
甘露醇 20g
注射用水 加至1000g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃， 作为水相；边剪切或搅拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.2μm聚碳酸酯膜挤出3次；加入配方量的蔗糖和甘露醇，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例5
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
BHT 0.1g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的BHT和美西替康溶解于配方量的无水乙醇中，在60℃下加热溶解，溶清后加入配方量的蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱，在60℃下加热溶解，溶清后作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切或搅拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.2μm聚碳酸酯膜挤出可得具有一定粒径大小和一定粒径分布的脂质体料液；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例6
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
依地酸二钠 0.1g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康溶解于配方量的无水乙醇中，在60℃下加热溶解，溶清后加入配方量的蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱，在60℃下加热溶解，溶清后作为有机相；取配方量的依地酸二钠和配制量的70％的注射用水，在60℃下加热溶解，作为水相； 边剪切或搅拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出可得具有一定粒径大小和一定粒径分布的脂质体料液；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例7
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
盐酸或氢氧化钠 适量
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康溶解于配方量的无水乙醇中，在60℃下加热溶解，溶清后加入配方量的蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱，在60℃下加热溶解，溶清后作为有机相；取配制量的70％的注射用水，60℃保温后作为水相；边剪切或搅拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.2μm聚碳酸酯膜挤出可得具有一定粒径大小和一定粒径分布的脂质体料液；加入配方量的蔗糖，并加盐酸或氢氧化钠调节pH至5，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例8
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康溶解于配方量的无水乙醇中，在60℃下加热溶解，溶清后加入配方量的蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱，在60℃下加热溶解，溶清后作为有机相；取配方量的蔗糖和配制量的70％的注射用水，60℃加热溶解，作为水相；边剪切或搅 拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出可得具有一定粒径大小和一定粒径分布的脂质体料液；再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例9
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化蛋黄磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康溶解于配方量的无水乙醇中，在60℃下加热溶解，溶清后加入配方量的蛋黄磷脂酰胆碱和氢化蛋黄磷脂酰胆碱，在60℃下加热溶解，溶清后作为有机相；取配制量的70％的注射用水，60℃保温后作为水相；边剪切或搅拌水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出可得具有一定粒径大小和一定粒径分布的脂质体料液；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例10
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 22.5g
氢化大豆磷脂酰胆碱 7.5g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：具体实施步骤同实施例2。用超滤法测得包封率＞99％。
实施例11
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 150g
氢化大豆磷脂酰胆碱 50g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：具体实施步骤同实施例2。用超滤法测得包封率＞99％。
实施例12
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 18g
蔗糖 60g
注射用水 加至1000g
制备工艺：具体实施步骤同实施例2。用超滤法测得包封率＞99％。
实施例13
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 100g
蔗糖 60g
注射用水 加至1000g
制备工艺：具体实施步骤同实施例2。用超滤法测得包封率＞99％。
实施例14：
配方：
1kg配制量 配方量
紫杉醇 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
备注：除了将配方中的美西替康换成紫杉醇以外，实施例2-13中的各配方和工艺也适用于此实施例。
制备工艺：将配方量的紫杉醇、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例15：
配方：
1kg配制量 配方量
多西他赛 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
备注：除了将配方中的美西替康换成多西他赛以外，实施例2-13中的各配方和工艺也适用于此实施例。
制备工艺：将配方量的多西他赛、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例16：
配方：
1kg配制量 配方量
他克莫司 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
备注：除了将配方中的美西替康换成他克莫司以外，实施例2-13中的各配方和工艺也适用于 此实施例。
制备工艺：将配方量的他克莫司、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
实施例17：
配方：
1kg配制量 配方量
阿普唑仑 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
备注：除了将配方中的美西替康换成阿普唑仑以外，实施例2-13中的各配方和工艺也适用于此实施例。
制备工艺：将配方量的阿普唑仑、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。用超滤法测得包封率＞99％。
对比实施例1：薄膜分散法
配方：
100g配制量 配方量
美西替康 0.2g
蛋黄磷脂酰胆碱 3g
氢化大豆磷脂酰胆碱 1g
无水乙醇 6g
蔗糖 6g
注射用水 加至100g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取有机相放于梨形烧瓶内并置于旋转蒸发 仪中，在60℃下减压旋转蒸发除去乙醇，使有机相形成一层薄膜；取配制量的70％的注射用水，加热至60℃，作为水相；将水相加入具有经旋蒸形成的薄膜的烧瓶内，水化后形成脂质体料液；将所得的脂质体料液经0.2μm聚碳酸酯膜挤出；加入配方量的蔗糖，再加注射用水稀释至100g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。
结果：不能顺利进行水化，难以形成均匀的脂质体料液，且所得脂质体料液无法通过聚碳酸酯膜挤出，并且静置后会沉降分层，因此薄膜分散法不适用于制备美西替康的脂质体。对比实施例2：微射流均质法
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经微射流均质处理；处理后加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中后冷冻干燥即得。
结果：所得脂质体样品难以复溶，复溶后粒径很大，粒径分布很宽。
对比实施例3：胶束制剂1
1kg配制量 配方量
美西替康 2g
聚氧乙烯蓖麻油 30g
甘油 10g
无水乙醇 58g
制备工艺：取配方量的美西替康、聚氧乙烯蓖麻油、甘油和无水乙醇，于45℃水浴中加热溶清，溶清后除菌过滤，分装即得。
对比实施例4：乳剂
1kg配制量 配方量
美西替康 1g
维生素E 50g
F68(泊洛沙姆188) 20g
水 1000
无水乙醇 10g
制备工艺：①取配方量的美西替康、维生素E和无水乙醇加热溶清；②取配方量的F68和配方量的水溶清；③取②所得溶液的一半，边剪切边将①所得溶液加入其中，充分剪切后，将②所得溶液的另一半加入其中，剪切混匀；④将③所得溶液经高压均质处理10次，分装即得。
对比实施例5:胶束制剂2
1kg配制量 配方量
美西替康 5g
叔丁醇加至 1000g
制备工艺：取配方量的美西替康和叔丁醇，加热溶清后除菌过滤，分装冻干后可得美西替康粉末。
1kg配制量 配方量
ELP(蓖麻油聚烃氧酯(35)) 315g
甘油 105g
无水乙醇(药用级) 610g
制备工艺：取配方量的ELP、甘油和无水乙醇，混匀后除菌过滤，分装即得专用溶媒。
用法：用100倍量的专用溶媒溶解美西替康粉末后得到注射液，即可进行稀释用药。
对比实施例6：高压均质法
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经高压均质处理；处理后样品粒径仍很大，在500nm以上，且高压均质机难以正常运行。
对比实施例7：
配方：
1kg配制量 配方量
美西替康 2g
蛋黄磷脂酰胆碱 30g
氢化大豆磷脂酰胆碱 10g
无水乙醇 60g
蔗糖 60g
注射用水 加至1000g
制备工艺：将配方量的美西替康、蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱溶解于配方量的无水乙醇中，在60℃下加热溶解，作为有机相；取配制量的70％的注射用水，加热至60℃，作为水相；边剪切水相，边将有机相加入水相中，得到脂质体料液；将所得的脂质体料液经0.1μm聚碳酸酯膜挤出3次；加入配方量的蔗糖，再加注射用水稀释至1000g；分装至中性硼硅玻璃管制注射剂瓶(西林瓶)中即得。结果所得样品在40℃下放置15天后性状发生了显著的变化，即，白色乳液变成了黄色乳液。
实施例18：稳定性试验
按照实施例1-7，对比实施例3、4、5和7中的配方和制备工艺，制备含有美西替康的药物组合物10批。将每批样品置于40℃下保存15天，检测各样品中的性状和有关物质并与第0天检测结果进行比较，结果如下。
Figure PCTCN2016108840-appb-000002
实施例19：长期稳定性试验
按照实施例1-7的配方和制备工艺，制备含有美西替康的药物组合物7批，将每批药物 组合物分别置于6℃和25℃条件下保存。在6℃下保存样品时分别于第3、6、9、12月取样检测有关物质，在25℃下保存样品时分别于第1、2、3、6月取样检测有关物质，并与第0天检测结果进行比较，结果如下：
Figure PCTCN2016108840-appb-000003
Figure PCTCN2016108840-appb-000004
实施例20：粒径检测
按照实施例1-9和对比实施例2的配方和制备工艺，制备含有美西替康的药物组合物10批，将每批药物组合物各取1支，加水复溶后取样，并用纳米粒度分析仪检测粒径，结果如下：
Figure PCTCN2016108840-appb-000005
Figure PCTCN2016108840-appb-000006
实施例21：毒性试验
按照实施例2的配方和制备工艺，制备含有美西替康的药物组合物1批；按照对比实施例5的配方和制备工艺，制备含有美西替康的胶束制剂1批。将所得的2批药物制剂分别以相同的剂量在小鼠和大鼠身上进行了小鼠的急性毒性试验、大鼠的急性毒性试验和大鼠给药4周的毒性试验。将两种药物制剂的毒性试验结果进行了对比，结果如下：
Figure PCTCN2016108840-appb-000007
实施例22：药效试验
按照实施例2的配方和制备工艺，制备含有美西替康的药物组合物1批；按照对比实施例5的配方和制备工艺，制备含有美西替康的胶束制剂1批。将所得的2批药物在NCI-H292肺癌裸小鼠身上进行了药效试验，即对异种移植肿瘤生长的抑制作用。两种药物制剂的药效试验结果如下：
Figure PCTCN2016108840-appb-000008
实施例23：长毒试验
按照实施例2的配方和制备工艺，制备含有美西替康的药物组合物1批；按照对比实施例5的配方和制备工艺，制备含有美西替康的胶束制剂1批。将所得的2批药物在大鼠身上进行了长期毒性试验。大鼠的给药剂量分别为60、30和10mg/kg。将两种药物制剂的试验结果进行了对比，结果如下：
Figure PCTCN2016108840-appb-000009
Figure PCTCN2016108840-appb-000010
实施例24：组织分布
按照实施例2的配方和制备工艺，制备含有美西替康的药物组合物1批。取SD大鼠18只，分为3组，每组6只，3雌3雄。向各组大鼠经尾静脉注射给予30mg/kg美西替康的药物组合物后，分别于给药后15min、2h和6h麻醉并采集血样和组织，血样和组织分别经处理后得到血浆和组织匀浆液样品，建立LC-MS/MS法以测定血浆和组织匀浆液中的美西替康及其活性代谢物SN-38(化学名：20(s)-7-乙基-10-羟基喜树碱)，结果见图1和图2。图1显示美西替康主要分布在直肠、肝、肺、血浆、结肠、肾、卵巢和心等器官中，图2显示SN-38主要分布在结肠、直肠、肝、肺、血浆、卵巢、空肠、回肠、十二指肠和肾等器官中。直肠中美西替康和SN-38浓度均非常高，而结肠中虽然美西替康浓度低于血浆，但是美西替康的活性代谢产物SN-38的浓度却最高，这表明本申请的药物组合物在特定的器官或组织中集中分布。美西替康和SN-38均在脑和睾丸中浓度最低。
实施例25：体内靶向性研究
按照实施例1的配方(需再加入约0.8％(w/w，以配方中的磷脂总量为100％计)的接有荧光探针IR623的DSPE(将其加入并溶解在有机相里))和制备工艺制备一批含有荧光探针(IR623)的美西替康药物组合物(粒径约100nm)。按照实施例3的配方(需再加入约0.8％(w/w，以配方中的磷脂总量为100％计)的接有荧光探针IR623的DSPE(将其加入并溶解在有机相里))和制备工艺制备一批含有荧光探针(IR623)的美西替康药物组合物(粒径约400nm)。两批药物组合物分别在HT29肠癌荷瘤裸小鼠身上采用近红外在体显影技术进行体内靶向性研究，并和荧光探针IR623的体内靶向性进行对比，结果见图3和图4。
结果显示：游离的荧光探针经尾静脉注射至体内后，0.5h时在腹部有明显的荧光信号。随着时间的推移，荧光信号逐渐减弱，并被代谢到体外(结果见图3)。注射了粒径为100nm的具有荧光探针的美西替康脂质体的小鼠，0.5h时全身遍布荧光信号，从4h开始荧光信号开始向肿瘤部位集中，8h时肿瘤部位的荧光信号强度最强，8h后肿瘤部位的荧光信号开始减弱，48h时肿瘤部位仍有荧光信号(结果见图3)。而注射了粒径为400nm的具有荧光探针的美西替康脂质体的小鼠，0.5h时在腹部有明显的荧光信号，4h的时候腹部荧光信号增强，而后荧光信号一直集中在腹部(结果见图3)。
在注射药物48h后，解剖荷瘤小鼠，取其体内各脏器(肿瘤、肝、脾、肾和肠)在活体成像仪中观察各脏器的荧光分布情况。从图4中，我们可以看到注射游离的荧光探针的小鼠的器官的荧光很弱，几乎代谢完全。在注射粒径为100nm的具有荧光探针的美西替康脂质体的小鼠的器官中，相比于其他器官，肿瘤有较强的荧光。而在注射粒径为400nm的具有荧光探 针的美西替康脂质体的小鼠的器官中，肝脏的荧光是最强的。
从而可以推断，粒径为100nm的具有荧光探针的美西替康脂质体有被动的肿瘤靶向性，而粒径为400nm的具有荧光探针的美西替康脂质体主要是在肝脏蓄积。荧光探针主要是通过肠道及肾脏代谢排出体外。

Claims (17)

  1. 脂质体的制备方法，其包括：
    (1)将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得脂质体料液；
    (2)将步骤(1)所得的脂质体料液经聚碳酸脂膜挤出；
    (3)冷冻干燥。
  2. 如权利要求1所述的制备方法，其中在步骤(1)的水相中或者在实施步骤(3)中的冷冻干燥之前加入冻干保护剂。
  3. 脂质体的制备方法，其包括：
    (1)将待包封的物质和磷脂溶解于有机溶剂中，得到有机相，然后与水相混合得脂质体料液；
    (2)将步骤(1)所得的脂质体料液经聚碳酸脂膜挤出；
    (3)加入注射用水，除菌过滤，分装和冷冻干燥；
    其中在步骤(1)的水相中或者在实施步骤(3)中的除菌过滤之前加入冻干保护剂。
  4. 如权利要求1-3中任一项所述的制备方法，其中所述待包封的物质选自美西替康、多西他赛、紫杉醇、阿霉素、两性霉素B、他克莫司、伊立替康、前列地尔、利培酮、西地那非、利多卡因、芬太尼、布比卡因、地塞米松、曲前列环素、阿柏西普、非布索坦、长春瑞滨、头孢匹胺钠、异环磷酰胺、氨柔比星、夫西地酸钠、头孢美唑钠、还原型谷胱甘肽、依达拉奉、加替沙星、盐酸氟西汀、阿苯达唑、米托蒽醌、阿普唑仑、万古霉素、头孢克洛、头孢克肟、盐酸氢溴索或阿托伐他汀。
  5. 如权利要求1-4中任一项所述的制备方法，其中所述磷脂选自蛋黄磷脂酰胆碱、氢化蛋黄磷脂酰胆碱、大豆磷脂酰胆碱、氢化大豆磷脂酰胆碱、双软脂酸磷脂酰胆碱、二癸酰基磷脂酰胆碱、二棕榈酰磷脂酰胆碱、磷酯酰丝氨酸、磷脂酰肌醇、磷脂酰乙醇胺、培化磷脂酰乙醇胺、磷脂酰甘油、磷脂酰胆碱、二鲸蜡磷酸酯、二肉豆蔻酰磷脂酰胆碱、二硬脂酰磷脂酰胆碱、二月桂酰磷脂酰胆碱、二油酰磷脂酰胆碱、二芥酰基磷脂酰胆碱、1-肉豆蔻酰基-2-棕榈酰基磷脂酰胆碱、1-棕榈酰基-2-硬脂酰基磷脂酰胆碱、1-棕榈酰基-2-肉豆蔻酰基磷脂酰胆碱、1-硬脂酰基-2-肉豆蔻酰基磷脂酰胆碱1-硬脂酰基-2-棕榈酰基磷脂酰胆碱、1-肉豆蔻酰基-2-油酰基磷脂酰胆碱、1-棕榈酰基-2-油酰基磷脂酰胆碱、1-硬脂酰基-2-油酰基磷脂酰胆碱、二肉豆蔻酰基磷脂酰乙醇胺、二棕榈酰基磷脂酰乙醇胺、二硬脂酰基磷脂酰乙醇胺、二油酰基磷脂酰乙醇胺、二芥酰基磷脂酰乙醇胺或1-棕榈酰基-2-油酰基磷脂酰乙醇胺中的一种或多种；优选地，所述磷脂选自蛋黄磷脂酰胆碱、氢化蛋黄磷脂酰胆碱、大豆磷脂酰胆 碱或氢化大豆磷脂酰胆碱中的一种或多种；更优选地，所述磷脂选自蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱的组合；还更优选地，所述磷脂选自蛋黄磷脂酰胆碱和氢化大豆磷脂酰胆碱的组合，其中蛋黄磷脂酰胆碱与氢化大豆磷脂酰胆碱的重量比为3:1。
  6. 如权利要求1-5中任一项所述的制备方法，其中在步骤(1)中所述有机溶剂选自无水乙醇、95％的乙醇、甲醇、丙醇、叔丁醇、正丁醇、丙酮、甲基吡咯烷酮、乙酸乙酯、异丙醚或乙醚中的一种或多种；优选地，所述有机溶剂选自无水乙醇、95％的乙醇或叔丁醇。
  7. 如权利要求1-6中任一项所述的制备方法，其中在步骤(1)中所述待包封的物质与所述磷脂的重量比为1:1-500；优选1:1-100；更优选1:15-50；还更优选1:20。
  8. 如权利要求1-7中任一项所述的制备方法，其中在步骤(1)中所述待包封的物质与所述有机溶剂的重量比为1:1-100；优选1:9-50；更优选1:30。
  9. 如权利要求1-8中任一项所述的制备方法，其中所述水相包含水作为主要组分或基本上由水组成，例如，去离子水、蒸馏水、纯化水、注射用水等，优选注射用水。
  10. 如权利要求1-9中任一项所述的制备方法，其中所述水相进一步包含金属离子螯合剂，所述金属离子螯合剂选自依地酸二钠、依地酸钙钠、1,2-二氨基环己烷四乙酸、二乙三胺五乙酸、N-(2-羟乙基)-乙二胺三乙酸三钠或N-二(2-羟乙基)甘氨酸。
  11. 如权利要求1-10中任一项所述的制备方法，其中在步骤(1)中所述有机相与所述水相混合时的温度为25-80℃；优选55-65℃。
  12. 如权利要求1-11中任一项所述的制备方法，其中所述聚碳酸酯膜的孔径为0.1微米或0.2微米。
  13. 如权利要求1-12中任一项所述的制备方法，其中在步骤(2)中控制所述脂质体料液的温度为25-80℃，优选55-65℃。
  14. 如权利要求2-13中任一项所述的制备方法，其中所述冻干保护剂选自甘露醇、葡萄糖、半乳糖，蔗糖、乳糖、麦芽糖或海藻糖中的一种或多种；优选地，所述冻干保护剂选自蔗糖、海藻糖或甘露醇中的一种或多种；更优选地，所述冻干保护剂选自蔗糖或蔗糖与甘露醇的组合；还更优选地，所述冻干保护剂选自蔗糖或蔗糖与甘露醇的组合，其中蔗糖与甘露醇的重量比为2:1。
  15. 如权利要求1-14中任一项所述的制备方法，其中在步骤(1)中所述有机相进一步包含抗氧化剂，所述抗氧化剂选自亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、维生素C、抗坏血酸棕榈酸酯，叔丁基对羟基茴香醚、二叔丁基对甲酚、维生素E醋酸酯、半胱氨酸、蛋氨酸中的一种或多种。
  16. 如权利要求1-15中任一项所述的制备方法，其中在步骤(3)中在实施冷冻干燥之前可加 入pH调节剂，所述pH调节剂选自盐酸、硫酸、乙酸、磷酸、枸橼酸、酒石酸、马来酸、氢氧化钠、碳酸氢钠、磷酸氢二钠、磷酸二氢钠或枸橼酸钠。
  17. 根据权利要求1-16中任一项所述的制备方法制备的脂质体，其特征在于，所述脂质体加水或水性溶剂后复溶，复溶后的脂质体粒径为50-400nm，优选为100-250nm。
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EP3388055A4 (en) 2019-07-31
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US20180361342A1 (en) 2018-12-20
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CN108289846B (zh) 2020-12-04

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