Liposomes of gemcitabine or its salts, and methods of making and using the same

Abstract

The present application provides a liposome of gemcitabine or its salt, and a preparation method and use thereof. Specifically, the present application provides a liposome containing a liposome membrane and an internal aqueous phase encapsulated inside the liposome membrane; wherein the internal aqueous phase contains gemcitabine or its salt and metal ions; the components constituting the liposome membrane include phospholipids, cholesterol and optional functional long-circulating materials; the phospholipids are neutral phospholipids or a combination of neutral phospholipids and negatively charged phospholipids; the weight ratio of the phospholipids to cholesterol is (1-125):(0.1-25), and the weight ratio of the phospholipids to long-circulating materials (if any) is (1-125):(0.1-25); and the molar ratio of the gemcitabine or its salt to metal ions is (0.25-20):1. The liposome has the characteristics of good stability, high encapsulation efficiency, prolonged in-vivo circulation time of gemcitabine, enhanced tumor targeting, improved therapeutic effect and reduced toxic side effects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, specifically to liposomes of gemcitabine or its salts, methods of preparation, and uses thereof. Background Technology

[0002] Gemcitabine is a pyrimidine nucleic acid analog that primarily acts on DNA synthesis and belongs to the antimetabolite class of anticancer drugs. After administration, it is phosphorylated intracellularly by deoxycytidine enzymes to difluorodeoxycytidine monophosphate (dFdCMP), and then metabolized by nucleoside kinases to active diphosphate (dFdCDP) and nucleoside triphosphate (dFdCTP). Gemcitabine mainly inhibits DNA synthesis by the insertion of active dFdCTP into the DNA strand; simultaneously, dFdCDP inhibits the activity of ribonucleotide reductase, thus inhibiting the production of deoxyribonucleotides (dCTP), essential for DNA synthesis. Furthermore, dFdCTP can interfere with DNA self-repair mechanisms, ultimately leading to tumor cell death. Gemcitabine, alone or in combination with other drugs, is a first-line chemotherapy regimen for various solid tumors (including pancreatic cancer, breast cancer, non-small cell lung cancer, ovarian cancer, etc.), exhibiting a broad anticancer spectrum and widespread clinical application.

[0003] Gemcitabine has a simple molecular structure and strong hydrophilicity. It is rapidly metabolized by cytidine deaminase in the liver, kidneys, blood, and other tissues, with a short half-life (42-97 min, age- and sex-related). Therefore, conventional injectable versions require high doses in clinical practice (recommended dose 1000 mg / m²). 2 Gemcitabine is used to maintain its blood concentration and kill tumor cells by prolonging the infusion time (more than 30 minutes). However, due to poor targeting, it often leads to serious toxic side effects, including hematologic toxicity (anemia, neutropenia, and thrombocytopenia), hair loss, gastrointestinal toxicity (such as nausea and vomiting), fatigue, and fever. Among these, hematologic toxicity is its dose-limiting toxicity. Gemcitabine produces bone marrow suppression after a single dose. Its active metabolites disrupt the proliferation and differentiation of bone marrow cells, reducing hematopoietic cells and subsequently decreasing platelet and neutrophil counts, thus limiting its clinical use. Currently, gemcitabine is only available in injectable formulations, including powder for injection, aqueous injection, and infusion.

[0004] Liposomes, as drug carriers, offer advantages such as safety, non-toxicity, and good biocompatibility. Encapsulating drugs in liposomes prevents degradation by external factors (pH, light, enzymes), improving drug stability. Once inside the body, they avoid damage from related enzymes and proteins in the bloodstream, while also prolonging their blood circulation time. Compared to normal tissues, tumor tissues have larger intervascular spaces and incomplete structures. Nanoscale liposomes can passively target tumor sites through the EPR effect, directly releasing drugs to kill tumor cells, thereby improving efficacy and reducing toxic side effects.

[0005] Literature reports that liposomes of gemcitabine can prevent rapid metabolism of the molecule and prolong its duration of action. However, the preparation methods used result in low encapsulation rates, instability, easy leakage in vivo, rapid release, complex processes, and difficulty in meeting clinical needs. CN102846547 B uses soybean lecithin, cholesterol, and polyoxyethylene ricinoleate to prepare gemcitabine liposomes, which improves encapsulation and particle size. However, the excipient polyoxyethylene ricinoleate used is prone to causing adverse reactions such as acute hypersensitivity, peripheral neurotoxicity, and cytotoxicity, posing a significant risk to injection safety and requiring further improvement in safety. CN 102716089 B uses gemcitabine hydrochloride, cholesterol, egg yolk phosphatidylinositol, lecithin, Tween 80, trehalose, and polyvinylpyrrolidone lyophilized to prepare gemcitabine hydrochloride liposome injection. However, the introduction of Tween 80 also poses a safety risk for in vivo injection. Summary of the Invention

[0006] The purpose of this invention is to provide gemcitabine liposomes with high encapsulation efficiency and high stability, as well as a method for their preparation. These liposomes can significantly prolong the blood half-life of gemcitabine, improve tissue distribution, enhance tumor targeting, improve efficacy, and reduce toxic side effects. Furthermore, the liposomes have safe components, a simple preparation process, and are easy to industrialize and translate into clinical applications.

[0007] In a first aspect, the present invention provides a liposome comprising a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane;

[0008] The aqueous phase contains gemcitabine or its salt and metal ions; the components constituting the liposome membrane include phospholipids, cholesterol and optional functional long-circulation materials; the phospholipids are neutral phospholipids or a combination of neutral phospholipids and negatively charged phospholipids;

[0009] The weight ratio of phospholipids to cholesterol is (1-125):(0.1-25), preferably (1-100):(0.1-20), more preferably (2-80):(0.1-15), for example (2-10):1, and even more preferably (4-8):1;

[0010] The weight ratio of the phospholipid to the long-cycle material (if any) is (1-125):(0.1-25); for example (1-20):1, or even (2-15):1.

[0011] Furthermore, the molar ratio of gemcitabine or its salt to the metal ion is (0.25–20):1.

[0012] The proportions mentioned above refer to the proportions of the components in the prepared liposomes. These proportions may differ from the actual feed ratios used in preparation due to variations in drug utilization.

[0013] The drug utilization rate described in this invention refers to the ratio of the amount of drug in the prepared liposomes to the amount of drug added during preparation. The amount of drug in the liposomes is determined using high-performance liquid chromatography (HPLC) according to the gemcitabine content determination method specified in the Chinese Pharmacopoeia.

[0014] In an embodiment of the present invention, the gemcitabine salt is gemcitabine hydrochloride.

[0015] In another aspect, the present invention provides a liposome prepared by preparation method 1 or preparation method 2:

[0016] Preparation method 1

[0017] (1) Preparation of blank liposomes: Using a solution of phospholipids and cholesterol as the oil phase and a solution containing metal ions as the aqueous phase, a primary emulsion is prepared by membrane hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation. The granules are then granulated, and optionally, one or more of the following treatments are performed: dialysis, centrifugation, ultrafiltration or concentration to obtain blank liposomes.

[0018] (2) Drug loading: Add a solution of gemcitabine or its salt to the blank liposomes prepared in step (1). Optionally, the solution of gemcitabine or its salt contains the metal ions in the aqueous phase of step (1). Heat and incubate above the phospholipid phase transition temperature to obtain drug-loaded liposomes.

[0019] (3) Removal of free drug: The drug-loaded liposomes obtained in step (2) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0020] In step (1), an optional functional long-circulating material is added to the oil phase, or in step (2), an optional functional long-circulating material is added to prepare liposomes containing the functional long-circulating material.

[0021] Preparation method 2

[0022] (1) Preparation of oil phase: Dissolve phospholipids and cholesterol in an organic solvent to prepare oil phase;

[0023] (2) Preparation of aqueous phase: Gemcitabine or its salt is dissolved in a solution containing metal ions to prepare an aqueous phase;

[0024] (3) Preparation of drug-loaded liposomes: The oil phase of step (1) and the aqueous phase of step (2) are prepared into liposomes by thin-film hydration, organic solvent injection, pipeline emulsification or reverse evaporation, or (2) is directly mixed with precursor liposomes (such as commercial Presome), granulated, and drug-loaded liposomes are obtained.

[0025] (4) Removal of free drug: The drug-loaded liposomes obtained in step (3) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0026] In step (1), functional long-circulating material is added when preparing the oil phase, or in step (3), functional long-circulating material is added after granulation, so as to prepare liposomes containing functional long-circulating material.

[0027] In another aspect, the present invention provides a liposome formulation comprising the liposomes described herein, and one or more pharmaceutical excipients.

[0028] In another aspect, the present invention provides the use of the liposomes described herein in the preparation of therapeutic antitumor drugs.

[0029] In another aspect, the present invention provides an antitumor method comprising administering an effective amount of the liposomes or liposomal formulations described herein to a subject in need of such treatment.

[0030] In another aspect, the present invention provides a method for preparing the liposomes described herein, which is selected from preparation method 1 and preparation method 2:

[0031] Preparation method 1

[0032] (1) Preparation of blank liposomes: A solution of phospholipids and cholesterol is used as the oil phase, and a solution containing metal ions is used as the aqueous phase. The primary emulsion is prepared by membrane hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation. The granules are then granulated, and optionally, one or more of the following treatments are performed: dialysis, centrifugation, ultrafiltration and concentration to obtain blank liposomes.

[0033] (2) Drug loading: Add a solution of gemcitabine or its salt to the blank liposomes prepared in step (1). Optionally, the solution of gemcitabine or its salt contains the metal ions in the aqueous phase of step (1). Heat and incubate above the phospholipid phase transition temperature to obtain drug-loaded liposomes.

[0034] (3) Removal of free drug: The drug-loaded liposomes obtained in step (2) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0035] In step (1), an optional functional long-circulating material is added to the oil phase, or in step (2), an optional functional long-circulating material is added to prepare liposomes containing the functional long-circulating material.

[0036] In some embodiments, the weight ratio of phospholipids 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), for example (2-10):1, and even more preferably (4-8):1.

[0037] In some embodiments, the weight ratio of gemcitabine added in step (2) to phospholipids in the blank liposomes prepared in step (1) is 1:(0.01-100), preferably 1:(0.02-80), more preferably 1:(0.05-50), for example 1:(0.5-5), and even more preferably 1:(0.5-2).

[0038] In some implementations, the weight ratio of gemcitabine added in step (2) to the optional functional long-cycle material is 1:(0 to 100), preferably 1:(0.05 to 50), more preferably 1:(0.1 to 25), for example 1:(0.1 to 1), and even more preferably 1:(0.1 to 0.5).

[0039] In some implementations, the phospholipids added in step (1) are neutral phospholipids.

[0040] In some embodiments, the phospholipids added in step (1) are a combination of neutral phospholipids and negatively charged phospholipids; preferably, the weight ratio between neutral phospholipids and negatively charged phospholipids is (0.1-20):1; more preferably (0.5-15):1, and even more preferably (1-10):1.

[0041] Preparation method 2

[0042] (1) Preparation of oil phase: Dissolve phospholipids and cholesterol in an organic solvent to prepare oil phase;

[0043] (2) Preparation of aqueous phase: Gemcitabine or its salt is dissolved in a solution containing metal ions to prepare an aqueous phase;

[0044] (3) Preparation of drug-loaded liposomes: The oil phase of step (1) and the aqueous phase of step (2) are prepared into liposomes by thin-film hydration, organic solvent injection, pipeline emulsification or reverse evaporation, or (2) is directly mixed with precursor liposomes (such as commercial Presome), granulated, and drug-loaded liposomes are obtained.

[0045] (4) Removal of free drug: The drug-loaded liposomes obtained in step (3) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0046] In step (1), functional long-circulating material is added when preparing the oil phase, or in step (3), functional long-circulating material is added after granulation, so as to prepare liposomes containing functional long-circulating material.

[0047] In some embodiments, the weight ratio of phospholipids 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), for example (2-10):1, and even more preferably (4-8):1.

[0048] In some implementations, the weight ratio of gemcitabine added in step (2) to phospholipid 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 even more preferably 1:(0.5 to 2).

[0049] In some implementations, the weight ratio of gemcitabine added in step (2) to the optional functional long-cycle material is 1:(0 to 100), preferably 1:(0.05 to 50), more preferably 1:(0.1 to 25), for example 1:(0.1 to 1), and even more preferably 1:(0.1 to 0.5).

[0050] In some implementations, the phospholipids added in step (1) are neutral phospholipids.

[0051] In some embodiments, the phospholipids added in step (1) are a combination of neutral phospholipids and negatively charged phospholipids; preferably, the weight ratio between neutral phospholipids and negatively charged phospholipids is (0.1-20):1; more preferably (0.5-15):1, and even more preferably (1-10):1.

[0052] Preparation methods 1 and 2 described in this article can encapsulate gemcitabine without involving freeze-thaw or repeated freeze-thaw operations. Invention Details

[0054] definition

[0055] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0056] As used herein, the term "liposome" refers to a self-assembled structure comprising one or more amphiphilic lipid bilayers, each lipid bilayer comprising two anti-oriented amphiphilic lipid monolayers. The amphiphilic lipids comprise one or more nonpolar (hydrophobic) acyl or alkyl chains covalently linked to polar (hydrophilic) head groups. The highly unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous medium causes the amphiphilic lipid molecules themselves to align such that the polar head groups are oriented towards the surface of the bilayer and the acyl chains towards the interior of the bilayer, effectively preventing the acyl chains from contacting the aqueous environment.

[0057] The liposomes used in this article can be a single lipid bilayer (monocompartment liposome) or multiple lipid bilayers (multicompartment liposomes) surrounding or encapsulating aqueous compartments. Various forms of liposomes are described, for example, in Cullis et al. Biochim. Biophys Acta, 559:399-420 (1987).

[0058] The term "effective amount" as used in this article refers to an amount sufficient to achieve the desired therapeutic effect, such as an amount that reduces symptoms associated with the disease to be treated.

[0059] The term "treatment" as used herein aims to alleviate or eliminate the targeted disease state or symptom. A subject is successfully "treated" if, after receiving a therapeutic amount of the crystal form or pharmaceutical composition thereof as described herein, one or more indications and symptoms exhibit an observable and / or detectable reduction or improvement. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0060] In one aspect, the present invention provides a liposome comprising a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane;

[0061] The aqueous phase contains gemcitabine or its salt and metal ions; the components constituting the liposome membrane include phospholipids, cholesterol and optional functional long-circulation materials; the phospholipids are neutral phospholipids or a combination of neutral phospholipids and negatively charged phospholipids;

[0062] The weight ratio of phospholipids to cholesterol is (1-125):(0.1-25), preferably (1-100):(0.1-20), more preferably (2-80):(0.1-15), for example (2-10):1, and even more preferably (4-8):1;

[0063] The weight ratio of the phospholipid to the long-cycle material (if any) is (1-125):(0.1-25); for example (1-20):1, or even (2-15):1.

[0064] Furthermore, the molar ratio of gemcitabine or its salt to the metal ion is (0.25–20):1.

[0065] In some embodiments, the liposomes comprise a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane;

[0066] The aqueous phase contains gemcitabine or its salt and metal ions; the components constituting the liposome membrane include phospholipids, cholesterol and optional functional long-circulation materials; the phospholipids are neutral phospholipids or a combination of neutral phospholipids and negatively charged phospholipids;

[0067] The weight ratio of gemcitabine or its salt, phospholipids, cholesterol, and long-circulating material is 1:(1-125):(0.1-25):(0-25);

[0068] Furthermore, the molar ratio of gemcitabine or its salt to the metal ion is (0.25–20):1.

[0069] In some embodiments, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine, or a combination thereof. In some embodiments, the neutral phospholipid is soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearate phosphatidylcholine, or any combination thereof.

[0070] 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, distearylphosphatidylglycerol, or a combination of both.

[0071] In some embodiments, the components constituting the liposome membrane include neutral phospholipids, cholesterol, and optional functional long-circulation materials.

[0072] In some preferred embodiments, the components constituting the liposome membrane include neutral phospholipids and cholesterol; more preferably, the neutral phospholipids are selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, and combinations thereof; preferably, the components constituting the liposome membrane include neutral phospholipids, cholesterol, and functional long-circulation materials; more preferably, the neutral phospholipids are selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, and any combination thereof; more preferably, the neutral phospholipids are selected from hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, and any combination thereof.

[0073] In some preferred embodiments, the components constituting the liposome membrane include neutral phospholipids and cholesterol, and the weight ratio of gemcitabine or its salt, neutral phospholipids and cholesterol is 1:(1-125):(0.1-25); more preferably, the neutral phospholipid is selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine and combinations thereof; more preferably, the weight ratio of gemcitabine or its salt, neutral phospholipids and cholesterol is 1:(1-100):(0.1-20); more preferably, the weight ratio of gemcitabine or its salt, neutral phospholipids and cholesterol is 1:(2-80):(0.1-15).

[0074] In some embodiments, the components constituting the liposome membrane include neutral phospholipids, cholesterol, and a functional long-circulating material, and the weight ratio of gemcitabine or its salt, neutral phospholipids, cholesterol, and the functional long-circulating material is 1:(1-125):(0.1-25):(0.1-25); more preferably, the weight ratio of gemcitabine or its salt, neutral phospholipids, cholesterol, and the functional long-circulating material is 1:(1-100):(0.1-20):(0.1-20); more preferably, the weight ratio of gemcitabine or its salt, neutral phospholipids, and cholesterol is 1:(2-80):(0.1-15):(0.1-15); more preferably, the neutral phospholipid is selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearate phosphatidylcholine, and any combination thereof.

[0075] In some embodiments, the liposomes comprise the following components:

[0076] 1g gemcitabine;

[0077] 5-50g of hydrogenated soybean phosphatidylcholine;

[0078] 0.1-10g cholesterol;

[0079] Copper sulfate, with a molar ratio of 1:(1-8) to gemcitabine.

[0080] In some embodiments, the liposomes comprise the following components by weight:

[0081] 1g gemcitabine;

[0082] 4-40g soybean phosphatidylcholine;

[0083] 1-10g cholesterol;

[0084] Copper gluconate-triethanolamine, wherein the molar ratio of copper ions to gemcitabine is 1:(0.25-20).

[0085] In some embodiments, the liposomes comprise the following components:

[0086] 1g gemcitabine;

[0087] 1-20g of hydrogenated soybean phosphatidylcholine;

[0088] 0.1-5g cholesterol;

[0089] 0.1-5g mPEG2000-DSPE;

[0090] Copper gluconate, with a molar ratio of 1:(2-10) to gemcitabine, for example 1:5.

[0091] In some embodiments, the liposomes comprise the following components by weight:

[0092] 1g gemcitabine;

[0093] 5-80g distearate phosphatidylcholine;

[0094] 1-15g cholesterol;

[0095] 1-15g mPEG2000-DSPE;

[0096] Zinc gluconate-triethanolamine, wherein the molar ratio of zinc ions to gemcitabine is 1:(0.25–10).

[0097] In some embodiments, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, cholesterol, and optionally functional long-circulating materials.

[0098] In some preferred embodiments, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, and cholesterol; more preferably, the neutral phospholipids are selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, and any combination thereof; more preferably, the negatively charged phospholipids are dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, and combinations thereof; preferably, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, cholesterol, and functional long-circulation materials; more preferably, the neutral phospholipids are selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, and any combination thereof; more preferably, the negatively charged phospholipids are selected from dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, and combinations thereof.

[0099] In some preferred embodiments, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, and cholesterol, and the weight ratio of gemcitabine or its salt, neutral phospholipids, negatively charged phospholipids, and cholesterol is 1:(1-100):(0.1-25):(0.1-25); more preferably, the neutral phospholipid is selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, and any combination thereof; more preferably, the negatively charged phospholipid is dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, and any combination thereof; more preferably, the weight ratio of gemcitabine or its salt, neutral phospholipids, negatively charged phospholipids, and cholesterol is 1:(1-80):(0.1-20):(0.1-20).

[0100] In some embodiments, the components constituting the liposome membrane include neutral phospholipids, negatively charged phospholipids, cholesterol, and functional long-circulating materials, and the weight ratio of gemcitabine or its salt, neutral phospholipids, negatively charged phospholipids, cholesterol, and functional long-circulating materials is 1:(1-100):(0.1-25):(0.1-25):(0.1-25); more preferably, the neutral phospholipids are selected from soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl phosphatidylcholine, and any combination thereof; more preferably, the negatively charged phospholipids are selected from dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, and combinations thereof; more preferably, the weight ratio of gemcitabine or its salt, neutral phospholipids, negatively charged phospholipids, cholesterol, and functional long-circulating materials is 1:(1-80):(0.1-20):(0.1-20):(0.1-20).

[0101] In some embodiments, the functional long-cycle material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-modified distearate phosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified myristoyl phosphatidylethanolamine (MPEG-DMPE), and polyethylene glycol-modified dipalmitoyl phosphatidylethanolamine (MPEG-DPPE), wherein the molecular weight of the polyethylene glycol is 500 Daltons to 10,000 Daltons, preferably 2,000 Daltons. In some embodiments, the functional long-cycle material is MPEG2000-DSPE.

[0102] In some embodiments, the liposomes comprise the following components:

[0103] 1g gemcitabine;

[0104] 2-30g distearylphosphatidylcholine;

[0105] 1-15g distearate phosphatidylglycerol;

[0106] 0.1-10g cholesterol;

[0107] Copper gluconate-triethanolamine, wherein the molar ratio of copper ions to gemcitabine is 1:(1-6).

[0108] In some embodiments, the liposomes comprise the following components by weight:

[0109] 1g gemcitabine;

[0110] 1-40g soybean phosphatidylcholine;

[0111] 1-15g distearate phosphatidylglycerol;

[0112] 0.5-10g cholesterol;

[0113] Copper gluconate, with a molar ratio of 1:(1-6) to gemcitabine.

[0114] In some embodiments, the liposomes comprise the following components:

[0115] 1g gemcitabine;

[0116] 1-40g distearate phosphatidylcholine;

[0117] 1-15g dipalmitoylphosphatidylglycerol;

[0118] 0.1-10g cholesterol;

[0119] 0.1-5g mPEG2000-DSPE;

[0120] Calcium acetate-acetic acid, wherein the molar ratio of calcium ions to gemcitabine is 1:(1-4).

[0121] In some embodiments, the liposomes comprise the following components by weight:

[0122] 1g gemcitabine;

[0123] 3-40g of hydrogenated soybean phosphatidylcholine;

[0124] 1-15g dipalmitoylphosphatidylglycerol;

[0125] 0.5-10g cholesterol;

[0126] 0.5-15g mPEG2000-DSPE;

[0127] Zinc gluconate, with a molar ratio of 1:(1-4) to gemcitabine.

[0128] In some embodiments, the liposomes comprise the following components by weight:

[0129] 1g gemcitabine;

[0130] 3-40g distearate phosphatidylcholine;

[0131] 1-15g distearate phosphatidylglycerol;

[0132] 0.5-10g cholesterol;

[0133] 0.5-10g mPEG2000-DSPE;

[0134] Copper gluconate, with a molar ratio of 1:(1-6) to gemcitabine.

[0135] In some embodiments, the liposomes comprise the following components by weight:

[0136] 1g gemcitabine;

[0137] 5-80g soybean phosphatidylcholine;

[0138] 1-25g distearate phosphatidylglycerol;

[0139] 1-15g cholesterol;

[0140] 0.5-15g mPEG2000-DSPE;

[0141] Copper gluconate-arginine, wherein the molar ratio of copper ions to gemcitabine is 1:(0.25-20).

[0142] In some embodiments, the metal ion is selected from calcium ions, magnesium ions, and transition metal ions. In some embodiments, the metal ion is selected from one or more of copper ions, manganese ions, nickel ions, iron ions, molybdenum ions, cobalt ions, calcium ions, and zinc ions. In some embodiments, the metal ion is selected from one or more of copper ions, cobalt ions, calcium ions, and zinc ions. In some embodiments, the molar ratio of the metal ion to gemcitabine or its salt is 1:(0.25–20), preferably 1:(0.5–10), for example 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0143] In some embodiments, the internal aqueous phase contains acid radicals, such as inorganic or organic acid radicals. In some embodiments, the acid radicals are selected from one or more of sulfate, halide, gluconate, nitrate, and acetate. In some embodiments, the acid radicals are selected from one or more of gluconate, acetate, nitrate, and sulfate.

[0144] In some embodiments, the internal aqueous phase further contains a pH-adjusting component, such as hydrochloric acid, acetic acid, triethanolamine, sodium hydroxide, or arginine. In some embodiments, the pH of the internal aqueous phase is 2-8, preferably 3-7.5, more preferably 4-7.5, for example 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.

[0145] In some embodiments, the liposome particle size is 20-500 nm, for example 50-300 nm, or even 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 150 nm, 200 nm, 250 nm or 300 nm.

[0146] In another aspect, the present invention provides a liposome prepared by preparation method 1 or preparation method 2:

[0147] Preparation method 1

[0148] (1) Preparation of blank liposomes: Using a solution of phospholipids and cholesterol as the oil phase and a solution containing metal ions as the aqueous phase, a primary emulsion is prepared by membrane hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation. The granules are then granulated, and optionally, one or more of the following treatments are performed: dialysis, centrifugation, ultrafiltration or concentration to obtain blank liposomes.

[0149] (2) Drug loading: Add a solution of gemcitabine or its salt to the blank liposomes prepared in step (1). Optionally, the solution of gemcitabine or its salt contains the metal ions in the aqueous phase of step (1). Heat and incubate above the phospholipid phase transition temperature to obtain drug-loaded liposomes.

[0150] (3) Removal of free drug: The drug-loaded liposomes obtained in step (2) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0151] In step (1), an optional functional long-circulating material is added to the oil phase, or in step (2), an optional functional long-circulating material is added to prepare liposomes containing the functional long-circulating material.

[0152] Preparation method 2

[0153] (1) Preparation of oil phase: Dissolve phospholipids and cholesterol in an organic solvent to prepare oil phase;

[0154] (2) Preparation of aqueous phase: Gemcitabine or its salt is dissolved in a solution containing metal ions to prepare an aqueous phase;

[0155] (3) Preparation of drug-loaded liposomes: The oil phase of step (1) and the aqueous phase of step (2) are prepared into liposomes by thin-film hydration, organic solvent injection, pipeline emulsification or reverse evaporation, or (2) is directly mixed with precursor liposomes (such as commercial Presome), granulated, and drug-loaded liposomes are obtained.

[0156] (4) Removal of free drug: The drug-loaded liposomes obtained in step (3) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0157] In step (1), functional long-circulating material is added when preparing the oil phase, or in step (3), functional long-circulating material is added after granulation, so as to prepare liposomes containing functional long-circulating material.

[0158] In some embodiments, the method for preparing the liposomes includes one or more of the following features:

[0159] 1) In step (1) of preparation method 1, phospholipids and cholesterol are dissolved in a solvent selected from alcohols (e.g., methanol, ethanol or isopropanol), halogenated hydrocarbons (e.g., dichloromethane or trichloromethane), water and any combination thereof as the oil phase;

[0160] 2) In step (2) of preparation method 1, the solution of gemcitabine or its salt is an aqueous solution of gemcitabine or its salt or the solution containing metal ions in step (1);

[0161] 3) In step (2) of preparation method 1, the incubation is carried out at 50-70°C (e.g., 60-70°C, or even 60-65°C);

[0162] 4) In step (2) of preparation method 1, the incubation time is 15 min to 2 h (e.g., 15 min to 1 h, e.g., 30 min);

[0163] 5) In step (1) of preparation method 2, the organic solvent is selected from alcohol solvents (e.g., methanol, ethanol or isopropanol), halogenated hydrocarbon solvents (e.g., dichloromethane or trichloromethane) and their mixed solvents with water;

[0164] 6) In preparation method 1 or preparation method 2, the weight ratio of phospholipids to cholesterol is (1-125):(0.1-25), preferably (1-100):(0.1-20), more preferably (2-80):(0.1-15), for example (2-10):1, and even more preferably (4-8):1;

[0165] 7) In step (2) of preparation method 1, the weight ratio of gemcitabine or its salt to phospholipid is 1:(0.01-100), preferably 1:(0.02-80), more preferably 1:(0.05-50), for example 1:(0.5-5), and even more preferably 1:(0.5-2);

[0166] 8) In preparation method 1 or preparation method 2, the phospholipid is a neutral phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine or a combination thereof; preferably, the neutral phospholipid is soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearate phosphatidylcholine or any combination thereof.

[0167] 9) In preparation method 1 or preparation method 2, the phospholipid is a combination of neutral phospholipid and negatively charged phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine, or a combination of both; preferably, the neutral phospholipid is soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl 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 dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, or a combination of both.

[0168] Preferably, the weight ratio between neutral phospholipids and negatively charged phospholipids is (0.1-20):1; more preferably (0.5-15):1, and even more preferably (1-10):1;

[0169] 10) In preparation method 1 or preparation method 2, the metal ion is selected from calcium ion, magnesium ion and transition metal ion; preferably, 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; preferably, the metal ion is selected from one or more of copper ion, cobalt ion and calcium ion.

[0170] Preferably, the molar ratio of gemcitabine or its salt to the metal ion is (0.25–20):1, more preferably (0.5–10):1;

[0171] 11) In preparation method 1 or preparation method 2, the solution containing the metal ions is a buffer salt solution containing the metal ions; preferably, the pH of the buffer salt solution is 4-8, more preferably 5-7.5, for example 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, copper sulfate-triethanolamine solution, copper gluconate-arginine buffer solution, copper gluconate-triethanolamine solution, copper gluconate-tris(hydroxymethyl)aminomethane solution, copper sulfate-tris(hydroxymethyl)aminomethane solution, copper sulfate-4-hydroxyethylpiperazine ethanesulfonic acid solution, zinc gluconate solution or calcium acetate-acetic acid buffer solution with a pH of 4-8;

[0172] 12) In preparation method 1 or preparation method 2, the functional long-cycle material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-modified distearate phosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified myristoyl phosphatidylethanolamine (MPEG-DMPE), and polyethylene glycol-modified dipalmitoyl phosphatidylethanolamine (MPEG-DPPE), wherein the molecular weight of polyethylene glycol is 500 Daltons to 10,000 Daltons, preferably 2,000 Daltons; preferably, the functional long-cycle material is MPEG2000-DSPE;

[0173] Preferably, in preparation method 1 or preparation method 2, the weight ratio of gemcitabine or its salt to the optional functional long-cycle material is 1:(0 to 100), more preferably 1:(0.05 to 50), more preferably 1:(0.1 to 25), for example 1:(0.1 to 1), and even more preferably 1:(0.1 to 0.5).

[0174] In some embodiments, the liposome particle size is 20-500 nm, for example 50-300 nm, or even 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 150 nm, 200 nm, 250 nm or 300 nm.

[0175] In another aspect, the present invention provides a liposome formulation comprising the liposomes described in any one of the first aspects, and one or more pharmaceutically acceptable carriers.

[0176] Pharmaceutically acceptable carriers may include, for example, physiological saline, isotonic glucose, isotonic sucrose, Green's solution, and Hanks' solution. Buffers may be added to obtain a preferred pH for storage stability, for example, between 6.0 and 8.0. In some embodiments, a pH of 6.0-7.5 is preferred, at which the liposome membrane exhibits better stability and drug encapsulation stability.

[0177] The formulation may also contain stabilizers and / or antioxidants. In some embodiments, the stabilizer is selected from one or more of ethylenediaminetetraacetic acid (EDTA), disodium EDTA, and dicalcium EDTA. In some embodiments, the stabilizer is added at an amount of 0-0.5% / 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, and L-cysteine. In some embodiments, the oil-soluble antioxidant is selected from one or more of α-tocopherol, α-tocopherol succinate, and α-tocopherol acetate. In some embodiments, the antioxidant is added at an amount of 0-0.5% / v%.

[0178] The dosage will depend on the amount of drug encapsulated in the liposomes, the disease state to be treated, the dosage form, and the judgment of the physician. Generally, the dosage should be sufficient to deliver a therapeutically effective amount of gemcitabine to the subject.

[0179] The amount of formulation necessary to deliver a therapeutically effective dose can be determined using conventional in vivo and in vitro methods in the field of pharmaceutical testing. See, for example, Handbook of Anticancer Drug's Development, DBBudman, AHCalvert, EKRowinsky (eds.), LWW, 2003. Typically, the dosage of the pharmaceutical compositions of the present invention is in the range of about 0.001 mg to about 500 mg of drug per kilogram of body weight, and generally in the range of about 0.01 mg to about 100 mg of drug per kilogram of body weight.

[0180] Typically, the liposomes of the present invention can be prepared as topical or injectable solutions, or as liquid solutions or suspensions. They can also be prepared in solid forms suitable for dissolving or suspending in a liquid carrier prior to injection. They can also be formulated into enterically coated tablets or gel capsules according to methods known in the art.

[0181] The liposomes of the present invention can be administered in any medically acceptable manner, depending on the disease being treated. Possible routes of administration include injection, parenteral routes such as intramuscular, subcutaneous, intravenous, intraarterial, intraperitoneal, intra-articular, intradural, intrathecal, or intrathecal routes, such as oral, nasal, ocular, rectal, vaginal, or pulmonary administration, 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 direct intracranial infusion of liposomes into the tumor (convection-enhanced delivery, or CED) is particularly advantageous. See Saito, et al., Cancer Research, Vol. 64, pp. 2572-2679, 2004; Mamot, et al., J. Neuro-Oncology, Vol. 68, pp. 1-9, 2004. The liposomes of the present invention can also be applied directly to tissue surfaces. Sustained-release administration, pH-dependent release administration, or other specific chemical or environmental condition-mediated release administration are also explicitly included in this invention, for example, through methods such as depot injection or bioerodible implant.

[0182] In another aspect, the present invention provides the use of the liposomes described in any of the first aspects in the preparation of therapeutic antitumor drugs. In some embodiments, the tumor is selected from pancreatic cancer, breast cancer, non-small cell lung cancer, and ovarian cancer.

[0183] In another invention, the present invention provides a method for preparing liposomes according to any one of the first aspects, wherein the liposomes are selected from preparation method 1 and preparation method 2:

[0184] Preparation method 1

[0185] (1) Preparation of blank liposomes: Using a solution of phospholipids and cholesterol as the oil phase and a solution containing metal ions as the aqueous phase, a primary emulsion is prepared by membrane hydration, organic solvent injection, pipeline emulsification, emulsification or reverse evaporation. The granules are then granulated, and optionally, one or more of the following treatments are performed: dialysis, centrifugation, ultrafiltration or concentration to obtain blank liposomes.

[0186] (2) Drug loading: Add gemcitabine or its salt solution to the blank liposomes prepared in step (1). Optionally, the above solution contains the metal ions in the aqueous phase of step (1). Heat and incubate above the phospholipid phase transition temperature to obtain drug-loaded liposomes.

[0187] (3) Removal of free drug: The drug-loaded liposomes obtained in step (2) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0188] In step (1), an optional functional long-circulating material is added to the oil phase, or in step (2), an optional functional long-circulating material is added to prepare liposomes containing the functional long-circulating material.

[0189] Preparation method 2

[0190] (1) Preparation of oil phase: Dissolve phospholipids and cholesterol in an organic solvent to prepare oil phase;

[0191] (2) Preparation of aqueous phase: Gemcitabine or its salt is dissolved in an aqueous solution containing metal ions to prepare an aqueous phase;

[0192] (3) Preparation of drug-loaded liposomes: The oil phase of step (1) and the aqueous phase of step (2) are prepared into liposomes by thin-film hydration, organic solvent injection, pipeline emulsification or reverse evaporation, or (2) is directly mixed with the prepared precursor liposomes (such as commercial Presome), granulated, and drug-loaded liposomes are obtained.

[0193] (4) Removal of free drug: The drug-loaded liposomes obtained in step (3) are subjected to one or more treatments such as dialysis, centrifugation and ultrafiltration to remove free drug and optional concentration treatment to obtain the liposomes.

[0194] In step (1), an optional functional long-circulating material is added when preparing the oil phase, or an optional functional long-circulating material is added after granulation in step (3) to prepare liposomes containing functional long-circulating materials.

[0195] In some embodiments, the method for preparing the liposomes includes one or more of the following features:

[0196] 1) In step (1) of preparation method 1, phospholipids and cholesterol are dissolved in a solvent selected from alcohols (e.g., methanol, ethanol or isopropanol), halogenated hydrocarbons (e.g., dichloromethane or trichloromethane), water and any combination thereof as the oil phase;

[0197] 2) In step (2) of preparation method 1, the solution of gemcitabine or its salt is an aqueous solution of gemcitabine or its salt or the solution containing metal ions in step (1);

[0198] 3) In step (2) of preparation method 1, the incubation is carried out at 50-70°C (e.g., 60-70°C, or even 60-65°C);

[0199] 4) In step (2) of preparation method 1, the incubation time is 15 min to 2 h (e.g., 15 min to 1 h, e.g., 30 min);

[0200] 5) In step (1) of preparation method 2, the organic solvent is selected from alcohol solvents (e.g., methanol, ethanol or isopropanol), halogenated hydrocarbon solvents (e.g., dichloromethane or trichloromethane) and their mixed solvents with water;

[0201] 6) In preparation method 1 or preparation method 2, the weight ratio of phospholipids to cholesterol is (1-125):(0.1-25), preferably (1-100):(0.1-20), more preferably (2-80):(0.1-15), for example (2-10):1, and even more preferably (4-8):1;

[0202] 7) In step (2) of preparation method 1, the weight ratio of gemcitabine or its salt to phospholipid is 1:(0.01-100), preferably 1:(0.02-80), more preferably 1:(0.05-50), for example 1:(0.5-5), and even more preferably 1:(0.5-2);

[0203] 8) In preparation method 1 or preparation method 2, the phospholipid is a neutral phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine or a combination thereof; preferably, the neutral phospholipid is soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearate phosphatidylcholine or any combination thereof.

[0204] 9) In preparation method 1 or preparation method 2, the phospholipid is a combination of neutral phospholipid and negatively charged phospholipid; preferably, the neutral phospholipid is phosphatidylcholine, phosphatidylethanolamine, or a combination of both; preferably, the neutral phospholipid is soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, distearyl 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 dipalmitoyl phosphatidylglycerol, distearyl phosphatidylglycerol, or a combination of both.

[0205] Preferably, the weight ratio between neutral phospholipids and negatively charged phospholipids is (0.1-20):1; more preferably (0.5-15):1, and even more preferably (1-10):1;

[0206] 10) In preparation method 1 or preparation method 2, the metal ion is selected from calcium ion, magnesium ion and transition metal ion; preferably, 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; preferably, the metal ion is selected from one or more of copper ion, cobalt ion and calcium ion.

[0207] Preferably, the molar ratio of gemcitabine or its salt to the metal ion is (0.25–20):1, more preferably (0.5–10):1;

[0208] 11) In preparation method 1 or preparation method 2, the solution containing the metal ions is a buffer salt solution containing the metal ions; preferably, the pH of the buffer salt solution is 4-8, more preferably 5-7.5, for example 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, copper sulfate-triethanolamine solution, copper gluconate-arginine buffer solution, copper gluconate-triethanolamine solution, copper gluconate-tris(hydroxymethyl)aminomethane solution, copper sulfate-tris(hydroxymethyl)aminomethane solution, copper sulfate-4-hydroxyethylpiperazine ethanesulfonic acid solution, zinc gluconate solution or calcium acetate-acetic acid buffer solution with a pH of 4-8;

[0209] 12) In preparation method 1 or preparation method 2, the functional long-cycle material is one or more of polyethylene glycol (PEG), vitamin E polyethylene glycol succinate (TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-modified distearate phosphatidylethanolamine (MPEG-DSPE), polyethylene glycol-modified myristoyl phosphatidylethanolamine (MPEG-DMPE), and polyethylene glycol-modified dipalmitoyl phosphatidylethanolamine (MPEG-DPPE), wherein the molecular weight of polyethylene glycol is 500 Daltons to 10,000 Daltons, preferably 2,000 Daltons; preferably, the functional long-cycle material is MPEG2000-DSPE;

[0210] Preferably, in preparation method 1 or preparation method 2, the weight ratio of gemcitabine or its salt to the optional functional long-cycle material is 1:(0 to 100), more preferably 1:(0.05 to 50), more preferably 1:(0.1 to 25), for example 1:(0.1 to 1), and even more preferably 1:(0.1 to 0.5).

[0211] Beneficial effects of the invention

[0212] This invention provides liposomes of gemcitabine or its salts, a method for their preparation, and their uses. The liposomes can achieve one or more of the following technical effects:

[0213] (1) Good stability; in some embodiments, the liposomes are stored at room temperature or under refrigeration for 3 months or more without significant changes in particle size and encapsulation efficiency.

[0214] (2) High encapsulation efficiency; in some embodiments, the encapsulation efficiency of the liposomes can reach more than 90%, for example more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, and more than 99%.

[0215] (3) It can prolong the circulation time of gemcitabine in vivo; in some embodiments, the liposomes prepared by the present invention can prolong the retention time of gemcitabine in vivo by more than 3 times compared with ordinary injections.

[0216] (4) It can enhance tumor targeting, improve efficacy, and reduce its toxic side effects. Attached Figure Description

[0217] Figure 1 The pharmacokinetic results of administration of gemcitabine liposomes (1.0 mg / kg, IV) and gemcitabine hydrochloride for injection (50 mg / kg, IV) to CD-1 mice are shown. Detailed Implementation

[0218] The following experimental examples and embodiments are only used to further illustrate the present invention, but should not be construed as limiting the invention to these. Those skilled in the art can make various modifications or improvements based on the basic ideas of the present invention, but as long as they do not depart from the basic ideas of the present invention, they are all within the scope of the present invention. Where specific conditions are not specified in the embodiments, they are performed under conventional conditions or conditions recommended by the manufacturer. Where the manufacturers of reagents or instruments are not specified, they are all commercially available conventional products.

[0219] In the following examples, unless otherwise specified, the encapsulation efficiency of liposomes was determined by dextran gel percolation. The specific steps of the gel percolation method are as follows: Take one pre-packed PD-10 desalting columns and rinse with 25 mL of deionized water to equilibrate. Load 0.2 mL of sample onto the column, hold for 1–2 min, then elute with 0.9% sodium chloride solution. Collect the first 7 mL of eluent in a 25 mL volumetric flask, dilute to volume with solvent, and use this as the encapsulated drug sample. Continue elution, collecting the next 15 mL of eluent in a 25 mL volumetric flask, and dilute to volume with solvent to use this as the free drug sample. The encapsulation efficiency was determined using UV-Vis spectrophotometry and calculated according to the following formula: Encapsulation efficiency = W / (W+Wf)×100%, where W is the amount of encapsulated drug and Wf is the amount of free drug.

[0220] In the following examples, unless otherwise specified, the particle size of the liposomes was determined by dynamic light scattering (DLS). The specific steps of DLS are as follows: an appropriate amount of liposome sample was taken, diluted with water, and then the particle size was determined using a Malvern Nano-ZS90 particle size analyzer. The particle size is expressed as intensity.

[0221] Example 1: Preparation of gemcitabine liposomes

[0222] (1) Organic phase preparation

[0223] Weigh out 6g of hydrogenated soybean phosphatidylcholine and 0.75g of cholesterol, heat at 60℃ and dissolve in 40ml of ethanol, then heat for later use.

[0224] (2) Aqueous phase preparation

[0225] Weigh 6g of gemcitabine hydrochloride and heat it to dissolve it in a copper sulfate-triethanolamine solution (200mM) with pH=7.4, wherein the molar ratio of gemcitabine to copper sulfate is 1.25:1. Heat the solution for later use.

[0226] (3) Preparation of drug-loaded liposomes

[0227] The organic phase from (1) was injected into the aqueous phase from (2) using a 50 ml syringe. After heating and stirring at 60°C for 30 min, the drug-loaded liposome proemulsion was obtained. The drug-loaded liposome proemulsion was granulated to 120 nm using a high-speed shear and high-pressure homogenizer, and then dialyzed using a tangential flow ultrafiltration device (the dialysate was a 10% sucrose-histidine solution with a pH of 6.5). After dialysis, gemcitabine liposomes were obtained. The average particle size of the obtained liposomes was approximately 116 nm, the gemcitabine concentration was 1.05 mg / ml, and the encapsulation efficiency was 95.7%.

[0228] Example 2: Preparation of gemcitabine liposomes

[0229] (1) Organic phase preparation

[0230] Weigh out 2.75g of hydrogenated soybean phosphatidylcholine, 0.36g of cholesterol, and 0.65g of MPEG2000-DSPE. Dissolve them in 20ml of anhydrous ethanol at 60℃ and heat for later use.

[0231] (2) Aqueous phase preparation

[0232] Weigh gemcitabine (7.5g) and heat to dissolve in a 100mM copper gluconate solution with pH=7.4, wherein the molar ratio of gemcitabine to copper ions is 5:1. Heat for later use.

[0233] (3) Preparation of drug-loaded liposomes

[0234] The organic phase in (1) was injected into the aqueous phase in (2) at a rate of 100 ml / min. After heating and stirring for 30 min, the drug-loaded liposome proemulsion was obtained. The drug-loaded liposome proemulsion was granulated to 48 nm by high-speed shearing and high-pressure homogenization. It was then subjected to ultrafiltration dialysis using a tangential flow ultrafiltration device (dialysis solution was a 10% sucrose-histidine solution with pH 6.5). After dialysis, gemcitabine liposomes were obtained. The average particle size of the obtained liposomes was about 52 nm, the gemcitabine concentration was 0.31 mg / ml, and the encapsulation efficiency was 91.3%.

[0235] Example 3: Preparation of polyethylene glycol-modified gemcitabine-containing phospholipid liposomes

[0236] (1) Organic phase preparation

[0237] Weigh out 2.2g of distearylphosphatidylcholine, 0.7g of dipalmitoylphosphatidylglycerol, and 0.37g of cholesterol. Dissolve them in 9ml of ethanol / water (16:1, v / v) at 60℃ and heat for later use.

[0238] (2) Aqueous phase preparation

[0239] Weigh gemcitabine (3.3g) and heat to dissolve in a 100mM calcium acetate-acetic acid buffer solution with pH=6.5, wherein the molar ratio of gemcitabine to calcium ions is 1.5:1. Heat for later use.

[0240] (3) Preparation of drug-loaded liposomes

[0241] The organic phase from (1) was injected into the aqueous phase from (2) at a rate of 150 ml / min. After heating and stirring at 70°C for 30 min, the drug-loaded liposome pre-emulsion was obtained. The drug-loaded liposome pre-emulsion was granulated to approximately 187 nm using a high-speed shear and high-pressure homogenizer. 0.5 mL of MPEG2000-DSPE (0.26 g) was added, and the mixture was heated and incubated at 60°C for 10 min, followed by immediate cooling to obtain polyethylene glycol-modified gemcitabine liposomes containing negatively charged phospholipids. 300 mM EDTA-disodium solution was added, and the mixture was stirred at room temperature for 20 min. Ultrafiltration was performed using a tangential flow ultrafiltration device. After dialysis, gemcitabine liposomes were obtained. The average particle size of the obtained liposomes was approximately 206 nm, the gemcitabine concentration was 3.6 mg / ml, and the encapsulation efficiency was 97.3%.

[0242] Example 4: Preparation of gemcitabine liposomes

[0243] (1) Thin film formation

[0244] Weigh 1.4g of distearylphosphatidylcholine, 0.7g of distearylphosphatidylglycerol, and 0.4g of cholesterol. Dissolve them in 30ml of dichloromethane / methanol / water (10:2:1, v / v / v) at 60℃. Place the solution in a round-bottom flask and evaporate it to dryness at 37℃ on a rotary evaporator to remove the organic solvent, thus obtaining a lipid film.

[0245] (2) Hydration

[0246] Weigh 3g of gemcitabine and heat to dissolve it in a 100mM copper gluconate-triethanolamine solution (pH=7.5), where the molar ratio of gemcitabine to copper ions is 1.5:1, to obtain an hydrated solution. Add the hydrated solution to a lipid membrane and hydrate for 30 min.

[0247] (3) Preparation of drug-loaded liposomes

[0248] After hydration, the solution was sheared and dispersed by a high-speed shearing machine and then homogenized to 115 nm by a homogenizer. It was then dialyzed using a tangential flow ultrafiltration device. After dialysis, gemcitabine liposomes were obtained. The average particle size of the obtained liposomes was about 135 nm, the gemcitabine concentration was 2.4 mg / ml, and the encapsulation efficiency was 93.0%.

[0249] Example 5: Preparation of polyethylene glycol-modified gemcitabine liposomes

[0250] (1) Thin film formation

[0251] Weigh out 2.0g of hydrogenated soybean phosphatidylcholine, 0.7g of dipalmitoylphosphatidylglycerol, 0.36g of cholesterol and 0.7g of MPEG2000-DSPE, heat at 60℃ and dissolve in 30ml of dichloromethane / methanol / water (10:1:1, v / v / v), place in a round-bottom flask and evaporate to dryness at 37℃ on a rotary evaporator to remove organic solvent and obtain lipid film.

[0252] (2) Hydration

[0253] Weigh gemcitabine (3.3 g) and heat to dissolve in a 100 mM zinc gluconate solution (pH 7.4), where the molar ratio of gemcitabine to zinc ions is 1.5:1, to obtain an hydrated solution. Add the hydrated solution to a lipid membrane and hydrate for 30 min to obtain the final product.

[0254] (3) Preparation of drug-loaded liposomes

[0255] After hydration, the solution was sheared and dispersed by a high-speed shearing machine, then granulated to 120 nm by an extruder, and finally dialyzed using a tangential flow ultrafiltration device. After dialysis, gemcitabine liposomes were obtained with an average particle size of 130 nm, a gemcitabine concentration of 0.71 mg / ml, and an encapsulation efficiency of 95.7%.

[0256] Example 6: Preparation of polyethylene glycol-modified gemcitabine liposomes

[0257] (1) Thin film formation

[0258] Weigh 1.8g of distearylphosphatidylcholine, 0.64g of distearylphosphatidylglycerol, and 0.32g of cholesterol. Dissolve them in 30ml of chloroform / methanol / water (10:5:0.1, v / v / v) at 60℃. Place the solution in a round-bottom flask and evaporate it to dryness at 37℃ on a rotary evaporator to remove the organic solvent, thus obtaining a lipid film.

[0259] (2) Hydration

[0260] Gemcitabine (2.7 g) was weighed and dissolved in a 100 mM copper gluconate solution at pH 7.4, with a molar ratio of gemcitabine to copper ions of 1:1, to obtain an hydrated solution. The hydrated solution was then added to a lipid membrane and hydrated for 30 min.

[0261] (3) Preparation of drug-loaded liposomes

[0262] After hydration, the solution was sheared and dispersed by a high-speed shearing machine and then granulated to 80 nm by an extruder. 0.3 g of MPEG2000-DSPE was weighed, heated (60 °C), dissolved in 0.5 ml of purified water, and added to the granulated drug-loaded liposomes. After heating and incubation at 60 °C for 10 min, the mixture was immediately cooled to obtain polyethylene glycol-modified drug-loaded liposomes. 150 mM EDTA-disodium solution was added and stirred for 20 min. The mixture was then dialyzed using a tangential flow ultrafiltration device (the dialysate was a pH 6.5, 10% sucrose-histidine-EDTA-disodium solution (1 mM)). After dialysis, gemcitabine liposomes were obtained. The average particle size of the obtained liposomes was 104 nm, the gemcitabine concentration was 2.0 mg / ml, and the encapsulation efficiency was 99.8%.

[0263] Example 7: Preparation of gemcitabine liposomes

[0264] (1) Thin film formation

[0265] Weigh out 1.5g of soybean phosphatidylcholine, 0.5g of distearate phosphatidylglycerol, and 0.25g of cholesterol. Dissolve them in 30ml of chloroform / methanol / water (10:5:0.1, v / v / v) at 60℃. Place the solution in a round-bottom flask and evaporate it to dryness at 37℃ on a rotary evaporator to remove the organic solvent, thus obtaining a lipid film.

[0266] (2) Hydration

[0267] Gemcitabine (2.3 g) was weighed and dissolved in a 100 mM copper gluconate solution at pH 7.4, with a molar ratio of gemcitabine to copper ions of 1.5:1, to obtain an hydrated solution. The hydrated solution was then added to a lipid membrane and hydrated for 30 min.

[0268] (3) Preparation of drug-loaded liposomes

[0269] After hydration, the solution was sheared and dispersed by a high-speed shear mill, then extruded to 82 nm, cooled, and then mixed with 150 mM EDTA-disodium solution for 20 min. The mixture was then dialyzed using a tangential flow ultrafiltration device (the dialysate was a pH 6.5, 10% sucrose-histidine-EDTA-disodium solution (1 mM)). After dialysis, gemcitabine liposomes were obtained with an average particle size of 97 nm, a gemcitabine concentration of 1.77 mg / ml, and an encapsulation efficiency of 99.7%.

[0270] Example 8

[0271] (1) Preparation of blank liposomes

[0272] 1.5 g of hydrogenated soybean phosphatidylcholine, 0.5 g of distearate phosphatidylglycerol, and 0.25 g of cholesterol were weighed and dissolved in 30 ml of chloroform / methanol / water (10:5:0.1, v / v / v) at 60 °C. The solution was placed in a round-bottom flask and evaporated to dryness at 37 °C on a rotary evaporator to remove the organic solvent, thus obtaining a lipid membrane. The lipid membrane was hydrated for 30 min using a 100 mM copper gluconate-arginine buffer solution (pH 7.4). After hydration, the solution was sheared and dispersed using a high-speed shear mill, then extruded to 90 nm. The liposome product was concentrated by dialysis using a tangential flow ultrafiltration device with histidine-sucrose buffer as the displacement medium to obtain blank liposomes.

[0273] (2) Preparation of drug-loaded liposomes

[0274] Dissolve 1g of gemcitabine hydrochloride and 0.2g of mPEG2000-DSPE in water, add to the blank liposomes prepared in (1), incubate at 60℃ for 30min, and then cool in an ice-water bath to obtain drug-loaded liposomes.

[0275] (3) Removal of free drugs

[0276] Using EDTA-histidine-sucrose buffer as the ultrafiltration medium, dialysis was performed using a tangential flow ultrafiltration device to remove external free drugs and copper ions. Gemcitabine liposomes were then concentrated to obtain a particle size of 95 nm and an encapsulation efficiency of 98.2%.

[0277] Example 9

[0278] (1) Preparation of blank liposomes

[0279] Weigh 5g of distearate phosphatidylcholine and 1g of cholesterol, and dissolve them in 60ml of anhydrous ethanol at 60℃ to form the organic phase. Use a copper gluconate-triethanolamine buffer solution (150mM) with pH=7.4 as the aqueous phase. Incubate the organic phase at 60℃ and then inject it into the aqueous phase. After the injection is complete, continue hydration for 10min to obtain the liposome colostrum. Use a 100nm extrusion membrane and a 50nm extrusion membrane to extrude the liposomes to a particle size of 75nm. Use histidine-sucrose buffer as the replacement medium and dialysis with a tangential flow ultrafiltration device to remove organic solvents and external copper ions. Concentrate the liposome product to obtain blank liposomes.

[0280] (2) Preparation of drug-loaded liposomes

[0281] Dissolve 3.5g of gemcitabine hydrochloride and 1.0g of mPEG2000-DSPE in water, add to the blank liposomes prepared in (1), incubate at 65℃ for 30min, and then cool in an ice-water bath to obtain drug-loaded liposomes.

[0282] (3) Removal of free drugs

[0283] Using EDTA-histidine-sucrose buffer as the ultrafiltration medium, dialysis was performed using a tangential flow ultrafiltration device to remove external free drugs and copper ions. Gemcitabine liposomes were then concentrated to obtain a particle size of 76 nm and an encapsulation efficiency of 97.9%.

[0284] Example 10

[0285] (1) Preparation of blank liposomes

[0286] 8g of hydrogenated soybean phosphatidylcholine and 2g of cholesterol were weighed and dissolved in 40ml of anhydrous ethanol at 60℃ to form the organic phase. A copper gluconate-triethanolamine buffer solution (100mM) with pH=7.4 was used as the aqueous phase. The organic phase was injected into the aqueous phase under 60℃ incubation. After injection, hydration was continued for 10min to obtain the liposome colostrum. The colostrum was extruded sequentially using a 100nm extrusion membrane and a 50nm extrusion membrane until the particle size was 79nm. A copper gluconate-triethanolamine buffer solution (100mM) with pH=7.4 was used as the displacement medium. The colostrum was dialyzed using a tangential flow ultrafiltration device to remove the organic solvent and concentrate the liposome product to obtain blank liposomes.

[0287] (2) Preparation of drug-loaded liposomes

[0288] Dissolve 10g of gemcitabine hydrochloride in a copper gluconate-triethanolamine buffer solution (100mM) at pH 7.4, add it to the blank liposomes prepared in (1), incubate at 60℃ for 30min, and then cool in an ice-water bath to obtain drug-loaded liposomes.

[0289] (3) Removal of free drugs

[0290] Using EDTA-histidine-sucrose buffer as the ultrafiltration medium, dialysis was performed using a tangential flow ultrafiltration device to remove external free drugs and copper ions. Gemcitabine liposomes were then concentrated to obtain a particle size of 82 nm and an encapsulation efficiency of 98.1%.

[0291] Example 11

[0292] (1) Preparation of blank liposomes

[0293] 8g of hydrogenated soybean phosphatidylcholine and 2g of cholesterol were weighed and dissolved in 40ml of anhydrous ethanol at 60℃ to form the organic phase. PBS buffer with pH=7.4 was used as the aqueous phase. The organic phase was incubated at 60℃ and then injected into the aqueous phase. After incubation, hydration was continued for 10min to obtain the liposome colostrum. The colostrum was extruded sequentially using a 100nm extrusion membrane and a 50nm extrusion membrane until the particle size was 81nm. PBS buffer with pH=6.0 was used as the replacement medium, and the liposome product was dialyzed using a tangential flow ultrafiltration device to remove the organic solvent. The liposome product was then concentrated to obtain blank liposomes.

[0294] (2) Preparation of drug-loaded liposomes

[0295] Dissolve 5g of gemcitabine hydrochloride in water and add it to the blank liposomes prepared in (1). Incubate at 60°C for 30 minutes and then cool in an ice-water bath to obtain drug-loaded liposomes.

[0296] (3) Removal of free drugs

[0297] Using histidine-sucrose buffer as the ultrafiltration medium, tangential flow ultrafiltration was used for dialysis to remove external free drugs. Gemcitabine liposomes were then concentrated to obtain a particle size of 80 nm and an encapsulation efficiency of 80.8%.

[0298] Example 12

[0299] (1) Preparation of blank liposomes

[0300] 8g of hydrogenated soybean phosphatidylcholine was weighed and dissolved in 40ml of anhydrous ethanol at 60℃ to form the organic phase. A 100mM copper gluconate-triethanolamine buffer solution (pH=7.4) was used as the aqueous phase. The organic phase was incubated at 60℃ and then injected into the aqueous phase. After the injection was completed, hydration was continued for 10min to obtain the liposome proemulsion. The proemulsion was extruded sequentially using a 100nm extrusion membrane and a 50nm extrusion membrane until the particle size was 79nm. A 100mM copper gluconate-triethanolamine buffer solution (pH=7.4) was used as the displacement medium. The proemulsion was dialyzed using a tangential flow ultrafiltration device to remove the organic solvent and concentrate the liposome product to obtain blank liposomes.

[0301] (2) Preparation of drug-loaded liposomes

[0302] Dissolve 10g of gemcitabine hydrochloride in water and add it to the blank liposomes prepared in (1). Incubate at 60°C for 30 minutes and then cool in an ice-water bath to obtain drug-loaded liposomes.

[0303] (3) Removal of free drugs

[0304] Using EDTA-histidine-sucrose buffer as the ultrafiltration medium, dialysis was performed using a tangential flow ultrafiltration device to remove external free drugs and copper ions. Gemcitabine liposomes were then concentrated to obtain a particle size of 79 nm and an encapsulation efficiency of 98.5%.

[0305] Experimental Example 1: Liposome Stability Study

[0306] The prepared liposomes were placed at room temperature (25°C) and refrigerated (2-8°C) for 0, 1, and 3 months, respectively. The particle size and encapsulation efficiency of the liposome compositions under different storage conditions and times were measured (as shown in Table 1 below). The results show that the liposome composition prepared in Example 11 had a stable particle size distribution at 25°C and 2-8°C, but slow leakage occurred over time, and the encapsulation efficiency decreased. The liposome composition prepared in Example 12 showed almost no change in particle size after 3 months of storage at room temperature and refrigeration, but the encapsulation efficiency decreased slightly, and stratification occurred. The liposome compositions prepared in Examples 7 and 9 were stable after 3 months of storage at room temperature and refrigeration, with particle size and encapsulation efficiency remaining almost unchanged over the 3 months.

[0307] Table 1. Stability study of liposome compositions

[0308]

[0309]

[0310] Example 2: Pharmacokinetic Study of Gemcitabine Conventional Injection and Gemcitabine Liposome

[0311] Mouse species: CD-1

[0312] Grouping: 12 mice per group (gemcitabine hydrochloride for injection) and 24 mice per group (liposome preparation details are in Example 9).

[0313] Blood collection point design and method: Liposome group: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h), 0.15ml blood was collected from the orbital vein (EDTA-K2 anticoagulation). Conventional injection group: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 0.15ml blood was collected from the orbital vein (EDTA-K2 anticoagulation).

[0314] Dosage: Conventional injection group: 50 mg / kg; Liposome group: 1 mg / kg;

[0315] Blood sample processing: Collected whole blood was immediately stored in crushed ice and centrifuged at 4000 rpm for 10 min within 1 hour to separate the plasma (4℃). The collected plasma was stored at -80℃ for testing.

[0316] Pharmacokinetic results: After being prepared into liposomes according to the method of the present invention (Example 9), when the dosage was 1 / 50 of that of ordinary injection, the blood clearance of gemcitabine was significantly reduced and the in vivo retention time was significantly prolonged, with the in vivo retention time being 30.8 times longer than that of ordinary injection.

[0317]

Claims

1. Liposomes, which contain a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane; in, The aqueous phase contains gemcitabine or its salt and metal ions; the components constituting the liposome membrane include phospholipids, cholesterol and functional long-circulation materials; the phospholipid is distearate phosphatidylcholine; The functional long-cycle material is polyethylene glycol-modified distearate phosphatidylethanolamine (MPEG-DSPE), wherein the molecular weight of polyethylene glycol is 500 Daltons to 10,000 Daltons. The weight ratio of the phospholipids to cholesterol is (4~8):1; The metal ion is selected from copper ions; The weight ratio of the phospholipid to the long-cycle material is (2~15):1; Furthermore, the molar ratio of gemcitabine or its salt to metal ions is (0.5~10):1; the liposome particle size is 50-300 nm; The liposomes are prepared in the following manner: (1) Preparation of blank liposomes: The solution of phospholipids and cholesterol is used as the oil phase, and the copper gluconate solution with pH 4-8 is used as the aqueous phase. The colostrum is prepared by organic solvent injection, granulated, dialyzed and concentrated to obtain blank liposomes. (2) Drug loading: Add a solution of gemcitabine or its salt to the blank liposomes prepared in step (1) and heat and incubate above the phospholipid phase transition temperature to obtain drug-loaded liposomes; (3) Removal of free drug: The drug-loaded liposomes obtained in step (2) are subjected to dialysis and ultrafiltration to remove free drug and to concentrate the liposomes. In step (2), the functional long-circulating material is added to prepare liposomes containing the functional long-circulating material; The weight ratio of gemcitabine or its salt to the functional long-cycle material is 1:(0.1~0.5).

2. The liposome of claim 1, wherein the polyethylene glycol has a molecular weight of 2000 Daltons.

3. The liposomes of claim 1, wherein, The internal aqueous phase also contains components for adjusting pH.

4. The liposomes of claim 1, wherein the preparation method comprises one or both of the following features: 1) In step (1), phospholipids and cholesterol are dissolved in a solvent selected from alcohols, halogenated hydrocarbons and any combination thereof as the oil phase; 2) In step (2), at 50-70 o C performs the incubation.

5. The liposomes of claim 1, wherein the particle size is 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 150 nm, 200 nm, 250 nm or 300 nm.

6. A liposome formulation comprising the liposomes according to any one of claims 1-5, and one or more pharmaceutically acceptable carriers.

7. Use of the liposomes according to any one of claims 1-5 in the preparation of antitumor drugs.

8. A method for preparing the liposomes according to any one of claims 1-5, comprising the following steps: (1) Preparation of blank liposomes: A solution of phospholipids and cholesterol was used as the oil phase, and a copper gluconate solution with pH 4-8 was used as the aqueous phase. The colostrum was prepared by organic solvent injection, granulated, and subjected to dialysis and ultrafiltration to obtain blank liposomes. (2) Drug loading: Add a solution of gemcitabine or its salt to the blank liposomes prepared in step (1) and heat and incubate above the phospholipid phase transition temperature to obtain drug-loaded liposomes; (3) Removal of free drug: The drug-loaded liposomes obtained in step (2) are subjected to dialysis and ultrafiltration to remove free drug and to concentrate the liposomes. In step (2), functional long-circulating materials are added to prepare liposomes containing functional long-circulating materials.

9. The preparation method according to claim 8, characterized in that... One or more of the following: 1) In step (1), phospholipids and cholesterol are dissolved in a solvent selected from alcohols, halogenated hydrocarbons and any combination thereof as the oil phase; 2) In step (2), at 50-70 o C performs the incubation; 3) In step (2), the incubation time is 15 min to 2 h.

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