WO2023051799A1 - 一种酒石酸长春瑞滨脂质体及其原料组合物、制备方法和应用 - Google Patents

一种酒石酸长春瑞滨脂质体及其原料组合物、制备方法和应用 Download PDF

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WO2023051799A1
WO2023051799A1 PCT/CN2022/123371 CN2022123371W WO2023051799A1 WO 2023051799 A1 WO2023051799 A1 WO 2023051799A1 CN 2022123371 W CN2022123371 W CN 2022123371W WO 2023051799 A1 WO2023051799 A1 WO 2023051799A1
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parts
liposome
drug
sucrose octasulfate
ammonium sulfate
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English (en)
French (fr)
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尧志凌
李太行
梅进
张兆利
林一峯
李增荣
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上海济煜医药科技有限公司
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Priority to CN202280053356.4A priority Critical patent/CN117750944A/zh
Publication of WO2023051799A1 publication Critical patent/WO2023051799A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to vinorelbine tartrate liposome and its raw material composition, preparation method and application.
  • Vinorelbine is a semi-synthetic vinblastine semi-synthetic derivative, which is commercially available in the form of tartrate. It is a cell cycle-specific drug that mainly acts on the late stage of DNA synthesis in tumor cells, preventing tubulin from polymerizing to form microtubules, and at the same time Induce the depolymerization of microtubules, stop the mitotic proliferation of tumor cells in the metaphase of mitosis, and achieve the purpose of anti-tumor.
  • the current commercially available product is vinorelbine tartrate injection (trade name: Navelbine) developed by Pierre Fabre Medicament Production in France in 1989. The approved indication is non-small cell lung cancer.
  • liposomes are recognized by researchers and companies.
  • the distribution behavior of the drug in the body can carry the packaged drug passively or actively targeted to the lesion site, thereby improving the effective utilization of the drug and reducing the damage to normal cells and tissues, and enhancing the efficacy of the clinical drug.
  • vinorelbine tartrate liposome As a drug delivery system and the clinical shortcomings of vinorelbine tartrate preparations, different companies have designed different prescriptions and drug loading methods to develop vinorelbine liposomes.
  • the vinorelbine tartrate liposome developed by Qilu [CN101933904] needs to be subpackaged into three independent units of blank liposome, sodium phosphate solution and vinorelbine tartrate because the preparation system is unstable. Intravenous injection can only be done after the unit is mixed and properly heated to load the drug, which brings great inconvenience to clinical use.
  • the vinorelbine tartrate liposome developed by CSPC [EP2494960A1] adopts sulfobutyl ether cyclodextrin to carry out drug loading. The encapsulation efficiency is lower than 80%.
  • the technical problem to be solved by the present invention is to overcome the defects of poor stability, inconvenient use, short half-life or high skin toxicity of vinorelbine liposomes in the prior art, for this reason, a kind of vinorelbine tartrate lipid is provided Body and its raw material composition, preparation method and application.
  • the liposome of the invention has the advantages of good stability, suitable drug release rate, less toxic and side effects, low skin toxicity, good pharmacokinetics and convenient use, and the preparation process of the invention is simple and suitable for industrial production.
  • the present invention solves the above-mentioned technical problems through the following technical solutions.
  • the first aspect of the present invention provides a liposome raw material composition, which comprises 1-8.3 parts of vinorelbine salt, 2-12 parts of phospholipids, 0.1-0.8 parts of long-circulation membrane material, 0.7-4.0 parts of cholesterol and drug-loaded salt;
  • the drug-loaded salt is an aqueous solution of sucrose octasulfate-ammonium sulfate, the concentration of the sucrose octasulfate in the described sucrose octasulfate-ammonium sulfate is 1-75mM, and the sucrose octasulfate The concentration of ammonium sulfate in the salt-ammonium sulfate is 100-500 mM.
  • the vinorelbine salt is preferably vinorelbine tartrate.
  • the number of parts of the vinorelbine salt is preferably 2-3 parts, such as 2.77 parts.
  • the number of parts of the phospholipid is preferably 2-3 parts, such as 2.8 parts.
  • the type of phospholipids can be conventional phospholipids forming liposomes, and can also be soybean phospholipids, hydrogenated soybean phospholipids (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine One or more of choline (DPPC), dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC), such as hydrogenated soybean lecithin and/or distearoylphosphatidylcholine .
  • HSPC hydrogenated soybean phospholipids
  • DSPC distearoylphosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • the number of parts of the long-cycle membrane material is preferably 0.1-0.5 parts, such as 0.34 parts.
  • the type of the long-circulation membrane material can be a conventional long-circulation membrane material in liposomes, preferably polyethylene glycol derivatized phospholipids, preferably polyethylene glycol-dipalmitoylphosphatidylethanolamine (mPEG- DPPE) and/or polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE), such as mPEG2000-DSPE.
  • the molecular weight of the polyethylene glycol in the polyethylene glycol-dipalmitoylphosphatidylethanolamine and the polyethylene glycol in the polyethylene glycol-distearoylphosphatidylethanolamine is preferably 500-5000Da, such as 2000 .
  • the number of parts of the cholesterol is preferably 0.7-2.5 parts.
  • the liposome raw material composition in the liposome raw material composition, it preferably comprises 1.4-8.3 parts (such as 2.77 parts or 5.53 parts) of vinorelbine salt, 7.59 parts of phospholipids, 0.15-0.49 parts The long-cycle membrane material and 2.53 parts of cholesterol.
  • the number of parts of the vinorelbine salt is preferably 1.4-2.8 parts.
  • the liposome raw material composition it more preferably comprises any group of raw materials in the following groups 1-5 in parts by weight:
  • Group 1 2.77 parts of vinorelbine salt, 7.59 parts of phospholipids, 0.49 parts of long-circulation membrane materials and 2.53 parts of cholesterol;
  • Group 2 1.38 parts of vinorelbine salt, 7.59 parts of phospholipids, 0.49 parts of long-circulation membrane materials and 2.53 parts of cholesterol;
  • Group 3 5.53 parts of vinorelbine salt, 7.59 parts of phospholipids, 0.49 parts of long-circulation membrane material and 2.53 parts of cholesterol.
  • Group 4 8.31 parts of vinorelbine salt, 7.59 parts of phospholipids, 0.49 parts of long-circulation membrane materials and 2.53 parts of cholesterol;
  • Group 5 2.77 parts of vinorelbine salt, 7.59 parts of phospholipids, 0.15 parts of long-circulation membrane material and 2.53 parts of cholesterol.
  • the concentration of ammonium sulfate in the sucrose octasulfate-ammonium sulfate is preferably 200-400, for example, 300 mM.
  • the sucrose octasulfate in the described sucrose octasulfate-ammonium sulfate is preferably sucrose octasulfate alkali metal salt, sucrose octasulfate alkaline earth metal salt, sucrose octasulfate ammonium and sucrose octasulfate
  • organic amine salts such as alkali metal sucrose octasulfate (eg, potassium sucrose octasulfate or sodium sucrose octasulfate).
  • the alkali metal sucrose octasulfate is preferably one or more of lithium sucrose octasulfate, sodium sucrose octasulfate and potassium sucrose octasulfate, such as potassium sucrose octasulfate.
  • the alkaline earth metal salt of sucrose octasulfate is preferably calcium sucrose octasulfate and/or magnesium sucrose octasulfate.
  • the organic amine salt of sucrose octasulfate is preferably one or more of sucrose octasulfate trimethylamine, sucrose octasulfate diethylamine and sucrose octasulfate triethylamine.
  • the aqueous solution of the sucrose octasulfate-ammonium sulfate is an aqueous solution of 1.5-30mM sucrose octasulfate alkali metal salt and 200-400mM ammonium sulfate (i.e. sucrose octasulfate-ammonium sulfate in the aqueous solution
  • concentration of potassium sulfate is 1.5-30 mM
  • the concentration of ammonium sulfate is preferably 300 mM), such as an aqueous solution of 15 mM potassium sucrose octasulfate and 300 mM ammonium sulfate.
  • the raw material composition of the liposome can also add other raw materials according to the conventional requirements in the art when preparing liposomes, such as ultrafiltration medium, and the ultrafiltration medium can be 9.0% (W/ W) sucrose solution as ultrafiltration medium.
  • the second aspect of the present invention also provides a liposome preparation method, which is prepared by using the above-mentioned raw material composition, which includes the following steps: using ammonium sulfate gradient method to carry out drug loading to obtain the liposome.
  • the conditions and operations of the drug loading can be conventional conditions and operations in the art, and the present invention preferably follows the conditions:
  • the temperature of the drug loading is preferably 42-52°C.
  • the time for loading the drug is preferably 5-30 min.
  • a cooling step may also be included, for example, rapidly cooling the obtained liposomes in an ice-water bath at 0-5°C to below 20°C.
  • the drug loading is preferably to carry out drug loading of vinorelbine salt and blank liposome to obtain the liposome;
  • Described blank liposome comprises the internal aqueous phase in liposome membrane and liposome membrane;
  • the liposome membrane includes the following components in parts by weight: 2-12 parts of phospholipids, 0.7-4.0 parts of cholesterol, and 0.1-0.8 parts of long-circulation membrane materials;
  • the inner water phase is an aqueous solution of sucrose octasulfate-ammonium sulfate, the concentration of the sucrose octasulfate is 1-75mM, and the concentration of the ammonium sulfate is 100-500mM.
  • Described blank liposome is more preferably made by following method;
  • the preparation method of described blank lipid comprises the steps:
  • Step 1 mixing the phospholipid, the cholesterol, the long-cycle membrane material and ethanol to obtain an oil phase solution;
  • Step 2 using the ethanol injection method, the oil phase solution obtained in step 1 is injected into the aqueous solution of sucrose octasulfate-ammonium sulfate to obtain liposome colostrum solution;
  • Step 3 the liposome colostrum solution obtained in step 2 is obtained by extrusion
  • Step 4 Using the isotonic solution as the medium, the mixture in step 3 is subjected to ultrafiltration to obtain blank liposomes.
  • the mixing temperature may be 55-70°C.
  • step 2 the incubation temperature in the ethanol injection method may be 55-65°C.
  • step 3 the temperature in the extrusion method may be 55-65°C.
  • the extrusion pore diameters in the extrusion method may be 100 nm and 80 nm in turn.
  • the isotonic solution is preferably an aqueous sodium chloride solution and/or an aqueous sucrose solution, such as an aqueous solution of 0.9% (W/W) sodium chloride and 9.0% sucrose (W/W).
  • an ultrafiltration step may be further included, for example, 9.0% (W/W) sucrose solution is used as an ultrafiltration medium for ultrafiltration.
  • the third aspect of the present invention provides a liposome, which is prepared by using the above-mentioned liposome raw material composition.
  • the liposome is preferably prepared according to the above liposome preparation method.
  • the liposome is preferably used to inhibit the growth of cancer cells.
  • the fourth aspect of the present invention provides a liposome, which comprises a liposome membrane and an aqueous phase in the liposome membrane; the aqueous phase in the liposome membrane comprises 1-8.3 parts by weight of the drug Entity, the drug entity contains sucrose octasulfate anion, sulfate anion and vinorelbine cation;
  • the liposome membrane comprises the following components in parts by weight: 2-12 parts of phospholipid, 0.7-4 part of cholesterol and 0.1-0.8 part of long-circulation membrane material.
  • the number of parts of the pharmaceutical entity is preferably 2-3 parts.
  • the drug entity is preferably obtained by the following method: in the presence of an aqueous solution of sucrose octasulfate-ammonium sulfate, vinorelbine salt is loaded with ammonium sulfate gradient method to obtain the drug entity; the Changchun Rebine salt (comprising kind and number of parts) and the aqueous solution of described sucrose octasulfate-ammonium sulfate (comprising kind and concentration) are all the same as previously described.
  • described phospholipid comprising the type and number of parts of phospholipid
  • the number of parts of described cholesterol comprising the type and number of parts of long-cycle membrane material
  • the aqueous phase in the liposome membrane may also include water.
  • the liposomes may also include a liposome extramembrane water phase, and the liposome extramembrane water phase is an isotonic solution.
  • the isotonic solution is preferably an aqueous sodium chloride solution and/or an aqueous sucrose solution, such as an aqueous solution of 0.9% (W/W) sodium chloride and 9.0% sucrose (W/W).
  • the liposome is preferably used to inhibit the growth of cancer cells.
  • the fifth aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned liposome and excipients, and the excipients are the above-mentioned isotonic solution and/or pharmaceutically acceptable buffer.
  • the concentration of the pharmaceutically acceptable buffer is preferably 0.01-1 mM, and the pH is preferably 5.5-8.0.
  • HEPES 4-hydroxyethylpiperazineethanesulfonic acid
  • the present invention also provides an application of a substance A in the preparation of a drug for inhibiting the growth of cancer cells;
  • the substance A is the above-mentioned liposome, the liposome obtained by the above-mentioned liposome preparation method or The above-mentioned pharmaceutical composition.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the liposome of the present invention has the advantages of high encapsulation efficiency, good stability, high drug activity, suitable drug release rate, small toxic and side effects, good pharmacokinetics and convenient use.
  • the invention has a simple preparation process and is suitable for industrialized production.
  • Figure 1 is a graph showing the effect of different types of drug-loaded salts on the in vitro release of preparations.
  • Figure 2 is a graph showing the effect of drug-to-lipid ratio on the in vitro release of the formulation.
  • Figure 3 is a comparison chart of body weight changes in mice administered with vinorelbine tartrate liposomes of different drug-loaded salts at a dosage of 7.5 mg/kg.
  • Figure 4 is a comparison chart of body weight changes in mice administered different drug-loaded salt vinorelbine tartrate liposomes at a dosage of 10.0 mg/kg.
  • Figure 5 is a comparison chart of the skin toxicity scores of vinorelbine tartrate liposomes administered with different drug-loaded salts at a dosage of 7.5 mg/kg.
  • Figure 6 is a comparison chart of the skin toxicity scores of vinorelbine tartrate liposomes administered with different drug-loaded salts at a dosage of 10.0 mg/kg.
  • Figure 7 is a comparison chart of blood drug concentration in rats administered with different drug-loaded salt vinorelbine tartrate liposomes at a dosage of 7.5 mg/kg.
  • in vitro release method 0.005M Ammonium Chloride/0.3M Glucose/0.01M L-Histidine as release solution, mix 1.6ml sample with 2.4ml release solution and transfer to a dialysis bag, place the dialysis bag at 37°C, 90rpm Incubated in a constant temperature water bath shaker, and samples were taken at 1.0h, 2.0h, 3.0h, and 4.5h to measure the release rate.
  • in vitro release rate % initial encapsulation rate % - encapsulation rate % after release test, calculate in vitro release rate.
  • Ammonium sulfate and vinorelbine tartrate cannot form a precipitate, which indicates that ammonium sulfate is not suitable as a drug-loading salt alone.
  • Sucrose octasulfate metal salts such as Li salts, Na salts, K salts, Ca salts, Mg salts or Al salts
  • the ammonium gradient cannot be used as a drug-loading salt alone.
  • ammonium sulfate with potassium sucrose octasulfate use ammonium sulfate to provide an ammonium gradient, and potassium sucrose octasulfate combines drugs to achieve effective drug loading.
  • changing the concentration of ammonium sulfate and potassium sucrose octasulfate can adjust the loading salt Binding rate with vinorelbine tartrate (API).
  • Oil phase preparation mix and dissolve hydrogenated soybean phosphatidylcholine, cholesterol, cultured phosphatidylethanolamine (mPEG2000-DSPE) and absolute ethanol at 55-70°C according to the formula in Table 2 below to obtain an oil phase solution .
  • mPEG2000-DSPE cultured phosphatidylethanolamine
  • step (3) Inject the oil phase solution obtained in step (1) into the water phase solution obtained in step (2), and complete the incubation at 55-65° C. to obtain a liposome colostrum solution.
  • step (4) Mix the blank liposome solution obtained in step (4) with the completely dissolved vinorelbine tartrate solution and incubate at 42-52°C for 5-30min, and place the hot mixture at 0-5°C Rapidly cool down to below 20°C in an ice-water bath to complete drug loading.
  • sucrose octasulfate triethylamine and water were mixed and dissolved at 55-65° C. to obtain a 75 mM aqueous solution of sucrose octasulfate triethylamine (SOSTEA).
  • Water phase preparation mix and dissolve ammonium sulfate, sodium dextran sulfate and water at 55-65°C to obtain an aqueous phase solution with a concentration of ammonium sulfate of 300mM and a concentration of dextran sulfate sodium (DS) of 3mM .
  • Example 2 The liposomes of Example 2 and reference liposomes 1-3 were tested for encapsulation efficiency and release in vitro, and the results are shown in Table 3 and Figure 1 below.
  • the drug-loaded salt is vinorelbine liposome (reference liposome 1) of 3mM DS/300mM AS, the encapsulation efficiency after drug loading is less than 90%, and the low encapsulation efficiency will affect the safety and effectiveness of the preparation.
  • the drug-loaded salt is the vinorelbine liposome (reference liposome 2) of 75mM SOSTEA, and the drug release rate is too slow, which will cause the drug encapsulated in the liposome to accumulate in a large amount in normal tissues, and cause serious toxic and side effects to the human body .
  • the drug-loaded salt is the vinorelbine liposome (reference liposome 3) of 300mM AS, and the drug release rate is too fast, which will cause a large amount of drug to be released in the blood circulation process, and the exposure of the drug at the tumor site is insufficient, and the drug effect Unable to achieve expected results.
  • the drug-loaded salt is the vinorelbine liposome (embodiment 2) of 15mM SOSK/300mM AS, which has a high encapsulation efficiency and a suitable drug release rate, which makes it between the best anticancer efficacy and the minimum toxic and side effects. Achieve balance.
  • Vinorelbine liposomes with different drug-loaded salt concentrations all have high encapsulation efficiency ( ⁇ 90%), and the drug-release behavior of the preparation can be regulated by changing the drug-loaded salt SOSK concentration.
  • Liposomes with different particle sizes were tested for encapsulation efficiency and in vitro release, and the results are shown in Table 5 below.
  • Vinorelbine tartrate liposomes with different particle sizes can be prepared by using different extrusion times and membrane combinations.
  • the vinorelbine tartrate liposome particle size varies in the range of 90-135nm, all of which have high encapsulation efficiency ( ⁇ 90%) and suitable in vitro release rate.
  • the blank liposome solution obtained in step (4) in Example 2 was mixed with 6.93 mg (the ratio of medicine to fat was 1.40:7.59), 27.7 mg (the ratio of medicine to fat was 5.54:7.59) and 41.58 mg (the ratio of medicine to fat was 5.54:7.59) respectively.
  • the vinorelbine tartrate solution with a ratio of 8.31:7.59) was mixed and incubated at 55-65°C for 20-40 minutes, and the hot mixed solution was placed in an ice-water bath at 0-5°C to quickly cool down to below 20°C, and then completed Drug-loaded.
  • Example 2 The other steps were consistent with Example 2 to obtain liposomes with different drug-to-lipid ratios.
  • Example 2 The liposomes (Example 2, Example 6, Example 7, Example 8) with different drug-to-lipid ratios were tested for encapsulation efficiency and in vitro release, and the results are shown in Table 6 and Figure 2 below.
  • Encapsulation efficiency after drug loading refers to the encapsulation efficiency measured in samples after drug loading is completed. After the drug loading is completed, the sample will be subjected to secondary ultrafiltration to remove free drug.
  • Product Encapsulation Efficiency refers to the encapsulation efficiency of the sample obtained after secondary ultrafiltration and weight determination.
  • the encapsulation efficiency decreased and the in vitro release increased after drug loading.
  • the release behavior of the preparation can be regulated, but if the drug-to-lipid ratio is too high, the SOS is not enough to lock all the drugs, which will lead to a decrease in the encapsulation efficiency of the preparation.
  • the drug-to-lipid ratio was adjusted to 2.77:7.59 (i.e., Example 2), and the preparation had a higher encapsulation efficiency and a suitable drug release rate.
  • Oil phase preparation According to the formula, distearate phosphatidylcholine (DSPC), cholesterol, cultured phosphatidylethanolamine and absolute ethanol are mixed and dissolved at 55-70°C to obtain an oil phase solution.
  • DSPC distearate phosphatidylcholine
  • cholesterol cholesterol
  • cultured phosphatidylethanolamine and absolute ethanol are mixed and dissolved at 55-70°C to obtain an oil phase solution.
  • the liposomes of different phospholipid types were tested for encapsulation efficiency and in vitro release, and the results are shown in the table below.
  • Oil phase preparation According to the formula, hydrogenated soybean phosphatidylcholine, cholesterol, and different amounts of 0.74mg (ie 0.15 parts), 12.25mg (ie 2.45 parts), cultured phosphatidylethanolamine (mPEG2000-DSPE) and no Mix and dissolve water and ethanol at 55-70°C to obtain an oil phase solution.
  • the liposomes with different drug-to-lipid ratios were tested for encapsulation efficiency and in vitro release, and the results are shown in Table 8 below.
  • Table 8 The effect of mPEG2000-DSPE content on the encapsulation efficiency and in vitro release of the preparation
  • mPEG2000-DSPE Increasing the dosage of mPEG2000-DSPE will reduce the encapsulation efficiency and in vitro release rate after drug loading, but if it is not added or the amount added is too small, liposomes will not be able to escape the phagocytosis of the reticuloendothelial system, prolong the circulation time in the body, and enhance tumor targeting. tropism. According to the above experimental results, the proper dosage of mPEG2000-DSPE is 0.49 phr.
  • Example 12 Comparative study on the toxic and side effects and myelosuppressive effects of vinorelbine liposomes with different drug-loaded salts
  • mice Female BALB/c nude mice were used to conduct a comparative study on the toxicity and side effects of different drug-loaded vinorelbine liposomes.
  • the experimental design is as follows in Table 8:
  • the maximum average body weight of the mice with reference liposome 1 changed by more than 15%, and many mice were euthanized during the experiment.
  • Example 2 self-made prescription
  • reference liposome 2 reference liposome 3 maximum body weight changes are all less than 15%.
  • the three drug-loaded salts of Example 2, reference liposome 2, and reference liposome 3 were equivalent to bone marrow suppression in mice, and the counts of red blood cells, hemoglobin, and platelets all decreased to a certain extent. .
  • reference liposome 2 erythrocyte, hemoglobin, platelet count obviously decline, and the bone marrow suppression degree is greater than embodiment 2 (self-made prescription), reference liposome 3.
  • Embodiment 13 comparative study of pharmacokinetic experiment
  • Rats were used to carry out a PK comparative study of vinorelbine liposomes with different drug-loaded salts, and the experimental design was as follows:
  • the liposome of the present invention (embodiment 2) has a suitable drug release rate, which can meet the ideal drug release rate. Requirements for the preparation of vinorelbine tartrate liposomes.
  • Example 13 Comparison of the efficacy of homemade vinorelbine tartrate liposome and vinorelbine tartrate injection on mice inoculated with colon cancer cells HT-29
  • Drug efficacy test results the drug efficacy comparison results of homemade vinorelbine tartrate liposome and vinorelbine tartrate injection on mice inoculated with colon cancer cells HT-29 are shown in Table 19
  • Embodiment 14 Comparison of the efficacy of homemade vinorelbine tartrate liposome and vinorelbine tartrate injection on mice inoculated with fibrosarcoma cells HT-1080
  • Test method every 5 ⁇ 10 6 cells were resuspended with 100ul medium containing 50% Matrigel, and inoculated in the right axilla of the mouse.
  • random grouping was performed by Excel according to tumor size and body weight, and group Intravenous administration was carried out Q4D*2, and the single administration dose was 10.0 mg/kg.
  • Table 20 shows the results of drug efficacy comparison between homemade vinorelbine tartrate liposome and vinorelbine tartrate injection on mice inoculated with fibrosarcoma HT-1080.
  • the inventors of the present invention have carried out in-depth and extensive research on the drug-loaded salt and the preparation process, and finally obtained the liposome of the present invention, which has the advantages of high encapsulation efficiency, good stability, small toxic and side effects, superior drug efficacy and convenient clinical use .

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Abstract

一种酒石酸长春瑞滨脂质体及其原料组合物、制备方法和应用。脂质体原料组合物包含1-8.3份的长春瑞滨盐、2-12份的磷脂、0.1-0.8份的长循环膜材、0.7-4.0份的胆固醇和载药盐。该脂质体具有良好的制剂学特点，应用前景较好。

Description

一种酒石酸长春瑞滨脂质体及其原料组合物、制备方法和应用
本申请要求申请日为2021/9/30的中国专利申请2021111625656的优先权以及申请日为2022/9/27的中国专利申请2022111852813的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种酒石酸长春瑞滨脂质体及其原料组合物、制备方法和应用。
背景技术
长春瑞滨是一种半合成长春碱半合成衍生物，以酒石酸盐的形式市售，属细胞周期特异性药物，主要作用于肿瘤细胞的DNA合成后期，阻止微管蛋白聚合形成微管，同时诱导微管解聚，使肿瘤细胞的有丝分裂增殖停止于有丝分裂的中期，达到抗肿瘤目的。目前市售产品是1989年法国皮尔法伯制药公司(Pierre Fabre Medicament Production)研发的酒石酸长春瑞滨注射液(商品名：Navelbine诺维本)上市，获批的适应症为非小细胞肺癌，1991年获批转移乳腺癌适应症，至今仍为FDA用于非小细胞肺癌一线用药。国内豪森药业的酒石酸长春瑞滨注射液(商品名：盖诺)1998年国内上市，该产品属于2002年新品种，2012年冻干粉FDA批准上市。另外，市售制剂产品中还有法国皮尔法伯制药公司开发的酒石酸长春瑞滨软胶囊。但这些市售产品中注射剂临床使用时易出现静脉炎，骨髓抑制等副作用明显，口服制剂生物利用度低。
脂质体作为较为成熟纳米药物递送系统的一种药物载体被研究学者和企业所认知，其主要特点可以保护被包封药物，增加药物的稳定性，降低临床使用时的血管刺激性，改变药物在体内的分布行为，可携载包裹药物被动或主动靶向到病灶部位，进而提高药物有效利用率和降低对正常细胞组织的伤害，增强临床药物疗效。
鉴于脂质体作为药物递送系统的特点和酒石酸长春瑞滨制剂临床缺点，不同企业设计不同的处方和载药方式开发长春瑞滨脂质体。齐鲁[CN101933904]开发的酒石酸长春瑞滨脂质体，因制剂体系不稳定，需分装成空白脂质体、磷酸钠溶液、酒石酸长春瑞滨三个独立单元，临床使用前将三个分装单元混合并进行适当加热载药后方可静脉注射，这给临床使用带来极大不便。中国台湾微脂体公司[US6465008B2]开发的酒石酸长春瑞滨脂质体采用的是硫酸铵和葡聚糖硫酸酯钠的混合盐进行载药，制得的酒石酸长春瑞滨脂质体半衰期较长＞30h，存在一定的皮肤毒性(20％左右)。而石药集团[EP2494960A1]开发的 酒石酸长春瑞滨脂质体采用的是磺丁基醚环糊精进行载药，制得的酒石酸长春瑞滨脂质体体内半衰期短约为5.4h，载药包封率低于80％。
因此要解决上述问题，制备出满足临床应用及工业化生产要求的酒石酸长春瑞滨脂质体，必须找到合适的载药盐及生产工艺。
发明内容
本发明所要解决的技术问题是为了克服现有技术中长春瑞滨脂质体的稳定性差、使用不方便、半衰期短或皮肤毒性高等缺陷，为此，而提供了一种酒石酸长春瑞滨脂质体及其原料组合物、制备方法和应用。本发明的脂质体具有稳定性好、合适的释药速率、毒副作用小、皮肤毒性低、药代动力学好和使用方便的优点，且本发明的制备制备工艺简单，适合工业化生产。
本发明是通过以下技术方案解决上述技术问题的。
本发明第一方面提供了一种脂质体原料组合物，以重量份计，其包含1-8.3份的长春瑞滨盐、2-12份的磷脂、0.1-0.8份的长循环膜材、0.7-4.0份的胆固醇和载药盐；
所述的载药盐为蔗糖八硫酸酯盐-硫酸铵的水溶液，所述的蔗糖八硫酸酯盐-硫酸铵中的蔗糖八硫酸酯盐的浓度为1-75mM，所述的蔗糖八硫酸酯盐-硫酸铵中的硫酸铵的浓度为100-500mM。
本发明中，所述的长春瑞滨盐优选为酒石酸长春瑞滨。
本发明中，所述的长春瑞滨盐的份数优选为2-3份，例如2.77份。
本发明中，所述的磷脂的份数优选为2-3份，例如2.8份。
本发明中，所述的磷脂的种类可以为形成脂质体常规的磷脂，还可以为大豆磷脂、氢化大豆磷脂(HSPC)、二硬质酰磷脂酰胆碱(DSPC)、二棕榈酰磷脂酰胆碱(DPPC)、二肉蔻酰磷脂酰胆碱(DMPC)和二油酰磷脂酰胆碱(DOPC)中一种或多种，例如氢化大豆磷脂和/或二硬质酰磷脂酰胆碱。
本发明中，所述的长循环膜材的份数优选为0.1-0.5份，例如0.34份。
本发明中，所述长循环膜材的种类可为脂质体中常规的长循环膜材，优选为聚乙二醇衍生化磷脂，优选聚乙二醇-二棕榈酰磷脂酰乙醇胺(mPEG-DPPE)和/或聚乙二醇-二硬脂酰磷脂酰乙醇胺(mPEG-DSPE)，例如mPEG2000-DSPE。所述的聚乙二醇-二棕榈酰磷脂酰乙醇胺中的聚乙二醇和所述的聚乙二醇-二硬脂酰磷脂酰乙醇胺中聚乙二醇的分子量优选为500-5000Da，例如2000。
本发明中，所述的胆固醇的份数优选为0.7-2.5份。
本发明中，所述的脂质体原料组合物中，以重量份计，其优选包含1.4-8.3份(例如2.77份或5.53份)的长春瑞滨盐、7.59份的磷脂、0.15-0.49份的长循环膜材和2.53份的胆固醇。
其中，所述的长春瑞滨盐的份数优选为1.4-2.8份。
其中，所述的脂质体原料组合物中，以重量份计，其更优选包含如下组1-5中任一组原料：
组1：2.77份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
组2：1.38份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
组3：5.53份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇。
组4：8.31份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
组5：2.77份的长春瑞滨盐、7.59份的磷脂、0.15份的长循环膜材和2.53份的胆固醇。
本发明中，所述的蔗糖八硫酸酯盐-硫酸铵中的硫酸铵的浓度优选为200-400，例如300mM。
本发明中，所述的蔗糖八硫酸酯盐-硫酸铵中的蔗糖八硫酸酯盐优选为蔗糖八硫酸酯碱金属盐、蔗糖八硫酸酯碱土金属盐、蔗糖八硫酸酯铵和蔗糖八硫酸酯有机胺盐中的一种或多种，例如蔗糖八硫酸酯碱金属盐(例如蔗糖八硫酸酯钾或蔗糖八硫酸酯钠)。
其中，所述的蔗糖八硫酸酯碱金属盐优选为蔗糖八硫酸酯锂、蔗糖八硫酸酯钠和蔗糖八硫酸酯钾中的一种或多种，例如蔗糖八硫酸酯钾。
其中，所述的蔗糖八硫酸酯碱土金属盐优选为蔗糖八硫酸酯钙和/或蔗糖八硫酸酯镁。
其中，所述的蔗糖八硫酸酯有机胺盐优选为蔗糖八硫酸酯三甲胺、蔗糖八硫酸酯二乙胺和蔗糖八硫酸酯三乙胺中的一种或多种。
本发明中，所述蔗糖八硫酸酯盐-硫酸铵的水溶液为1.5-30mM蔗糖八硫酸酯碱金属盐和200-400mM硫酸铵的水溶液(即蔗糖八硫酸酯盐-硫酸铵的水溶液中蔗糖八硫酸酯钾的浓度为1.5-30mM，硫酸铵的浓度优选为300mM)，例如15mM蔗糖八硫酸酯钾和300mM硫酸铵的水溶液。
本发明中，所述的脂质体的原料组合物还可以根据本领域在制备脂质体时的常规需 求添加其他原料，例如超滤介质，所述的超滤介质可以为9.0％(W/W)蔗糖溶液作为超滤介质。
本发明第二方面还提供了一种脂质体的制备方法，采用上述的原料组合物制得，其包括如下步骤：采用硫酸铵梯度法进行载药得到所述的脂质体，即可。
本发明中，所述的载药的条件和操作可以为本领域常规的条件和操作，本发明优选如下条件：
其中，所述的载药的温度优选为42-52℃。
其中，所述的载药的时间优选为5-30min。
其中，所述的载药后还可包括冷却步骤，例如将得到的脂质体在0-5℃的冰水浴中快速降温至20℃以下。
本发明中，所述的载药优选为将长春瑞滨盐和空白脂质体进行载药得到所述的脂质体；
所述的空白脂质体包含脂质体膜和脂质体膜内的内水相；
所述的脂质体膜包括如下以重量份计的组分：2-12份的磷脂、0.7-4.0份的胆固醇、0.1-0.8份的长循环膜材；
所述的内水相为蔗糖八硫酸酯盐-硫酸铵的水溶液，所述的蔗糖八硫酸酯盐的浓度为1-75mM，所述的硫酸铵的浓度为100-500mM。
所述的空白脂质体更优选通过如下方法制得；所述的空白脂质的制备方法包括如下步骤：
步骤1：将所述的磷脂、所述的胆固醇、所述的长循环膜材和乙醇进行混合得到油相溶液；
步骤2：采用乙醇注入法，将步骤1得到的油相溶液注入所述的蔗糖八硫酸酯盐-硫酸铵的水溶液得到脂质体初乳溶液；
步骤3：将步骤2得到脂质体初乳溶液通过用挤出法得到混合物；
步骤4：以等渗溶液为介质，将步骤3混合物进行超滤，得到空白脂质体。
步骤1中，所述的混合的温度可为55-70℃。
步骤2中，所述的乙醇注入法中的孵化的温度可为55-65℃。
步骤3中，所述的挤出法中的温度可为55-65℃。
步骤3中，所述的挤出法中的挤出的孔径依次可为100nm和80nm。
步骤4中，所述的等渗溶液优选为氯化钠水溶液和/或蔗糖水溶液，例如0.9％(W/W)氯化钠和9.0％蔗糖的水溶液(W/W)。
本发明中，所述的载药后，还可进一步包括超滤步骤，例如采用9.0％(W/W)蔗糖溶液作为超滤介质进行超滤。
本发明第三方面提供了一种脂质体，所述的脂质体采用上述的脂质体原料组合物制得。
本发明中，所述的脂质体优选按照上述的脂质体的制备方法制得。
本发明中，所述的脂质体优选用于抑制癌细胞生长。
本发明第四方面提供了一种脂质体，其包含脂质体膜和脂质体膜内水相；所述的脂质体膜内水相包含以重量份计的1-8.3份的药物实体，所述的药物实体含有蔗糖八硫酸酯阴离子、硫酸根阴离子和长春瑞滨阳离子；
所述的脂质体膜包含如下以重量份计的组分：2-12份的磷脂、0.7-4份的胆固醇和0.1-0.8份的长循环膜材。
本发明中，所述的药物实体的份数优选为2-3份。
本发明中，所述的药物实体优选通过如下方法得到：在蔗糖八硫酸酯盐-硫酸铵的水溶液存在下，将长春瑞滨盐通过硫酸铵梯度法进行载药得到药物实体；所述的长春瑞滨盐(包括种类和份数)和所述的蔗糖八硫酸酯盐-硫酸铵的水溶液(包括种类和浓度)均同前所述。
其中，所述的载药的条件和操作均可同前所述。
本发明中，所述的磷脂(包括磷脂的种类和份数)、所述的胆固醇的份数和所述的长循环膜材(包括长循环膜材的种类和份数)均可同前所述。
本发明中，所述的脂质体膜内水相还可包括水。
本发明中，所述的脂质体还可包括脂质体膜外水相，所述的脂质体膜外水相为等渗溶液。所述的等渗溶液优选为氯化钠水溶液和/或蔗糖水溶液，例如0.9％(W/W)氯化钠和9.0％蔗糖的水溶液(W/W)。
本发明中，所述的脂质体优选用于抑制癌细胞生长。
本发明第五方面提供了一种药物组合物，其包括上述的脂质体和辅料，所述的辅料为上述的等渗溶液和/或药学可接受的缓冲液。
所述的药物组合物中，所述的药学可接受的缓冲液的浓度优选为0.01-1mM，pH优选为5.5-8.0。
所述的药物组合物中，所述的药学可接受的缓冲液优选为4-羟乙基哌嗪乙磺酸(HEPES)、组氨酸溶液和磷酸盐溶液中的一种或多种，例如组氨酸缓冲液(例如pH＝6.5，用量为处方量)。
本发明还提供了一种物质A在制备用于抑制癌细胞生长的药物中的应用；所述的物质A为上述的脂质体、通过上述的脂质体的制备方法得到的脂质体或上述的药物组合物。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明的脂质体具有包封率高、稳定性好、药物活性高、合适的释药速率、毒副作用小、药代动力学好和使用方便的优点，且本发明的制备制备工艺简单，适合工业化生产。
附图说明
图1为不同载药盐种类对制剂体外释放影响的曲线图。
图2为药脂比对制剂体外释放影响的曲线图。
图3为7.5mg/kg给药剂量下，给药不同载药盐的酒石酸长春瑞滨脂质体的小鼠体重变化对比图。
图4为10.0mg/kg给药剂量下，给药不同载药盐酒石酸长春瑞滨脂质体的小鼠体重变化对比图。
图5为7.5mg/kg给药剂量下，给药不同载药盐酒石酸长春瑞滨脂质体的皮肤毒性评分对比图。
图6为10.0mg/kg给药剂量下，给药不同载药盐酒石酸长春瑞滨脂质体的皮肤毒性评分对比图。
图7为7.5mg/kg给药剂量下，给药不同载药盐酒石酸长春瑞滨脂质体在大鼠中的血药浓度对比图。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
包封率的测定方法：量取100ul脂质体，使用Sepharose CL-4B琼脂糖凝胶分离脂质体和游离药部分，加入6ml 0.9％NaCl溶液洗脱脂质体部分，再加入7ml 0.9％NaCl溶液洗脱游离药物部分，使用液相色谱法分别测定游离药物和脂质体部分的含量，按公式包封率＝脂质体部分/(脂质体部分+游离药物部分)*100％，计算包封率。
体外释放方法：0.005M氯化铵/0.3M葡萄糖/0.01M L-组氨酸作为释放液，将1.6ml样品与2.4ml释放液混合转移到透析袋中，透析袋置于37℃、90rpm转速的恒温水浴振荡器内孵育，分别在1.0h、2.0h、3.0h、4.5h取样测定释放率。按公式体外释放率％＝初始包封率％－释放试验后的包封率％，计算体外释放率。
实施例1 药物与载药盐结合实验
配制60mg/ml酒石酸长春瑞滨水溶液与不同浓度的载药盐溶液按照体积比为1：2充分搅拌混合后，离心处理得上清液，检测酒石酸长春瑞滨含量，计算酒石酸长春瑞滨与载药盐的结合效率。数据下表1所示：
沉淀百分率计算公式：沉淀百分比＝100％-C 1/C 2，其中C 1为离心后上层清液API浓度，C 2为上述混合溶液中API的理论浓度，即20mg/ml。
表1 不同载药盐溶液浓度与酒石酸长春瑞滨沉淀结合试验
Figure PCTCN2022123371-appb-000001
结果：
硫酸铵与酒石酸长春瑞滨不能形成沉淀，这表明硫酸铵不适合单独作为载药盐。蔗糖八硫酸酯金属盐(例如Li盐、Na盐、K盐、Ca盐、Mg盐或者Al盐)可与酒石酸长春瑞滨形成稳定的沉淀，但因其不能为脂质体内外水相提供有效的铵梯度，也无法单独作为载药盐。
将硫酸铵与蔗糖八硫酸酯钾混合，利用硫酸铵提供铵梯度，蔗糖八硫酸酯钾结合药物，可实现有效的药物装载，同时改变硫酸铵、蔗糖八硫酸酯钾浓度，可调节载药盐与酒石酸长春瑞滨(API)的结合率。
实施例2
(1)油相配制：根据下表2的配方将氢化大豆磷脂酰胆碱、胆固醇、培化磷脂酰乙醇胺(mPEG2000-DSPE)与无水乙醇在55-70℃下混合溶解，得到油相溶液。
(2)水相配制：根据配方将硫酸铵，蔗糖八硫酸酯钾盐与水在55-65℃下混合溶解，得到硫酸铵浓度为300mM和蔗糖八硫酸酯钾浓度为15mM的水相溶液。
(3)将步骤(1)得到的油相溶液注入到步骤(2)得到水相溶液中，在55-65℃下完成孵化，得到脂质体初乳溶液。
(4)将步骤(3)得到的脂质体初乳溶液，在55-65℃条件下经100nm和80nm聚碳酸酯膜完成挤出得到合适粒径。使用0.9％(W/W)氯化钠溶液和蔗糖溶液9.0％(W/W)做为超滤介质对脂质体溶液进行超滤，以便除掉脂质体外水相中的硫酸铵和蔗糖八硫酸酯钾，水相中的乙醇，从而构建脂质体内外水相盐梯度，得到空白脂质体溶液。
(5)将步骤(4)得到的空白脂质体溶液，与完全溶解的酒石酸长春瑞滨溶液混合后在42-52℃条件下孵化保持5-30min，将热混合液置于0-5℃的冰水浴中快速降温至20℃以下，进而完成载药。
(6)使用蔗糖溶液9.0％(W/W)对步骤(5)完成载药的脂质体样品进行二次超滤，除去游离药物并加入处方量的组氨酸缓冲液(pH＝6.5)进行定容。
表2
Figure PCTCN2022123371-appb-000002
Figure PCTCN2022123371-appb-000003
参比脂质体1
1)水相配制：将蔗糖八硫酸酯三乙胺与水在55-65℃下混合溶解，得到浓度为75mM的蔗糖八硫酸酯三乙胺(SOSTEA)水相溶液。
其他步骤同与上述的步骤一致。
参比脂质体2
1)水相配制：将硫酸铵，葡聚糖硫酸酯钠与水在55-65℃下混合溶解，得到硫酸铵浓度为300mM和葡聚糖硫酸酯钠(DS)浓度为3mM的水相溶液。
其他步骤与上述的步骤一致。
参比脂质体3
1)水相配制：将硫酸铵与水在55-65℃下混合溶解，得到浓度为300mM的水相溶液。
其他步骤与上述的步骤一致。
将实施例2脂质体和参比脂质体1-3进行包封率和体外释放测试，其结果见下表3和图1。
表3：不同载药盐种类对制剂包封率、体外释放影响
Figure PCTCN2022123371-appb-000004
结果与讨论：
载药盐为3mM DS/300mM AS的长春瑞滨脂质体(参比脂质体1)，载药后包封率小于90％，包封率偏低会影响制剂安全性及有效性。
载药盐为75mM SOSTEA的长春瑞滨脂质体(参比脂质体2)，释药速率过慢，这将导致脂质体包裹的药物在正常组织大量累积，对人体产生严重的毒副作用。
载药盐为300mM AS的长春瑞滨脂质体(参比脂质体3)，释药速率过快，这将导致药物在血液循环过程中大量释放，药物在肿瘤部位暴露量不足，药效无法达到预期效果。
载药盐为15mM SOSK/300mM AS的长春瑞滨脂质体(实施例2)，具有高包封率、合适的释药速率，这使得其能在最佳抗癌功效与最小毒副作用之间达到平衡。
实施例3-4
1)水相配制：在55-65℃温度下，分别配制8、30mM的蔗糖八硫酸酯钾盐/300mM硫酸铵水溶液。
其他步骤同实施例2一致，得到实施例3和4脂质体。
将实施例3和4脂质体进行包封率和体外释放测试，其结果见下表4。
表4：不同载药盐浓度对制剂包封率、体外释放影响
Figure PCTCN2022123371-appb-000005
结果与讨论：
不同载药盐浓度的长春瑞滨脂质体，均有较高的包封率(≥90％)，通过改变载药盐SOSK浓度，可对制剂释药行为进行调控。
实施例5
按照实施例2中步骤(3)得到的脂质体初乳溶液，在55-65℃温度下，采用不同模组合、挤压遍数，制备不同粒径的空白脂质体。
其他步骤同实施例2一致，得到不同粒径脂质体。
将不同粒径的脂质体进行包封率和体外释放测试，其结果见下表5。
表5：不同粒径脂质体的对制剂包封率、体外释放影响
Figure PCTCN2022123371-appb-000006
Figure PCTCN2022123371-appb-000007
结果：
采用不同挤压遍数和膜组合，可制备得到不同粒径的酒石酸长春瑞滨脂质体。酒石酸长春瑞滨脂质体粒径在90-135nm范围内变化，均具有较高的包封率(≥90％)、合适的体外释放速率。
实施例6-8
1)将实施例2中步骤(4)得到的空白脂质体溶液，分别与6.93mg(药脂比为1.40：7.59)、27.7mg(药脂比为5.54：7.59)和41.58mg(药脂比为8.31：7.59)的酒石酸长春瑞滨溶液混合后在55-65℃条件下孵化保持20-40min，将热混合液置于0-5℃的冰水浴中快速降温至20℃以下，进而完成载药。
其他步骤同实施例2一致，得到不同药脂比的脂质体。
将不同药脂比的脂质体(实施例2、实施例6、实施例7、实施例8)进行包封率和体外释放测试，其结果见下表6和图2。
表6：药脂比对制剂包封率及体外释放影响
Figure PCTCN2022123371-appb-000008
备注：“载药后包封率”是指载药完成后样品测得的包封率。载药完成后会对样品进行二次超滤除去游离药物。“产品包封率”是指经二次超滤、定重后获得的样品包封率。
结果与讨论：
随着药脂比提高，载药后包封率呈下降、体外释放呈上升趋势。通过改变药脂比，可对制剂释药行为进行调控，但药脂比过高，SOS不足以锁定所有药物，会导致制剂包封率下降。药脂比调整到2.77：7.59(即实施例2)，制剂具有较高的包封率及合适的释药速率。
实施例9
1)油相配制：根据配方将二硬脂酸磷脂酰胆碱(DSPC)，胆固醇，培化磷脂酰乙醇胺与无水乙醇在55-70℃下混合溶解，得到油相溶液。
其他步骤与同实施例2一致。
将不同磷脂种类的脂质体进行包封率和体外释放测试，其结果见下表。
表7 磷脂种类对制剂包封率、体外释放影响
Figure PCTCN2022123371-appb-000009
结果与讨论：
结合上述数据及实施例2可知：采用不同磷脂(HSPC、DSPC)制备的酒石酸长春瑞滨脂质体，载药后包封率均≥90％，且体外释放无明显差异。这表明HSPC、DSPC两者具有可替代性。
实施例10-11
1)油相配制：根据配方将氢化大豆磷脂酰胆碱，胆固醇，与不同量0.74mg(即0.15份)、12.25mg(即2.45份)、的培化磷脂酰乙醇胺(mPEG2000-DSPE)与无水乙醇在55-70℃下混合溶解，得到油相溶液。
其他步骤与同实施例2一致，得到不同mPEG2000-DSPE的脂质体。
将不同药脂比的脂质体进行包封率和体外释放测试，其结果见下表8。
表8：mPEG2000-DSPE含量对制剂包封率、体外释放影响
Figure PCTCN2022123371-appb-000010
结果与讨论：
mPEG2000-DSPE投料量增加，会降低载药后包封率及体外释放速率，但若不加入或加入量过少，脂质体将无法逃避网状内皮系统吞噬，延长体内循环时间，增强肿瘤靶向性。依据上述实验结果，mPEG2000-DSPE合适的投料量为0.49份。
实施例12：对不同载药盐长春瑞滨脂质体的毒副作用和骨髓抑制作用的对比研究
1、毒副作用的研究
采用雌性BALB/c裸小鼠，对不同载药盐长春瑞滨脂质体进行毒副作用对比研究，实验设计如下表8：
表9
Figure PCTCN2022123371-appb-000011
Figure PCTCN2022123371-appb-000012
研究期间对下表10中的项目进行观察：
表10
Figure PCTCN2022123371-appb-000013
其中皮肤毒性评分标准如下表11：
表11
Figure PCTCN2022123371-appb-000014
体重变化结果如表12以及图3和4所示。
表12
Figure PCTCN2022123371-appb-000015
Figure PCTCN2022123371-appb-000016
在7.5mg/kg、10mg/kg给药剂量下，参比脂质体1的小鼠最大平均体重变化超过15％，且实验过程中有多只小鼠被处以安乐死。实施例2(自制处方)、参比脂质体2、参比脂质体3最大体重变化均小于15％。停止给药后，各组别小鼠体重出现缓慢恢复，给药第20天，所有小鼠体重均恢复到正常水平。
皮肤毒性结果如表13以及图5和6所示。
表13
Figure PCTCN2022123371-appb-000017
7.5mg/kg、10mg/kg给药剂量下，实施例2皮肤毒性显著小于参比脂质体1、参比脂质体2。7.5mg/kg给药剂量下实施例2与参比脂质体3皮肤毒性评分在统计学上无显著差异，10mg/kg给药剂量下参比脂质体3皮肤毒性评分略优于实施例2。
2、骨髓抑制的研究
最后1次给药96h后血常规结果见表14：
表14
Figure PCTCN2022123371-appb-000018
Figure PCTCN2022123371-appb-000019
从血常规结果可以看出，骨髓抑制具有剂量相关性，所有处方10mg/kg给药剂量的骨髓抑制程度均高于7.5mg/kg。
在7.5mg/kg、10mg/kg给药剂量下，参比脂质体1小鼠的红细胞、血红蛋白、血小板计数均出现明显下降，SOSTEA作为载药盐会引起严重的骨髓抑制。
在7.5mg/kg给药剂量下，实施例2、参比脂质体2、参比脂质体3三种载药盐对小鼠骨髓抑制相当，红细胞、血红蛋白、血小板计数均出现一定程度下降。
在10mg/kg给药剂量下，参比脂质体2红细胞、血红蛋白、血小板计数出现明显下降，骨髓抑制程度大于实施例2(自制处方)、参比脂质体3。
第一次给药至第20天血常规结果表15：
表15：
Figure PCTCN2022123371-appb-000020
Figure PCTCN2022123371-appb-000021
给药后第20天，实施例2、参比脂质体2、参比脂质体3血小板计数迅速恢复到正常水平，红细胞、血红蛋白也出现缓慢恢复迹象。参比脂质体1血小板计数迅速恢复到正常水平，但红细胞未出现任何恢复进展。
结论见表16：
表16
Figure PCTCN2022123371-appb-000022
实施例13：药代动力学实验对比研究
采用大鼠对不同载药盐长春瑞滨脂质体进行PK对比研究，实验设计如下：
1、酒石酸长春瑞滨脂质体动物PK实验基本信息如表17
表17
Figure PCTCN2022123371-appb-000023
Figure PCTCN2022123371-appb-000024
2、酒石酸长春瑞滨脂质体动物PK实验样品采集信息如表18所示：
表18
Figure PCTCN2022123371-appb-000025
3、酒石酸长春瑞滨脂质体动物PK实验血样检测结果如表19所示以及图7所示：
表19
Figure PCTCN2022123371-appb-000026
结果与讨论：酒石酸长春瑞滨注射液进入大鼠体内，被快速消除；不同载药盐酒石酸长春瑞滨脂质体在体内均存在缓慢释药行为。
药物在血液循环过程中释放过快，将导致药物在肿瘤部位暴露量不足，药效无法达到预期效果；药物释放过慢，药物在正常组织大量累积如皮肤，将产生严重的毒副作用如手足综合症，从上述PK结果可知，参比脂质体1释放过慢，参比脂质体3释放过快，本发明的脂质体(实施例2)具有合适的释药速率，可满足理想的酒石酸长春瑞滨脂质体制备要求。
实施例13：自制酒石酸长春瑞滨脂质体与酒石酸长春瑞滨注射液对接种结肠癌细胞HT-29小鼠的药效对比
试验方法：每1×10 7个细胞用100ul含50％Matrigel的培养基重悬，接种于小鼠右侧腋下。当肿瘤体积均值达到约139mm 3时(肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长和宽)，根据肿瘤大小和体重通过Excel进行随机分组，按组进行Q3D*4静脉给药，单次给药剂量为7.5mg/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瘤作用，肿瘤体积的计算公式为V＝0.5a×b 2，其中a和b分别表示肿瘤的长和宽,实验结果采用GraphPad Prism 5进行分析。
药效试验结果：自制酒石酸长春瑞滨脂质体与酒石酸长春瑞滨注射液对接种结肠癌细胞HT-29小鼠的药效对比结果见表19
表19
Figure PCTCN2022123371-appb-000027
结论：由药效试验结果可见，与空白组相比，酒石酸长春瑞滨脂质体、酒石酸长春瑞滨注射液均有抑瘤作用。酒石酸长春瑞滨脂质体比酒石酸长春瑞滨注射液对HT-29肿瘤生长的抑制更强。
实施例14：自制酒石酸长春瑞滨脂质体与酒石酸长春瑞滨注射液对接种纤维肉瘤细 胞HT-1080小鼠的药效对比
试验方法：每5×10 6个细胞用100ul含50％Matrigel的培养基重悬，接种于小鼠右侧腋下。当肿瘤体积均值达到约144mm 3时(肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长和宽)，根据肿瘤大小和体重通过Excel进行随机分组，按组进行Q4D*2静脉给药，单次给药剂量为10.0mg/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瘤作用，肿瘤体积的计算公式为V＝0.5a×b 2，其中a和b分别表示肿瘤的长和宽,实验结果采用GraphPad Prism 5进行分析。
药效试验结果：自制酒石酸长春瑞滨脂质体与酒石酸长春瑞滨注射液对接种纤维肉瘤HT-1080小鼠的药效对比结果见表20。
表20
Figure PCTCN2022123371-appb-000028
结论：由药效试验结果可见，与空白组相比，酒石酸长春瑞滨脂质体、酒石酸长春瑞滨注射液均有抑瘤作用。酒石酸长春瑞滨脂质体比酒石酸长春瑞滨注射液对HT-1080肿瘤生长的抑制更强。
本领域均知，理想的酒石酸长春瑞滨脂质体应具备以下几个特征：1)较高的药物装 载效率；2)合适的体内半衰期，以实现最佳抑瘤效果与最小毒副作用之间平衡；3)存储过程中相对稳定，不发生药物泄漏；4)制备工艺简单，适合工业化生产。
本发明的发明人通过对载药盐及制备工艺进行深入广泛的研究，最终得到本发明的脂质体具有包封率高、稳定性好、毒副作用小、药效优越以及临床使用方便的优点。

Claims (13)

  1. 一种脂质体原料组合物，其特征在于，以重量份计，其包含1-8.3份的长春瑞滨盐、2-12份的磷脂、0.1-0.8份的长循环膜材、0.7-4.0份的胆固醇和载药盐；
    所述的载药盐为蔗糖八硫酸酯盐-硫酸铵的水溶液，所述的蔗糖八硫酸酯盐-硫酸铵中的蔗糖八硫酸酯盐的浓度为1-75mM，所述的蔗糖八硫酸酯盐-硫酸铵中的硫酸铵的浓度为100-500mM。
  2. 如权利要求1所述的脂质体原料组合物，其特征在于，所述的脂质体原料组合物满足如下1个或多个条件：
    (1)所述的长春瑞滨盐为酒石酸长春瑞滨，例如2.77份；
    (2)所述的磷脂的份数为2-3份，例如2.8份；
    (3)所述的磷脂的种类为大豆磷脂、氢化大豆磷脂、二硬质酰磷脂酰胆碱、二棕榈酰磷脂酰胆碱、二肉蔻酰磷脂酰胆碱和二油酰磷脂酰胆碱中一种或多种，例如氢化大豆磷脂和/或二硬质酰磷脂酰胆碱；
    (4)所述的长循环膜材的份数为0.1-0.5份，例如0.34份；
    (5)所述长循环膜材的种类为聚乙二醇衍生化磷脂，优选聚乙二醇-二棕榈酰磷脂酰乙醇胺和/或聚乙二醇-二硬脂酰磷脂酰乙醇胺，例如mPEG2000-DSPE；
    (6)所述的胆固醇的份数为0.7-2.5份；
    (7)所述的蔗糖八硫酸酯盐-硫酸铵中的蔗糖八硫酸酯盐的浓度为1-50mM，例如30mM；
    (8)所述的蔗糖八硫酸酯盐-硫酸铵中的硫酸铵的浓度为200-400，例如300mM；
    (9)所述的蔗糖八硫酸酯盐-硫酸铵中的蔗糖八硫酸酯盐为蔗糖八硫酸酯碱金属盐、蔗糖八硫酸酯碱土金属盐、蔗糖八硫酸酯铵和蔗糖八硫酸酯有机胺盐中的一种或多种，例如蔗糖八硫酸酯碱金属盐。
  3. 如权利要求2所述的脂质体原料组合物，其特征在于，所述蔗糖八硫酸酯盐-硫酸铵的水溶液为1.5-30mM蔗糖八硫酸酯碱金属盐和200-400mM硫酸铵的水溶液，例如15mM蔗糖八硫酸酯钾和300mM硫酸铵的水溶液；
    和/或，所述的脂质体原料组合物中，以重量份计，其包含1.4-8.3份的长春瑞滨盐、7.59份的磷脂、0.15-0.49份的长循环膜材和2.53份的胆固醇；所述的长春瑞滨盐的份数优选为1.4-2.8份。
  4. 如权利要求3所述的脂质体原料组合物，其特征在于，所述的脂质体原料组合物中，以重量份计，其选包含如下组1-5中任一组原料：
    组1：2.77份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
    组2：1.38份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
    组3：5.53份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
    组4：8.31份的长春瑞滨盐、7.59份的磷脂、0.49份的长循环膜材和2.53份的胆固醇；
    组5：2.77份的长春瑞滨盐、7.59份的磷脂、0.15份的长循环膜材和2.53份的胆固醇。
  5. 一种脂质体的制备方法，其特征在于，采用如权利要求1-4中任一下相的脂质体原料组合物制得，其包括如下步骤：采用硫酸铵梯度法进行载药得到所述的脂质体，即可。
  6. 如权利要求5所述的脂质体的制备方法，其特征在于，
    (1)所述的载药的温度为42-52℃；
    (2)所述的载药的时间为5-30min；
    (3)所述的载药后，还进一步包括超滤步骤，例如采用9.0％蔗糖溶液作为超滤介质进行超滤；
    (4)所述的载药为将长春瑞滨盐和空白脂质体进行载药得到所述的脂质体；
    所述的空白脂质体包含脂质体膜和脂质体膜内的内水相；
    所述的脂质体膜包括如下以重量份计的组分：2-12份的磷脂、0.7-4.0份的胆固醇、0.1-0.8份的长循环膜材；
    所述的内水相为蔗糖八硫酸酯盐-硫酸铵的水溶液，所述的蔗糖八硫酸酯盐的浓度为1-75mM，所述的硫酸铵的浓度为100-500mM；
    优选通过选通过如下方法制得；所述的空白脂质的制备方法包括如下步骤：
    步骤1：将所述的磷脂、所述的胆固醇、所述的长循环膜材和乙醇进行混合得到油相溶液；
    步骤2：采用乙醇注入法，将步骤1得到的油相溶液注入所述的蔗糖八硫酸酯盐-硫酸铵的水溶液得到脂质体初乳溶液；
    步骤3：将步骤2得到脂质体初乳溶液通过用挤出法得到混合物；
    步骤4：以等渗溶液为介质，将步骤3混合物进行超滤，得到空白脂质体。
  7. 如权利要求6所述的脂质体的制备方法，其特征在于，(1)所述的空白脂质的制备方法中的步骤1中，所述的混合的温度为55-70℃；
    (2)步骤2中，所述的乙醇注入法中的孵化的温度为55-65℃；
    (3)步骤3中，所述的挤出法中的温度为55-65℃；
    (4)步骤3中，所述的挤出法中的挤出的孔径依次为100nm和80nm；
    (5)步骤4中，所述的等渗溶液为氯化钠水溶液和/或蔗糖水溶液，例如0.9％氯化钠和9.0％蔗糖的水溶液。
  8. 一种脂质体，其特征在于，所述的脂质体采用如权利要求1-4中的任一项所述的脂质体原料组合物制得。
  9. 如权利要求8所述的脂质体，其特征在于，所述的脂质体按照如权利要求5-8中任一项所述的脂质体的制备方法制得；
    和/或，所述的脂质体用于抑制癌细胞生长。
  10. 一种脂质体，其特征在于，其包含脂质体膜和脂质体膜内水相；所述的脂质体膜内水相包含以重量份计的1-8.3份的药物实体，所述的药物实体含有蔗糖八硫酸酯阴离子、硫酸根阴离子和长春瑞滨阳离子；
    所述的脂质体膜包含如下以重量份计的组分：2-12份的磷脂、0.7-4份的胆固醇和0.1-0.8份的长循环膜材。
  11. 如权利要求10所述的脂质体，其特征在于，所述的脂质体满足如下1个或多个条件：
    (1)所述的药物实体的份数为2-3份；
    (2)所述的药物实体通过如下方法得到：在蔗糖八硫酸酯盐-硫酸铵的水溶液存在下，将长春瑞滨盐通过硫酸铵梯度法进行载药得到药物实体；所述的长春瑞滨盐和所述的蔗糖八硫酸酯盐-硫酸铵的水溶液均如权利要求1-4中任一项所述；所述的载药的条件和操作均如权利要求6或7所述；
    (3)所述的磷脂、所述的胆固醇的份数和所述的长循环膜材均如权利要求1-4中任一项所述；
    (4)所述的脂质体膜内水相还包括水；
    (5)所述的脂质体还包括脂质体膜外水相，所述的脂质体膜外水相为等渗溶液；所述的等渗溶液优选为氯化钠水溶液和/或蔗糖水溶液，例如0.9％氯化钠和9.0％蔗糖的水溶液；
    (6)所述的脂质体用于抑制癌细胞生长。
  12. 一种药物组合物，其包括如权利要求8-11中任一项所述的脂质体和辅料，所述的辅料为等渗溶液和/或药学可接受的缓冲液，所述的等渗溶液如权利要求7所述；
    所述的药学可接受的缓冲液的浓度优选为0.01-1mM，pH优选为5.5-8.0；
    所述的药学可接受的缓冲液优选为4-羟乙基哌嗪乙磺酸、组氨酸溶液和磷酸盐溶液中的一种或多种，例如组氨酸缓冲液。
  13. 一种物质A在制备用于抑制癌细胞生长的药物中的应用；所述的物质A为如权利要求8-11中任一项所述的脂质体或如权利要求12所述的药物组合物。
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