CN114306240A

CN114306240A - Gemcitabine or liposome of salt thereof, preparation method and application thereof

Info

Publication number: CN114306240A
Application number: CN202011057224.8A
Authority: CN
Inventors: 张瑞霞; 郝斐; 刘厦; 苏正兴; 李明; 赵栋; 王晶翼
Original assignee: Sichuan Kelun Pharmaceutical Research Institute Co Ltd
Current assignee: Sichuan Kelun Pharmaceutical Research Institute Co Ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2022-04-12

Abstract

The invention provides a liposome of gemcitabine or a salt thereof, a preparation method and application thereof, in particular to a liposome, which comprises a liposome membrane and an internal water phase encapsulated in the liposome membrane; wherein the inner aqueous phase contains gemcitabine or a salt thereof and metal ions; the components constituting the liposome membrane comprise phospholipids, cholesterol and optionally a functional long-circulating material; the phospholipid is neutral phospholipid or the combination of neutral phospholipid and negative-charge phospholipid; the weight ratio of the phospholipid to the cholesterol is (1-125): (0.1-25), the weight ratio of the phospholipid to the long-circulating material (if any) is (1-125): (0.1-25); and the molar ratio of the gemcitabine or the salt thereof to the metal ions is (0.25-20): 1. The liposome has the characteristics of good stability, high encapsulation rate, capability of prolonging the in vivo circulation time of gemcitabine, enhancing the tumor targeting property, improving the curative effect and reducing the toxic and side effects of the gemcitabine.

Description

Gemcitabine or liposome of salt thereof, preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical preparations, in particular to gemcitabine or gemcitabine salt liposome and a preparation method and application thereof.

Background

Gemcitabine is a pyrimidine nucleic acid analogue, mainly acts on a DNA synthesis phase, and belongs to an antimetabolite anticancer drug. It is phosphorylated intracellularly by deoxycytidine enzyme to difluorodeoxycytidine monophosphate (dFdCMP) after administration, and then metabolized to active diphosphate (dFdCDP) and nucleoside triphosphate (dFdCTP) by nucleoside kinase. Gemcitabine inhibits DNA synthesis primarily by intercalation of active dFdCTP into the DNA strand; at the same time, dFdCDP can inhibit the activity of ribonucleotide reductase, so that the generation of deoxyribonucleotide (dCTP) necessary for synthesizing DNA is inhibited. In addition, dFdCTP can interfere with the self-repair mechanisms of DNA, ultimately leading to tumor cell death. Gemcitabine is used as a first-line chemotherapy scheme for various solid tumors (including pancreatic cancer, breast cancer, non-small cell lung cancer, ovarian cancer and the like) by single or combined with other medicines, and has wide anticancer spectrum and wide clinical application.

Gemcitabine has simple molecular structure, strong hydrophilicity, rapid metabolism by cytidine deaminase in liver, kidney, blood and other tissues, and short half-life (42-9)7min, age and sex related), so that the general injection needs to be used clinically in a large dose (recommended dose is 1000 mg/m)²) And prolonging the infusion time (more than 30min) to maintain the blood concentration to kill tumor cells, which often causes serious toxic and side effects due to poor targeting, including blood toxicity (anemia, neutropenia and thrombocytopenia), alopecia, gastrointestinal toxicity (such as nausea and vomiting), fatigue, fever, etc. Among them, the hematologic toxicity is its dose-limiting toxicity. Gemcitabine produces myelosuppression after a single administration, and active metabolites can destroy the proliferation and differentiation of bone marrow cells, so that hematopoietic cells are reduced, the number of platelets and neutrophils is reduced, and the clinical application is limited. At present, gemcitabine is only available in the market as common injection, including powder injection, water injection and infusion solution.

The liposome has the advantages of safety, no toxicity and good biocompatibility as a drug carrier, and after the drug is wrapped by the liposome, the damage of external factors (pH, light and enzyme) can be avoided, and the stability of the drug is improved; after entering into the body, the damage of related enzyme, protein and the like in the blood to the medicine can be avoided, and the blood circulation time of the medicine can be prolonged. Compared with normal tissues, the tumor tissues have large vascular gaps and incomplete structures, and the nano-sized liposome can be passively targeted to tumor parts through an EPR effect and directly release drugs to kill tumor cells, so that the aims of improving the curative effect and reducing toxic and side effects are fulfilled.

It is reported in the literature that gemcitabine can be formulated into liposomes to avoid rapid metabolism of gemcitabine molecules and prolong the duration of action. But the preparation method has low encapsulation efficiency on gemcitabine liposome, is unstable, is easy to leak in vivo, has quick release, has complex process and is difficult to meet clinical requirements. CN 102846547B utilizes soybean phospholipid, cholesterol and polyoxyethylene ricinoleate to prepare gemcitabine liposome, the encapsulation and the particle size are improved, but the adopted auxiliary material polyoxyethylene ricinoleate is easy to cause adverse reactions such as acute hypersensitivity, peripheral neurotoxicity, cytotoxicity and the like, the injection safety risk is large, and the safety needs to be improved. CN 102716089B utilizes gemcitabine hydrochloride, cholesterol, egg yolk phosphatidylinositol, lecithin, tween 80, trehalose and polyvinylpyrrolidone to freeze-dry and prepare gemcitabine hydrochloride liposome injection, and the introduction of tween 80 also brings the safety risk of in vivo injection.

Disclosure of Invention

The invention aims to provide gemcitabine liposome with high encapsulation efficiency and high stability and a preparation method thereof. The liposome can remarkably prolong the blood half-life of gemcitabine, improve tissue distribution, improve tumor targeting property, improve curative effect and reduce toxic and side effects. Meanwhile, the liposome has safe components and simple preparation process, and is easy for industrial production and clinical transformation.

In a first aspect, the present invention provides a liposome comprising a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane;

wherein the inner aqueous phase contains gemcitabine or a salt thereof and metal ions; the components constituting the liposome membrane comprise phospholipids, cholesterol and optionally a functional long-circulating material; the phospholipid is neutral phospholipid or the combination of neutral phospholipid and negative-charge phospholipid;

the weight ratio of the phospholipid to the cholesterol is (1-125): (0.1-25), preferably (1-100): 0.1-20), more preferably (2-80): 0.1-15), such as (2-10): 1, for example (4-8): 1;

the weight ratio of the phospholipid to the long circulating material (if any) is (1-125): 0.1-25); for example, (1 to 20):1, for example (2-15): 1;

and the molar ratio of the gemcitabine or the salt thereof to the metal ions is (0.25-20): 1.

The above ratios refer to the ratio of the ingredients in the prepared liposomes. This ratio may differ from the dosing ratio in the preparation, depending on the availability of the drug.

The drug utilization rate of the invention is the ratio of the amount of the traditional Chinese medicine in the liposome to the amount of the medicine added during preparation. Wherein the content of liposome traditional Chinese medicine is determined by high performance liquid chromatography by adopting gemcitabine content determination method specified in Chinese pharmacopoeia.

In an embodiment of the invention, the salt of gemcitabine is the hydrochloride salt of gemcitabine.

In another aspect, the present invention provides a liposome prepared by preparation method 1 or preparation method 2, wherein:

preparation method 1

(1) Preparing blank liposome: preparing colostrum by using phospholipid and cholesterol solution as oil phase and metal ion-containing solution as water phase, and performing membrane hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation, grading, and optionally performing dialysis, centrifugation, ultrafiltration or concentration to obtain blank liposome;

(2) carrying out medicine loading: adding a solution of gemcitabine or a salt thereof into the blank liposome prepared in the step (1), optionally, adding the metal ions in the water phase in the step (1) into the solution of gemcitabine or the salt thereof, and heating and incubating above the phospholipid phase transition temperature to obtain a drug-loaded liposome;

(3) removing free drugs: carrying out one or more treatments of dialysis, centrifugation and ultrafiltration on the drug-loaded liposome prepared in the step (2) to remove free drugs and optionally carrying out concentration treatment to obtain the liposome;

wherein, adding optional functional long-circulating material into the oil phase in the step (1) or adding optional functional long-circulating material into the oil phase in the step (2) to prepare liposome containing the functional long-circulating material;

preparation method 2

(1) Preparing an oil phase: dissolving phospholipid and cholesterol in an organic solvent to prepare an oil phase;

(2) preparing a water phase: dissolving gemcitabine or a salt thereof in a solution containing metal ions to prepare an aqueous phase;

(3) preparing a drug-loaded liposome: preparing the liposome from the oil phase in the step (1) and the water phase in the step (2) by thin film hydration, organic solvent injection, pipeline emulsification or reverse evaporation, or directly mixing the (2) with proliposome (such as commercial Presome), and grading to obtain drug-loaded liposome;

(4) removing free drugs: carrying out one or more treatments of dialysis, centrifugation and ultrafiltration on the drug-loaded liposome prepared in the step (3) to remove free drugs and optionally carrying out concentration treatment to obtain the liposome;

wherein, the functional long-circulating material is added when the oil phase is prepared in the step (1), or the functional long-circulating material is added after the size of the particles is finished in the step (3) to prepare the liposome containing the functional long-circulating material.

In another aspect, the invention provides a liposome formulation comprising a liposome as described herein, and one or more pharmaceutical excipients.

In another aspect, the invention provides the use of a liposome as described herein in the preparation of a medicament for the treatment of an antineoplastic agent.

In another aspect, the invention provides an anti-tumor method comprising administering to a subject in need thereof an effective amount of a liposome or liposome formulation described herein.

In another aspect, the invention provides a method of making a liposome described herein, selected from the group consisting of method 1 and method 2:

preparation method 1

(1) Preparing blank liposome: using the solution of phospholipid and cholesterol as oil phase, using the solution containing metal ions as water phase, preparing colostrum by thin film hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation, grading, optionally, performing one or more treatments of dialysis, centrifugation, ultrafiltration and concentration to obtain blank liposome;

wherein, an optional functional long-circulating material is added into the oil phase in the step (1), or an optional functional long-circulating material is added into the oil phase in the step (2), so as to prepare the liposome containing the functional long-circulating material.

In some embodiments, the weight ratio of phospholipid and cholesterol added in step (1) is (1-125): 0.1-25), preferably (1-100): 0.1-20, more preferably (2-80): 0.1-15), such as (2-10): 1, for example (4-8): 1.

in some embodiments, the weight ratio of gemcitabine added in step (2) to phospholipids in the blank liposomes prepared in step (1) is 1: (0.01 to 100), preferably 1: (0.02 to 80), more preferably 1: (0.05 to 50), for example, 1: (0.5 to 5), and further example is 1: (0.5-2).

In some embodiments, the weight ratio of gemcitabine to optional functional long-circulating material added in step (2) is 1: (0 to 100), preferably 1: (0.05 to 50), more preferably 1 (0.1 to 25), for example, 1: (0.1-1), and further example 1: (0.1-0.5).

In some embodiments, the phospholipid added in step (1) is a neutral phospholipid.

In some embodiments, the phospholipid added in step (1) is a combination of neutral phospholipids and negatively charged phospholipids; preferably, the weight ratio between the neutral phospholipid and the negative-charged phospholipid is (0.1-20): 1; more preferably (0.5-15): 1, and even more preferably (1-10): 1.

preparation method 2

(3) preparing a drug-loaded liposome: preparing the liposome from the oil phase in the step (1) and the water phase in the step (2) by thin film hydration, organic solvent injection, pipeline emulsification or reverse evaporation, or directly mixing the (2) with proliposome (such as commercial Presome), and grading to obtain liposome carrying medicine;

in some embodiments, the weight ratio of gemcitabine added in step (2) to phospholipid of step (1) is 1: (0.01 to 100), preferably 1: (0.02 to 80), more preferably 1: (0.05 to 50), for example, 1: (0.5 to 5), and further example is 1: (0.5-2).

the preparation methods 1 and 2 described herein do not involve freeze-thawing or repeated freeze-thawing to achieve encapsulation of gemcitabine.

Detailed Description

Definition of

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "liposome" refers to a self-assembled structure comprising one or more amphiphilic lipid bilayers, each comprising two amphiphilic lipid monolayers containing inverted orientation. Amphiphilic lipids comprise a polar (hydrophilic) head group covalently linked to one or two or more non-polar (hydrophobic) acyl or alkyl chains. The very unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous medium results in the amphiphilic lipid molecules aligning themselves such that the polar head groups are oriented towards the surface of the bilayer and the acyl chains are oriented towards the interior of the bilayer, effectively preventing the acyl chains from coming into contact with the aqueous environment.

Liposomes, as used herein, can have a single lipid bilayer (unilamellar liposomes) or multiple lipid bilayers (multilamellar liposomes) surrounding or encapsulating an aqueous compartment (component). Various forms of liposomes are described, for example, in the following: cullis et al Biochim Biophys Acta 559: 399-.

The term "effective amount" as used herein refers to an amount sufficient to achieve a desired therapeutic effect, e.g., to achieve alleviation of symptoms associated with the disease being treated.

The term "treatment" as used herein is intended to reduce or eliminate the disease state or condition for which it is directed. A subject is successfully "treated" if the subject, following the methods described herein, receives a therapeutic amount of the crystalline form or pharmaceutical composition thereof and the subject exhibits an observable and/or detectable decrease or improvement in one or more of the indications and symptoms. It is also understood that treatment of the disease state or condition described includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.

In one aspect, the present invention provides a liposome comprising a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane;

In some embodiments, the liposomes comprise a liposomal membrane and an internal aqueous phase encapsulated within the liposomal membrane;

the weight ratio of gemcitabine or salt thereof, phospholipid, cholesterol and the long-circulating material is 1: (1-125): (0.1-25): (0-25);

in some embodiments, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine, or a combination of both. In some embodiments, the neutral phospholipid is soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine, or any combination thereof.

In some embodiments, the negatively charged phospholipid is selected from one or more of Phosphatidylglycerol (PG), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidic Acid (PA), and cardiolipin. In some embodiments, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, or a combination of both.

In some embodiments, the components making up the liposomal membrane include neutral phospholipids, cholesterol, and optionally functional long-circulating materials.

In some preferred embodiments, the components that make up the liposomal membrane include neutral phospholipids and cholesterol; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, and combinations thereof; preferably, the components constituting the liposome membrane include neutral phospholipids, cholesterol and functional long-circulating materials; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the neutral phospholipid is selected from the group consisting of hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof.

In some preferred embodiments, the ingredients constituting the liposome membrane include a neutral phospholipid and cholesterol, and the gemcitabine or a salt thereof, the neutral phospholipid and the cholesterol are present in a weight ratio of 1: (1-125): (0.1 to 25); further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, and combinations thereof; further preferably, the weight ratio of gemcitabine or its salt, neutral phospholipid and cholesterol is 1: (1-100): (0.1 to 20); further preferably, the weight ratio of gemcitabine or its salt, neutral phospholipid and cholesterol is 1: (2-80): (0.1-15).

In some embodiments, the ingredients comprising the liposomal membrane comprise a neutral phospholipid, cholesterol, and a functional long-circulating material, and the gemcitabine, or a salt thereof, the neutral phospholipid, cholesterol, and the functional long-circulating material are in a weight ratio of 1: (1-125): (0.1-25): (0.1 to 25); further preferably, the weight ratio of gemcitabine or a salt thereof, neutral phospholipid, cholesterol and functional long-circulating material is 1: (1-100): (0.1-20): (0.1 to 20); further preferably, the weight ratio of gemcitabine or its salt, neutral phospholipid and cholesterol is 1: (2-80): (0.1-15): (0.1-15), further preferably, the neutral phospholipid is selected from the group consisting of soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof.

In some embodiments, the liposome comprises the following components:

1g gemcitabine;

5-50g hydrogenated soy phosphatidylcholine;

0.1-10g cholesterol;

copper sulfate in a 1 to gemcitabine molar ratio: (1-8).

In some embodiments, the liposome comprises the following ingredients in parts by weight:

1g gemcitabine;

4-40g soybean phosphatidylcholine;

1-10g cholesterol;

copper gluconate-triethanolamine, wherein the molar ratio of copper ions to gemcitabine is 1: (0.25 to 20).

In some embodiments, the liposome comprises the following components:

1g gemcitabine;

1-20g hydrogenated soy phosphatidylcholine;

0.1-5g cholesterol;

0.1-5g mPEG2000-DSPE；

copper gluconate in a 1 (2-10), e.g., 1:5, molar ratio to gemcitabine.

1g gemcitabine;

5-80g distearoyl phosphatidylcholine;

1-15g cholesterol;

1-15g mPEG2000-DSPE；

zinc gluconate-triethanolamine in a molar ratio of zinc ions to gemcitabine of 1: (0.25-10).

In some embodiments, the components that make up the liposomal membrane include neutral phospholipids, negatively charged phospholipids, cholesterol, and optionally functional long-circulating materials.

In some preferred embodiments, the components that make up the liposomal membrane include neutral phospholipids, negatively charged phospholipids, and cholesterol; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the negatively charged phospholipid is dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and combinations thereof; preferably, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, cholesterol, and functional long-circulating materials; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and combinations thereof.

In some preferred embodiments, the ingredients constituting the liposome membrane include a neutral phospholipid, a negatively charged phospholipid and cholesterol, and the gemcitabine or a salt thereof, the neutral phospholipid, the negatively charged phospholipid and the cholesterol are present in a weight ratio of 1: (1-100): (0.1-25): (0.1 to 25); further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the negatively charged phospholipid is dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, or any combination thereof; further preferably, the weight ratio of gemcitabine or a salt thereof, neutral phospholipid, negatively charged phospholipid and cholesterol is 1: (1-80): (0.1-20): (0.1 to 20).

In some embodiments, the ingredients comprising the liposomal membrane comprise a neutral phospholipid, a negatively charged phospholipid, cholesterol, and a functional long-circulating material, and the gemcitabine, or a salt thereof, the neutral phospholipid, the negatively charged phospholipid, the cholesterol, and the functional long-circulating material are in a weight ratio of 1: (1-100): (0.1-25): (0.1-25): (0.1 to 25); further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and combinations thereof; further preferably, the weight ratio of gemcitabine or a salt thereof, neutral phospholipid, negatively charged phospholipid, cholesterol and functional long circulating material is 1: (1-80): (0.1-20): (0.1-20): (0.1 to 20).

In some embodiments, the functional long circulating material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol 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), wherein the polyethylene glycol has a molecular weight of 500 to 10,000 daltons, preferably 2000 daltons. In some embodiments, the functional long-circulating material is MPEG 2000-DSPE.

In some embodiments, the liposome comprises the following components:

1g gemcitabine;

2-30g distearoyl phosphatidylcholine;

1-15g distearoyl phosphatidyl glycerol;

0.1-10g cholesterol;

copper gluconate-triethanolamine, wherein the molar ratio of copper ions to gemcitabine is 1: (1-6).

1g gemcitabine;

1-40g soybean phosphatidylcholine;

1-15g distearoyl phosphatidyl glycerol;

0.5-10g cholesterol;

copper gluconate in a 1 to gemcitabine molar ratio: (1-6).

In some embodiments, the liposome comprises the following components:

1g gemcitabine;

1-40g distearoyl phosphatidylcholine;

1-15g dipalmitoyl phosphatidylglycerol;

0.1-10g cholesterol;

0.1-5g mPEG2000-DSPE；

calcium acetate-acetic acid, wherein the molar ratio of calcium ions to gemcitabine is 1 (1-4).

1g gemcitabine;

3-40g hydrogenated soy phosphatidylcholine;

1-15g dipalmitoyl phosphatidylglycerol;

0.5-10g cholesterol;

0.5-15g mPEG2000-DSPE；

the molar ratio of the zinc gluconate to the gemcitabine is 1 (1-4).

1g gemcitabine;

3-40g distearoyl phosphatidylcholine;

1-15g distearoyl phosphatidyl glycerol;

0.5-10g cholesterol;

0.5-10g mPEG2000-DSPE；

and the molar ratio of the copper gluconate to the gemcitabine is 1 (1-6).

1g gemcitabine;

5-80g soybean phosphatidylcholine;

1-25g distearoyl phosphatidyl glycerol;

1-15g cholesterol;

0.5-15g mPEG2000-DSPE；

the molar ratio of copper ions to gemcitabine is 1 (0.25-20).

In some embodiments, the metal ion is selected from the group consisting of calcium ion, magnesium ion, and transition metal ion. In some embodiments, the metal ion is selected from one or more of copper ion, manganese ion, nickel ion, iron ion, molybdenum ion, cobalt ion, calcium ion, and zinc ion. In some embodiments, the metal ion is selected from one or more of copper ion, cobalt ion, calcium ion, and zinc ion. In some embodiments, the molar ratio of the metal ion to gemcitabine or a salt thereof is 1 (0.25-20), preferably 1 (0.5-10), such as 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1: 10.

In some embodiments, the internal aqueous phase contains an acid ion, such as an inorganic acid ion or an organic acid ion. In some embodiments, the acid ion is selected from one or more of sulfate, halide, gluconate, nitrate, and acetate. In some embodiments, the acid ion is selected from one or more of gluconate, acetate, nitrate, and sulfate.

In some embodiments, the internal aqueous phase also contains a pH adjusting ingredient, such as hydrochloric acid, acetic acid, triethanolamine, sodium hydroxide, or arginine. In some embodiments, the internal aqueous phase has a pH of 2 to 8, preferably 3 to 7.5, more preferably 4 to 7.5, such as 4.0, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.1, 6.2, 6.5, 6.8, 6.9, 7.0, 7.3, 7.4, 7.5, 7.8, 7.9 or 8.0.

In some embodiments, the liposome particle size is 20-500nm, such as 50-300nm, further such as 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 150nm, 200nm, 250nm, or 300 nm.

preparation method 1

preparation method 2

In some embodiments, the method of preparing the liposome comprises one or more of the following features:

1) in the preparation method 1, step (1), phospholipid and cholesterol are dissolved in a solvent selected from alcohols (e.g., methanol, ethanol or isopropanol), halogenated hydrocarbons (e.g., dichloromethane or chloroform), water and any combination thereof as an oil phase;

2) in step (2) of preparation method 1, the solution of gemcitabine or a salt thereof is an aqueous solution of gemcitabine or a salt thereof or the metal ion-containing solution in step (1);

3) in step (2) of preparation method 1, the incubation is performed at 50 to 70 ℃ (e.g., 60 to 70 ℃, further e.g., 60 to 65 ℃);

4) in step (2) of preparation method 1, the incubation time is 15min-2h (e.g., 15min-1h, e.g., 30 min);

5) in step (1) of preparation method 2, the organic solvent is selected from an alcohol solvent (e.g., methanol, ethanol or isopropanol), a halogenated hydrocarbon solvent (e.g., dichloromethane or chloroform), and a mixed solvent thereof with water;

6) in the preparation method 1 or the preparation method 2, the weight ratio of the phospholipid to the cholesterol is (1-125): (0.1-25), preferably (1-100): (0.1-20), more preferably (2-80): 0.1-15), such as (2-10): 1, for example (4-8): 1;

7) in preparation method 1, step (2), the weight ratio of gemcitabine or a salt thereof to phospholipids is 1: (0.01 to 100), preferably 1: (0.02 to 80), more preferably 1: (0.05 to 50), for example, 1: (0.5 to 5), and further example is 1: (0.5 to 2);

8) in the preparation method 1 or the preparation method 2, the phospholipid is a neutral phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine or a combination of both; preferably, the neutral phospholipid is soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine or any combination thereof;

9) in the production method 1 or the production method 2, the phospholipid is a combination of a neutral phospholipid and a negatively charged phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine or a combination of both; preferably, the neutral phospholipid is soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine or any combination thereof; preferably, the negatively charged phospholipid is selected from one or more of Phosphatidylglycerol (PG), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidic Acid (PA) and cardiolipin; preferably, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, or a combination of both;

preferably, the weight ratio between the neutral phospholipid and the negative-charged phospholipid is (0.1-20): 1; more preferably (0.5-15): 1, and even more preferably (1-10): 1;

10) in production method 1 or production method 2, the metal ion is selected from the group consisting of a calcium ion, a magnesium ion, and a transition metal ion; preferably, the metal ions are selected from one or more of copper ions, manganese ions, nickel ions, iron ions, molybdenum ions, cobalt ions, calcium ions, and zinc ions; preferably, the metal ion is selected from one or more of copper ion, cobalt ion and calcium ion;

preferably, the molar ratio of the gemcitabine or the salt thereof to the metal ion is (0.25 to 20):1, preferably (0.5-10): 1;

11) in the preparation method 1 or the preparation method 2, the solution containing the metal ions is a buffered salt solution containing the metal ions; preferably, the buffered salt solution has a pH of 4 to 8, preferably 5 to 7.5, such as 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0; preferably, the solution containing metal ions is a copper gluconate solution, a copper sulfate-triethanolamine solution, a copper gluconate-arginine buffer solution, a copper gluconate-triethanolamine solution, a copper gluconate-tris (hydroxymethyl) aminomethane solution, a copper sulfate-4-hydroxyethylpiperazine ethanesulfonic acid solution, a zinc gluconate solution or a calcium acetate-acetic acid buffer solution with a pH of 4 to 8;

12) in the preparation method 1 or the preparation method 2, the functional long-circulating material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol modified distearoyl phosphatidyl ethanolamine (MPEG-DSPE), polyethylene glycol modified dimyristoyl phosphatidyl ethanolamine (MPEG-DMPE), and polyethylene glycol modified dipalmitoyl phosphatidyl ethanolamine (MPEG-DPPE), wherein the molecular weight of the polyethylene glycol is 500 to 10,000 daltons, preferably 2000 daltons; preferably, the functional long-circulating material is MPEG 2000-DSPE;

preferably, gemcitabine or a salt thereof and the optional functional long-circulating material are added in preparation method 1 or preparation method 2 in a weight ratio of 1: (0 to 100), preferably 1: (0.05 to 50), more preferably 1 (0.1 to 25), for example, 1: (0.1-1), and further example 1: (0.1-0.5).

In another aspect, the present invention provides a liposome formulation comprising a liposome according to any one of the first aspect, and one or more pharmaceutically acceptable carriers.

Pharmaceutically acceptable carriers can be, for example, physiological saline, isotonic dextrose, isotonic sucrose, gellin's solution, and Hanks' solution. Buffer substances may be added to obtain a storage stable preferred pH, e.g. a pH between 6.0 and 8.0. In some embodiments, a pH of 6.0-7.5 is preferred at which the liposome membrane has better stability, as well as encapsulation stability for the drug.

The preparation may also contain stabilizer and/or antioxidant. In some embodiments, the stabilizing agent is selected from one or more of ethylenediaminetetraacetic acid, disodium salts of ethylenediaminetetraacetic acid, and dicalcium salts of ethylenediaminetetraacetic acid. In some embodiments, the stabilizing agent is added in an amount of 0 to 0.5%/v%. In some embodiments, the antioxidant is a water-soluble or oil-soluble antioxidant. In some embodiments, the water-soluble antioxidant is selected from one or more of ascorbic acid, sodium bisulfite, sodium sulfite, sodium metabisulfite, L-cysteine. In some embodiments, the oil-soluble antioxidant is selected from one or more of alpha-tocopherol, alpha-tocopherol succinate, and alpha-tocopherol acetate. In some embodiments, the antioxidant is added in an amount of 0 to 0.5%/v%.

The dosage administered will depend on the amount of drug encapsulated in the liposome, the disease state to be treated, and the dosage form and the judgment of the skilled practitioner. Generally, the dose administered will be sufficient to deliver a therapeutically effective amount of gemcitabine to the subject.

The amount of formulation necessary to deliver a therapeutically effective amount of the dose can be determined by conventional in vivo and in vitro methods in the field of pharmaceutical testing. See, e.g., Handbook of Anticancer Drug's Development, d.b. budman, a.h. calvert, e.k.rowinsky (editor), LWW, 2003. Typically, the dosage of the pharmaceutical composition of the present invention is in the range of about 0.001mg to about 500mg of drug per kilogram of body weight, usually in the range of about 0.01mg to about 100mg of drug per kilogram of body weight.

Typically, the liposomes of the invention are prepared as topical medicaments or as injection solutions, or as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid carriers prior to injection can also be prepared. Enteric-coated tablets or gel capsules may also be formulated according to methods known in the art.

The liposomes of the invention may be administered in any manner that is medically acceptable, depending on the condition being treated. Possible routes of administration include injection, parenteral routes, such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intraarticular, intracerebral, intrathecal, or other routes, such as oral, nasal, ocular, rectal, vaginal or pulmonary, for example by inhalation. For the delivery of liposomal drugs formulated according to the present invention to tumors of the central nervous system, slow and continuous intracranial infusion of liposomes directly into tumors (convection enhanced delivery) or CED) is particularly advantageous. See Saito, et al, Cancer Research, Vol.64, p.2572-2679,2004; mamot, et al, j.neuro-Oncology, vol.68, p.1-9, 2004. Liposomes of the invention may also be applied directly to the surface of a tissue. Sustained release administration, pH-dependent release administration or other specific chemical or environmental condition mediated release administration is also expressly encompassed by the present invention, for example by such means as depot injection (depot injection) or bioerodible implantation (bioerodible implant).

In another aspect, the invention provides the use of a liposome according to any one of the first aspect in the preparation of a medicament for the treatment of an antineoplastic agent. In some embodiments, the tumor is selected from pancreatic cancer, breast cancer, non-small cell lung cancer, and ovarian cancer.

In another aspect, the present invention provides a method for preparing the liposome of any one of the first aspect, which is selected from the group consisting of preparation method 1 and preparation method 2:

preparation method 1

(1) Preparing blank liposome: preparing colostrum by using phospholipid and cholesterol solution as oil phase and metal ion-containing solution as water phase, performing membrane hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation, grading, and optionally performing dialysis, centrifugation, ultrafiltration or concentration to obtain blank liposome;

(2) carrying out medicine loading: adding gemcitabine or a salt solution thereof into the blank liposome prepared in the step (1), optionally, adding the metal ions in the water phase in the step (1) into the solution, and heating and incubating the solution above the phospholipid phase transition temperature to obtain a drug-loaded liposome;

wherein, an optional functional long-circulating material is added in the oil phase in the step (1), or an optional functional long-circulating material is added in the step (2), so as to prepare the liposome containing the functional long-circulating material.

Preparation method 2

(2) preparing a water phase: dissolving gemcitabine or a salt thereof in an aqueous solution containing metal ions to prepare an aqueous phase;

(3) preparing a drug-loaded liposome: preparing the liposome from the oil phase in the step (1) and the water phase in the step (2) by adopting a film hydration method, an organic solvent injection method, a pipeline emulsification method or a reverse evaporation method, or directly mixing the (2) and the prepared proliposome (such as commercialized Presome), and finishing granules to prepare a drug-loaded liposome;

wherein, optional functional long-circulating material is added when the oil phase is prepared in the step (1), or optional functional long-circulating material is added after the step (3) is finished, so as to prepare the liposome containing the functional long-circulating material.

Advantageous effects of the invention

The invention provides a liposome of gemcitabine or a salt thereof, and a preparation method and application thereof. The liposome can achieve one or more of the following technical effects:

(1) the stability is good; in some embodiments, the liposome has no significant change in particle size and encapsulation efficiency when stored for 3 months or more at normal temperature or in a refrigerated environment.

(2) The encapsulation rate is high; in some embodiments, the liposome encapsulation efficiency can be greater than 90%, e.g., greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%.

(3) Can prolong the circulation time of gemcitabine in vivo; in some embodiments, the liposomes prepared by the present invention can prolong gemcitabine retention time in vivo by more than 3 times compared to conventional injections.

(4) Can enhance tumor targeting property, improve therapeutic effect, and reduce toxic and side effects.

Drawings

FIG. 1 shows the results of pharmacokinetic experiments with gemcitabine liposomes (1.0mg/kg, iv) and gemcitabine hydrochloride (50mg/kg, iv) administered to CD-1 mice.

Detailed Description

The following experimental examples and examples are only for further illustrating the present invention, but it should not be construed that the present invention is limited thereto. Various modifications and improvements may be made by those skilled in the art based on the basic idea of the invention, but they are within the scope of the invention as long as they do not depart from the basic idea of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the following examples, unless otherwise specified, the encapsulation efficiency of liposomes was determined by the Sephadex column chromatography. The gel column passing method comprises the following specific steps: 1 PD-10 desaling columns are taken to be pre-packed, and 25mL deionized water is used for washing and balancing. Sampling 0.2mL, loading on a column, keeping for 1-2 min, eluting with 0.9% sodium chloride solution, placing 7mL before 1 mL/collection in a 25mL volumetric flask, diluting with a solvent to a constant volume, taking the solution as an encapsulated drug sample, continuing to elute, collecting subsequent 15mL of eluate in the 25mL volumetric flask, and taking the solution as a free drug sample to a constant volume with the solvent. The encapsulation efficiency is calculated according to the following formula by measuring with an ultraviolet-visible spectrophotometry. The encapsulation efficiency is W/(W + Wf) × 100%. Wherein W is the amount of encapsulated drug and Wf is the amount of free drug.

In the following examples, the particle size of liposomes was measured by Dynamic Light Scattering (DLS), unless otherwise specified. The dynamic light scattering method comprises the following specific steps: an appropriate amount of the liposome sample was taken, diluted with water and subjected to particle size measurement using a Malvern particle sizer (Malvern Nano-ZS 90). Particle size is expressed as Intensity diameter (Intensity).

EXAMPLE 1 preparation of Gemcitabine liposomes

(1) Preparation of organic phase

Weighing 6g of hydrogenated soybean phosphatidylcholine and 0.75g of cholesterol, heating at 60 ℃, dissolving in 40ml of ethanol, and heating for later use.

(2) Preparation of aqueous phase

Gemcitabine hydrochloride (6 g) was weighed out and dissolved in a copper sulfate-triethanolamine solution (200mM) at pH 7.4 in a molar ratio of gemcitabine to copper sulfate of 1.25:1, and heated for use.

(3) Preparation of drug-loaded liposomes

Injecting the organic phase in the step (1) into the water phase in the step (2) through a 50ml injector, and heating and stirring at 60 ℃ for 30min to obtain the drug-loaded liposome primary emulsion. And (3) granulating the primary drug-loaded liposome emulsion to 120nm by a high-speed shearing and high-pressure homogenizer, and dialyzing by a tangential flow ultrafiltration device (the dialysate is a sucrose-histidine solution with the pH of 6.5 and the concentration of 10%) to obtain gemcitabine liposome after dialysis, wherein the average particle size of the obtained liposome is about 116nm, the concentration of gemcitabine is 1.05mg/ml, and the entrapment rate is 95.7%.

EXAMPLE 2 preparation of Gemcitabine liposomes

(1) Preparation of organic phase

Weighing hydrogenated soybean phosphatidylcholine 2.75g, cholesterol 0.36g, MPEG2000-DSPE 0.65g, heating at 60 deg.C to dissolve in 20ml anhydrous ethanol, and heating for use.

(2) Preparation of aqueous phase

Gemcitabine (7.5g) was weighed out and heated to a molar ratio of gemcitabine to copper ions of 5:1 in a copper gluconate solution (100mM) at pH 7.4 for use.

(3) Preparation of drug-loaded liposomes

Injecting the organic phase in the step (1) into the water phase in the step (2) at a speed of 100ml/min, heating and stirring for 30min to obtain drug-loaded liposome colostrum, carrying out high-speed shearing and high-pressure homogenizer granulation to 48nm, carrying out ultrafiltration and dialysis treatment by using a tangential flow ultrafiltration device, wherein a dialysate is a sucrose-histidine solution with a pH of 6.5 and a concentration of 10%, and obtaining gemcitabine liposome after dialysis, wherein the average particle size of the obtained liposome is about 52nm, and the gemcitabine concentration is: 0.31mg/ml, and the encapsulation efficiency is 91.3 percent.

EXAMPLE 3 preparation of polyethylene glycol modified negatively charged phospholipid Gemcitabine liposomes

(1) Preparation of organic phase

2.2g of distearoyl phosphatidylcholine, 0.7g of dipalmitoyl phosphatidylglycerol and 0.37g of cholesterol were weighed, dissolved in 9ml of ethanol/water (16:1, v/v) at 60 ℃ and heated for use.

(2) Preparation of aqueous phase

Gemcitabine (3.3g) was weighed out and heated to a molar ratio of gemcitabine to calcium ions of 1.5:1 in a calcium acetate-acetic acid buffer solution (100mM) at pH6.5 for use.

(3) Preparation of drug-loaded liposomes

And (2) injecting the organic phase in the step (1) into the aqueous phase in the step (2) at a speed of 150mL/min, heating and stirring at 70 ℃ for 30min to obtain the primary drug-loaded liposome emulsion, carrying out high-speed shearing and high-pressure homogenizer size stabilization to about 187nm, adding 0.5mL of MPEG2000-DSPE (0.26g), heating and incubating at 60 ℃ for 10min, and immediately cooling to obtain the preparation of the polyethylene glycol modified gemcitabine liposome containing the negatively charged phospholipid. Adding 300mM EDTA-disodium solution, stirring at normal temperature for 20min, performing ultrafiltration dialysis treatment by using a tangential flow ultrafiltration device, and obtaining gemcitabine liposome after dialysis, wherein the average particle size of the obtained liposome is about 206nm, and the gemcitabine concentration is as follows: 3.6mg/ml, the encapsulation efficiency is 97.3 percent.

EXAMPLE 4 preparation of Gemcitabine liposomes

(1) Thin film formation

1.4g of distearoyl phosphatidylcholine, 0.7g of distearoyl phosphatidylglycerol and 0.4g of cholesterol were weighed, heated at 60 ℃ and dissolved in 30ml of dichloromethane/methanol/water (10:2:1, v/v/v), placed in a round-bottomed flask, and then subjected to rotary evaporation at 37 ℃ on a rotary evaporator to remove the organic solvent, thereby obtaining a lipid thin film.

(2) Hydration of water

A hydrated solution was prepared by weighing 3g of gemcitabine and heating it in a solution of copper gluconate-triethanolamine (100mM) dissolved at pH 7.5 with a molar ratio of gemcitabine to copper ions of 1.5: 1. Adding the hydration solution into the lipid film for hydration for 30 min.

(3) Preparation of drug-loaded liposomes

Shearing and dispersing the hydrated solution by a high-speed shearing machine, granulating to 115nm by a homogenizer, dialyzing by a tangential flow ultrafiltration device to obtain gemcitabine liposome after dialysis, wherein the average particle size of the obtained liposome is about 135nm, the gemcitabine concentration is 2.4mg/ml, and the entrapment rate is 93.0%.

Example 5 preparation of liposomes of Gemcitabine modified with polyethylene glycol

(1) Thin film formation

Weighing 2.0g of hydrogenated soybean phosphatidylcholine, 0.7g of dipalmitoyl phosphatidylglycerol, 0.36g of cholesterol and 0.7g of MPEG2000-DSPE, heating at 60 ℃, dissolving in 30ml of dichloromethane/methanol/water (10:1:1, v/v/v), placing in a round-bottomed flask, and performing rotary evaporation at 37 ℃ on a rotary evaporator to remove the organic solvent to obtain the lipid film.

(2) Hydration of water

Gemcitabine (3.3g) was weighed and dissolved in a zinc gluconate solution (100mM) at pH 7.4 with a molar ratio of gemcitabine to zinc ions of 1.5:1 to obtain a hydrated solution. Adding the hydration solution into lipid film, and hydrating for 30 min.

(3) Preparation of drug-loaded liposomes

Shearing and dispersing the hydrated solution by a high-speed shearing machine, granulating the solution to 120nm by an extruder, dialyzing by a tangential flow ultrafiltration device, and obtaining gemcitabine liposome after dialysis, wherein the average particle size of the obtained liposome is 130nm, the gemcitabine concentration is 0.71mg/ml, and the entrapment rate is 95.7%.

EXAMPLE 6 preparation of liposomes of Gemcitabine modified with polyethylene glycol

(1) Thin film formation

1.8g of distearoylphosphatidylcholine, 0.64g of distearoylphosphatidylglycerol and 0.32g of cholesterol were weighed, dissolved in 30ml of chloroform/methanol/water (10:5:0.1, v/v/v) at 60 ℃ and placed in a round-bottomed flask, and then evaporated to dryness at 37 ℃ on a rotary evaporator to remove the organic solvent, thereby obtaining a lipid film.

(2) Hydration of water

Gemcitabine (2.7g) was weighed and dissolved in a copper gluconate solution (100mM) at pH 7.4 in a molar ratio of 1:1 of gemcitabine to copper ions to give a hydrated solution. Adding the hydration solution into the lipid film for hydration for 30 min.

(3) Preparation of drug-loaded liposomes

Shearing and dispersing the hydrated solution by a high-speed shearing machine, granulating to 80nm by an extruder, weighing 0.3g of MPEG2000-DSPE, heating (60 ℃) to dissolve in 0.5ml of purified water, adding the purified water into the granulated drug-loaded liposome, heating and incubating at 60 ℃ for 10min, immediately cooling to obtain the polyethylene glycol modified drug-loaded liposome, adding 150mM EDTA-disodium solution, stirring for 20min, performing dialysis treatment by a tangential flow ultrafiltration device (wherein the dialysate is 10% sucrose-histidine-EDTA-disodium solution (1mM) with pH 6.5), and obtaining gemcitabine liposome after the dialysis is finished, wherein the obtained liposome has the average particle size of 104nm, the gemcitabine concentration of 2.0mg/ml and the entrapment rate of 99.8%.

Example 7 preparation of Gemcitabine liposomes

(1) Thin film formation

Weighing 1.5g of soybean phosphatidylcholine, 0.5g of distearoyl phosphatidyl glycerol and 0.25g of cholesterol, heating at 60 ℃, dissolving in 30ml of chloroform/methanol/water (10:5:0.1, v/v/v), placing in a round-bottom flask, and rotary evaporating at 37 ℃ on a rotary evaporator to remove the organic solvent to obtain the lipid film.

(2) Hydration of water

Gemcitabine (2.3g) was weighed and heated to a solution of copper gluconate (100mM) at pH 7.4 with a molar ratio of gemcitabine to copper ions of 1.5:1 to give a hydrated solution. Adding the hydration solution into the lipid film for hydration for 30 min.

(3) Preparation of drug-loaded liposomes

Shearing and dispersing the hydrated solution by a high-speed shearing machine, granulating to 82nm by an extruder, cooling, adding 150mM EDTA-disodium solution, stirring for 20min, dialyzing by a tangential flow ultrafiltration device (wherein the dialysate is a 10% sucrose-histidine-EDTA-disodium solution (1mM) with pH 6.5), and obtaining gemcitabine liposome after dialysis, wherein the average particle size of the obtained liposome is 97nm, the gemcitabine concentration is 1.77mg/ml, and the entrapment rate is 99.7%.

Example 8

(1) Blank liposome preparation

1.5g of hydrogenated soybean phosphatidylcholine, 0.5g of distearoyl phosphatidyl glycerol and 0.25g of cholesterol were weighed, dissolved in 30ml of chloroform/methanol/water (10:5:0.1, v/v/v) at 60 ℃ and placed in a round-bottomed flask, and then evaporated to dryness at 37 ℃ on a rotary evaporator to remove the organic solvent, thereby obtaining a lipid film. The lipid film was hydrated for 30min with a buffered solution of copper gluconate-arginine (100mM) at pH 7.4. Shearing and dispersing the hydrated solution by a high-speed shearing machine, granulating to 90nm by an extruder, carrying out dialysis treatment by using a tangential flow ultrafiltration device by using a histidine-sucrose buffer solution as a displacement medium, and concentrating a liposome product to obtain a blank liposome.

(2) Preparation of drug-loaded liposomes

Dissolving 1g of gemcitabine hydrochloride and 0.2g of mPEG2000-DSPE in water, adding the gemcitabine hydrochloride and the mPEG2000-DSPE into the blank liposome prepared in the step (1), incubating for 30min at 60 ℃, and then cooling in an ice water bath to obtain the drug-loaded liposome.

(3) Removal of free drug

And (2) taking EDTA-histidine-sucrose buffer solution as an ultrafiltration medium, performing dialysis treatment by adopting a tangential flow ultrafiltration device, removing external free drugs and copper ions, and concentrating to obtain the gemcitabine liposome with the particle size of 95nm and the entrapment rate of 98.2%.

Example 9

(1) Blank liposome preparation

Weighing 5g of distearoyl phosphatidylcholine and 1g of cholesterol, heating at 60 ℃, and dissolving in 60ml of absolute ethanol to obtain an organic phase; copper gluconate-triethanolamine buffer (150mM) at pH 7.4 was used as the aqueous phase; injecting the organic phase into the water phase under incubation at 60 ℃, continuously hydrating for 10min after the injection is finished to obtain liposome primary emulsion, sequentially extruding the primary emulsion by adopting a 100nm extrusion film and a 50nm extrusion film until the particle size is 75nm, taking histidine-sucrose buffer solution as a displacement medium, carrying out dialysis treatment by adopting a tangential flow ultrafiltration device, removing the organic solvent and external copper ions, and concentrating the liposome product to obtain a blank liposome.

(2) Preparation of drug-loaded liposomes

Dissolving gemcitabine hydrochloride 3.5g and mPEG2000-DSPE 1.0g in water, adding into the blank liposome prepared in the step (1), incubating for 30min at 65 ℃, and then cooling in ice-water bath to obtain the drug-loaded liposome.

(3) Removal of free drug

And (2) taking EDTA-histidine-sucrose buffer solution as an ultrafiltration medium, performing dialysis treatment by adopting a tangential flow ultrafiltration device, removing external free drugs and copper ions, and concentrating to obtain the gemcitabine liposome with the particle size of 76nm and the entrapment rate of 97.9%.

Example 10

(1) Blank liposome preparation

Weighing 8g of hydrogenated soybean phosphatidylcholine and 2g of cholesterol, heating at 60 ℃, and dissolving in 40ml of absolute ethanol to obtain an organic phase; copper gluconate-triethanolamine buffer solution (100mM) with pH 7.4 was used as aqueous phase; injecting the organic phase into the water phase under incubation at 60 ℃, continuously hydrating for 10min after the injection is finished to obtain liposome primary emulsion, sequentially extruding the primary emulsion by adopting a 100nm extrusion film and a 50nm extrusion film until the particle size is 79nm, adopting a copper gluconate-triethanolamine buffer solution (100mM) with the pH value of 7.4 as a displacement medium, carrying out dialysis treatment by adopting a tangential flow ultrafiltration device, removing the organic solvent, and concentrating the liposome product to obtain a blank liposome.

(2) Preparation of drug-loaded liposomes

Dissolving 10g of gemcitabine hydrochloride in a copper gluconate-triethanolamine buffer solution (100mM) with the pH value of 7.4, adding the gemcitabine hydrochloride into the blank liposome prepared in the step (1), incubating at 60 ℃ for 30min, and cooling in an ice-water bath to obtain the drug-loaded liposome.

(3) Removal of free drug

And (2) taking EDTA-histidine-sucrose buffer solution as an ultrafiltration medium, performing dialysis treatment by adopting a tangential flow ultrafiltration device, removing external free drugs and copper ions, and concentrating to obtain the gemcitabine liposome with the particle size of 82nm and the entrapment rate of 98.1%.

Example 11

(1) Blank liposome preparation

Weighing 8g of hydrogenated soybean phosphatidylcholine and 2g of cholesterol, heating at 60 ℃, and dissolving in 40ml of absolute ethanol to obtain an organic phase; PBS buffer at pH 7.4 was used as the aqueous phase; injecting the organic phase into the water phase under incubation at 60 ℃, continuously hydrating for 10min after the injection is finished to obtain liposome primary emulsion, sequentially extruding the primary emulsion by adopting a 100nm extrusion membrane and a 50nm extrusion membrane until the particle size is 81nm, adopting a PBS (phosphate buffer solution) with the pH value of 6.0 as a replacement medium, carrying out dialysis treatment by adopting a tangential flow ultrafiltration device, removing the organic solvent, and concentrating the liposome product to obtain a blank liposome.

(2) Preparation of drug-loaded liposomes

Dissolving gemcitabine hydrochloride 5g in water, adding the gemcitabine hydrochloride into the blank liposome prepared in the step (1), incubating for 30min at 60 ℃, and then cooling in an ice water bath to obtain the drug-loaded liposome.

(3) Removal of free drug

And (3) taking histidine-sucrose buffer solution as an ultrafiltration medium, performing dialysis treatment by adopting a tangential flow ultrafiltration device, removing external free drugs, and concentrating to obtain gemcitabine liposome with the particle size of 80nm and the entrapment rate of 80.8%.

Example 12

(1) Blank liposome preparation

Weighing 8g of hydrogenated soybean phosphatidylcholine, heating at 60 ℃, and dissolving in 40ml of absolute ethanol to obtain an organic phase; copper gluconate-triethanolamine buffer solution (100mM) with pH 7.4 was used as aqueous phase; injecting the organic phase into the water phase under incubation at 60 ℃, continuously hydrating for 10min after the injection is finished to obtain liposome primary emulsion, sequentially extruding the primary emulsion by adopting a 100nm extrusion film and a 50nm extrusion film until the particle size is 79nm, adopting a copper gluconate-triethanolamine buffer solution (100mM) with the pH value of 7.4 as a displacement medium, carrying out dialysis treatment by adopting a tangential flow ultrafiltration device, removing the organic solvent, and concentrating the liposome product to obtain a blank liposome.

(2) Preparation of drug-loaded liposomes

Dissolving 10g of gemcitabine hydrochloride in water, adding the gemcitabine hydrochloride into the blank liposome prepared in the step (1), incubating for 30min at 60 ℃, and then cooling in an ice-water bath to obtain the drug-loaded liposome.

(3) Removal of free drug

And (2) taking EDTA-histidine-sucrose buffer solution as an ultrafiltration medium, performing dialysis treatment by adopting a tangential flow ultrafiltration device, removing external free drugs and copper ions, and concentrating to obtain the gemcitabine liposome with the particle size of 79nm and the entrapment rate of 98.5%.

Test example 1 Liposome stability examination

The liposomes thus prepared were left at room temperature (25 ℃) and refrigerated (2-8 ℃) for 0, 1 and 3 months, respectively, and the particle size and encapsulation efficiency of the liposome composition were measured under different conditions and times (as shown in table 1 below), and it was found from the results that the liposome composition prepared in example 11 had stable particle size distribution at 25 ℃ and 2-8 ℃ but the encapsulation efficiency was decreased with the slow leakage over time. The liposome composition prepared in example 12 was stored at room temperature and in a refrigerated environment for 3 months, the particle size was hardly changed, the encapsulation efficiency was slightly decreased, and the delamination occurred. The liposome compositions prepared in examples 7 and 9 can be stored stably for 3 months at normal temperature and in a refrigerated environment, and the particle size and the encapsulation efficiency are almost kept unchanged within 3 months.

Table 1 stability study of liposome compositions

Test example 2 Gemcitabine general injection and Gemcitabine liposome pharmacokinetic test

Mouse species: CD-1

Grouping: the general injection group (gemcitabine hydrochloride for injection) and the liposome (see example 9 for liposome preparation details) group were 12 in each group and 24 in total

Blood sampling point design and blood sampling method: liposome group: 0.083h,0.25h,0.5h,1h,2h,4h,6h,8h,24h,48h,72h,96h), and 0.15ml of orbital vein blood is collected (EDTA-K2 anticoagulation). General injection group: 0.083h,0.25h,0.5h,1h,2h,4h,6h,8h,24h, orbital vein blood 0.15ml (EDTA-K2 anticoagulation)

Administration dose: general injection group: 50 mg/kg; liposome group: 1 mg/kg;

blood sample treatment: the collected whole blood was immediately stored in crushed ice and centrifuged at 4000rpm for 10min within 1h to separate plasma (4 ℃), and the collected plasma was stored in a refrigerator at-80 ℃ to be tested.

And (4) medicine substitution results: after being prepared into liposomes according to the method of the present invention (example 9), gemcitabine was significantly reduced in blood clearance and significantly prolonged in vivo retention time at the administration dose of 1/50, which is a common injection, and prolonged in vivo retention time by 30.8 times as compared to that of the common injection.

Claims

1. Liposomes comprising a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane;

2. The liposome of claim 1, wherein the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine, or a combination of both;

preferably, the neutral phospholipid is soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine or any combination thereof.

3. The liposome of claim 1 or 2, wherein the negatively charged phospholipid is selected from one or more of Phosphatidylglycerol (PG), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidic Acid (PA) and cardiolipin;

preferably, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol or a combination of both.

4. The liposome according to any one of claims 1 to 3, wherein the components constituting the liposome membrane comprise neutral phospholipids, cholesterol and optionally functional long-circulating materials;

preferably, the components constituting the liposome membrane include neutral phospholipids and cholesterol; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, and combinations thereof;

preferably, the components constituting the liposome membrane include neutral phospholipids, cholesterol and functional long-circulating materials; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the neutral phospholipid is selected from the group consisting of hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof.

5. The liposome according to any one of claims 1 to 3, wherein the components constituting the liposome membrane comprise neutral phospholipids, negatively charged phospholipids, cholesterol and optionally functional long-circulating materials;

preferably, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids and cholesterol; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the negatively charged phospholipid is dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and combinations thereof;

preferably, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, cholesterol, and functional long-circulating materials; further preferably, the neutral phospholipid is selected from the group consisting of soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine and any combination thereof; further preferably, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, and combinations thereof.

6. The liposome according to any of claims 1 to 5, wherein the functional long circulating material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol 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), wherein the polyethylene glycol has a molecular weight of 500 to 10,000 daltons, preferably 2000 daltons; preferably, the functional long-circulating material is MPEG 2000-DSPE.

7. The liposome of any one of claims 1-6, wherein the metal ion is selected from the group consisting of calcium ion, magnesium ion, and transition metal ion; preferably, the metal ions are selected from one or more of copper ions, manganese ions, nickel ions, iron ions, molybdenum ions, cobalt ions, calcium ions, and zinc ions; preferably, the metal ion is selected from one or more of copper ion, cobalt ion and calcium ion;

preferably, the molar ratio of gemcitabine or a salt thereof to the metal ion is (0.5 to 10): 1.

8. the liposome according to any of claims 1 to 7, wherein the internal aqueous phase contains an acid ion, such as an inorganic acid ion or an organic acid ion; preferably, the acid radical ion is selected from one or more of sulfate radical, halogen ion, gluconate ion, nitrate ion and acetate ion; preferably, the acid ion is selected from one or more of gluconate, acetate, nitrate and sulfate.

9. The liposome according to any of claims 1 to 8, wherein the internal aqueous phase further comprises a pH adjusting component, such as hydrochloric acid, acetic acid, triethanolamine, sodium hydroxide or arginine;

preferably, the pH of the inner aqueous phase is 2 to 8, preferably 3 to 7.5, more preferably 4 to 7.5, such as 4.0, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.1, 6.2, 6.5, 6.8, 6.9, 7.0, 7.3, 7.4, 7.5, 7.8, 7.9 or 8.0.

10. A liposome prepared by preparation method 1 or preparation method 2:

preparation method 1

preparation method 2

11. The liposome of claim 10, prepared by a method comprising one or more of the following features:

4) in step (1) of preparation method 2, the organic solvent is selected from an alcohol solvent (e.g., methanol, ethanol or isopropanol), a halogenated hydrocarbon solvent (e.g., dichloromethane or chloroform), and a mixed solvent thereof with water;

5) in the preparation method 1 or the preparation method 2, the weight ratio of the phospholipid to the cholesterol is (1-125): (0.1-25), preferably (1-100): (0.1-20), more preferably (2-80): 0.1-15), such as (2-10): 1, for example (4-8): 1;

6) in preparation method 1, step (2), the weight ratio of gemcitabine or a salt thereof to phospholipids is 1: (0.01 to 100), preferably 1: (0.02 to 80), more preferably 1: (0.05 to 50), for example, 1: (0.5 to 5), and further example is 1: (0.5 to 2);

7) in the preparation method 1 or the preparation method 2, the phospholipid is a neutral phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine or a combination of both; preferably, the neutral phospholipid is soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine or any combination thereof;

8) in the production method 1 or the production method 2, the phospholipid is a combination of a neutral phospholipid and a negatively charged phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine or a combination of both; preferably, the neutral phospholipid is soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, distearoyl phosphatidylcholine or any combination thereof; preferably, the negatively charged phospholipid is selected from one or more of Phosphatidylglycerol (PG), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidic Acid (PA) and cardiolipin; preferably, the negatively charged phospholipid is selected from dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, or a combination of both;

9) in production method 1 or production method 2, the metal ion is selected from the group consisting of a calcium ion, a magnesium ion, and a transition metal ion; preferably, the metal ions are selected from one or more of copper ions, manganese ions, nickel ions, iron ions, molybdenum ions, cobalt ions, calcium ions, and zinc ions; preferably, the metal ion is selected from one or more of copper ion, cobalt ion and calcium ion;

10) in the preparation method 1 or the preparation method 2, the solution containing the metal ions is a buffered salt solution containing the metal ions; preferably, the buffered salt solution has a pH of 4 to 8, preferably 5 to 7.5, such as 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0; preferably, the solution containing metal ions is a copper gluconate solution, a copper sulfate-triethanolamine solution, a copper gluconate-arginine buffer solution, a copper gluconate-triethanolamine solution, a zinc gluconate solution or a calcium acetate-acetic acid buffer solution with a pH of 6 to 8;

11) in the preparation method 1 or the preparation method 2, the functional long-circulating material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol modified distearoyl phosphatidyl ethanolamine (MPEG-DSPE), polyethylene glycol modified dimyristoyl phosphatidyl ethanolamine (MPEG-DMPE), and polyethylene glycol modified dipalmitoyl phosphatidyl ethanolamine (MPEG-DPPE), wherein the molecular weight of the polyethylene glycol is 500 to 10,000 daltons, preferably 2000 daltons; preferably, the functional long-circulating material is MPEG 2000-DSPE;

preferably, gemcitabine or a salt thereof and the optional functional long-circulating material are added in the preparation method 1 and the preparation method 2 in a weight ratio of 1: (0 to 100), preferably 1: (0.05 to 50), more preferably 1 (0.1 to 25), for example, 1: (0.1-1), and further example 1: (0.1-0.5).

12. The liposome according to any of claims 1 to 11, having a particle size of 20-500nm, such as 50-300nm, further such as 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 150nm, 200nm, 250nm or 300 nm.

13. A liposomal formulation comprising the liposome of any one of claims 1-12, and one or more pharmaceutically acceptable carriers.

14. Use of a liposome according to any one of claims 1 to 12 in the manufacture of a medicament for the treatment of an antineoplastic drug; preferably, the tumor is selected from pancreatic cancer, breast cancer, non-small cell lung cancer and ovarian cancer.

15. A method for preparing a liposome as claimed in any one of claims 1 to 9, which is selected from the group consisting of preparation method 1 and preparation method 2:

preparation method 1

wherein, optional functional long-circulating materials are added in the oil phase in the step (1), or optional functional long-circulating materials are added in the step (2) to prepare the liposome containing the functional long-circulating materials;

preparation method 2

(4) removing free drugs: carrying out one or more treatments of dialysis, centrifugation and ultrafiltration on the drug-loaded liposome colostrum prepared in the step (3) to remove free drugs and optionally carrying out concentration treatment to obtain the liposome;

16. The method of claim 15, characterized by one or more of the following:

5) in the step (1) of the preparation method 2, the organic solvent is selected from an alcohol solvent (such as methanol, ethanol or isopropanol), a halogenated hydrocarbon solvent (such as dichloromethane or trichloromethane) and a mixed solvent 6 of the alcohol solvent and the halogenated hydrocarbon solvent with water), and in the preparation method 1 or the preparation method 2, the weight ratio of the phospholipid to the cholesterol is (1-125): 0.1-25, preferably (1-100): 0.1-20, more preferably (2-80): 0.1-15), such as (2-10): 1, for example (4-8): 1;

11) in the preparation method 1 or the preparation method 2, the solution containing the metal ions is a buffered salt solution containing the metal ions; preferably, the buffered salt solution has a pH of 4 to 8, preferably 5 to 7.5, such as 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0; (ii) a Preferably, the solution containing metal ions is a copper gluconate solution, a copper sulfate-triethanolamine solution, a copper gluconate-arginine buffer solution, a copper gluconate-triethanolamine solution, a copper gluconate-tris (hydroxymethyl) aminomethane solution, a copper sulfate-4-hydroxyethylpiperazine ethanesulfonic acid solution, a zinc gluconate solution or a calcium acetate-acetic acid buffer solution with a pH of 4 to 8;

preferably, the weight ratio of gemcitabine or a salt thereof to the optional functional long circulating material is 1: (0 to 100), preferably 1: (0.05 to 50), more preferably 1 (0.1 to 25), for example, 1: (0.1-1), and further example 1: (0.1-0.5).