Pharmaceutical composition of gemcitabine liposomes, its preparation method and use

Abstract

Pharmaceutical composition of gemcitabine liposome, its preparation method and its use. According to the preparation method of liposome, by optimizing and controlling the ratio of phospholipid to cholesterol, the ratio of total lipid to optional long-circulating functional substance, the ratio of total lipid to drug, and the type and concentration of aqueous inner salt solution, liposomes are prepared, which have high encapsulation efficiency, good storage stability and high filtration ability, do not leak easily, have stable and controllable release, significantly extend half-life, improve therapeutic effect, and reduce toxic and side effects.In addition, the formulation and preparation process of the provided liposomes is simple, and it is easy to be produced on a large scale and to be widely used and applied in clinical practice.

Description

[Technical field]

[0001] This application claims priority to CN Application No. 202210300041.7 and filed March 25, 2022. The disclosure of the CN application is hereby incorporated in its entirety into this application.

[0002] [Technical field] The present invention relates to the field of pharmaceutical preparations, in particular to a liposomal composition of gemcitabine or its pharma-ceutically acceptable salts and its preparation method and use.

[0003] [Background technology] Gemcitabine is a metabolic anti-cancer drug with a wide range of clinical applications. Gemcitabine is used as a first-line chemotherapy regimen, alone or in combination with other drugs, for various solid tumors (including pancreatic cancer, breast cancer, non-small cell lung cancer, ovarian cancer, etc.). Due to its simple molecular structure and strong hydrophilicity, gemcitabine is rapidly metabolized and excreted by the liver and kidney after entering the body, resulting in its extremely short half-life, weak targeting, and large clinical dose, which often causes severe toxicity in the blood system, limiting its clinical use.

[0004] As a nanoscale drug carrier, liposomes have the advantages of safety, non-toxicity, good biocompatibility, sustained release, and targeting to tumor tissues / cells through enhanced vascular permeability and retention (EPR). After drugs are encapsulated in liposomes, they can avoid rapid clearance of drugs, prolong blood circulation time, enhance targeting to tumor tissues / cells, and reduce distribution to normal tissues, thereby achieving the purpose of reducing toxicity and increasing efficacy.

[0005] Up to now, there is no liposomal formulation of gemcitabine available on the market. Current research on gemcitabine liposomes faces problems of fast release, instability, low encapsulation efficiency, complicated process, difficulty in industrial scale-up production, easy leakage in the body, and high safety risk, while current research also faces shortcomings of insufficient research on the type and formulation ratio of drugs, as well as phospholipid excipients and buffer salt systems. China Patent No. 106474067 discloses polyethylene glycol-modified gemcitabine liposomes, the liposomes have low encapsulation efficiency (about 70%) and low stability (approximately 30 days of stability), making them difficult for industrial production and clinical application. China Patent No. 102784107 discloses gemcitabine liposomes containing injection oil, the encapsulation efficiency of liposomes is improved, but lyophilization is required to maintain the stability of liposomes, resulting in complicated process and high cost, so liposomes are not conducive to industrial production. Chinese Patent No. 102846547 discloses gemcitabine liposomes containing emulsifiers, which improves the stability of liposomes, but the liposomes contain polyoxyethylene ricinoleate, which tends to cause high safety risks and acute hypersensitivity reactions, neurotoxicity, etc. Some documents (e.g., Chinese Patent Application Publication No. 111035616, Chinese Patent Application Publication No. 111437259, Chinese Patent Application Publication No. 108348538, Chinese Patent Application Publication No. 111632030) record the composition and preparation method of gemcitabine liposomes, and the individual protocols of liposomes show that some indicators are better. However, there is still insufficient information on the study of key parameters related to liposome stability, encapsulation efficiency, release, filterability, attenuation and synergy (e.g., selection of excipients constituting the liposome, excipient ratio, excipient to drug ratio, control of the aqueous internal environment of the liposome, etc.).

[0006] In conclusion, it is of great significance to prepare gemcitabine liposomes that have stable and controllable release, less leakage during storage, high encapsulation efficiency, high stability and high drug loading capacity, while at the same time can significantly extend the serum half-life of gemcitabine, improve efficacy, reduce toxic effects and side effects, and have a simple composition that is practical for industrial production, thereby meeting clinical needs.

[0007] [Contents of the present invention] The purpose of the present application is to provide a gemcitabine liposome or its pharma- ceutical acceptable salt, which has high drug loading capacity, high encapsulation efficiency, good storage stability and filterability, stable and controllable release, is suitable for industrial scale-up production, has low toxic and side effects, and / or has better drug efficacy.Compared with the traditional injection of gemcitabine, the liposome of the present application can avoid the rapid metabolism of gemcitabine or its pharma-ceutical acceptable salt in the body, prolong the blood circulation time, improve tumor targeting, improve therapeutic effect, and reduce the severe hematological toxicity of traditional injection, thereby meeting the need for clinical improvement.

[0008] In one aspect, the present invention relates to a gemcitabine liposome comprising a liposomal membrane and an aqueous interior encapsulated by the liposomal membrane, The components that make up the liposome membrane include phospholipids, cholesterol, and optionally, a long-circulating functional substance; the aqueous interior includes gemcitabine or a pharma- ceutically acceptable salt thereof, and a salt solution; and The weight ratio of phospholipid to cholesterol is from 2:1 to 20:1; Long-circulating functional substances account for 0% to 30% by mass of total lipids; the weight ratio of total lipid in the liposome to gemcitabine or a pharma- ceutically acceptable salt thereof is 1:1 to 100:1; The molarity of the salt solution is 50-800 mM.

[0009] In the liposomes of the present invention, the aqueous interior contains a salt solution, and the molarity of the salt solution refers to the molar concentration of the salt molecules in the salt solution; when the aqueous interior contains different salts, the molarity refers to the total molar concentration of the different salt molecules in the salt solution.

[0010] In the liposomes, interactions are observed between gemcitabine and lipids (including phospholipids, cholesterol and long-circulating functional substances), between gemcitabine and the aqueous internal salt solution, between lipids and components contained in the aqueous internal environment, and among lipids themselves, thereby forming liposomes with good stability, high encapsulation efficiency, uniform particle size, stable and controllable release, and good filterability, which can significantly extend the circulation time of gemcitabine in the body, improve efficacy, and reduce toxic effects and side effects.

[0011] In some embodiments, the weight ratio of phospholipid to cholesterol is from 2:1 to 15:1, preferably from 2:1 to 10:1, preferably from 2.5:1 to 10:1, more preferably from 2:1 to 8:1, for example from 2:1 to 3:1 or from 7:1 to 8:1.

[0012] In some embodiments, the long circulating functional substance comprises 0% to 20% by weight of the total lipids (including phospholipids, cholesterol and optional long circulating functional substances), preferably 0% to 15% by weight, for example 0% or 0.1% to 15% by weight, as further examples 0.1% to 0.5%, 0.1% to 5%, 0.1% to 10%, 0.1% to 12%, 0.1% to 15%, 0.5% to 5%, 0.5% to 10%, 0.5% to 12%, 0.5% to 15%, 5% to 10%, 5% to 12%, 5% to 15%, 10% to 12%, 10% to 15%, or 12% to 15% by weight.

[0013] In some embodiments, the weight ratio of total lipids (including phospholipids, cholesterol, and optional long-circulating functional substances) to gemcitabine or a pharma- ceutically acceptable salt thereof is from 5:1 to 80:1, preferably from 10:1 to 50:1, more preferably from 10:1 to 40:1, such as from 10:1 to 15:1, 10:1 to 20:1, 10:1 to 25:1, 10:1 to 30 ... 5:1, 10:1~40:1, 15:1~20:1, 15:1~25:1, 15:1~30:1, 15:1~35:1, 15:1~40:1, 20:1~25:1, 20:1~30:1, 20:1~35:1, 20:1~40:1, 25:1~30:1, 25:1~35:1, 25:1~40:1, 30:1~35:1, 30:1~40:1, 35:1~40:1.

[0014] In some embodiments, gemcitabine or a pharma- ceutically acceptable salt thereof is present in the aqueous interior, and the salt solution in the aqueous interior is a sulfate solution, or a combination of a sulfate solution and a buffered salt solution (e.g., a phosphate buffer solution), or a combination of a chloride salt solution and a buffered salt solution (e.g., a phosphate buffer solution).

[0015] In some embodiments, the molarity of the aqueous internal salt solution is between 100 and 800 mM, preferably between 200 and 600 mM.

[0016] In some embodiments, the aqueous internal salt solution is an ammonium sulfate solution, and preferably the concentration of the ammonium sulfate solution is 100-800 mM, preferably 200-600 mM, more preferably 200-400 mM, more preferably 300-400 mM.

[0017] In some embodiments, the aqueous internal salt solution is a mixed solution of sodium sulfate and phosphate buffer salt, or a mixed solution of sodium chloride and phosphate buffer salt, or a mixed solution of ammonium sulfate and phosphate buffer salt, and preferably, the concentration of sodium sulfate, sodium chloride or ammonium sulfate in the mixed solution is 100 to 800 mM (preferably 200 to 600 mM, more preferably 200 to 500 mM, more preferably 250 to 500 mM), and the concentration of phosphate buffer salt is 100 to 200 mM.

[0018] In some embodiments, the liposomes are unilamellar and have an average particle size of 10 to 200 nm, preferably 30 to 150 nm, and more preferably 50 to 100 nm.

[0019] In some embodiments, the concentration of gemcitabine or a pharma- ceutically acceptable salt thereof in the liposome is 0.1 to 3 mg / ml, more preferably 0.1 to 2.5 mg / ml, 0.1 to 2 mg / ml, for example, 0.1 to 0.5 mg / ml, 0.1 to 1.0 mg / ml, 0.1 to 1.5 mg / ml, 0.1 to 2.0 mg / ml, 0.1 to 2.5 mg / ml, 0.1 to 3.0 mg / ml, 0.5 to 1.0 mg / ml, 0.5 ~1.5mg / ml, 0.5~2.0mg / ml, 0.5~2.5mg / ml, 0.5~3.0mg / ml, 1.0~1.5mg / ml, 1.0~2.0mg / ml, 1.0~2.5mg / ml, 1.0 ~3.0mg / ml, 1.5~2.0mg / ml, 1.5~2.5mg / ml, 1.5~3.0mg / ml, 2.0~2.5mg / ml, 2.0~3.0mg / ml, 2.5~3.0mg / ml.

[0020] In some embodiments, the phospholipid is one or two selected from natural phospholipids and their derivatives, including lecithin, cephalin, soybean lecithin, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, sphingomyelin, cardiolipin, etc. (including hydrogenated phospholipids obtained by hydrogenating the above phospholipids). When the liposome contains one type of phospholipid, it is preferably one selected from the group consisting of neutral phospholipids, negatively charged phospholipids, and sphingomyelin, and is preferably phosphatidylcholine or sphingomyelin, such as hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), hydrogenated sphingomyelin (HSM). When two kinds of phospholipids are contained, it is preferably a combination of a neutral phospholipid and a negatively charged phospholipid, more preferably a combination of a phosphatidylcholine and a phosphatidylglycerol, such as DSPC and distearoylphosphatidylglycerol (DSPG), HSPC and DSPG, or HSPC and dipalmitoylphosphatidylglycerol (DPPG).When the phospholipid contains one or more of sphingomyelin, hydrogenated sphingomyelin, and a negatively charged phospholipid, preferably, the liposome membrane does not contain a long-circulating functional substance, i.e., the mass ratio of the long-circulating functional substance is 0%.

[0021] In some preferred embodiments, the phospholipid is one or more of hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), sphingomyelin (SM) and hydrogenated sphingomyelin (HSM), and the phospholipid is preferably hydrogenated soy phosphatidylcholine (HSPC).

[0022] When distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), sphingomyelin (SM) or hydrogenated sphingomyelin (HSM) are used as the phospholipid, the liposome membrane does not contain long-circulating functional substances.

[0023] In some preferred embodiments, the phospholipid is one or two selected from the group consisting of hydrogenated soy phosphatidylcholine (HSPC), dipalmitoyl phosphatidylglycerol (DPPG), sphingomyelin (SM) and hydrogenated sphingomyelin (HSM), and when dipalmitoyl phosphatidylglycerol (DPPG), sphingomyelin (SM) or hydrogenated sphingomyelin (HSM) is used as the phospholipid, the liposome membrane does not contain long-circulating functional substances.

[0024] In some preferred embodiments, the phospholipid is selected from the group consisting of hydrogenated soy phosphatidylcholine, sphingomyelin, and a combination of hydrogenated soy phosphatidylcholine and dipalmitoyl phosphatidylglycerol.

[0025] In some embodiments, the long-circulating functional substance is one or more selected from the group consisting of polyethylene glycol (PEG), polyethylene glycol-vitamin E succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-modified distearoylphosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified dimyristoylphosphatidylethanolamine (MPEG-DMPE) and polyethylene glycol-modified dipalmitoylphosphatidylethanolamine (MPEG-DPPE), preferably a polyethylene glycol-modified phospholipid of the same type as the phospholipid constituting the liposome membrane. The molecular weight of the polyethylene glycol is 500 to 10,000 daltons, preferably 2000 to 5000 daltons.

[0026] In some preferred embodiments, the long-circulating functional agent is polyethylene glycol-modified distearoylphosphatidylethanolamine (MPEG-DSPE), such as MPEG2000-DSPE.

[0027] The advantages of liposome compositions in reducing toxicity and enhancing efficacy are closely related to the release of liposome compositions in blood. Further studies on liposome compositions have found that the type and amount of salt solution affect the drug release behavior of liposomes in blood. In some embodiments, the salt is Na + , K + , NH4 + , or H + The acid ions constituting the salt may be one or more selected from the group consisting of sulfate, carbonate, chloride, hydrogen phosphate, dihydrogen phosphate, sucrose octasulfate, citrate, gluconate, methanesulfonate, acetate, oxalate, 4-hydroxyethylpiperazine ethanesulfonate, and may be one or more organic or inorganic acid radicals, including, but not limited to, sulfate, carbonate, chloride, hydrogen phosphate, dihydrogen phosphate, sucrose octasulfate, citrate, gluconate, methanesulfonate, acetate, oxalate, 4-hydroxyethylpiperazine ethanesulfonate, and may be one or more selected from the group consisting of chloride, sulfate, sucrose octasulfate, acetate, hydrogen phosphate, and dihydrogen phosphate, and more preferably sulfate, or a combination of sulfate, chloride, phosphate, hydrogen phosphate, and dihydrogen phosphate.

[0028] In some embodiments, the salt is preferably ammonium sulfate, or a combination of sodium sulfate and a phosphate buffer salt, or a combination of sodium chloride and a phosphate buffer salt, or a combination of ammonium sulfate and a phosphate buffer salt.

[0029] In some embodiments, the present invention provides a liposome of gemcitabine or a pharma- ceutically acceptable salt thereof, the liposome comprising a liposomal membrane and an aqueous interior encapsulated by the liposomal membrane; The components constituting the liposome membrane include hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and polyethylene glycol-modified distearoylphosphatidylethanolamine (MPEG2000-DSPE), the aqueous interior contains gemcitabine hydrochloride, and a salt solution, the salt solution being an ammonium sulfate solution, or a mixed solution of sodium sulfate and a phosphate buffer salt, or a mixed solution of sodium chloride and a phosphate buffer salt, or a mixed solution of ammonium sulfate and a phosphate buffer salt; the weight ratio of HSPC to cholesterol is 2:1 to 20:1, preferably 2:1 to 10:1, more preferably 2:1 to 8:1, for example 2:1 to 3:1 or 7:1 to 8:1; MPEG2000-DSPE accounts for 0% by mass to 30% by mass, preferably 0% by mass to 20% by mass, preferably 10% by mass to 20% by mass, preferably 0% by mass to 15% by mass, preferably 0.01% by mass to 15% by mass of the total lipids, The weight ratio of the total lipid in the liposome to gemcitabine hydrochloride is 10:1 to 50:1; The molarity of the aqueous inner salt solution is between 50 and 800 mM.

[0030] In some embodiments, the aqueous internal salt solution is an ammonium sulfate solution, and the ammonium sulfate solution has a concentration of 300-400 mM.

[0031] In some embodiments, the liposome of gemcitabine or a pharma- ceutically acceptable salt thereof provided by the present invention can be an injectable solution, the aqueous exterior of which optionally comprises an osmolality modifier, the osmolality modifier being one or more selected from the group consisting of glucose, sucrose, sorbitol, mannitol, sodium chloride, phosphate, sulfate, acetate, glycerol, histidine and its hydrochloride, glycine and its hydrochloride, lysine, serine, glutamic acid, arginine, or valine.

[0032] In some embodiments, the liposomes have an encapsulation efficiency of greater than 85%, such as greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%.

[0033] In a second aspect, the present application further provides a method for preparing gemcitabine liposomes as described in any one of the first aspects, the method comprising the steps of: S1, preparation of aqueous phase: dissolving gemcitabine or a pharma- ceutically acceptable salt thereof in a salt solution having a concentration of 50-800 mM to obtain an aqueous phase; S2, preparation of lipid phase: dissolving phospholipids and cholesterol in a weight ratio of 2:1 to 20:1 in a solvent to obtain a lipid phase, the solvent being one or more selected from the group consisting of ethanol, methanol, ethyl acetate, chloroform, dichloromethane and diethyl ether; S3, Preparation of drug-loaded liposomes: mixing the aqueous phase obtained in S1 with the lipid phase obtained in S2 by organic solvent injection, thin film hydration, inverse phase evaporation, emulsification, freeze-thaw cycles, pipeline mixing, or microfluidic methods to obtain a primary emulsion of liposomes containing gemcitabine or a pharma- ceutically acceptable salt thereof and an aqueous inner salt solution; S4, Granulation: Granulating the primary emulsion of drug-loaded liposomes using one or more selected from the group consisting of a shearer, homogenizer and extruder to reach a target particle size; S5, Removal of free drug: Removing the free drug by one or more processes selected from filtration, centrifugation, dialysis, ultrafiltration, concentration, etc., to obtain drug-loaded liposomes comprising gemcitabine or a pharma- ceutically acceptable salt thereof; Optionally, adding a long-circulating functional substance when preparing the lipid phase in step S2.

[0034] In some embodiments, the salt solution in step S1 is preferably an ammonium sulfate solution. In some embodiments, the molarity of the ammonium sulfate solution is 50-800 mM, preferably 50-600 mM, preferably 50-400 mM, preferably 50-200 mM, preferably 50-100 mM. In some embodiments, the molarity of the ammonium sulfate solution is 100-800 mM, preferably 200-600 mM, more preferably 200-400 mM, more preferably 300-400 mM.

[0035] In some embodiments, a pH adjuster can be added to adjust the pH of the aqueous phase in step S1, hi some embodiments, the pH adjuster is selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and others.

[0036] In some embodiments, the phospholipid in step S2 is HSPC.

[0037] In some embodiments, the long-circulating functional substance in step S2 is MPEG2000-DSPE.

[0038] In some embodiments, in step S2, the weight ratio of phospholipid to cholesterol is from 2:1 to 15:1, preferably from 2:1 to 10:1, preferably from 2.5:1 to 10:1, more preferably from 5:1 to 8:1.

[0039] In some embodiments, the long-circulating functional substance added in step S2 accounts for 0% to 30% by mass of the total lipids, preferably 0% to 20% by mass, preferably 10% to 20% by mass, preferably 0% to 15% by mass, preferably 0.01% to 15% by mass.

[0040] In some embodiments, in step S3, drug-loaded liposomes are obtained by mixing the aqueous phase prepared in S1 with the lipid phase prepared in S2 in a lipid to water ratio of 1:1 to 1:20, preferably the lipid to water ratio is 1:3, 1:5, 1:7, 1:10 or 1:20.

[0041] In some embodiments, step S3 is carried out at or above the lipid phase transition temperature.

[0042] In some embodiments, step S3 is carried out at 30 to 70° C. (eg, 30 to 40° C., 50 to 60° C., 60 to 70° C., or 60 to 65° C.).

[0043] In some embodiments, in step S4, the primary emulsion of drug-loaded liposomes is granulated to reach an average particle size of 10-200 nm, e.g., 30-200 nm, 50-200 nm, 150-200 nm, 10-150 nm, 30-150 nm, 50-150 nm, 100-150 nm, 10-100 nm, 30-100 nm, 50-100 nm, 10-50 nm, 30-50 nm, or 10-30 nm, preferably 50-100 nm.

[0044] In some embodiments, the drug-loaded liposomes obtained in step S4 are diluted with a sucrose-histidine solution and ultrafiltered to remove free gemcitabine; preferably, the sucrose-histidine solution is 10% sucrose-0.155% histidine (w / v, pH 6.5).

[0045] The concentration of gemcitabine or a pharma- ceutically acceptable salt thereof in the gemcitabine liposome prepared by the method of the present invention is 0.1 to 3 mg / ml, preferably 0.1 to 2 mg / ml, for example, 0.1 to 1.5 mg / ml, 0.1 to 1 mg / ml, or 0.1 to 0.5 mg / ml.

[0046] The encapsulation efficiency of gemcitabine liposomes prepared by the methods of the present invention is greater than 85%, more preferably greater than 95%, such as greater than 96%, greater than 97%, greater than 98%, greater than 99%.

[0047] In the gemcitabine liposome prepared by the method of the present invention, the weight ratio of total lipid to gemcitabine or a pharma- ceutically acceptable salt thereof is 1:1 to 100:1, preferably 5:1 to 80:1, more preferably 10:1 to 50:1, more preferably 15:1 to 40:1.

[0048] In a third aspect, the present application provides another method for preparing gemcitabine liposomes as described in any one of the first aspects, the method comprising the steps of: SI, Preparation of aqueous phase: preparing a salt solution having a concentration of 50-800 mM as an aqueous phase; SII, Preparation of lipid phase: dissolving phospholipids and cholesterol in a weight ratio of 2:1 to 20:1 in a solvent to obtain a lipid phase, the solvent being one or more selected from the group consisting of water, ethanol, methanol, ethyl acetate, chloroform, dichloromethane and diethyl ether; SIII, preparation of blank liposome primary emulsion: mixing the aqueous phase obtained in SI and the lipid phase obtained in SII in appropriate amounts by organic solvent injection, thin film hydration, inverse phase evaporation, emulsification, freeze-thaw cycle, pipeline mixing, or microfluidic method to obtain blank liposome primary emulsion; SIV, Granulation: granulating the blank liposome primary emulsion by one or more selected from the group consisting of shear, homogenizer and extruder to reach the target particle size; SV, Removal of organic solvent: removing the organic solvent by one or more processes selected from the group consisting of filtration, centrifugation, dialysis, ultrafiltration, concentration, etc., to obtain blank liposomes; SVI, Preparation of API solution: preparing an API solution by dissolving gemcitabine or a pharma- ceutically acceptable salt thereof in water or in the aqueous phase of step SI, diluted or undiluted; SVII. Preparation of drug-loaded liposomes: heating and incubating the obtained blank liposomes and API solution at 30°C-75°C for 5-60 minutes to obtain drug-loaded liposomes; SVIII, Removal of free drug: Removing the free drug by one or more processes selected from filtration, centrifugation, dialysis, ultrafiltration, concentration, etc., to obtain drug-loaded liposomes containing gemcitabine or a pharma- ceutically acceptable salt thereof; Optionally, adding a long-circulating functional agent when preparing the API solution in step SVI or when preparing the drug-loaded liposomes in step SVII.

[0049] In some embodiments, the long-circulating functional agent optionally added during preparation of the API solution in step SVI or during preparation of the drug-loaded liposomes in step SVII is MPEG2000-DSPE.

[0050] In some embodiments, the salt solution in step SI is an ammonium sulfate solution, a mixed solution of sodium sulfate and a phosphate buffer salt, a mixed solution of sodium chloride and a phosphate buffer salt, or a mixed solution of ammonium sulfate and a phosphate buffer salt. In some embodiments, the concentration of the ammonium sulfate solution is 100 to 800 mM, preferably 200 to 600 mM, more preferably 200 to 400 mM, more preferably 300 to 400 mM. In some embodiments, the concentration of sodium sulfate, sodium chloride, or ammonium sulfate in the mixed solution is 100 to 800 mM (preferably 200 to 600 mM, more preferably 200 to 500 mM, more preferably 250 to 500 mM), and the concentration of the phosphate buffer salt is 100 to 200 mM.

[0051] In some embodiments, the phospholipid in step SII is selected from the group consisting of sphingomyelin, hydrogenated soy phosphatidylcholine, and a combination of hydrogenated soy phosphatidylcholine and dipalmitoyl phosphatidylglycerol. In some embodiments, the weight ratio of phospholipid to cholesterol in step SII is 2:1 to 15:1, preferably 2:1 to 10:1, more preferably 2:1 to 8:1, such as 2:1 to 3:1 or 7:1 to 8:1.

[0052] In some embodiments, the solvent in step SII is ethanol or a mixture of ethanol and water.

[0053] In some embodiments, in step SIII, the lipid phase prepared in step SII is added to an appropriate amount of the aqueous phase prepared in step SI in an oil to water ratio of 1:1 to 1:20 (e.g., 1:1 to 1:10, particularly, e.g., 1:3, 1:5, 1:10, or 1:20) and stirred at or above the lipid phase transition temperature to obtain a blank liposome primary emulsion.

[0054] In some embodiments, step SIII includes the following steps: stirring and emulsifying at 30 to 75°C (e.g., 30 to 40°C, 50 to 60°C, or 60 to 70°C) to obtain a primary emulsion of blank liposomes.

[0055] In some embodiments, the primary emulsion of blank liposomes prepared in step SIV has an average particle size of 10 to 200 nm, for example, 30 to 200 nm, 50 to 150 nm, 50 to 100 nm, 60 to 80 nm, specifically, for example, 60 nm, 65 nm, 70 nm, 75 nm, or 80 nm.

[0056] In some embodiments, the concentration of the API solution in step SVI is between 0.01-0.5 g / ml, such as between 0.01-0.2 g / ml, such as between 0.05-0.2 g / ml.

[0057] In some embodiments, a pH adjuster can be added in step SVI to adjust the pH of the API solution, hi some embodiments, the pH adjuster is selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, and others.

[0058] In some embodiments, in step SVII, the weight ratio of gemcitabine or a pharma- ceutically acceptable salt thereof to total lipid in the blank liposome is 2:1 to 1:10, preferably 2:1 to 1:5, for example 1:1 to 1:5, for example 1:1 to 1:3, for example 1:1 to 1:2, particularly for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0.

[0059] In some embodiments, the incubation temperature in step SVII is 50 to 75° C. (e.g., 65 to 70° C.).

[0060] In some embodiments, the incubation time in step SVII is between 10 minutes and 45 minutes.

[0061] In some embodiments, the long circulating functional substance added in step SVI or SVII accounts for 0% to 15%, such as 0%, or 0.01% to 15%, in particular for example 0.01%, 0.05%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% of the total lipid mass added in steps SII and SVI or SVII.

[0062] In some embodiments, in step SVIII, the drug-loaded liposomes obtained in step SVII are added to an aqueous phase and / or a sucrose-histidine solution as described in the SI and ultrafiltered to remove free gemcitabine; preferably, the sucrose-histidine solution is 10% sucrose - 0.155% histidine (w / v, pH 6.5).

[0063] The concentration of gemcitabine or a pharma- ceutically acceptable salt thereof in the gemcitabine liposome prepared by the method of the present invention is 0.1 to 3 mg / ml, preferably 0.5 to 3 mg / ml, for example, 0.5 to 2.5 mg / ml.

[0064] The encapsulation efficiency of gemcitabine liposomes prepared by the methods of the present invention is greater than 85%, more preferably greater than 95%, such as greater than 96%, greater than 97%, greater than 98%, greater than 99%.

[0065] In the gemcitabine liposome prepared by the method of the present invention, the weight ratio of total lipid to gemcitabine or a pharma- ceutically acceptable salt thereof is 1:1 to 100:1, preferably 5:1 to 80:1, more preferably 10:1 to 50:1, more preferably 15:1 to 40:1.

[0066] In a fourth aspect, the present invention provides a liposomal formulation comprising gemcitabine liposomes as described in any one of the first aspects and one or more pharma- ceutically acceptable carriers.

[0067] In some embodiments, the liposome formulation comprises a osmolality adjusting agent, such as a sucrose-histidine solution, preferably a 10% sucrose-histidine solution, preferably a 10% sucrose-0.155% histidine solution.

[0068] In some embodiments, the liposomal formulation is an injectable.

[0069] In another aspect, the present application provides the use of a gemcitabine liposome as described in any one of the first aspect, or a liposomal formulation as described in any one of the fourth aspect, in the manufacture of a medicament for treating a tumor.

[0070] In another aspect, the present invention provides the use of gemcitabine liposomes according to any one of the first aspect, or a liposomal formulation according to any one of the fourth aspect, in the manufacture of an antitumor medicament.

[0071] In some embodiments, the tumor is selected from the group consisting of pancreatic cancer, breast cancer, non-small cell lung cancer, and ovarian cancer.

[0072] In another aspect, the present application provides a method for treating a tumor, comprising administering to a subject in need thereof an effective amount of gemcitabine liposome according to any one of the first aspect or a liposomal formulation according to any one of the fourth aspect.

[0073] In some embodiments, the tumor is selected from the group consisting of pancreatic cancer, breast cancer, non-small cell lung cancer, and ovarian cancer.

[0074] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terms used herein in describing the present invention are for the purpose of describing specific embodiments only and are not intended to be limiting of the present invention.

[0075] As used herein, the term "phosphatidylethanolamine pegol" refers to polyethylene glycol-modified phosphatidylethanolamine, such as polyethylene glycol-modified distearoylphosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified dimyristoylphosphatidylethanolamine (MPEG-DMPE), polyethylene glycol-modified dipalmitoylphosphatidylethanolamine (MPEG-DPPE), etc. In the present invention, MPEG-DSPE, such as MPEG2000-DSPE, is particularly preferred.

[0076] As used herein, the term "lipid phase" may also be referred to as "oil phase" or "organic phase."

[0077] As used herein, the term "API" refers to "gemcitabine or a pharma- ceutically acceptable salt thereof," and in some embodiments, specifically to gemcitabine hydrochloride.

[0078] As used herein, the term "total lipids" includes phospholipids, cholesterol, and long-circulating functional substances contained in the liposome.

[0079] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0080] As used herein, the term "treatment" refers to a method carried out to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction of disease extent, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, improvement or alleviation of disease state, and elimination of symptoms (whether partial or complete). In addition, "treatment" can also refer to prolonging survival compared to the expected survival if no treatment was received.

[0081] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) has a tumor or is at risk of suffering from a disease as described above.

[0082] As used herein, the term "effective amount" refers to an amount sufficient to at least partially obtain a desired effect. For example, a prophylactically effective amount refers to an amount sufficient to prevent, stop, or prolong the occurrence of a disease (e.g., a tumor), and a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent an existing disease and its complications in a patient suffering from the disease. It is well within the capabilities of a person skilled in the art to determine such an effective amount. For example, an effective amount for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the overall condition of the patient, such as age, weight, and sex, the manner in which the drug is administered, and other treatments administered in parallel, etc.

[0083] The terms "cancer" and "tumor" are used interchangeably and refer to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors, i.e., cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes blood tumors, particularly hematological malignancies.

[0084] Beneficial Effects of the Invention The present invention provides a method for preparing liposomes of gemcitabine or its pharma- ceutically acceptable salts, in which the liposomes are prepared by optimizing and controlling the ratio of phospholipid to cholesterol, the ratio of long-circulating functional substances in total lipids, the ratio of total lipid to drug, and the type and concentration of aqueous internal salt solution, and have high encapsulation efficiency, good storage stability, strong filterability, low leakage, stable and controllable release, significantly extended half-life, improved therapeutic effect, and reduced toxic effects and side effects.After intravenous injection of liposomes, the liposomes can avoid the rapid metabolic degradation of free gemcitabine, significantly extend the circulation time of gemcitabine in the body, enhance targeting to tumor tissues / cells, and at the same time reduce the dose of gemcitabine, improve efficacy, reduce toxic effects and side effects, thereby meeting the clinical need of improved efficacy and reduced toxicity.In addition, the liposomes provided by the present invention have a simple formulation and preparation process, making it feasible for scale-up production and clinical promotion and application.

[0085] The drawings described in this specification are used to provide a further understanding of the present invention and constitute a part of this application. The illustrative examples of the present invention and the description thereof are used to explain the present invention and do not constitute undue limitations to the present invention. The accompanying drawings are as follows: [Brief description of the drawings]

[0086] [Figure 1] FIG. 1 shows drug-time curves of liposome and conventional injection groups in tumor-bearing female Balb / c mice. [Diagram 2] FIG. 1 shows a diagram of changes in tumor volume in liposome and conventional injection groups in a 4T1 Balb / c mouse pharmacodynamics study. [Diagram 3] FIG. 1 shows a diagram of efficacy results for BxPC-3.

[0087] [Specific model for implementing the present invention] The technical solutions in the embodiments of the present invention are clearly and completely described below in the embodiments of the present invention with reference to the accompanying drawings. Obviously, the described embodiments are only some of the examples, not all of the examples of the present invention. The following description of at least one illustrative example is merely exemplary in nature and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, any other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention.

[0088] Example 1: Preparation of Gemcitabine Liposomes The preparation steps were as follows: 1. Lipid Phase Preparation 16.6 g of hydrogenated soy phosphatidylcholine, 2.0 g of cholesterol, and 4.3 g of MPEG2000-DSPE were weighed and dissolved in 20 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0089] 2. Preparation of Aqueous Phase 1.56 g of disodium hydrogen phosphate and 0.15 g of sodium dihydrogen phosphate monohydrate were weighed and dissolved in 3000 g of water to obtain 4 mM phosphate buffer at pH 7.78, heated to 60° C. and stored at that temperature for later use.

[0090] 3. Preparation of Blank Liposomes At 60°C, the lipid phase was injected into the aqueous phase at a rate of 60ml / min. After the injection was completed, stirring was continued for 30 minutes to obtain the primary emulsion of liposomes, which was then extruded through a homogeneous extruder so that the primary emulsion of liposomes had an average particle size of 68nm, and then ethanol was removed by an ultrafiltration system to obtain 134g of blank liposomes that did not contain drugs.

[0091] 4. Preparation of Drug-loaded Liposomes 4.1 Preparation of PBS buffer 20.41 g of sodium chloride, 2.88 g of disodium hydrogen phosphate, and 0.51 g of sodium dihydrogen phosphate monohydrate were weighed out and dissolved in 234 g of water to obtain a PBS buffer solution.

[0092] 4.2 Preparation of API solutions 3.25 g of gemcitabine hydrochloride and 0.22 g of sodium hydroxide were weighed and dissolved in 19.0 g of water for injection, mixed with 13.50 g of PBS buffer prepared in (4.1), then heated to 70° C. and stored at that temperature for later use.

[0093] 4.3 Drug filling 35.4g of blank liposome prepared in (3) and 0.22g of sodium hydroxide were weighed, heated to 70℃, then heated with API solution prepared in (4.2) for 10 minutes to load the drug, immediately cooled to 40℃, diluted by adding 1016mM sucrose / 37mM histidine solution, then subjected to ultrafiltration dialysis by using 275mM sucrose / 10mM histidine, and the aqueous exterior was replaced by ultrafiltration dialysis to obtain the target liposome. The obtained liposome had an average particle size of 77.7nm, a gemcitabine hydrochloride concentration of 1.12mg / ml, an encapsulation efficiency of 95.23%, a total lipid to API mass ratio of 34:1, and MPEG2000-DSPE accounted for 18.9% by mass of the total lipid.

[0094] Example 2: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 2.75 g of hydrogenated soy phosphatidylcholine, 0.91 g of cholesterol, and 0.86 g of MPEG2000-DSPE were weighed and dissolved in 20 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0095] (2) Preparation of aqueous phase 1.86 g of gemcitabine hydrochloride and 0.25 g of sodium hydroxide were weighed and dissolved in 188.73 g of ammonium sulfate solution (73 mM, obtained by dissolving 1.82 g of ammonium sulfate in 188.73 g of water) and stored at a temperature of 60° C. for future use.

[0096] (3) Preparation of drug-loaded liposomes Under the same temperature conditions, with a total flow rate of 12 ml / min and an oil-to-water ratio of 1:7, the lipid phase and the aqueous phase were mixed separately through a microfluidic pipeline to obtain drug-loaded liposomes, which were diluted 10-fold by adding a 10% sucrose-0.155% histidine (w / v, adjusted to pH 6.5 by adding diluted hydrochloric acid) solution, and then subjected to ultrafiltration dialysis and concentration using a tangential flow ultrafiltration device (the dialysis solution was a 10% sucrose-0.155% histidine solution at pH 6.5). After dialysis, gemcitabine liposomes were obtained. The obtained liposomes had an average particle size of 152.20 nm, a gemcitabine hydrochloride concentration of 0.38 mg / ml, an encapsulation efficiency of 97.79%, a total lipid to API mass ratio of 38:1, and MPEG2000-DSPE accounted for 19% by mass of the total lipid.

[0097] Example 3: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 12.29 g of sphingomyelin and 4.49 g of cholesterol were weighed and dissolved in 81.72 g of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0098] (2) Preparation of aqueous phase 225.88 g of ammonium sulfate was weighed out and dissolved in 5000 g of water to prepare a 342 mM ammonium sulfate buffered saline solution and stored at 60° C. for future use.

[0099] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 800 ml of the aqueous phase prepared in (2) at a rate of 100 ml / min, and heated and stirred constantly for 45 minutes to obtain a primary emulsion of liposomes, which was then subjected to extrusion, reaching an average particle size of about 75 nm, and the organic solvent was removed by dialysis using a tangential flow ultrafiltration device to obtain blank liposomes with an aqueous interior containing 342 mM ammonium sulfate buffer salt solution.

[0100] (4) Preparation of drug-loaded liposomes 4.11 g of gemcitabine hydrochloride and 5.50 g of sodium hydroxide were weighed and dissolved in 52.28 g of water by heating to 65°C, then added to 100 g of blank liposomes prepared in (3) incubated at 65°C, heated for 30 minutes to obtain drug-loaded liposomes, diluted 5-fold by adding 10% sucrose-0.155% histidine (w / v, pH 6.5), and then dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had an average particle size of 74.37 nm, a gemcitabine hydrochloride concentration of 1.39 mg / ml, an encapsulation efficiency of 98.89%, and a total lipid to API mass ratio of 40:1.

[0101] Example 4: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 8.29 g of hydrogenated soy phosphatidylcholine and 1.10 g of cholesterol were weighed and dissolved in 60 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0102] (2) Preparation of aqueous phase 112.00 g of sodium sulfate, 23.01 g of disodium hydrogen phosphate, and 5.24 g of sodium dihydrogen phosphate (monohydrate) were weighed and dissolved in 2000.5 g of water by heating to 60° C. to obtain a 394 mM sodium sulfate-100 mM phosphate buffer solution, and the buffer was stored at 60° C. for future use.

[0103] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 300 ml of the aqueous phase prepared in (2) at an injection rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes, which was extruded to reach an average particle size of 70 nm, and then dialyzed using a tangential flow ultrafiltration device to remove the organic solvent. After concentration, 64 g of blank liposomes with an aqueous interior containing 394 mM sodium sulfate-100 mM phosphate buffer solution was obtained.

[0104] (4) Preparation of drug-loaded liposomes 2.88 g of gemcitabine hydrochloride, 0.49 g of sodium hydroxide, and 0.44 g of MPEG2000-DSPE were weighed out, heated to 60°C, dissolved in 27.00 g of a 3-fold diluted solution of the aqueous phase prepared in (2), and then added to 30 g of the blank liposomes prepared in (3) that had been incubated at 70°C. To load the drug, the mixture was heated at 70°C for 10 minutes to obtain drug-loaded liposomes. The liposomes were diluted 5-fold by adding the aqueous phase prepared in (2), and dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine, yielding gemcitabine liposomes. The resulting liposomes had a mean particle size of 82.30 nm, a gemcitabine hydrochloride concentration of 2.06 mg / ml, an encapsulation efficiency of 99.12%, a total lipid to API mass ratio of 15:1, and MPEG2000-DSPE accounted for 12.5% ​​by mass of the total lipid.

[0105] Example 5: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 8.23 g of hydrogenated soy phosphatidylcholine and 1.03 g of cholesterol were weighed and dissolved in 60 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0106] (2) Preparation of aqueous phase 74.00 g of ammonium sulfate, 22.99 g of disodium hydrogen phosphate, and 5.25 g of sodium dihydrogen phosphate (monohydrate) were weighed and dissolved in 2000 g of purified water to obtain a 280 mM ammonium sulfate-100 mM phosphate buffer solution, and the buffer solution was stored at 60° C. for later use.

[0107] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into the aqueous phase prepared in (2) at an injection rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes. The primary emulsion of liposomes obtained was subjected to extrusion, whereby the average particle size reached about 75 nm. After removal of the organic solvent by dialysis using a tangential flow ultrafiltration device and concentration, 92 g of blank liposomes having 280 mM ammonium sulfate-100 mM phosphate buffer solution as the aqueous interior was obtained.

[0108] (4) Preparation of drug-loaded liposomes 1.16 g of gemcitabine hydrochloride, 0.16 g of sodium hydroxide, and 38.34 mg of MPEG2000-DSPE were weighed and dissolved in 18.18 g of the aqueous phase prepared in (2) by heating to 70°C, then added to 40 g of blank liposomes prepared in (3) incubated at 70°C, heated for 10 minutes to load the drug to obtain drug-loaded liposomes, and the liposomes were dialyzed by a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had an average particle size of 79.38 nm, a gemcitabine hydrochloride concentration of 0.94 mg / ml, an encapsulation efficiency of 98.35%, a total lipid to API ratio of 33:1, and MPEG2000-DSPE accounted for 1.7% by mass of the total lipids.

[0109] Example 6: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 28.43 g of hydrogenated soy phosphatidylcholine and 3.61 g of cholesterol were weighed and dissolved in 200 ml of absolute ethanol by heating to 60° C. and stored at that temperature for future use.

[0110] (2) Preparation of aqueous phase 108.00 g of NaCl, 46.00 g of disodium hydrogen phosphate, and 10.49 g of sodium dihydrogen phosphate (monohydrate) were weighed and dissolved in 4000 g of water to prepare a 462 mM NaCl-100 mM phosphate buffer solution, and the buffer solution was stored at 60° C. for later use.

[0111] (3) Preparation of blank liposomes The lipid phase prepared in (1) was poured into 1045.5 g of the aqueous phase prepared in (2), and heated and stirred for 10 minutes to obtain a primary emulsion of liposomes. The primary emulsion of liposomes was granulated to about 75 nm through an extruder, and then ethanol was removed by ultrafiltration to obtain blank liposomes having a 462 mM NaCl-100 mM phosphate buffer solution as the aqueous interior.

[0112] (4) Preparation of drug-loaded liposomes 23.05 g of gemcitabine hydrochloride, 3.43 g of MPEG2000-DSPE, and 3.08 g of sodium hydroxide were weighed, dissolved in 328.00 g of NaCl 462 mM-phosphate 100 mM buffer solution prepared in (2) by heating to 70° C., and then added to 310 g of blank liposomes prepared in (3) incubated at 65° C., and then heated for 10 minutes to obtain drug-loaded liposomes. After the drug-loaded liposomes were subjected to ultrafiltration to remove free drug, the resulting liposomes had an average particle size of 86.31 nm, a gemcitabine hydrochloride concentration of 2.34 mg / ml, an encapsulation efficiency of 95.14%, a total lipid to API mass ratio of 27:1, and MPEG2000-DSPE accounted for 12% by mass of the total lipids.

[0113] Example 7: Preparation of Gemcitabine Liposomes The preparation methods of (1) lipid phase, (2) aqueous phase, and (3) blank liposomes were the same as those in Example 5.

[0114] (4) Preparation of drug-loaded liposomes 1.16 g of gemcitabine hydrochloride, 0.16 g of sodium hydroxide, and 7.67 mg of MPEG2000-DSPE were weighed out, dissolved in 18.18 g of the aqueous phase prepared in (2) by heating to 70°C, and then added to 40 g of the blank liposomes prepared in (3) incubated at 70°C, heated for 10 minutes to load the drug, and obtained drug-loaded liposomes, which were dialyzed by a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The obtained liposomes had an average particle size of 80.20 nm, a gemcitabine hydrochloride concentration of 0.85 mg / ml, an encapsulation efficiency of 98.17%, a total lipid to API ratio of 35:1, and MPEG2000-DSPE accounted for 0.3% by mass of the total lipids.

[0115] Example 8: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 6.60 g of hydrogenated soybean phosphatidylcholine, 2.12 g of dipalmitoyl phosphatidylglycerol, and 1.09 g of cholesterol were weighed out, dissolved in a mixed solvent of 60 ml of absolute ethanol and 6 ml of water by heating to 60° C., and stored at that temperature for later use.

[0116] (2) Preparation of aqueous phase 65.00 g of ammonium sulfate, 23.00 g of disodium hydrogen phosphate, and 5.25 g of sodium dihydrogen phosphate (monohydrate) were weighed and dissolved in 2000 g of water to prepare a 246 mM ammonium sulfate-100 mM phosphate buffer solution, and the buffer solution was stored at 60° C. for later use.

[0117] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 300 ml of the aqueous phase prepared in (2) at a rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes. When the obtained primary emulsion of liposomes was subjected to extrusion, the average particle size reached 65 nm, and the organic solvent was removed by dialysis using a tangential flow ultrafiltration device, blank liposomes with an aqueous interior containing 246 mM ammonium sulfate-100 mM phosphate buffer solution were obtained.

[0118] (4) Preparation of drug-loaded liposomes 1.24 g of gemcitabine hydrochloride and 0.167 g of sodium hydroxide were weighed and dissolved in 17.45 g of water by heating to 70°C, then added to 30 g of blank liposomes prepared in (3) incubated at 70°C, heated for 10 minutes to obtain drug-loaded liposomes, diluted 5-fold by adding 10% sucrose-0.155% histidine (w / v, pH 6.5), and then dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had an average particle size of 73.83 nm, a gemcitabine hydrochloride concentration of 0.75 mg / ml, an encapsulation efficiency of 98.39%, and a total lipid to API mass ratio of 25:1.

[0119] Example 9: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 13.72 g of hydrogenated soy phosphatidylcholine and 1.81 g of cholesterol were weighed and dissolved in 100 ml of absolute ethanol by heating to 60° C. and stored at that temperature for future use.

[0120] (2) Preparation of aqueous phase 97.54 g of ammonium sulfate, 68.99 g of disodium hydrogen phosphate, and 15.74 g of sodium dihydrogen phosphate (monohydrate) were weighed and dissolved in 3000 g of water to prepare a 246 mM ammonium sulfate-200 mM phosphate buffer solution, and the buffer solution was stored at 60° C. for later use.

[0121] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 500 ml of the aqueous phase prepared in (2) at a rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes. The primary emulsion of liposomes obtained was subjected to extrusion, and the average particle size reached 72 nm. After removing the organic solvent by dialysis using a tangential flow ultrafiltration device, 110 g of blank liposomes containing 246 mM ammonium sulfate-200 mM phosphate buffer solution as the aqueous interior were obtained.

[0122] (4) Preparation of drug-loaded liposomes 1.35 g of gemcitabine hydrochloride, 0.18 g of sodium hydroxide, and 36.0 mg of MPEG2000-DSPE were weighed out, heated to 70°C and dissolved in 26.82 g of water, then added to 25 g of blank liposomes prepared in (3) that had been incubated at 70°C and heated for 10 minutes to obtain drug-loaded liposomes, which were then diluted two-fold by adding 10% sucrose-0.155% histidine (w / v, pH 6.5), and dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had a mean particle size of 74.76 nm, a gemcitabine hydrochloride concentration of 1.21 mg / ml, an encapsulation efficiency of 99.01%, a total lipid to API mass ratio of 22:1, and MPEG2000-DSPE accounted for 1.6% by mass of the total lipid.

[0123] Example 10: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 13.73 g of hydrogenated soy phosphatidylcholine and 1.81 g of cholesterol were weighed and dissolved in 100 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0124] (2) Preparation of aqueous phase 135.33 g of ammonium sulfate was weighed out and dissolved in 3000 g of water to prepare a 341 mM ammonium sulfate solution, and the solution was stored at 60° C. for future use.

[0125] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 537.17 g of the aqueous phase prepared in (2) at a rate of 100 ml / min, and heated and stirred constantly for 30 minutes to obtain a primary emulsion of liposomes, which was then subjected to extrusion, reaching an average particle size of 71.86 nm, and was then dialyzed using a tangential flow ultrafiltration device to remove the organic solvent, yielding 126 g of blank liposomes containing 341 mM ammonium sulfate solution as the aqueous interior.

[0126] (4) Preparation of drug-loaded liposomes 5.29 g of gemcitabine hydrochloride, 7.16 g of sodium hydroxide, and 0.8007 g of MPEG2000-DSPE were weighed and dissolved in 58.68 g of water by heating to 70° C., then added to 53 g of blank liposomes prepared in (3) incubated at 70° C., and heated for 10 minutes to obtain drug-loaded liposomes, and the liposomes were diluted 5-fold by adding 10% sucrose-0.155% histidine (w / v, pH 6.5), and then dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had a mean particle size of 96.24 nm, a gemcitabine hydrochloride concentration of 1.87 mg / ml, an encapsulation efficiency of 97.21%, a total lipid to API mass ratio of 25:1, and MPEG2000-DSPE accounted for 12% by mass of the total lipid.

[0127] Example 11: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 8.26 g of hydrogenated soy phosphatidylcholine and 1.09 g of cholesterol were weighed and dissolved in 60 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0128] (2) Preparation of aqueous phase 112.0 g of ammonium sulfate, 23.03 g of disodium hydrogen phosphate, and 5.25 g of sodium dihydrogen phosphate monohydrate were weighed and dissolved in 2000 g of water to prepare a 424 mM ammonium sulfate-100 mM phosphate buffer solution, and the buffer solution was stored at 60° C. for later use.

[0129] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 308.5 g of the aqueous phase prepared in (2) at a rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes. The primary emulsion of liposomes obtained was subjected to extrusion, and the average particle size reached 76.41 nm. After dialysis using a tangential flow ultrafiltration device to remove the organic solvent, 61.5 g of blank liposomes containing 424 mM ammonium sulfate-100 mM phosphoric acid solution as the aqueous interior was obtained.

[0130] (4) Preparation of drug-loaded liposomes 2.27 g of gemcitabine hydrochloride, 0.31 g of sodium hydroxide, and 0.337 g of MPEG2000-DSPE were weighed out and dissolved in 13.26 g of the aqueous phase prepared in (2) by heating to 70°C, then added to 30 g of blank liposomes prepared in (3) incubated at 70°C, heated for 10 minutes to load the drug to obtain drug-loaded liposomes, diluted 5-fold by adding a 1.5-fold diluted solution of the aqueous phase prepared in (2), and dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had a mean particle size of 85.86 nm, a gemcitabine hydrochloride concentration of 1.68 mg / ml, an encapsulation efficiency of 98.83%, a total lipid to API mass ratio of 15:1, and MPEG2000-DSPE accounted for 12.5% ​​by mass of the total lipid.

[0131] Example 12: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 6.87 g of hydrogenated soy phosphatidylcholine and 0.90 g of cholesterol were weighed and dissolved in 50 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0132] (2) Preparation of aqueous phase 275 g of glucose, 28.75 g of disodium hydrogen phosphate, and 6.56 g of sodium dihydrogen phosphate monohydrate were weighed and dissolved in 2500 g of water to prepare a 610 mM glucose-100 mM phosphate solution, and the solution was stored at 60° C. for later use.

[0133] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 258 g of the aqueous phase prepared in (2) at a rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes, which was then subjected to extrusion, reaching an average particle size of 69.03 nm, and was then dialyzed using a tangential flow ultrafiltration device to remove the organic solvent, yielding 57.5 g of blank liposomes containing 610 mM glucose-100 mM phosphate solution as the aqueous interior.

[0134] (4) Preparation of drug-loaded liposomes 1.73 g of gemcitabine hydrochloride, 0.23 g of sodium hydroxide, and 0.257 g of MPEG2000-DSPE were weighed out and dissolved in 16.9 g of the aqueous phase prepared in (2) by heating to 70°C, then added to 20 g of blank liposomes prepared in (3) incubated at 70°C, heated for 10 minutes to load the drug to obtain drug-loaded liposomes, diluted 5-fold by adding a 1.5-fold diluted solution of the aqueous phase prepared in (2), and dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had a mean particle size of 81.9 nm, a gemcitabine hydrochloride concentration of 1.70 mg / ml, an encapsulation efficiency of 94.32%, a total lipid to API mass ratio of 16:1, and MPEG2000-DSPE accounted for 12.5% ​​by mass of the total lipid.

[0135] Example 13: Preparation of gemcitabine liposomes (comparative example, using mannitol-phosphate buffer solution as aqueous interior) (1) Preparation of lipid phase 6.87 g of hydrogenated soy phosphatidylcholine and 0.90 g of cholesterol were weighed and dissolved in 50 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0136] (2) Preparation of aqueous phase 220 g of mannitol, 23.00 g of disodium hydrogen phosphate, and 5.24 g of sodium dihydrogen phosphate monohydrate were weighed and dissolved in 2000 g of water to prepare a 604 mM mannitol-100 mM phosphate solution, and the solution was stored at 60° C. for later use.

[0137] (3) Preparation of blank liposomes Under the same temperature conditions, the lipid phase prepared in (1) was injected into 258.5 g of the aqueous phase prepared in (2) at a rate of 60 ml / min, and heated and stirred constantly for 10 minutes to obtain a primary emulsion of liposomes, which was then subjected to extrusion, reaching an average particle size of 71.68 nm, and then dialyzed using a tangential flow ultrafiltration device to remove the organic solvent, yielding 52.0 g of blank liposomes containing 604 mM mannitol-100 mM phosphate solution as the aqueous interior.

[0138] (4) Preparation of drug-loaded liposomes 1.52 g of gemcitabine hydrochloride, 0.27 g of sodium hydroxide, and 0.230 g of MPEG2000-DSPE were weighed out, heated to 70° C. and dissolved in 12.4 g of the aqueous phase prepared in (2), then added to 20 g of blank liposomes prepared in (3) incubated at 70° C., heated for 10 minutes to load the drug to obtain drug-loaded liposomes, diluted 5-fold by adding a 1.5-fold dilution of the aqueous phase prepared in (2), and dialyzed using a tangential flow ultrafiltration device to remove free gemcitabine to obtain gemcitabine liposomes. The resulting liposomes had a mean particle size of 80.49 nm, a gemcitabine hydrochloride concentration of 0.79 mg / ml, an encapsulation efficiency of 98.60%, a total lipid to API mass ratio of 25:1, and MPEG2000-DSPE accounted for 12.5% ​​by mass of the total lipid.

[0139] Example 14: Preparation of Gemcitabine Liposomes (1) Preparation of lipid phase 8.23 g of hydrogenated soy phosphatidylcholine, 1.08 g of cholesterol, and 1.30 g of MPEG2000-DSPE were weighed and dissolved in 60 ml of absolute ethanol by heating to 60° C. and stored at that temperature for later use.

[0140] (2) Preparation of aqueous phase 13.8 g of gemcitabine hydrochloride and 1.85 g of sodium hydroxide were weighed and dissolved in 300 g of ammonium sulfate solution (340 mM, obtained by dissolving 13.5 g of ammonium sulfate in 300 g of water) and stored at 60° C. for future use.

[0141] (3) Preparation of drug-loaded liposomes Under the same temperature conditions, with a total flow rate of 36 ml / min and an oil-to-water ratio of 1:5, the lipid phase was injected into the aqueous phase to obtain a primary emulsion of drug-loaded liposomes, and the primary emulsion was extruded through a microjet high-pressure homogenizer to reach a particle size of 90 nm, and then diluted 5-fold by adding 10% sucrose-0.155% histidine (w / v, pH 6.5) solution, and subjected to ultrafiltration, dialysis and concentration using a tangential flow ultrafiltration device to remove free drug and ethanol (the dialysis solution was 10% sucrose-0.155% histidine solution at pH 6.5). After the dialysis was completed, gemcitabine liposomes were obtained. The resulting liposomes had a mean particle size of 95.23 nm, a gemcitabine hydrochloride concentration of 1.21 mg / ml, an encapsulation efficiency of 95.98%, a total lipid to API mass ratio of 23:1, and MPEG2000-DSPE accounted for 12.3% by mass of the total lipid.

[0142] Experimental Example 1: Stability study of gemcitabine liposomes The gemcitabine liposomes prepared in Examples 5, 8 and 9 were placed in a refrigerator at 2-8°C for 0 days and 150 days, respectively, to investigate the changes in encapsulation efficiency and content. As shown in Table 1 below, the changes in encapsulation efficiency and content of the drug-loaded liposomes were less than 5% within 150 days, indicating that the prepared gemcitabine liposomes have good stability.

[0143] [Table 1]

[0144] Experimental Example 2: Pharmacokinetic study of conventional injection of gemcitabine and gemcitabine liposome Mouse species: tumor-bearing female Balb / c mice. Group division: conventional injection group, liposome group, 4 animals in each group, total 4 groups, total 16 animals. Sample preparation: Conventional injection (GEM): Gemcitabine for injection (0.2 g / bottle, Zefei (Gemcitabine hydrochloride injection)), which was diluted by adding 5 ml of 0.9% saline to make the gemcitabine concentration 38 mg / ml. Liposome group: The method of preparing liposomes was detailed in Example 5, Example 6 and Example 7. Blood sampling point design and blood sampling method: Liposome group: 0.15 ml of blood (anticoagulated with EDTA EDTA-K2) was collected from the orbital venous plexus at 0.083 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, and 72 h after administration. Conventional injection group: 0.15 ml of blood (anticoagulated with EDTA) was collected from the orbital venous plexus at 0.5 h, 1 h, 3 h, 8 h, 24 h, and 48 h after administration. Dosage: conventional injection: 200mg / kg; liposome group: 4mg / kg (i.e., 4mpk). Blood sample processing: Collected whole blood was immediately stored in crushed ice and centrifuged at 4000 rpm for 10 min to separate plasma within 1 h (4°C). Collected plasma was stored in a -80°C refrigerator until testing. Results: The pharmacokinetic results are shown in Figure 1. Compared with conventional injection of gemcitabine, both gemcitabine liposome groups showed significantly extended serum half-life of gemcitabine. The conventional injection group showed that the amount of gemcitabine was below the detection limit after 8 hours, while the liposome group showed that gemcitabine was still detectable in the blood after 48 hours.

[0145] Experimental Example 3: Pharmacodynamic study of conventional injection and liposomal gemcitabine in tumor-bearing 4T1 mice Mouse species: Balb / c mice. Grouping: vehicle group (5% glucose injection), gemcitabine conventional injection group (GEM), liposome group (Example 10), 6 animals in each group, total 3 groups, total 18 animals were required. Sample preparation: Conventional injection: Gemcitabine for injection (0.2 g / bottle, Zefei (Gemcitabine Hydrochloride Injection)), which was diluted by adding 5 ml of 0.9% saline to make the gemcitabine concentration 38 mg / ml. Liposome group: The preparation method of liposomes was detailed in Example 10.

[0146] cell culture 4T1 cells were cultured in monolayer in vitro. Culture conditions included: RPMI1640 medium plus 10% fetal bovine serum, cultured in an incubator at 37°C and 5% CO2. Trypsin-EDTA was used for degradative digestion and passaging twice a week. When cells were in exponential growth phase, culture medium was taken for mycoplasma detection, and cells were harvested and counted.

[0147] Inoculation, grouping and administration Before tumor cell inoculation, the hair at the inoculation site of the test mice was removed using an electric razor, and the animals were numbered sequentially using mouse-specific numbering earrings. Each mouse received 2 × 10 4T1 cells. 5 The mice were subcutaneously inoculated with 0.1 ml of PBS into the right scapula. The average tumor volume of the mice was approximately 100–150 mm 3 When the tumors grew to 100 mm, mice with tumors that were too small or too large were eliminated. The remaining mice were randomly divided into groups based on the tumor volume and the weight of the animals and the medication given. Blank group: 0.9% saline, conventional injection of gemcitabine (GEM): 120 mg / kg (i.e., 120 mpk); Liposome group (Example 10): 4 mg / kg (i.e., 4 mpk); administered once a week for three consecutive weeks; The longest and shortest diameters of the tumors were measured using calipers, and the changes in tumor volume were observed and recorded. Here, tumor volume = longest diameter x shortest diameter x shortest diameter ÷ 2

[0148] Results: As shown in Figure 2, at a very low dose (4 mpk, 1 / 30 of the dose of the conventional injection group), the gemcitabine liposome group had higher efficacy than the conventional injection group (120 mpk), indicating that gemcitabine liposome has potential synergistic advantages.

[0149] Experimental Example 4: Pharmacodynamic study of gemcitabine conventional injection (GEM group) and gemcitabine liposome group (Examples 1 and 6) in tumor-bearing BxPC-3 mice Mouse species: Balb / c nu mice. Grouping: vehicle group (5% glucose injection), gemcitabine conventional injection group (GEM), liposome group (Example 1 and Example 6), 6 animals in each group, total 5 groups, total 30 animals were required. Sample preparation: Conventional injection: Gemcitabine for injection (0.2 g / bottle, Zefei (Gemcitabine hydrochloride injection)), which was diluted by adding 5 ml of 0.9% saline to make the gemcitabine concentration 38 mg / ml. Liposome group: The preparation method of liposome was described in detail in Example 1 and Example 6.

[0150] cell culture BxPC-3 cells were cultured in vitro in monolayer at 37°C and 5% CO2 in an incubator. Trypsin-EDTA was used once or twice a week for degradative digestion and passaging. When the cells were in the exponential growth phase, the culture medium was taken for mycoplasma detection, and the cells were harvested and counted.

[0151] Inoculation, grouping and administration Each mouse received 5 × 10 BxPC-3 cells. 6 The mice were subcutaneously inoculated with 0.1 ml of PBS into the right scapula. The average tumor volume of the mice was approximately 200 mm 3 When the tumors grew to 100 mm, mice with tumors that were too small or too large were eliminated, and the remaining mice were randomly divided into groups based on the tumor volume and the weight of the animals and the medicine given. Vehicle group: 0.9% sodium chloride solution; conventional injection: 240 mg / kg; liposome group: Example 1: 4 mg / kg; Example 6: 1 mg / kg and 4 mg / kg; once a week for three consecutive weeks; the longest and shortest diameters of the tumors were measured using calipers, and the changes in tumor volume and mouse weight were observed and recorded. Wherein, tumor volume = longest diameter x shortest diameter x shortest diameter ÷ 2 Tumor inhibition rate TGI (%) (腫瘍容積) =[1-(T Vt -TV0 ) / (C Vt -C V0 )] × 100% Where: T V0 represents the mean tumor volume of the test group at the time of grouping and administration; T Vt represents the mean tumor volume of the test group on day t after administration; C V0 represents the mean tumor volume in the vehicle group at the time of grouping and administration; C Vt represented the mean tumor volume of the vehicle group on day t after administration. At the time of tumor regression, TGI (%) (腫瘍容積) =100%-(T Vt -T V0 ) / T V0 ×100%.

[0152] Results: At a dose of 4 mg / kg, the gemcitabine conventional injection group (GEM, 240 mg / kg) and the liposome group-Example 1 showed intolerance, hunchback, diarrhea, and death occurred starting on the 4th day after the first administration, 4 / 6 animals in the GEM group died on the 6th day after administration, all animals (6 / 6) died in the liposome group-Example 1 (4 mg / kg), and no deaths occurred in the liposome group-Example 6. After 6 weeks of administration, the liposome group-Example 6 showed a TGI of 69.74% in gemcitabine-insensitive tumors at 4 mg / kg. Compared with the vehicle group, p<0.01 was a statistically significant difference. This indicated that the liposome of Example 6 exhibited antitumor efficacy and high tolerability (Figure 3).

[0153] Experimental Example 5: Effect of buffer salt systems in aqueous interiors on API impurity formation Methods: The gemcitabine liposomes prepared in Example 1, Example 4, Example 11, Example 12, and Example 13 were placed at 2-8°C and 25°C, respectively, to investigate the effect on the formation of buffer salt-based impurities in the aqueous interior at 0 days, 10 days, and 30 days. The results are shown in Table 2 below: Results: Compared with glucose-phosphate and mannitol-phosphate buffer systems, the sulfate-phosphate buffer system produced fewer impurities. The API in liposomes containing the sulfate buffer system had better stability, especially after being placed at room temperature.

[0154] [Table 2]

[0155] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that, based on all the teachings disclosed, various modifications and substitutions can be made to those details, and that these variations are within the scope of the present invention, the full scope of which is given by the appended claims and any equivalents thereof.

Claims

1. 1. A gemcitabine liposome comprising a liposomal membrane and an aqueous interior encapsulated by said liposomal membrane, The components constituting the liposome membrane include phospholipids, cholesterol, and optionally a long-circulating functional substance; the aqueous interior includes gemcitabine or a pharma- ceutically acceptable salt thereof, and a salt solution; and the weight ratio of said phospholipid to cholesterol is from 2:1 to 20:1; The long-circulating functional substance accounts for 0% by mass to 30% by mass of the total lipids; the weight ratio of gemcitabine or a pharma- ceutically acceptable salt thereof to total lipid in said liposome is 1:1 to 100:1; The gemcitabine liposome, wherein the molarity of said salt solution is 50-800 mM.

2. One or more of the following: (1) The phospholipid is one or more selected from the group consisting of lecithin, cephalin, soybean lecithin, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylserine, sphingomyelin, cardiolipin, and derivatives thereof (including hydrogenated phospholipids obtained by hydrogenating the above phospholipids); (2) When one type of phospholipid is contained, it is one selected from the group consisting of neutral phospholipids, negatively charged phospholipids and sphingomyelin (SM), preferably phosphatidylcholine or sphingomyelin, such as hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), sphingomyelin, hydrogenated sphingomyelin (HSM); When two types of phospholipids are included, it is a combination of a neutral phospholipid and a negatively charged phospholipid, preferably a combination of phosphatidylcholine and phosphatidylglycerol, such as DSPC and distearoylphosphatidylglycerol (DSPG), HSPC and DSPG, or HSPC and dipalmitoylphosphatidylglycerol (DPPG); (3) The long-circulating functional substance is one or more selected from polyethylene glycol (PEG), polyethylene glycol-vitamin E succinate (TPGS), polyethylene glycol-cholesterol (PEG-CHOL), polyethylene glycol-modified distearoylphosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified dimyristoylphosphatidylethanolamine (MPEG-DMPE), polyethylene glycol-modified dipalmitoylphosphatidylethanolamine (MPEG-DPPE), and polyethylene glycol-modified lipid, and is preferably a phospholipid modified with polyethylene glycol and of the same type as the phospholipid constituting the liposome membrane, and preferably the polyethylene glycol has a molecular weight of 500 to 10,000 daltons, more preferably 2000 to 5000 daltons; (4) The salt solution is one or more selected from the group consisting of a sodium salt solution, a potassium salt solution, and an ammonium salt solution, and the acid radical ion contained in the salt solution is selected from the group consisting of a sulfate radical, a chloride ion, a phosphate radical, a hydrogen phosphate radical, a dihydrogen phosphate radical, a sucrose octasulfate radical, a citrate radical, a gluconate radical, a methanesulfonate radical, an acetate radical, an oxalate radical, and a 4-hydroxyethylpiperazineethanesulfonate radical, preferably one or more selected from the group consisting of a sulfate radical, a chloride ion, a hydrogen phosphate radical, a dihydrogen phosphate radical, a sucrose octasulfate radical, and an acetate radical, more preferably a sulfate radical, or a combination of a sulfate radical, a chloride ion, a phosphate radical, a hydrogen phosphate radical, and a dihydrogen phosphate radical; more preferably, the salt solution is an ammonium sulfate solution, a solution of a combination of sodium sulfate and a phosphate buffer salt, a solution of a combination of sodium chloride and a phosphate buffer salt, or a solution of a combination of ammonium sulfate and a phosphate buffer salt; (5) The weight ratio of the phospholipid to cholesterol is from 2:1 to 15:1, preferably from 2:1 to 10:1, preferably from 2.5:1 to 10:1; (6) The long-circulating functional substance accounts for 0% by mass to 20% by mass of the total lipids; (7) When the phospholipid is sphingomyelin or a negatively charged phospholipid, the liposome membrane does not contain the long-circulating functional substance; (8) The weight ratio of total lipid to gemcitabine or a pharma- ceutically acceptable salt thereof in the liposome is 5:1 to 80:1, preferably 10:1 to 50:1; (9) The molarity of the salt solution in the aqueous interior is 100 to 800 mM, preferably 200 to 600 mM; 2. The gemcitabine liposome of claim 1, characterized by:

3. One or more of the following: 1) The gemcitabine liposomes have an average particle size of 10-200 nm, preferably 30-150 nm, more preferably 50-100 nm; 2) The concentration of gemcitabine or a pharma- ceutically acceptable salt thereof in the liposome is 0.1-3 mg / ml, preferably 0.1-2 mg / ml; 3) The encapsulation efficiency is greater than 85%, for example, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%; 3. The gemcitabine liposome of claim 1 or 2, characterized by:

4. Steps below: S1, Preparation of aqueous phase: Dissolving gemcitabine or a pharma- ceutically acceptable salt thereof in a salt solution having a concentration of 50-800 mM to obtain an aqueous phase; S2, Preparation of lipid phase: dissolving phospholipids and cholesterol in a weight ratio of 2:1 to 20:1 in a solvent to obtain a lipid phase, the solvent being one or more selected from the group consisting of ethanol, methanol, ethyl acetate, chloroform, dichloromethane and diethyl ether; S3, Preparation of drug-loaded liposomes: mixing the aqueous phase obtained in S1 with the lipid phase obtained in S2 by organic solvent injection, thin film hydration, inverse phase evaporation, emulsification, freeze-thaw cycles, pipeline mixing, or microfluidic methods to obtain a primary emulsion of liposomes containing gemcitabine or its pharma- ceutically acceptable salt and an aqueous interior salt solution; S4, Granulation: Granulating the primary emulsion of drug-loaded liposomes using one or more selected from the group consisting of shears, homogenizers and extruders to reach the target particle size; S5, Removal of free drug: Removing the free drug by one or more processes selected from filtration, centrifugation, dialysis, ultrafiltration, concentration, etc., to obtain drug-loaded liposomes comprising gemcitabine or a pharma-ceutically acceptable salt thereof; Optionally, adding a long-circulating functional substance when preparing the lipid phase in step S2. A method for preparing the gemcitabine liposome according to any one of claims 1 to 3, comprising:

5. One or more of the following: (A) the salt solution in step S1 is an ammonium sulfate solution, preferably the molarity of the ammonium sulfate solution is 50-800 mM, preferably 50-600 mM, preferably 50-400 mM, preferably 50-200 mM, preferably 50-100 mM; (B) the phospholipid in step S2 is HSPC; (C) the long-circulating functional substance in step S2 is MPEG2000-DSPE; (D) in step S2, the weight ratio of said phospholipid to cholesterol is from 2:1 to 15:1, preferably from 2:1 to 10:1, preferably from 2.5:1 to 10:1, more preferably from 5:1 to 8:1; (E) In step S2, the added long-circulating functional substance accounts for 0% to 30% by weight, preferably 0% to 20% by weight, preferably 10% to 20% by weight of the total lipids; (F) In step S3, the aqueous phase prepared in S1 is mixed with the lipid phase prepared in S2 in a lipid to water ratio of 1:1 to 1:20 to obtain drug-loaded liposomes, preferably in a lipid to water ratio of 1:3, 1:5, 1:7, 1:10 or 1:20; (G) performing step S3 at a temperature at or above the phase transition temperature of the lipid, preferably between 30° C. and 70° C. (e.g., between 30° C. and 40° C., between 50° C. and 60° C., between 60° C. and 70° C., or between 60° C. and 65° C.); (H) In step S4, the primary emulsion of drug-loaded liposomes is granulated to reach an average particle size of 10-200 nm, e.g., 30-200 nm, 50-200 nm, 150-200 nm, 10-150 nm, 30-150 nm, 50-150 nm, 100-150 nm, 10-100 nm, 30-100 nm, 50-100 nm, 10-50 nm, 30-50 nm, or 10-30 nm, preferably 50-100 nm; The method of claim 4, characterized by:

6. Steps below: SI, Preparation of aqueous phase: preparing a salt solution having a concentration of 50-800 mM as an aqueous phase; SII. Preparation of lipid phase: dissolving phospholipids and cholesterol in a weight ratio of 2:1 to 20:1 in a solvent to obtain a lipid phase, the solvent being one or more selected from the group consisting of water, ethanol, methanol, ethyl acetate, chloroform, dichloromethane and diethyl ether; SIII, preparation of blank liposome primary emulsion: mixing the aqueous phase obtained in SI and the lipid phase obtained in SII in appropriate amounts by organic solvent injection, thin film hydration, inverse phase evaporation, emulsification, freeze-thaw cycle, pipeline mixing, or microfluidic method to obtain blank liposome primary emulsion; SIV, Granulation: Granulating the blank liposome primary emulsion by one or more selected from the group consisting of shear, homogenizer and extruder to reach the target particle size; SV, Removal of organic solvent: Removing the organic solvent by one or more processes selected from the group consisting of filtration, centrifugation, dialysis, ultrafiltration, concentration, etc. to obtain blank liposomes; SVI. Preparation of API solution: dissolving gemcitabine or a pharma- ceutically acceptable salt thereof in water or the aqueous phase of step SI, diluted or undiluted, to prepare an API solution; SVII. Preparation of drug-loaded liposomes: heating and incubating the obtained blank liposomes and API solution at 30°C-75°C for 5-60 minutes to obtain drug-loaded liposomes; SVIII, Free Drug Removal: Removing free drug by one or more processes selected from filtration, centrifugation, dialysis, ultrafiltration, concentration, etc. to obtain drug-loaded liposomes containing gemcitabine or a pharma-ceutically acceptable salt thereof; Optionally, adding a long-circulating functional agent when preparing the API solution in step SVI or when preparing the drug-loaded liposomes in step SVII; A method for preparing the gemcitabine liposome according to any one of claims 1 to 3, comprising:

7. One or more of the following: (a) the salt solution in step SI is an ammonium sulfate solution, a mixed solution of sodium sulfate and a phosphate buffer salt, a mixed solution of sodium chloride and a phosphate buffer salt, or a mixed solution of ammonium sulfate and a phosphate buffer salt, preferably the concentration of the ammonium sulfate solution is 100-800 mM, preferably 200-600 mM, more preferably 200-400 mM, more preferably 300-400 mM, preferably the concentration of sodium sulfate, sodium chloride or ammonium sulfate in the mixed solution is 100-800 mM (preferably 200-600 mM, more preferably 200-500 mM, more preferably 250-500 mM), and the concentration of the phosphate buffer salt is 100-200 mM; (b) the phospholipid in step SII is selected from the group consisting of sphingomyelin, hydrogenated soy phosphatidylcholine, and a combination of hydrogenated soy phosphatidylcholine and dipalmitoyl phosphatidylglycerol, preferably the weight ratio of the phospholipid to cholesterol in step SII is from 2:1 to 15:1, preferably from 2:1 to 10:1, more preferably from 2:1 to 8:1, such as from 2:1 to 3:1 or from 7:1 to 8:1; (c) the solvent in step SII is ethanol or a mixed solution of ethanol and water; (d) in step SIII, adding the lipid phase prepared in step SII to an appropriate amount of the aqueous phase prepared in step SI in a lipid to water ratio of 1:1 to 1:20 (e.g., 1:3, 1:5, 1:10, 1:20) and stirring at or above the lipid phase transition temperature to obtain a blank liposome primary emulsion; (e) the long-circulating functional substance is MPEG2000-DSPE; The method of claim 6, characterized by:

8. One or more of the following: (i) step SIII includes the following steps: stirring and emulsifying at 30° C. to 75° C. (e.g., 30° C. to 40° C., 50° C. to 60° C., or 60° C. to 70° C.) to obtain a primary emulsion of blank liposomes; (ii) the primary emulsion of blank liposomes prepared in step SIV has an average particle size of 10-200 nm, e.g., 30-200 nm, 50-150 nm, 50-100 nm, 60-80 nm, specifically, e.g., 60 nm, 65 nm, 70 nm, 75 nm, or 80 nm; (iii) in step SVII, the weight ratio of gemcitabine or a pharma- ceutically acceptable salt thereof to the total lipid in the blank liposome is 2:1 to 1:10, preferably 2:1 to 1:5, for example 1:1 to 1:5, as another example 1:1 to 1:3, as another example 1:1 to 1:2, specifically, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0; (iv) the temperature of incubation in step SVII is between 50°C and 75°C (e.g., between 65°C and 70°C); (v) the incubation time in step SVII is between 10 minutes and 45 minutes; (vi) the mass fraction of the long-circulating functional substance added in step SVI or SVII in the liposome is 0% to 15%, for example 0% or 0.01% to 15%, specifically 0.01%, 0.05%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%; 8. The method according to claim 6 or 7, characterized in that

9. A liposomal formulation comprising gemcitabine liposomes according to any one of claims 1 to 3 or prepared by the method according to any one of claims 4 to 8, and one or more pharma- ceutically acceptable carriers; preferably, said liposomal formulation comprises an osmolality adjusting agent, such as a sucrose-histidine solution, preferably a 10% sucrose-histidine solution, preferably a 10% sucrose-0.155% histidine solution; Preferably, the liposome preparation is an injectable preparation.

10. 10. Use of the gemcitabine liposome according to any one of claims 1 to 3 or the liposome formulation according to claim 9 in the manufacture of an antitumor medicament, preferably wherein the tumor is selected from the group consisting of pancreatic cancer, breast cancer, non-small cell lung cancer and ovarian cancer.

11. A method for treating a tumor comprising administering to a subject in need thereof an effective amount of the gemcitabine liposome of any one of claims 1 to 3 or the liposomal formulation of claim 9, preferably wherein the tumor is selected from the group consisting of pancreatic cancer, breast cancer, non-small cell lung cancer and ovarian cancer.

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