WO2011050710A1 - 具有磺丁基醚环糊精盐内水相的脂质体 - Google Patents

具有磺丁基醚环糊精盐内水相的脂质体 Download PDF

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WO2011050710A1
WO2011050710A1 PCT/CN2010/078115 CN2010078115W WO2011050710A1 WO 2011050710 A1 WO2011050710 A1 WO 2011050710A1 CN 2010078115 W CN2010078115 W CN 2010078115W WO 2011050710 A1 WO2011050710 A1 WO 2011050710A1
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liposome
sulfobutyl ether
salt
drug
cyclodextrin
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PCT/CN2010/078115
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English (en)
French (fr)
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李春雷
张兰
王彩霞
张莉
申东民
李彦辉
修宪
梁敏
李永丰
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石药集团中奇制药技术（石家庄）有限公司
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Priority to JP2012534533A priority Critical patent/JP5785175B2/ja
Priority to AU2010312078A priority patent/AU2010312078B2/en
Priority to EP10826073.8A priority patent/EP2494960B1/en
Priority to RU2012119799/15A priority patent/RU2575793C2/ru
Priority to ES10826073.8T priority patent/ES2665033T3/es
Priority to US13/502,776 priority patent/US8871253B2/en
Application filed by 石药集团中奇制药技术（石家庄）有限公司 filed Critical 石药集团中奇制药技术（石家庄）有限公司
Priority to CN201080039899.8A priority patent/CN102596178B/zh
Priority to BR112012009660A priority patent/BR112012009660B8/pt
Priority to CA2776925A priority patent/CA2776925C/en
Priority to KR1020127013410A priority patent/KR101772399B1/ko
Publication of WO2011050710A1 publication Critical patent/WO2011050710A1/zh
Priority to US14/496,215 priority patent/US11357728B2/en

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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
    • 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
    • A61K31/435—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
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/724—Cyclodextrins
    • 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
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • 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—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5089—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a liposome having an aqueous phase in a sulfobutyl ether cyclodextrin salt and a process for the preparation thereof, and a process for the preparation thereof Use in medicines for treating tumor diseases.
  • liposome As a drug carrier, liposome has the characteristics of improving drug efficacy, reducing adverse drug reactions, targeting and sustained release, especially as a carrier for anti-tumor drugs, which can target drugs to tumor areas and reduce toxicity and increase Effect.
  • cytotoxic drugs account for the largest market share, which can be divided into the following five categories according to the mechanism of action: (1) drugs acting on the chemical structure of DNA, such as: alkylating agents and platinum; (2) drugs that affect nucleic acid synthesis, Such as: methotrexate, fluorouracil, etc.; (3) drugs that act on nucleic acid transcription, such as: doxorubicin, epirubicin, etc.; (4) drugs that act on tubulin synthesis, such as: taxanes, Vinblastines; drugs that act on topoisomerases, such as: camptothecins; (5) other cellular poisons.
  • the second and fourth types of drugs have cell cycle specificity, and they only kill a certain period of cells of the malignant tumor cell proliferation cycle.
  • the vinorelbine and topotecan which are mainly studied in the present invention are such drugs.
  • the cell cycle-specific antitumor drug is prepared into a liposome to achieve the effect of attenuating and enhancing the effect, and it is necessary to control the release rate of the drug in the liposome.
  • the release of liposome drugs is influenced by a variety of factors, including particle size, lipid membrane composition, internal aqueous phase, and drug loading methods.
  • the drug-loading methods of liposomes are divided into active drug loading and passive drug loading.
  • Lipid-soluble drugs generally adopt passive drug-loading technology, and water-soluble drugs adopt active drug-loading technology. Since vinorelbine and topotecan are both water-soluble weakly basic drugs, active drug-loading techniques are selected to prepare liposomes.
  • pH Gradient Method This method was invented by Canadian researchers in the 1880s. They found that alkaloids such as doxorubicin can achieve active transport due to the presence of pH gradient, specific to liposomes. Gather. The preparation process is to first select the internal aqueous phase buffer and the external phase buffer according to the nature of the drug. This step is very critical because it directly determines the stability of the drug during storage and the release behavior of the drug in the body. Then, the blank liposome is prepared by hydration using the internal aqueous phase buffer, and after the prepared blank liposome is further processed to reduce the particle size to the desired range, cross-flow dialysis, column chromatography and pH can be used.
  • the value adjustment and other means replace the outer phase of the liposome, resulting in a pH gradient inside and outside the phospholipid membrane.
  • the loading of the drug can be accomplished at a suitable temperature.
  • an ionophore to create a transmembrane pH gradient.
  • a divalent ion salt such as manganese sulfate
  • ion-loaded - A buffer such as A23187 and EDTA replaces the outer phase of the liposome.
  • the ionophore can specifically transport divalent ions to the membrane while transferring H+ into the interior of the liposome.
  • ammonium sulfate gradient method was invented by Israeli scholars in the early 1990s. The preparation of this method is similar to the conventional pH gradient method. First, a blank liposome was prepared using an ammonium sulfate buffer, and then ammonium bisulfate in the liposome external phase was removed by means of cross-flow dialysis to cause an ammonium sulfate gradient inside and outside the lipid film, and then the drug was loaded under heating. Initial studies have shown that the ammonium sulfate gradient method can achieve drug loading, which may be related to the diffusion of free ammonia across the membrane, resulting in a difference in pH between the inside and outside of the phospholipid membrane.
  • ammonium sulfate gradient method to complete drug loading may be a complicated process of Han-Yang diffusion, and the formation of pH gradient may be only one of the influencing factors.
  • the advantage of the ammonium sulfate gradient method is that in the process of preparing blank liposomes, the near neutral ammonium sulfate aqueous solution does not cause excessive hydrolysis of phospholipid molecules. Because if liposomes are prepared using saturated phospholipids, they need to be done at higher temperatures. Using conventional pH gradient methods, it is possible to make phospholipids more susceptible to hydrolysis. Further, liposomes prepared using an ammonium sulfate gradient method may have different behaviors for releasing drugs in vivo.
  • Sulfobutyl ether- ⁇ -cyclodextrin is an ionized ⁇ -cyclodextrin ( ⁇ -CD) derivative developed by Cydex Corporation in the United States in the 1990s. It is ⁇ -CD and 1 The product of the substitution reaction of 4-butane sultone. The substitution reaction can occur on the 2, 3, and 6 carbon hydroxyl groups of the ⁇ -CD glucose unit.
  • SBE- ⁇ -CD has the advantages of good water solubility, low nephrotoxicity, and low hemolytic property, and is an excellent pharmaceutical excipient. It has been approved by the US FDA for use as an injection aid.
  • SBE- ⁇ -CD Since its introduction, SBE- ⁇ -CD has been used for inclusion and solubilization of poorly soluble drugs, and is widely used in various preparations such as injections, oral preparations, and topical preparations.
  • Chakraborty used it for the liposome study of amphotericin B, and its purpose was to utilize SBE-P-CD for the insolubilization of poorly soluble drugs (Therapeutic and hemolytic evaluation of in-situ liposomal preparation containing amphotericin - ⁇ Complexed with different chemically modified ⁇ -cyclodextrins. J Pharm Pharmaceut Sci. 2003 Vol.6, No.2).
  • cyclodextrin inclusion complex liposome is a poorly soluble drug into a water-soluble cyclodextrin inclusion complex, and the inclusion compound is encapsulated in the inner aqueous phase of the liposome.
  • poorly soluble drugs it is not easy to enter the aqueous phase of the liposome, and the water is encapsulated by cyclodextrin. As the solubility increases, it is easily encapsulated by liposomes.
  • the main purpose of making a drug into a cyclodextrin inclusion liposome is to solve the solubility of a poorly soluble drug and increase the drug loading.
  • vinorelbine and topotecan have been the international hotspots for the study of liposome preparations.
  • the drug-loading technology has been studied in many ways, but most of them have some problems, which are detailed below.
  • Inex Corporation of Canada used sphingomyelin and cholesterol as a lipid membrane at a molar ratio of 55:45, and prepared a blank liposome with a magnesium sulfate solution as the internal aqueous phase, and then transported magnesium ions to the membrane by ionophore A23187, while transferring H+ to Inside the liposome, a pH gradient is formed to achieve drug loading.
  • the encapsulation efficiency of vinorelbine liposomes prepared by this method is greater than 90%, and can be stably stored for one year at 2-8 °C. (Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity.
  • Method 2 uses DSPC and cholesterol as lipid membranes, and copper sulfate solution as internal water phase to prepare blank liposomes, but the loading of topotecan can be achieved without adding A23187, which is due to the formation of copper ions and topotecan.
  • a stable complex, the principle is the above complex gradient method.
  • the drawback of this method is that the residual metal ions in the preparation may be poisonous in the blood.
  • DSPC stearic acid lecithin
  • DSPE-mPEG methoxypolyethylene glycol-distearoylphosphatidylethanolamine
  • white lipid prepared from triethylamine (TA) salt of sucrose octasulfate as internal water phase body.
  • TA triethylamine
  • the principle is basically the same as the ammonium sulfate gradient method, but since sucrose octasulfate carries 8 acid groups per molecule, it can form a dense bond with vinorelbine to achieve better retention.
  • the plasma half-life of vinorelbine liposomes prepared by this method can reach 9.2 hours. (Improved pharmacokinetics and efficacy of a highly stable nanoliposomal vinorelbine. The journal of pharmacology and experimental therapeutics. 2009 Vol. 328 No.1.)
  • sucrose 3 ⁇ 4 acid ester is an active substance in the body, which can be activated.
  • the principle is similar to the ammonium sulfate gradient method, in order to stabilize the retention by using a more compact combination of a polyvalent anionic polymer and a topotecan.
  • a disadvantage of this method is that the polyanionic polymers all have certain physiological activities, and such polymers are difficult to metabolize in vivo, and their safety needs further study.
  • the materials used have certain safety hazards: (1)
  • the polyanionic salts used by the above researchers include sucrose octasulfate triethylamine salt, sulfuric acid polymer, etc. These chemicals have certain physiological activities and do not meet the auxiliary materials. Inertness means no physiological and pharmacological activity;
  • the above complexation gradient method uses copper ions, nickel ions, manganese ions, etc., all of which are heavy metal ions, which remain in the formulation and have different degrees of damage to the human body, and It is not easy to cure, the medication cycle is usually very long, and the accumulation of heavy metal ions in the body will inevitably lead to the patient's intolerance. Therefore, it is still necessary to develop new liposomes and corresponding drug-loading methods.
  • One aspect of the present invention provides a liposome comprising a Han molecular layer and an internal aqueous phase, wherein the internal aqueous phase contains sulfobutyl ether cyclodextrin or a salt thereof and an active compound.
  • the sulfobutyl ether cyclodextrin may be sulfobutylether-cyclodextrin, sulfobutylether-beta-cyclodextrin or sulfobutylether-gamma- Cyclodextrin.
  • each molecule of the sulfobutyl ether cyclodextrin contains on average about 6.5 sulfo groups.
  • the salt of the sulfobutyl ether cyclodextrin is formed by one or more of a sulfobutyl ether cyclodextrin and an amine, a metal ion and an ammonium ion salt.
  • the salt of sulfobutyl ether cyclodextrin is sulfobutyl ether cyclodextrin with ammonia (NH 3 ), triethylamine (TA ), triethanolamine (TEA ), a salt formed by one or more of sodium ions, potassium ions, and calcium ions.
  • the active compound is a weakly basic compound
  • the active compound is preferably one of vinorelbine, vincristine, topotecan, and irinotecan. Or more.
  • the Han molecular layer contains a lipid substance modified with phosphorus, cholesterol, and a hydrophilic polymer.
  • Another aspect of the present invention provides a method of preparing the above liposome according to the present invention, comprising the steps of:
  • the carrier of the metal ion is added to the outer phase of the blank liposome of step (2) to form a pH gradient;
  • the carrier of the metal ion is an ionophore ⁇ 23187»
  • a liposome pharmaceutical preparation comprising the liposome according to the present invention according to any one of the above And a pharmaceutically acceptable carrier and/or excipient.
  • Still another aspect of the present invention provides the use of the liposome according to the present invention for the preparation of a medicament for treating a tumor patient, wherein the active compound in the liposome is vinorelbine, vincristine, extension One or more of Parkituk and Irinotecan.
  • the active compound in the liposome is vinorelbine, vincristine, extension One or more of Parkituk and Irinotecan.
  • the discovery of new methods relies on the discussion of the mechanisms of traditional drug-loading techniques.
  • the ammonium sulfate gradient method is analyzed, which includes the following process: The high concentration of the drug in the liposome phase is driven by the concentration difference and the pH difference, firstly overcomes the resistance of the lipid membrane (ie, the phospholipid layer) into the aqueous phase of the liposome, and enters the internal water.
  • the drug of the phase protonates and forms a precipitate with so 4 2- , and the drug is stably retained in the liposome in the form of a precipitate.
  • the release of the drug needs to be first dissociated from the precipitate and then diffused across the membrane. Therefore, the microstructure and solvency of the precipitate determine the rate at which the drug is released from the liposome, which in turn determines the safety and effectiveness of the formulation.
  • the microstructure and complexity of the drug and so 4 2 - precipitate formation are related to the spatial structure of the drug molecule and its alkaline strength.
  • Some drugs such as doxorubicin hydrochloride, have a strong basicity, easily form a precipitate with S0 4 2 -, and their molecular spatial structure is close to the plane, and the molecules can be mutually stacked, and the microstructure can be formed inside the liposome. Dense long strips of precipitation, so doxorubicin hydrochloride can be well retained in liposomes, and its liposomes are prescribed in KM - The half-life t 1/2 in mice is greater than 15 hours.
  • Some drugs, such as vinorelbine and topotecan have weaker basicity, have poor precipitation ability with SO-binding, and have a non-planar molecular structure, and cannot be stacked between molecules.
  • the weak alkalinity of vinorelbine and topotecan cannot be changed, so finding an anion that can bind to it and form a dense precipitate becomes the key to solve the problem, and the more complex polyanionic compound may produce a more stable complex with it. .
  • the experiment proves that the invention can realize the high-efficiency encapsulation of weakly basic drugs, such as vinorelbine and topotecan; the results of in vitro release and pharmacokinetic tests show that the release of drugs can be significantly delayed compared with the conventional aqueous phase prescription of ammonium sulfate. rate.
  • Antineoplastic drugs such as vincristine and irinotecan have weak alkalinity similar to those of vinorelbine and topotecan, and are also suitable for the technical solution of the present invention.
  • the inventors have broken through the conventional thinking, and have not utilized the SBE-P-CD for the cooperation of the capsule, but have utilized the polyanionic property as the aqueous phase in the liposome to carry out the active drug loading.
  • the salt of the sulfobutyl ether cyclodextrin is the same as the internal phase of the aqueous phase of the ammonium sulfate.
  • the anion in the inner aqueous phase forms a precipitate with the drug molecule to delay the release of the drug.
  • each sulfobutyl ether molecule contains an average of 6.5 S0 3 2 —, which can combine multiple drug molecules to form a more complex precipitation structure, so that high encapsulation efficiency can be achieved, and aqueous liposome with ammonium sulfate. In comparison, it can significantly extend the drug retention time.
  • the preparation of liposomes using sulfobutylcyclodextrin in the present invention is completely different from the usual cyclodextrin inclusion liposomes, and the present invention is not intended to solve the solubility of poorly soluble drugs, but to extend
  • the residence time of the weakly basic drug in the liposome increases the drug encapsulation efficiency, and the examples of the present invention also demonstrate that the encapsulation rate of the liposome obtained by the inclusion only by sulfobutylcyclodextrin is low. Can not meet the clinical drug needs.
  • the method adopted by the present invention includes:
  • (A) Preparation of a salt of sulfobutyl ether cyclodextrin An aqueous solution of a sulfobutyl ether cyclodextrin is prepared, which is salted with triethylamine, triethanolamine, ammonia, sodium hydroxide, potassium hydroxide or calcium hydroxide.
  • the particle size of the blank liposome is reduced by a microfluidic device or a high-pressure extrusion device, and then the sulfobutyl ether cyclodextrin salt of the liposome external phase is removed by dialysis or column chromatography to form an anion inside and outside the lipid film. gradient.
  • the salt of the sulfobutyl ether cyclodextrin used is a metal ion salt, then a metal ionophore needs to be added.
  • the metal ionophore can be inserted into the phospholipid membrane to exchange the metal ions and hydrogen ions inside and outside the liposome to form a pH gradient.
  • the drug solution and the liposome suspension are then incubated to obtain a liposomal drug.
  • the sulfobutyl ether cyclodextrin used in the present invention needs to be imported at present, it has been mass-produced, has sufficient supply, and has excellent quality, and can fully meet the requirements of large-scale production.
  • the salt of sulfobutyl ether cyclodextrin in this patent is completely feasible as the aqueous phase in the liposome from the aspects of drug encapsulation, retention effect and economical utility.
  • BEST MODE FOR CARRYING OUT THE INVENTION The following examples are intended to better illustrate the present invention, but should not be construed as limiting the scope of the invention.
  • the "drug-to-lipid ratio” means the weight ratio of the drug to the phospholipid
  • the “content of DSPE-mPEG” means the percentage of the total number of moles of the phospholipid component in the liposome Han molecular layer.
  • Example 1 General method for preparing sulfobutyl ether cyclodextrin aqueous phase liposome (prescription is SBE-CD) HSPC, cholesterol and DSPE-mPEG2000 are mixed in a mass ratio of 3:1:1, dissolved in 95 In % tert-butanol, the organic solvent is lyophilized to obtain a loose lipid powder, and the lipid powder is hydrated with a sulfobutylether- ⁇ -cyclodextrin aqueous solution, and incubated at 50-60 ° C for 1 hour to obtain unevenness. Chamber liposomes.
  • the microcapsule device was used to reduce the particle size of the liposomes, and then the anion of the blank liposome phase was removed using an ultrafiltration device to form a transmembrane dynamic gradient. According to the appropriate ratio of drug to lipid, the aqueous solution of the drug was added to the blank liposome and incubated at 60 ° C for 1 hour to obtain.
  • Example 2 General method for preparing liposomes in aqueous phase of sulfobutyl ether cyclodextrin triethylamine salt (prescription is SBE-CD/TA) HSPC, cholesterol and DSPE-mPEG2000 at 3:1:1 The mass ratio is mixed, dissolved in 95% t-butanol, lyophilized to remove the organic solvent to obtain a loose lipid powder, and hydrated the lipid powder with a sulfobutylether- ⁇ -cyclodextrin triethylamine salt aqueous solution, 50-60 Incubation at °C for 1 hour resulted in non-uniform multi-compartment liposomes.
  • the particle size of the liposomes was reduced using a high pressure extrusion apparatus, and then the anion of the blank phase of the blank lipid was removed using an ultrafiltration apparatus to form a transmembrane dynamic gradient. According to the appropriate ratio of drug to lipid, add the aqueous solution of the drug to the blank liposome at 60 ° C. - - After 1 hour of breeding, you will get.
  • Example 3 General method for preparing aqueous phase liposomes in sodium salt of sulfobutyl ether cyclodextrin (prescription is SBE-CD/Na) Mixing HSPC, cholesterol and DSPE-MPEG2000 in a mass ratio of 3:1:1 Dissolved in 95% t-butanol, lyophilized to remove the organic solvent to obtain a loose lipid powder, hydrated the lipid powder with a sulfobutylether- ⁇ -cyclodextrin sodium salt solution, and incubate at 50-60 ° C for 1 hour. To obtain uneven multi-chamber liposomes. The particle size of the liposomes is reduced using a high pressure extrusion apparatus.
  • the anion of the blank liposome external phase was then removed using column chromatography, followed by the addition of an appropriate amount of nikkomycin alcohol solution in an amount of 20 ng nikkomycin per mg of HSPC. Incubation at 60 ° C for 10 minutes exchanged hydrogen ions and sodium ions inside and outside the membrane to form a pH gradient. Then, the aqueous solution of the drug is added to the blank liposome at 60 ° C for 1 hour according to a suitable ratio of the drug to the lipid.
  • Example 4 Lipid entrapment ratios of different internal aqueous phases were compared with a drug-to-lipid ratio of 2: 9.58, and the lipids of the three internal aqueous phases of different drugs were prepared according to the methods of Examples 1, 2 and 3, respectively. Body (see Table 1). Table 1: Encapsulation efficiency of different aqueous phase liposomes in different aqueous phases and different drug liposomes (%)
  • Sample The aqueous phase vincristine liposome (method according to Example 2) and ⁇ 2 2 4 internal phase vincristine were prepared separately at a drug-to-lipid ratio of 3: 9.58. Platinum (in accordance with the method of Example 2, sulfobutylether- ⁇ -cyclodextrin triethylamine salt was replaced with ammonium sulfate).
  • the sample was diluted 10 times with the release solution into the dialysis bag, and the drug-containing dialysis bag was immersed in the dissolution cup containing the dialysate, and the dissolution cup was The volume of dialysate was 200 times the volume in the dialysis bag, and the release test was carried out at 37 ° C and 75 rpm, and samples were taken from the dialysis bag at 1, 2, 4, 6, 8, and 24 h, respectively.
  • the sample preparation ratio of the drug to the fat is 2: 9.58, and the internal aqueous phase is prepared as (NH 4 ) 2 S0 4 (the sulfobutyl ether- ⁇ -cyclodextrin triethylamine salt is replaced by the method of Example 2).
  • Ammonium sulfate), SBE-CD/TA prepared according to the method of Example 2), SBE-CD/NH 3 (in accordance with the method of Example 2, replacing sulfobutylether- ⁇ -cyclodextrin triethylamine salt with SBE-CD/NH 3 ) of vinorelbine, vincristine and irinotecan liposomes.
  • Formulation 1 Sulfocyl ether cyclodextrin / TA internal phase (prepared according to the method of Example 2).
  • Prescription 2 Ammonium sulphate aqueous phase (The sulfobutylether- ⁇ -cyclodextrin triethylamine salt was replaced with ammonium sulphate according to the method of Example 2).
  • the ratio of drug to fat was 3: 9.58, and the content of DSPE-mPEG2000 was 0.5%.
  • Test method Collect LLC lung cancer cells, dilute in DMEM medium and count 2.0 x 10 6
  • the inoculation volume is 0.2 ml, and the tumor-containing cells are about 4 x l0 5 .
  • the animals were grouped according to the size of the tumor, and were administered intravenously in groups of 10 mg/kg. After the administration, the animals were normally reared, and the antitumor effect of the test drug was dynamically observed by measuring the tumor diameter.
  • the formula for calculating tumor volume (TV) is:
  • T/C % TRTV I CRTV x 100%.
  • RTV treatment group RTV; CRTV: negative control group RTV).
  • Relative tumor volume (RTV): RTV Vt I Vo. Vo was measured for the tumor volume obtained when the cage was administered (ie, do), and Vt was the tumor volume at each measurement.
  • the relative tumor volume proliferation rate T/C% of the aqueous phase of ammonium oleate and the aqueous phase of sulfobutyl ether cyclodextrin is 51.8% and 31.1%, respectively, ie, sulfobutyl ether cyclodextrin.
  • the anti-tumor effect of aqueous liposomes on LLC lung cancer is greater than that of aqueous ammonium liposomes.
  • Example 9 Comparison of the effects of different internal aqueous phase topotecan liposomes on mice vaccinated with prostate cancer RM-1 - -
  • Formulation 1 Sulfocyl ether cyclodextrin / TA internal phase (prepared according to the method of Example 2).
  • Formulation 2 Sucrose octasulfate internal aqueous phase (in accordance with the method of Example 2, sulfobutylether- ⁇ -cyclodextrin triethylamine salt was replaced with sucrose octasulfate).
  • the ratio of drug to fat was 3: 9.58, and the content of DSPE-mPEG2000 was 0.5%.
  • Test method RM-1 lung cancer cells were collected, diluted in 1640 medium and counted at 2.0 x 10 6 /ml, and inoculated into the subcutaneous subcutaneous tissue of the forelimb of C57 female mice under aseptic conditions, the inoculation volume was 0.2 ml.
  • the tumor-bearing cells are about 4 x l0 5 .
  • Twelve days after the inoculation the animals were grouped according to the size of the tumor, and were administered intravenously in groups of 10 mg/kg. After the administration, the animals were normally reared, and the antitumor effect of the test drug was dynamically observed by measuring the tumor diameter.
  • Prescription 1 SBE-CD/TA internal aqueous phase (prepared according to the method of Example 2).
  • Formulation 2 Sucrose octasulfate internal aqueous phase (in accordance with the method of Example 2, sulfobutylether- ⁇ -cyclodextrin triethylamine salt was replaced with sucrose octasulfate).
  • the ratio of drug to fat was 3: 9.58, and the content of DSPE-mPEG2000 was 0.5%.
  • Test method 40.6 mg/kg was selected as the maximum dose, and the dose was reduced by 1.25, ie, 40.6, 32.5, 26.0, 20.8, 16.6, 13.3 and 10.6 mg/kg, three liposomes.
  • Two KM female mice were administered to each dose group of drug and free drug. The general state of the mice was observed daily and body weight was measured, and the observation period was 14 days. - Comparison of the toxicity of topotecan liposomes in different internal water phases
  • the drug toxicity sequence shown in the table is free drug ⁇ sulfobutyl ether cyclodextrin / ⁇ aqueous phase liposome ⁇ sucrose octasulfate internal aqueous liposome, in which sucrose octasulfate aqueous phase liposome Lower doses result in animal death.
  • the inventors of the present invention also prepared vinorelbine, vincristine and irinotecan liposomes, and compared the toxicity of KM mice according to a similar method, and obtained the same result as the topotecan liposome, that is, drug toxicity.
  • the order is free drug ⁇ sulfobutyl ether cyclodextrin / TA aqueous phase liposome ⁇ sucrose ⁇ 5 grams of acid ester aqueous phase liposome, wherein the sucrose 3 ⁇ 4 acid aqueous phase liposome at a lower dose Lead to animal death.

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一 一 具有磺丁基醚环糊精盐内水相的脂质体 技术领域 本发明涉及一种具有磺丁基醚环糊精盐内水相的脂质体及其制 备方法， 及其在制备用于治疗肿瘤疾病的药物中的用途。 背景技术 脂质体作为药物载体，具有提高药物疗效、减轻药物不良反应及 靶向、緩释等特点，尤其是作为抗肿瘤药物的载体时可以使药物靶向 肿瘤区而起到减毒、 增效作用。 目前临床上应用的抗肿瘤药物众多， 大致可分为细胞毒类、激素 类、 生物反应调节剂、单克隆抗体以及其他抗肿瘤药物这五类。 其中 细胞毒类药物占市场份额最大， 其按照作用机制可以分为以下五类： ( 1 )作用于 DNA化学结构的药物， 如： 烷化剂和铂类； （2 )影响 核酸合成的药物， 如： 甲氨蝶呤、 氟尿嘧啶等； （3 )作用于核酸转录 的药物， 如： 阿霉素、表阿霉素等；（4 )作用于微管蛋白合成的药物， 如： 紫杉类、 长春碱类； 作用于拓朴异构酶的药物， 如： 喜树碱类； ( 5 )其他细胞毒药。 其中第二、 四类药物具有细胞周期特异性， 它 们只对恶性肿瘤细胞增殖周期的某一间期细胞有杀灭作用，本发明重 点研究的长春瑞滨和拓朴替康即为此类药物。 将细胞周期特异性抗肿瘤药物制备成脂质体，从而达到减毒增效 的作用， 需要控制脂质体中药物的释放速度。若药物从脂质体中释放 过快则会导致如下结果： ( 1 )一部分药物在到达瘤区之前即从脂质体 中释放出来， 接着在血液中被快速清除， 而不能进入肿瘤区； （2 )由 - - 于同一时间肿瘤细胞所处的生长周期各不相同，因此即使进入瘤区的 药物也无法对处于非特定周期的肿瘤细胞产生杀伤作用，这样药物对 肿瘤细胞的暴露大大减少， 不利于药效的发挥，反而引起对正常组织 细胞的毒性反应。 所以， 对于细胞周期特异性药物来说， 控制药物从 脂质体中的释放就显得尤为重要。 脂质体药物的释放受到多种因素的影响， 包括粒度、 脂膜组成、 内水相以及载药方法等。脂质体的载药方法分为主动载药和被动载药 两种，脂溶性药物一般选择被动载药技术， 水溶性药物采用主动载药 技术。 由于长春瑞滨和拓朴替康均为水溶性弱碱性药物，所以选择主 动载药技术来制备脂质体。目前常用的主动载药技术主要有三种， pH 值梯度法、 硫酸铵梯度法以及络合梯度法：
( 1 ) pH值梯度法 该方法在 19世纪 80年代由加拿大学者发明，他们通过实验发现 阿霉素等生物碱类药物由于 pH值梯度的存在， 可以实现主动转运， 特异性的向脂质体内聚集。其制备过程为首先根据药物的性质选择内 水相緩冲液和外相緩冲液，这一步骤非常关键， 因为它直接决定了药 物在储存期内的稳定性和药物在体内的释放行为。然后使用内水相緩 冲液水化制备空白脂质体，将制得的空白脂质体经过进一步处理使其 粒度降低到所需的范围后， 则可以使用交叉流透析、 柱层析以及 pH 值调整等手段置换脂质体的外相， 造成磷脂膜内外的 pH梯度。 形成 跨膜梯度后， 可以在适宜的温度完成药物的装载。 也可以使用离子载体制造跨膜 pH梯度。在制备空白脂质体的过 程中， 将 2价离子盐， 如硫酸锰包封在脂质体中， 之后用含有离子载 - - 体如 A23187和 EDTA的緩冲液置换脂质体的外相。离子载体可以特 异性的向膜外转运 2价离子， 同时将 H+转移到脂质体内部。 使用上 述方法， 同样可以制造膜内外的 pH值梯度。 对于 pH梯度法载药的机理， 已经进行了较系统的研究。 目前上 市的 3个蒽环类抗生素脂质体制剂中，有 2个产品使用了 pH值梯度 法载药技术。
( 2 )硫酸铵梯度法 硫酸铵梯度法在 20世纪 90年代初期由以色列学者发明。该方法 的制备过程和传统的 pH值梯度法有相似之处。首先使用硫酸铵緩冲 液制备空白脂质体，之后采用交叉流透析等手段除去脂质体外相的硫 酸铵，造成脂膜内外的硫酸铵梯度， 然后在加热的条件下完成药物的 装载。 起初的研究表明， 硫酸铵梯度法之所以能够实现药物的装载， 可能和游离的氨跨膜扩散， 造成磷脂膜内外的 pH值的差异有关。但 是严谨的理论推导表明，使用硫酸铵梯度法完成药物的装载，可能是 一个比较复杂的汉向扩散的过程， pH值梯度的形成可能只是其中 的一个影响因素。 硫酸铵梯度法的优势在于制备空白脂质体的过程中 ,接近中性的 硫酸铵水溶液不会引起过多的磷脂分子水解。因为如果使用饱和磷脂 制备脂质体， 需要在较高的温度下完成。 使用传统的 pH值梯度法， 可能使得磷脂更容易发生水解。此夕卜，使用硫酸铵梯度法制备的脂质 体， 其在体内释放药物的行为可能也有所不同。
( 3 )络合梯度法 - - 该方法使用过渡态金属离子的盐，如硫酸铜、硫酸镍作为内水相 緩冲液制备空白脂质体,之后采用交叉流透析等手段除去脂质体外相 的金属离子，造成脂膜内外的金属离子梯度， 然后在加热的条件下完 成药物的装载。其载药原理为药物在脂质体内水相中与过渡态金属离 子结合生成稳定的络合物从而滞留在脂质体中。 磺丁基醚 - β -环糊精（SBE- P -CD )是 20世纪 90年代由美国 Cydex公司开发成功的离子化 β -环糊精（ β -CD )衍生物， 是 β -CD 与 1,4-丁烷磺内酯发生取代反应的产物。取代反应可发生在 β -CD 葡 萄糖单元的 2、 3、 6位碳羟基上， SBE- β -CD具有水溶性好、 腎毒 性低及溶血性小等优点， 是性能优良的药用辅料， 并已通过了美国 FDA的批准可作为注射用辅料。
SBE- β -CD 自面世以来一直被应用于难溶性药物的包合增溶， 广泛用于注射剂、口服制剂、局部给药制剂等各种制剂。 Chakraborty 将其用于两性霉素 B的脂质体研究， 其目的也是利用了 SBE- P -CD 对难溶性药物的包合增溶作用 （ Therapeutic and hemolytic evaluation of in-situ liposomal preparation containing amphotericin - β complexed with different chemically modified β-cyclodextrins. J Pharm Pharmaceut Sci . 2003 Vol.6, No.2 )。 王志宣，邓英杰等人 (环糊精包合物脂质体给药系统的研究进展， 沈阳药科大学学报， 2006 Vol23 )对环糊精包合物脂质体的国内外研 究情况进行了总结分析，环糊精包合物脂质体是将难溶性的药物制成 水溶性的环糊精包合物，再将包合物包封在脂质体的内水相中。对于 难溶性药物， 不容易进入脂质体内水相中， 利用环糊精包合后， 其水 溶性增加，则容易被脂质体包封。将药物制成环糊精包合物脂质体的 主要目的是解决难溶性药物溶解度， 提高载药量。 长春瑞滨和拓朴替康作为抗肿瘤领域的一线药物，其脂质体制剂 的研究一直是国际上的热点，目前已经有许多国家的研究小组对长春 瑞滨和拓朴替康脂质体的载药技术进行了多种研究，但大都存在一些 问题， 下面详述。 加拿大的 Inex公司使用鞘磷脂和胆固醇以摩尔比 55: 45作为脂 膜， 以硫酸镁溶液为内水相制备空白脂质体， 然后利用离子载体 A23187向膜外转运镁离子， 同时将 H+转移到脂质体内部， 形成 pH 梯度，从而实现药物的装载。用该方法制备得到的长春瑞滨脂质体包 封率大于 90% , 2-8 °C条件下可稳定储存一年。 （Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity. Journal of pharmaceutical sciences, 2005 Vol.94 No.5. ) 加拿大以 Bally为核心的研究小组则使用了两种方法制备得到了 高包封率的拓朴替康脂质体。 方法一以 DSPC和胆固醇作为脂膜， 以硫酸锰溶液为内水相制备空白脂质体， 然后利用离子载体 A23187 形成 pH梯度， 实现药物装载。 该法与 Inex公司的方法机理相同。 方法二以 DSPC 和胆固醇作为脂膜， 以硫酸铜溶液为内水相制备空 白脂质体， 但是不需要加入 A23187即可实现拓朴替康的装载， 这是 由于铜离子与拓朴替康形成了稳定的络合物，该原理即为上述络合梯 度法。该方法的缺陷在于制剂中残留的金属离子在血液中可能会产生 毒'! "生。 ( An evaluation of transmembrane ion gradient-mediated - - encapsulation of topotecan within liposomes. Journal of controlled release. 96(2004); Copper-topotecan complexation mediates drug accumulation into liposomes. Journal of controlled release 114(2006). ) 美国的研究者使用汉硬脂酸卵磷脂 （DSPC )、 胆固醇和甲氧基 聚乙二醇-二硬脂酰磷脂酰乙醇胺（DSPE-mPEG )作为脂膜， 以蔗 糖八硫酸酯的三乙胺（TA )盐为内水相制备空白脂质体。 之后采用 交叉流透析等手段除去脂质体外相的蔗糖八硫酸酯 TA, 造成脂膜内 外的蔗糖八硫酸酯 TA梯度， 然后完成药物的装载。 原理与硫酸铵梯 度法基本相同，但由于蔗糖八硫酸酯每一个分子携带 8个酸根所以能 够与长春瑞滨形成致密的结合， 达到更好的滞留作用。该方法制备的 长春瑞滨脂质体血浆半衰期能达到 9.2 小时。 （ Improved pharmacokinetics and efficacy of a highly stable nanoliposomal vinorelbine. The journal of pharmacology and experimental therapeutics. 2009 Vol.328 No.l. )但是该方法存在非常严重的问题是 蔗糖 ¾酸酯为体内活性物质，它可以激活体内成纤维细胞生长因子 ( Structural basis for activation of fibroblast growth factor signaling by sucrose octasulfate. MOLECULAR AND CELLULAR BIOLOGY, Oct. 2002， Vol. 22， No. 20 ), 产生一系列生理学效应， 因此其作为注 射用辅料使用存在很大风险。 美国的 Alza 公司使用氢化卵磷脂 （ HSPC )、 胆固醇和 DSPE-mPEG作为脂膜，以多阴离子聚物为内水相，如：硫酸葡聚糖、 硫酸蛋白聚糖、硫酸纤维素等等。之后采用交叉流透析置换外相形成 - - 多聚物梯度， 完成载药。 其原理与硫酸铵梯度法类似， 目的是利用多 价阴离子聚合物与拓朴替康更紧密的结合从而稳定滞留。该方法的缺 陷也是所述多阴离子聚合物均具有一定生理活性，并且此类聚合物在 体内很难代谢， 其安全性有待进一步研究。 （ Liposome-entrapped topoisomerase inhibitors.US6465008Bl )。 由以上研究实例分析可知目前长春瑞滨、拓朴替康等弱碱性药物 脂质体研究的方向主要有 pH梯度法，广义硫酸铵梯度法和络合梯度 法，但都只是实验室阶段，并且所用材料均存在一定的安全隐患：（1 ) 以上研究者使用的多阴离子盐有蔗糖八硫酸酯三乙胺盐、硫酸多聚物 等，这些化学物质均具有一定生理活性， 不符合辅料应惰性即无生理 及药理活性的原则； （2 )以上络合梯度法使用铜离子、镍离子、 锰离 子等， 均为重金属离子， 残留在制剂中均对人体有不同程度的损害， 且由于肿瘤不易治愈，用药周期通常很长，重金属离子在体内的不断 蓄积必然导致患者无法耐受。 因此， 仍然有必要发展新的脂质体和相应的载药方法。 发明内容 本发明的一个方面提供一种脂质体， 包含汉分子层和内水相，其 中所述内水相含有磺丁基醚环糊精或其盐和活性化合物。 根据本发明的脂质体的一些实施方式，其中磺丁基醚环糊精可以 是磺丁基醚- -环糊精、 磺丁基醚- β -环糊精或磺丁基醚- γ -环糊精。 根据本发明的脂质体的一些实施方式，其中磺丁基醚环糊精的每 个分子平均含有约 6.5个磺基基团。 - - 根据本发明的脂质体的一些实施方式，其中磺丁基醚环糊精的盐 是磺丁基醚环糊精与胺、金属离子和铵离子中的一种或更多种形成的 盐。 根据本发明的脂质体的一些实施方式，其中磺丁基醚环糊精的盐 是磺丁基醚环糊精与氨（NH3 )、 三乙胺（TA )、 三乙醇胺（TEA )、 钠离子、 钾离子和钙离子中的一种或更多种形成的盐。 根据本发明的脂质体的一些实施方式，其中所述活性化合物是弱 碱性的化合物， 所述活性化合物优选为长春瑞滨、 长春新碱、拓朴替 康和伊立替康中的一种或更多种。 根据本发明的脂质体的一些实施方式，其中所述汉分子层含有磷 月旨、 胆固醇和亲水性聚合物修饰的脂类物质。 本发明的另一方面提供一种制备上述根据本发明的脂质体的方 法， 包括如下步骤：
( 1 )用磺丁基醚环糊精或其盐的水溶液水化脂相粉末以形成含 有磺丁基醚环糊精或其盐的水溶液作为内水相的空白脂质体；
( 2 )除去步骤（ 1 )的空白脂质体的外相中的磺丁基醚环糊精的 盐以形成阴离子梯度；
( 3 )任选地， 当磺丁基醚环糊精的盐是金属离子盐时， 在步骤 ( 2 )的空白脂质体的外相中加入该金属离子的载体以形成 pH梯度； 和
( 4 )将步骤（2 )或 （3 ) 的空白脂质体与活性化合物的水溶液 - - 一起孵育以将活性化合物包封在脂质体内。 根据本发明的脂质体的制备方法，所述金属离子的载体是离子载 体 Α23187» 本发明的再另一方面提供一种脂质体药物制剂，含有上述任意一 项根据本发明的脂质体和药学上可接受的载体和 /或赋型剂。 根据本发明的脂质体药物制剂的一些实施方式，其中所述载体和 /或赋型剂中含有渗透压调节剂和 /或抗氧剂。 本发明的再另一方面提供上述任意一项根据本发明的脂质体在 制备用于治疗肿瘤患者的药物中的用途，其中脂质体中的活性化合物 是长春瑞滨、 长春新碱、 拓朴替康和伊立替康中的一种或更多种。 新方法的发现依赖于对传统载药技术机理的探讨。首先分析硫酸 铵梯度法， 其包括如下过程： 脂质体外相中高浓度药物在浓度差和 pH差驱使下， 首先克服脂膜（即磷脂汉分子层） 阻力进入脂质体内 水相， 进入内水相的药物质子化并与 so4 2-形成沉淀， 药物以沉淀形 式得以在脂质体内稳定的滞留。而药物的释放则需要首先从沉淀上解 离下来， 然后跨膜扩散出来， 因此， 沉淀的微观结构和溶解能力决定 了药物从脂质体中释放的速度， 进而决定了处方的安全性和有效性。 药物和 so4 2-形成沉淀的微观结构和复杂程度与药物分子的空间 结构及其碱性强弱有关。有些药物例如盐酸多柔比星，由于碱性较强， 易与 S04 2-结合形成沉淀， 并且其分子空间结构近平面， 分子间能够 相互堆垒， 微观结构显示在脂质体内部能够形成致密的长条状沉淀， 所以盐酸多柔比星在脂质体中能达到很好滞留，其脂质体处方在 KM — — 小鼠体内的半衰期 t1/2 大于 15小时。 而有些药物例如长春瑞滨和拓 朴替康， 由于碱性较弱， 与 SO —结合形成沉淀能力差， 且分子结构 非平面，分子间不能堆垒，所以当采用与上述盐酸多柔比星同样的脂 类组成和制备方法得到的脂质体在 KM小鼠体内 t1/2小于 5小时。 而 如此短的半衰期使得脂质体在血液循环的过程中，药物已有大部分渗 漏，不能进入瘤区；即便是少量进入瘤区的脂质体药物，也很快释放， 极其不利于细胞周期特异性抗肿瘤药物疗效的发挥。可见， 药物与阴 离子形成沉淀的复杂程度是影响药物释放的关键因素。 长春瑞滨和拓朴替康等药物的弱碱性无法改变，所以寻找能与其 结合并形成致密沉淀的阴离子成为解决问题的关键，而结构较复杂的 多阴离子化合物可能与其产生较稳定的复合物。 实验证明本发明能够实现弱碱性药物，如长春瑞滨、拓朴替康的 高效包封；体外释放和药代试验结果表明与传统硫酸铵内水相处方相 比， 能够显著延緩药物的释放速率。 长春新碱、伊立替康等抗肿瘤药 物，具有与长春瑞滨和拓朴替康分子类似的弱碱性，也适用于本发明 的技术方案。 本发明中发明人突破传统思维， 没有利用 SBE- P -CD的包合作 用， 而利用其多阴离子特性将其做为脂质体内水相， 进行主动载药。 磺丁基醚环糊精的盐做内水相与硫酸铵内水相载药的原理相同 ,都是 利用内水相中的阴离子与药物分子形成沉淀从而延緩药物释放。但是 每个磺丁基醚分子平均含有 6.5个 S03 2—, 能够同时结合多个药物分 子形成更为复杂的沉淀结构，所以可以实现高包封率，并且与硫酸铵 内水相脂质体相比， 能够显著延长药物滞留时间。 - - 本发明中采用磺丁基迷环糊精制备脂质体与通常的环糊精包合 物脂质体是完全不同的， 本发明不是为了解决难溶性药物的溶解性， 而是为了延长弱碱性药物在脂质体内的滞留时间， 提高药物包封率， 并且本发明实施例也证实了仅仅依靠磺丁基迷环糊精的包合作用所 得的脂质体包封率很低， 不能满足临床用药需求。 为了得到具有优良性质的脂质体制剂 ,首先需要制备磺丁基醚环 糊精的盐，然后采用合适的方法制备脂质体。本发明采用的方法包括：
( A )磺丁基醚环糊精的盐的制备： 配制磺丁基醚环糊精的水溶 液， 与三乙胺、三乙醇胺、氨、氢氧化钠、氢氧化钾或氢氧化钙成盐。 ( B )脂质体的制备： 将所用的脂类辅料溶解于有机溶剂中， 冻 干除去有机溶剂，得到疏松的脂质粉末，之后使用磺丁基醚环糊精的 盐溶液水化脂相粉末，形成空白脂质体。然后用微射流设备或者高压 挤出设备降低空白脂质体的粒度,之后采用透析或者柱层析等手段除 去脂质体外相的磺丁基醚环糊精的盐，从而使脂膜内外形成阴离子梯 度。如果所用磺丁基醚环糊精的盐是金属离子盐，则需要再加入金属 离子载体。金属离子载体可以插入磷脂膜， 实现脂质体内外金属离子 和氢离子的交换， 从而形成 pH梯度。 然后再将药物溶液和脂质体混 悬液孵育， 即得到脂质体药物。 本发明所使用的磺丁基醚环糊精目前虽然需要进口，但其已实现 批量生产， 货源充足， 品质优良， 完全能满足大生产的要求。 综上所述，本专利中以磺丁基醚环糊精的盐作为脂质体内水相无 论从药物包封、 滞留效果上还是经济实用方面是完全可行的。 - - 具体实施方式 下面所述实施例的目的是为了更好的说明本发明，但不应对本发 明的范围构成限定。 下文中所述 "药脂比" 指药物与磷脂的重量比， "DSPE-mPEG 的含量" 指其在脂质体汉分子层中磷脂成分总摩尔数的百分率。 实施例 1: 制备磺丁基醚环糊精内水相脂质体的一般方法 （处方为 SBE-CD ) 将 HSPC、 胆固醇和 DSPE-mPEG2000以 3: 1: 1的质量比混 合， 溶解于 95%叔丁醇中， 冻干除去有机溶剂得到疏松的脂质粉末， 以磺丁基醚- β -环糊精水溶液水化脂质粉末， 50-60°C孵育 1小时，得 到不均匀的多室脂质体。使用微射流设备降低脂质体的粒度， 然后使 用超滤装置移去空白脂质体外相的阴离子， 以便形成跨膜动力梯度。 按照合适的药脂比， 在空白脂质体中加入药物水溶液 60°C孵育 1小 时， 即得。 实施例 2: 制备磺丁基醚环糊精三乙胺盐内水相的脂质体的通用方法 (处方为 SBE-CD/TA ) 将 HSPC、 胆固醇和 DSPE-mPEG2000以 3: 1: 1的质量比混 合， 溶解于 95%叔丁醇中， 冻干除去有机溶剂得到疏松的脂质粉末， 以磺丁基醚 - β -环糊精三乙胺盐水溶液水化脂质粉末， 50-60°C孵育 1 小时，得到不均匀的多室脂质体。用高压挤出设备降低脂质体的粒度， 然后使用超滤装置移去空白脂质体外相的阴离子，以便形成跨膜动力 梯度。 按照合适的药脂比， 在空白脂质体中加入药物水溶液 60°C孵 - - 育 1小时， 即得。 实施例 3: 制备磺丁基醚环糊精钠盐内水相脂质体的一般方法（处方 为 SBE-CD/Na ) 将 HSPC、 胆固醇和 DSPE-MPEG2000以 3: 1: 1的质量比混 合， 溶解于 95%叔丁醇中， 冻干除去有机溶剂得到疏松的脂质粉末， 以磺丁基醚 - β -环糊精钠盐水溶液水化脂质粉末， 50-60°C孵育 1 小 时，得到不均匀的多室脂质体。使用高压挤出设备降低脂质体的粒度。 然后使用柱层析移去空白脂质体外相的阴离子，然后加入适量的尼可 霉素醇溶液，加入量为 20ng尼可霉素 /lmg HSPC。60°C孵育 10分钟， 使膜内外的氢离子与钠离子发生交换， 从而形成 pH梯度。 然后按照 合适的药脂比， 在空白脂质体中加入药物水溶液 60°C孵育 1小时， 即得。 实施例 4: 不同内水相的脂质体包封率比较 以药脂比为 2: 9.58, 分别才艮据实施例 1、 2和 3的方法制备不同 药物的 3种内水相的脂质体（见表 1 )。 表 1: 不同内水相、 不同药物脂质体的包封率 不同内水相脂质体的包封率（％ )
药物
SBE-CD SBE-CD/TA ~~ SBE-CD/Na 盐酸米托蒽醌 7.6 48.5 77.6
盐酸拓朴替康 4.8 63.6 74.6
盐酸伊立替康 5.3 64.1 96.1
盐酸阿霉素 11.3 63.5 91.8
酒石酸长春瑞滨 4.7 38.2 75.9
硫酸长春新碱 3.8 47.8 79.7
结论： 从包封率结果可见， SBE-CD内水相脂质体包封率极低, — —
SBE-CD/TA和 SBE-CD/Na包封率较高， 说明只有在离子转运形成 pH梯度的条件下， 才能有较好的包封， 药物进入脂质体内水相后首 先质子化， 再与 SBE-CD作用， 而仅靠 SBE-CD的包合作用药物几 乎没有装载。 实施例 5: 磺丁基醚环糊精 /TA内水相和硫酸铵内水相长春新碱脂质 体体外释放比较
1、 样品： 以药脂比为 3: 9.58, 分别制备 SBE-CD/TA内水相长春新碱脂 质体（根据实施例 2的方法）和^ )2804内水相长春新碱脂质体（依 据实施例 2的方法， 将磺丁基醚- β -环糊精三乙胺盐替换成硫酸铵）。
2、 释放条件：
5mM NH4Cl/10mM组氨^ /260mM葡萄糖 pH7.0作为释放外液， 样品用释放液稀释 10倍移入透析袋中， 装有药物的透析袋浸入盛有 透析液的溶出杯中， 溶出杯中透析液体积是透析袋中体积的 200倍， 37 °C , 75 rpm进行释放试验， 分别在 1、 2、 4、 6、 8、 24h从透析袋 中取样， 测定。
3、 测定结果： 表 2: 不同内水相长春新碱脂质体释放比较 内水相 不同取样时间的释放率（ % )
Figure imgf000015_0001
SBE-CD/TA 22 31 44 52 61 94 7.2 硫酸铵 26 62 91 97 98 99 1.1 结论： 与硫酸铵内水相脂质体相比， SBE-CD/TA内水相脂质体 - - 能够非常显著的延长药物在脂质体内水相的滞留。 实施例 6: 磺丁基醚环糊精 /NH3-硫酸铵混合内水相长春瑞滨脂质体 体外释放
1. 样品： 药脂比为 3: 9.58 (依据实施例 2的方法， 将磺丁基醚 - β - 环糊精三乙胺盐替换成表 3中 A-F不同处方所示的磺丁基醚环糊 精 /ΝΗ3-硫酸铵混合溶液）。 表 3: 磺丁基醚环糊精 /ΝΗ3-硫酸铵混合内水相不同处方脂质体 编号 SBE-CD的 [Η+】浓度 ( mM ) 硫酸铵浓度 ( mM )
A 280.8 86.4
B 236.7 108.9
C 204.3 126.0
D 180.0 138.6
E 160.2 148.5
Γ 0 225.0
2. 释放条件： 2mM NH4Cl/10mM组氨^ /250mM葡萄糖 pH7.5作为释放外液， 样品用释放液稀释 10倍移入透析袋中， 装有药物的透析袋浸入盛有 透析液的溶出杯中， 溶出杯中透析液体积是透析袋中体积的 200倍， 37 °C , 75rpm进行释放试验， 分别在 1、 2、 4、 8h从透析袋中取样， 测定。 3. 测定结果： 表 4: 不同内水相长春瑞滨脂质体体外释放测定结果
取样时间 （h ) ~~不同内水相脂质体释放率 （％ ) - -
A B C D E Γ
1 34.9 25.1 33.2 36.0 39.1 68.3
2 56.6 51.8 59.0 63.1 67.7 91.5
4 83.6 83.5 89.3 90.2 93.4 98.6
8 97.4 97.2 98.0 98.5 98.6 99.3 结论： 当混合内水相中磺丁基醚环糊精 /NH3所占比例较高时药 物释放较慢， 说明 SBE-CD的铵盐的确能够延緩药物释放。 实施例 7: 硫酸铵内水相、 磺丁基醚环糊精不同铵盐内水相药物脂质 体的药代比较
1、 样品制备 药脂比为 2: 9.58, 分别制备内水相为 （NH4 ) 2S04 (依据实施 例 2 的方法， 将磺丁基醚 - β -环糊精三乙胺盐替换成硫酸铵）、 SBE-CD/TA (依据实施例 2的方法制备）、 SBE-CD/NH3 (依据实施 例 2的方法， 将磺丁基醚- β -环糊精三乙胺盐替换成 SBE-CD/NH3 ) 的长春瑞滨、 长春新碱和伊立替康脂质体。
2、 药代试验动物及给药剂量 雄性 DBA/2小鼠， 给药剂量： 10mg/kg
3、 试验结果： 表 5: 不同内水相脂质体药代血样测定结果 不同药物脂质体的半衰期 （h )
内水相
长春瑞滨 长春新碱 伊立替康
SBE-CD/TA 4.4 67.3 8.6
SBE-CD/NH3 5.4 46.2 11.3
Figure imgf000017_0001
结论：由药代结果可见磺丁基醚环糊精铵盐与硫酸铵内水相脂质 - - 体相比能够显著延长半衰期。 实施例 8: 不同内水相长春瑞滨脂质体对接种肺癌细胞 LLC小鼠的 药效比较
1、 处方： 处方 1:磺丁基醚环糊精 /TA内水相（根据实施例 2的方法制备）。 处方 2: 硫酸铵内水相 （依据实施例 2的方法， 将磺丁基醚 - β - 环糊精三乙胺盐替换成硫酸铵）。 药脂比均为 3: 9.58, DSPE-mPEG2000含量均为 0.5%。
2、 试验方法： 收集 LLC肺癌细胞， 以 DMEM培养基稀释并计数 2.0 x 106个
/ml, 于无菌条件下接种于 C57雌性小鼠前肢腋下皮下组织， 接种体 积为 0.2 ml, 含瘤细胞约 4 x l05个。 接种 14天后， 按肿瘤体积大小 将动物分组， 分别按组单次静脉给药， 给药剂量为 10 mg/kg。 给药后动物正常饲养，使用测量瘤径的方法，动态观察受试药的 抗肿瘤作用。 肿瘤体积（TV ) 的计算公式为：
TV = 1/2 X a X b2„ 其中 a和 b分别表示肿瘤长和宽。 根据测量结果计算出肿瘤体积。 实验结果采用 SPSS 11.5统计软 件， 实验结果采用 SPSS 11.5统计软件进行统计分析。
3、 药效试验结果 表 6: 不同内水相长春瑞滨脂质体对 LLC小鼠的药效比较（n=10, - -
肿瘤体积（mm 3 )
给药后 磺丁基醚环糊精 /TA 5%葡萄糖注射液 硫酸铵内水相
时间 内水相 对照组
0 785.0 + 343.0 692.2 + 259.3 780.8 + 353.3
1 1214.5土 732.4 979.7 + 507.3 1154.8 + 618.0
2 1179.6土 730.0 940.7土 415.1 1378.2 + 753.2
3 1420.5土 716.3 1116.8土 503.5 1964.3 + 1004.2
4 1591.6 + 1056.1 1091.6 + 562.3** 2456.5土 1170.1
6 1665.2土 1121.3* 1353.7 + 631.6** 3173.9 ± 1591.2
7 2034.7土 1233.8* 1846.7 + 1051.5** 4117.7土 2022.8
9 1939.0 + 1171.0** 2086.5土 1446.8** 4715.0土 2203.6
11 2605.2 + 1683.3** 3142.4土 1643.0* 6307.6 + 3194.9
12 2893.5 + 1656.5** 3650.4 + 1931.8** 7562.9 + 3819.7
14 3793.5土 26Ί1.Ί** 5106.1 土 2465.1** 9464.8 ± 4151.7
**P<0.01 , *P<0.05, 与 5%葡萄糖注射液对照组比较。 与 5%葡萄糖注射液对照组比较， 硫酸铵内水相脂质体自给药后 第 4天起，磺丁基醚环糊精内水相脂质体自第 6天起均显著抑制肿瘤 的生长。 计算相对肿瘤增殖率 T/C( % ): T/C % = TRTV I CRTV x 100%。 ( TRTV: 治疗组 RTV ； CRTV: 阴性对照组 RTV )。 相对肿瘤体 积（ RTV ): RTV = Vt I Vo。 Vo为分笼给药时（即 do)测量所得肿瘤体 积， Vt为每一次测量时的肿瘤体积。）石 酸铵内水相和磺丁基醚环糊 精内水相脂质体的相对肿瘤体积增殖率 T/C%最低点分别是 51.8% 和 31.1% , 即磺丁基醚环糊精内水相脂质体对 LLC肺癌的抑瘤疗效 大于硫酸铵内水相脂质体。 实施例 9: 不同内水相拓朴替康脂质体对接种前列腺癌细胞 RM-1小 鼠的药效比较 - -
1、 处方： 处方 1:磺丁基醚环糊精 /TA内水相（根据实施例 2的方法制备）。 处方 2: 蔗糖八硫酸酯内水相（依据实施例 2的方法， 将磺丁基 醚- β -环糊精三乙胺盐替换成蔗糖八硫酸酯）。 药脂比均为 3: 9.58, DSPE-mPEG2000含量均为 0.5%。
2、 试验方法： 收集 RM-1肺癌细胞，以 1640培养基稀释并计数 2.0 x 106个 /ml, 于无菌条件下接种于 C57雌性小鼠前肢腋下皮下组织， 接种体积为 0.2 ml, 含瘤细胞约 4 x l05个。 接种 12天后， 按肿瘤体积大小将动 物分组， 分别按组单次静脉给药， 给药剂量为 10 mg/kg。 给药后动物正常饲养，使用测量瘤径的方法，动态观察受试药的 抗肿瘤作用。 肿瘤体积 (tumor volume, TV)的计算公式为： TV = 1/2 x a x b2 。其中 a和 b分别表示肿瘤长和宽。根据测量结果计算出肿 瘤体积。 实验结果采用 SPSS 11.5统计软件进行统计分析。 3、 药效试验结果 表 7: 不同内水相拓朴替康脂质体对 RM-1小鼠的药效比较（n=10,
肿瘤体积（mm3 )
给药后 磺丁基醚环糊精 蔗糖 ¾fu酸酯内 5%葡萄糖注射
, , 游离药
天数 /TA内 目 水相 液对照组
0 220.1 ± 70.1 218.8±67.3 223.0 ± 65.7 219.6 ± 60.2
2 339.2 ± 145.0* 336.8±96.3* 484.0 ± 154.7 468.9 ± 137.7 - -
4 397.3 ± 234.4* 347.0±117.8** 606.0 ± ： 183.1 765.3 ± 415.2
6 483.1 ± 253.6** 500.3±165.5** 1060.7 ± 393.0 1376.9 ± 689.3
8 690.2 ± 656.7* 640.7±280.7** 1301.8 ± 563.7 2082.9 ± 1508.7
9 914.0 ± 691.4* 734.2±343.6* 1628.5 ± 835.4 2598.7土 2148.2
13 1876.2 ± 1931.9* 1247.8±858.7** 3592.9 ± 1523.5 4499.4 ± 2946.5
15 2833.9 ± 3016.7* 2571.1±2844.9** 6639.3 ± 2388.2 7504.9 ± 4335.9
**P<0.01, *P<0.05, 与 5%葡萄糖注射液对照组比较。 与 5%葡萄糖注射液对照组比较， 游离药无明显抑制肿瘤生长作 用 （P > 0.05 ), 两种不同内水相脂质体均显著抑制肿瘤的生长， 且与 等剂量的游离药组相比差异显著，但两脂质体处方对 RM-1肿瘤的抑 制作用无显著性差异。 实施例 10 不同内水相拓朴替康脂质体对 KM小鼠毒性比较
1、 处方： 处方 1: SBE-CD/TA内水相 (依据实施例 2的方法制备）。 处方 2: 蔗糖八硫酸酯内水相（依据实施例 2的方法， 将磺丁基 醚- β -环糊精三乙胺盐替换成蔗糖八硫酸酯）。 药脂比均为 3: 9.58, DSPE-mPEG2000含量均为 0.5%。
2、 试验方法： 选取 40.6 mg/kg作为最大给药剂量，以 1.25比率递减给药剂量， 即为 40.6、 32.5、 26.0、 20.8、 16.6、 13.3和 10.6 mg/kg剂量组， 三 种脂质体药物和游离药每个剂量组分别给予 2只 KM雌性小鼠。 每 天观察小鼠一般状态并测定体重， 观察期为 14天。 - - 不同内水相拓朴替康脂质体毒性比较
Figure imgf000022_0001
如表所示药物毒性顺序为游离药 <磺丁基醚环糊精 /ΤΑ 内水相脂 质体 <蔗糖八硫酸酯内水相脂质体， 其中蔗糖八硫酸酯内水相脂质体 在较低剂量就导致动物死亡。 本发明发明人还制备长春瑞滨、长春新碱和伊立替康脂质体，依 据类似方法对 KM小鼠进行了毒性比较， 得到了与拓朴替康脂质体 同样的结果， 即药物毒性顺序为游离药 <磺丁基醚环糊精 /TA 内水相 脂质体 <蔗糖 ^5克酸酯内水相脂质体， 其中蔗糖 ¾酸酯内水相脂质 体在较低剂量就导致动物死亡。

Claims

权利要求
1、 一种脂质体， 包含汉分子层和内水相， 其中所述内水相含有 磺丁基醚环糊精的盐和活性化合物。
2、根据权利要求 1的脂质体， 其中磺丁基醚环糊精是磺丁基醚- ot -环糊精、 磺丁基醚 - β -环糊精或磺丁基醚 - γ -环糊精。
3、 根据上述任意一项权利要求的脂质体， 其中磺丁基醚环糊精 的每个分子平均含有约 6.5个磺基基团。
4、 根据上述任意一项权利要求的脂质体， 其中磺丁基醚环糊精 的盐是磺丁基醚环糊精与胺、金属离子和铵离子中的一种或更多种形 成的盐。
5、 根据上述任意一项权利要求的脂质体， 其中磺丁基醚环糊精 的盐是磺丁基醚环糊精与氨水、 三乙胺、 三乙醇胺、 钠离子、 钾离子 和钙离子中的一种或更多种形成的盐。
6、 根据上述任意一项权利要求的脂质体， 其中所述活性化合物 是长春瑞滨、 长春新碱、 拓朴替康和伊立替康中的一种或更多种。
7、 根据上述任意一项权利要求的脂质体， 其中所述汉分子层含 有磷脂、 胆固醇和亲水性聚合物修饰的脂类物质。
8、 一种制备根据权利要求 1-7中任意一项的脂质体的方法， 包 括如下步骤： ( 1 )用磺丁基醚环糊精或其盐的水溶液水化脂相粉末以形成含 有磺丁基醚环糊精或其盐的水溶液作为内水相的空白脂质体； ( 2 )除去步骤（ 1 )的空白脂质体的外相中的磺丁基醚环糊精的 盐以形成阴离子梯度；
( 3 )任选地， 当磺丁基醚环糊精的盐是金属离子盐时， 在步骤 ( 2 )的空白脂质体的外相中加入该金属离子的载体以形成 pH梯度； 和
( 4 )将步骤（2 )或 （3 ) 的空白脂质体与活性化合物的水溶液 一起孵育以将活性化合物包封在脂质体内。
9、 一种脂质体药物制剂， 含有根据权利要求 1-7中任意一项的 脂质体和药学上可接受的载体和 /或赋型剂。
10、 根据权利要求 9的脂质体药物制剂， 其中所述载体和 /或赋 型剂中含有渗透压调节剂和 /或抗氧剂。
11、根据权利要求 1-7中任意一项的脂质体在制备用于治疗肿瘤 患者的药物中的用途，其中脂质体中的活性化合物是长春瑞滨、长春 新碱、 拓朴替康和伊立替康中的一种或更多种。
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