Liposome as well as preparation method and application thereof

Abstract

When the liposome and the composition thereof are used as a drug carrier, through a specific drug loading condition process, 1) high drug loading capacity (the highest drug loading capacity is not less than 18%) is obtained; and 2) when the drug loading capacity is lower than the highest drug loading capacity (18%), the encapsulation efficiency is not lower than 85%. The preparation method of the liposome and the composition thereof and the application of the liposome and the composition thereof as a drug carrier can be used for preparing various drug liposomes.

Description

Liposome and its preparation method and application

[0001] The present invention claims:

[0002] Priority to the prior application, patent application number 202211216387.5, filed with the State Intellectual Property Office of China on September 30, 2022, entitled “A liposome, a pharmaceutical composition, a preparation method and use thereof”;

[0003] Priority to the prior application, patent application number 202311241194.X, filed with the State Intellectual Property Office of China on September 22, 2023, entitled “A liposome, its preparation method and application”.

[0004] The entire contents of said prior application are incorporated into the present application by reference. Technical Field

[0005] The present invention belongs to the field of medicine, and in particular relates to a liposome and a preparation method and application thereof. Background Art

[0006] Liposomes are vesicles formed by a lipid bilayer containing an internal aqueous medium. Liposomes have been used as carriers for various therapeutic agents to provide improved delivery characteristics, such as increased drug circulation time in the blood, reduced cytotoxicity, sustained drug release, and targeted drug delivery to selected tissues. When using liposomes for therapeutic drug delivery, high drug encapsulation efficiency and drug content are desirable to reduce potential toxicity of the phospholipid excipient.

[0007] The previous technologies of US8932627B2 and US20190022004A1 use acetate or bicarbonate to load drugs in molecular form into liposomes. The drugs are stored in the internal aqueous phase in ionic form and the release rate of the drugs is regulated to treat respiratory diseases. However, the drug loading capacity of drug liposomes obtained by using acetate or bicarbonate prescription loading drugs is limited. Some studies have suggested that the use of bicarbonate in liposome preparations will lead to gas accumulation, which will destroy and destabilize the liposomes and cause premature drug release. Due to its destructive and destabilizing effects on liposomes, it is recommended to avoid using bicarbonate as a loading agent.

[0008] There are still many unresolved issues in the development of liposome preparations of weakly acidic drugs. The present invention provides a liposome that further increases the drug loading capacity of the liposome, in order to improve patient compliance and therapeutic effects in clinical applications.

[0009] Summary of the Invention

[0010] In order to improve the above technical problems, the present invention is implemented through the following technical solutions:

[0011] The first aspect of the present invention is to provide a liposome.

[0012] The present invention provides a liposome comprising phospholipids and an internal aqueous phase; the internal aqueous phase comprises meglumine and acetic acid; the pH of the internal aqueous phase is 4.0-10.5, for example 4.5-10.0, 6.5-10.5, preferably 4.97-9.52.

[0013] According to an embodiment of the present invention, the pH of the internal aqueous phase is 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, preferably 8.5.

[0014] According to an embodiment of the present invention, the meglumine in the internal aqueous phase provides the cationic portion of the internal aqueous phase, and the acetic acid provides the anionic portion of the internal aqueous phase.

[0015] According to an embodiment of the present invention, the concentration of meglumine in the internal aqueous phase is 0.1 to 0.8 M, such as 0.2 to 0.6 M, preferably 0.3 to 0.4 M, such as 0.3 M, 0.35 M, 0.4 M;

[0016] According to an embodiment of the present invention, the concentration of acetic acid in the internal aqueous phase is 0.1 to 0.8 M, for example, 0.2 to 0.6 M, such as 0.2 M, 0.211 M, 0.268 M, 0.286 M, 0.3 M, 0.35 M, 0.40 M, 0.5 M, 0.6 M;

[0017] According to an embodiment of the present invention, the preferred concentration of meglumine and acetic acid in the internal aqueous phase is 0.30-0.40M.

[0018] According to an embodiment of the present invention, the phospholipid has a bilayer lipid structure, and the bilayer lipid is composed of hydrogenated soy lecithin (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), with a molar ratio of 3: (1-3): (0.025-0.225), for example, 3: (2-3): (0.025-0.075), 3: (1.5-2.5): (0.025-0.075).

[0019] According to an embodiment of the present invention, the bilayer lipid is composed of hydrogenated soy lecithin (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), with a molar ratio of 3:2:(0.025-0.225), for example 3:2:(0.025-0.15), preferably 3:2:(0.025-0.075).

[0020] According to an embodiment of the present invention, the concentration of the phospholipid in the liposome composition is 1 to 100 mg / mL, for example, 1 to 50 mg / mL, 1 to 20 mg / mL, and in the preparation process of the liposomes of the present invention, the concentration of the phospholipid is also, for example, 1 to 15 mg / mL, 1.2 to 5.0 mg / mL, preferably 1.3 to 4.5 mg / mL, 1.4 to 4.18 mg / mL.

[0021] According to an embodiment of the present invention, the particle size of the liposome is 50-500 nm, for example, 100-200 nm, preferably 100-150 nm, 120-180 nm, 130-160 nm, or 140-190 nm.

[0022] The second aspect of the present invention is to provide a liposome composition comprising the liposome and an external aqueous phase.

[0023] The present invention also provides a liposome composition, characterized in that the liposome composition comprises the liposome described in the first aspect and an external aqueous phase; the internal aqueous phase in the liposome comprises meglumine and acetic acid, and the external aqueous phase suspends the liposome.

[0024] According to an embodiment of the present invention, the external aqueous phase is a buffer solution with a pH of 4.5 to 6.5.

[0025] According to an embodiment of the present invention, the buffer solution for achieving the corresponding pH value of the external aqueous phase is well known in the art and is not particularly limited. Commonly used buffer solutions in the art are selected from one or more of HEPES, sodium chloride, sucrose, citrate buffer, phosphate buffer, and Tris buffer. According to a preferred embodiment of the present invention, the buffer solution can be selected from citrate buffer; the concentration of the buffer solution is 0.05M to 0.25M, for example, 0.05M to 0.2M.

[0026] According to an embodiment of the present invention, the pH of the internal aqueous phase is 4.0-10.5, for example 4.5-10.0, 6.5-10.5, 7.5-9.5, such as 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, preferably 8.5.

[0027] According to an embodiment of the present invention, in the internal aqueous phase, the concentration of meglumine is 0.1 to 0.8 M, such as 0.2 to 0.6 M, preferably 0.30 to 0.4 M, such as 0.3 M, 0.35 M, or 0.4 M;

[0028] According to an embodiment of the present invention, in the internal aqueous phase, the concentration of acetic acid in the internal aqueous phase is 0.1-0.8M, for example, 0.2-0.6M, such as 0.2M, 0.211M, 0.268M, 0.286M, 0.3M, 0.35M, 0.40M, 0.5M, and 0.6M.

[0029] According to an embodiment of the present invention, the liposome has a pH gradient characteristic with a high pH in the inner aqueous phase and a low pH in the outer aqueous phase.

[0030] According to an embodiment of the present invention, the phospholipid has a bilayer lipid structure, and the bilayer lipid is composed of hydrogenated soy lecithin (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), with a molar ratio of 3: (1-3): (0.025-0.225), for example, 3: (2-3): (0.025-0.075), 3: (1.5-2.5): (0.025-0.075).

[0031] According to an embodiment of the present invention, the bilayer lipid is composed of hydrogenated soy lecithin (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), with a molar ratio of 3:2:(0.025-0.225), for example 3:2:(0.025-0.15), and preferably a molar ratio of 3:2:(0.025-0.075);

[0032] According to an embodiment of the present invention, the concentration of the phospholipid in the liposome composition is 1 to 100 mg / mL, for example, 1 to 50 mg / mL, 1 to 20 mg / mL, and in the preparation process of the liposomes of the present invention, the concentration of the phospholipid is also, for example, 1 to 15 mg / mL, 1.2 to 5.0 mg / mL, preferably 1.3 to 4.5 mg / mL, 1.4 to 4.18 mg / mL.

[0033] According to an embodiment of the present invention, the particle size of the liposome is 50-500 nm, for example, 100-200 nm, preferably 100-150 nm, 120-180 nm, 130-160 nm, or 140-190 nm.

[0034] According to an embodiment of the present invention, the concentration of meglumine and acetic acid in the internal aqueous phase is 0.30-0.40M.

[0035] The third aspect of the present invention is to provide a method for preparing liposomes.

[0036] The present invention also provides a method for preparing the liposome, comprising the following steps:

[0037] (1) Preparation of oil phase: Weigh hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol in parts by weight and dissolve them in ethanol to obtain an oil phase;

[0038] (2) Preparation of aqueous phase: After dissolving meglumine in water, acetic acid was added to adjust the pH to obtain an aqueous phase, i.e., the inner aqueous phase;

[0039] (3) Emulsification-extrusion: The oil phase and the aqueous phase are mixed to obtain colostrum, which is then extruded through a polycarbonate membrane to obtain the liposome.

[0040] According to an embodiment of the present invention, the preparation method further comprises the step of ultrafiltration: the liposomes obtained in step (3) are ultrafiltered with a citric acid-sodium citrate buffer solution to replace the liposome external phase medium and remove the organic solvent to obtain the liposomes.

[0041] The present invention also provides a method for preparing the liposome composition, comprising dispersing the liposome in an external aqueous phase to obtain the liposome composition;

[0042] According to an embodiment of the present invention, the external aqueous phase is a buffer solution with a pH of 4.5 to 6.5;

[0043] According to an embodiment of the present invention, the buffer solution for achieving the corresponding pH value of the external aqueous phase is well known in the art and is not particularly limited. Commonly used buffer solutions in the art are selected from one or more of HEPES, sodium chloride, sucrose, citrate buffer, phosphate buffer, and Tris buffer, preferably citrate buffer; the concentration of the buffer solution is 0.05M to 0.25M, for example, 0.08M to 0.2M.

[0044] The fourth aspect of the present invention is to provide a use of liposomes as drug carriers.

[0045] The present invention also provides use of the liposome or liposome composition as a drug carrier.

[0046] According to an embodiment of the present invention, the drug is a weakly acidic drug.

[0047] Preferably, the pKa of the weakly acidic drug is between 2 and 7;

[0048] According to an embodiment of the present invention, the weakly acidic drug is encapsulated in the inner aqueous phase of the liposome, and the liposome encapsulating the weakly acidic drug is suspended in the outer aqueous phase.

[0049] According to an embodiment of the present invention, the weakly acidic drug may be selected from at least one drug selected from the group consisting of prostaglandins, antipyretics and analgesics, quinoline carboxylic acid antibiotics, and stimulator of interferon genes (STING) receptors;

[0050] According to an embodiment of the present invention, the prostaglandin drugs such as treprostinil, beraprost, iloprost, carboprost, limaprost, epoprostenol, alprostadil, unoprostone, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs), the antipyretic and analgesic drugs such as aspirin, ibuprofen, naproxen, diclofenac sodium, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); the quinoline carboxylic acid antibacterial drugs such as nalidixic acid, pyrrolopyrimic acid, fluoroquinolinic acid, sitafloxacin, ciprofloxacin, enoxacin, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); the STING drugs such as MSA-2, STING agonist-7, Vadimezan, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs). Beneficial effects

[0051] The liposomes provided by the present invention can be used as drug carriers and loaded with drugs through specific drug loading conditions and processes: 1) high drug loading capacity (maximum drug loading capacity of not less than 18%); 2) when the drug loading capacity is below the maximum drug loading capacity (18%), the encapsulation efficiency is not less than 85%, preferably not less than 90%. Preferably, the liposomes of the present invention also have the effects of high drug loading capacity (e.g., not less than 18%) and high encapsulation efficiency (e.g., not less than 85%, preferably not less than 90%). Compared with the existing technology, the liposomes provided by the present invention can achieve higher drug loading capacity. DETAILED DESCRIPTION

[0052] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0053] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0054] The drug loading (LE) of the present invention is expressed as a mass percentage. The definitions and explanations of encapsulation efficiency (EE) and drug loading (LE) are as follows:

[0055] Encapsulation efficiency EE%=(1–W f / W t )×100% (Formula 1)

[0056] Where W f represents the amount of free drug in the external medium; W t Represents the total amount of drug in the liposome suspension.

[0057] The drug loading of the liposome pharmaceutical composition was calculated using the following formula 2: LE% = [W e / W m ]×100%=[(W t ×EE%) / (W l +W t ×EE%)]×100% (Formula 2)

[0058] Where LE represents the percentage of drug loading in liposomes; W e represents the amount of drug encapsulated in liposomes; W m Represents the total weight of drug-loaded liposomes (including the amount of liposome carrier and loaded drug). t is the total amount of drug in the liposome suspension; EE% is the encapsulation efficiency; W l is the total lipid content of the liposome carrier.

[0059] Experimental equipment: pH meter (Mettler, S220K); heat-collecting constant temperature heating magnetic stirrer (Shanghai Yukang Science and Education Instrument Equipment Co., Ltd., DF-101S); small-scale liposome extruder (ATS EX200, nitrogen source power); ultracentrifuge (Backman, MAX-XP); HPLC (Agilent1260, USA); nano-laser particle size analyzer (Malvern, ZS90); tangential flow ultrafiltration equipment (Millipore Pellicon);

[0060] Experimental materials: treprostinil sodium (Porton); hydrogenated soy phosphatidylcholine (HSPC) (Lipoid); distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000) (Lipoid); cholesterol (CHOL) (Nippon Seika); sodium bicarbonate (NaHCO3), sodium acetate (NaAc), meglumine (MEG) (merck); other materials are commonly used injection-grade excipients.

[0061] General experimental procedures

[0062] 1. Preparation of Treprostinil Liposome Composition

[0063] Example 1: Preparation of Treprostinil Liposomes

[0064] (1) Preparation of blank liposomes: First, 2.367 g of HSPC, 0.78 g of CHOL, and 0.21 g of DSPE-mPEG2000 (HSPC / CHOL / DSPE-mPEG2000 = 3 / 2 / 0.075 (molar ratio)) were weighed and added to a clean, dry container. 11.3 g of anhydrous ethanol was added, the container was sealed, and dissolved at 60-65°C to obtain a lipid solution. Then, the lipid solution is injected into 86.7g of sodium bicarbonate solution (taking 0.3M, pH=8.5 sodium bicarbonate solution as the internal aqueous phase as an example), and stirred and hydrated at 60-65°C for 20min; after emulsification, the colostrum is extruded through a small-scale liposome extruder at 60-65°C, and extruded through a 200nm and / or 100nm polycarbonate membrane multiple times at a pressure of 0.6-1.0MPa to obtain a sample particle size of 100-200nm and a PDI of less than 0.2. Then, the sample is cooled to room temperature, and the liposome external phase medium is replaced with a 0.05M citric acid-sodium citrate buffer at pH 5.5 using a tangential flow TFF filtration device. The blank liposomes after ultrafiltration are stored at 2-8°C for later use. Wherein, when the internal aqueous phase is meglumine-acetic acid, meglumine is dissolved in water, and acetic acid is added to adjust the pH (e.g., 8.5) to obtain an internal aqueous phase (aqueous phase).

[0065] (2) Preparation of Treprostinil liposomes: Treprostinil sodium is dissolved in 0.05 M sodium citrate solution, and then the Treprostinil sodium solution is added to the above-mentioned blank liposomes according to a certain theoretical dosage (for example, the theoretical drug loading is 22% (w / w)). An appropriate amount of citric acid-sodium citrate buffer is added to adjust the citric acid-sodium citrate concentration in the liposome external phase to 0.09-0.12 M, and the mixture is incubated at 35-45°C for 30-60 min to obtain a Treprostinil liposome composition.

[0066] General analytical methods

[0067] 1. Quantitative Characterization of Treprostinil Liposomal Composition

[0068] a. Preparation of reference solution:

[0069] Accurately weigh about 10 mg of treprostinil sodium bulk drug substance into a 50 ml volumetric flask, add 2.5 ml of purified water to dissolve, and dilute to the scale with methanol. Shake well to prepare the treprostinil sodium reference solution.

[0070] b. Free and total treprostinil concentrations:

[0071] Accurately measure 1 ml of the liposomal pharmaceutical composition solution into a 20 ml volumetric flask, add approximately 3 ml of methanol, and gently shake to remove any bubbles. Dilute to the mark with methanol and shake well. Samples are taken for HPLC analysis to determine the total treprostinil content in the composition.

[0072] Determination of free treprostinil: 1 ml of treprostinil liposomes was transferred to an ultracentrifuge tube and centrifuged in an ultracentrifuge at the following parameters (temperature: 4°C, speed: 100,000 rpm, time: 30 min). After 30 min, the sample was removed and the supernatant was analyzed by HPLC.

[0073] The specific HPLC chromatographic conditions are as follows:

[0074] c. Encapsulation efficiency (EE) and drug loading (LE):

[0075] The definitions and explanations of the encapsulation efficiency (EE) and drug loading (LE) described in the present invention have been described above. Combined with the specific analysis method, the formula is supplemented as follows:

[0076] Encapsulation efficiency

[0077] Where C f represents the amount of free drug in the external medium; C t Represents the total amount of drug in the liposome suspension; A c is the peak area of ​​the total drug content; A b is the peak area of ​​free drug; a is the dilution factor of total drug content, 20.

[0078] The concentration of the liposomal drug composition was calculated using the following formula 3:

[0079] C is the concentration of the liposome composition, mg / ml; W std is the sample weight of the reference substance, mg; A std The average peak area of ​​the five-point reference solution; T is the content of the main drug in the reference solution, %; D is the dilution volume of the reference solution; A sample is the peak area of ​​the total liposome drug content solution; a is the dilution volume of the total liposome drug content solution.

[0080] d. Average particle size and polydispersity index (PdI):

[0081] Take 30 μl of the treprostinil liposome composition, dilute it to 2 ml with purified water, mix well, and use the dynamic light scattering principle to detect the particle size distribution and polydispersity index of the composition using a Malvern nano-laser particle size analyzer (parameters: temperature 25°C, refractive index 1.33, equilibrium time 120 s, 3 cycles).

[0082] The cations and anions described in the examples represent the material concentrations that provide the corresponding ions, not the ion concentrations after the material is ionized.

[0083] Example 1. Treprostinil liposome compositions with different drug-loaded salt types and different feed ratios

[0084] In order to study the effect of different types of drug-loading salts on the drug loading of treprostinil liposomes, treprostinil liposome compositions containing different internal aqueous phases (sodium bicarbonate (NaHCO3), sodium acetate (NaAc), meglumine-acetic acid (MEG-HAc), pH about 8.5) and different drug loading amounts were prepared according to the procedure described in "1. Preparation of Treprostinil Liposome Compositions" in the previous paragraph titled "General Experimental Procedures", which have specific treprostinil liposomes as shown in Table 1.

[0085] The quantitative characterization of the treprostinil liposome composition, including the theoretical feed ratio (i.e., theoretical drug loading), encapsulation efficiency, and drug loading of the treprostinil liposome, was performed according to the procedure described in "1. Quantitative Characterization of Treprostinil Liposome Composition" in the previous paragraph entitled "General Analytical Methods".

[0086] As can be seen from the results in the table below: Due to the limited volume of the aqueous phase in the liposomes, as the feed amount increases, the drug loading rate first increases (from 8.22% to 19.72%), and then remains essentially unchanged. Therefore, when the feed amount is 21%, the encapsulation efficiency is 87.55% and the drug loading is 19.72%. However, as the feed amount increases, the drug loading remains essentially unchanged, resulting in a significant decrease in the encapsulation efficiency. Secondly, at high feed amounts, the maximum drug loading rate of the treprostinil liposomes in the meglumine formulation is 19.72%, while other formulations (such as sodium bicarbonate or sodium acetate) cannot exceed 18%; at low feed amounts, the encapsulation efficiency of the meglumine formulation can also be no less than 85%, or even no less than 90%. Taking Example C001 as an example, its drug loading is only 8.22%, but the encapsulation efficiency can reach 97.86%. Even when the drug loading of other formulations was 17.39%, the encapsulation efficiency was only 75.65%, and the encapsulation efficiency in most comparative examples was significantly lower than 60%.

[0087] As can be seen from the table below, compared to Comparative Examples A001 or B001, which both use the same 21% feed ratio, C003 achieves an encapsulation efficiency of 87.55% for meglumine, exceeding the 75.65% and 79.59% for sodium bicarbonate and sodium acetate, respectively. Furthermore, the drug loading is 19.72%, significantly higher than the 17.39% and 17.84% for sodium bicarbonate and sodium acetate, respectively. Similarly, under other conditions with the same feed ratio, the meglumine formulation of the present invention outperforms the sodium bicarbonate and sodium acetate formulations.

[0088] Table 1 Treprostinil liposome compositions prepared with different drug-loaded salts

[0089] Note: *The theoretical feed ratio indicates the theoretical drug loading (error within ±5%). The particle size range of liposomes is between 100 and 150 nm.

[0090] Example 2. Treprostinil liposome compositions containing meglumine-acetic acid drug-loaded salts of different particle sizes

[0091] Treprostinil liposome compositions containing different particle sizes (100-150 nm, 150-200 nm) were prepared according to the above-mentioned "General Experimental Procedures" and analyzed according to the above-mentioned general analytical methods.

[0092] Results: The encapsulation efficiency and drug loading of the treprostinil liposome compositions with different particle sizes are shown in Table 2. As can be seen from the table, within the particle size range of 140 to 190 nm, the encapsulation efficiency and drug loading of the treprostinil liposomes are comparable.

[0093] Table 2 Treprostinil compositions of different particle sizes

[0094] Note: The drug-loaded salt cation is MEG (0.30M); the drug-loaded salt anion is HAc (0.29M); the theoretical feed ratio is 21% (error within ±5%);

[0095] Example 3 Treprostinil liposome compositions with different internal aqueous phase concentrations

[0096] Treprostinil liposome compositions containing different concentrations of meglumine-acetic acid (meglumine concentration ranged from 0.20 to 0.60 M, pH 8.5) were prepared according to the aforementioned "General Experimental Procedure" and analyzed according to the aforementioned general analytical methods.

[0097] Results: As can be seen from the table, when the theoretical feed ratio is as high as 21%, the Treprostinil liposomes prepared at an aqueous phase concentration of 0.30-0.40 M have a high drug loading capacity (not less than 18%); when the theoretical feed ratio is lower than 18%, the Treprostinil liposomes prepared at an aqueous phase concentration between 0.30 and 0.40 M can obtain a high encapsulation efficiency (encapsulation efficiency is not less than 90%).

[0098] Table 3-1 High theoretical feed ratio - Treprostinil liposome composition with different internal aqueous phase (meglumine) concentrations

[0099] Note: The drug-loaded salt anion is HAc; the theoretical feed ratio is 21% (error within ±5%); the particle size range of the liposome is between 100 and 150 nm;

[0100] Table 3-2 Low theoretical feed ratio - Treprostinil liposome composition with different internal aqueous phase (meglumine) concentrations

[0101] Note: The anion of the drug-loaded salt is HAc; the theoretical feed ratio is 15% (error within ±5%); the particle size range of the liposome is between 100 and 150 nm;

[0102] Example 4 Treprostinil liposome compositions with different phospholipid prescriptions

[0103] Treprostinil liposome compositions containing different phospholipid formulations were prepared according to the above-mentioned "General Experimental Procedures" and analyzed according to the above-mentioned general analytical methods.

[0104] Results: As can be seen from the table, at a high theoretical feed ratio, when the molar ratio of HSPC, CHOL and DSPE-mPEG2000 is 3 / 2 / (0.025-0.150), the prepared treprostinil liposomes have a high drug loading capacity (not less than 18%); when the molar ratio of HSPC, CHOL and DSPE-mPEG2000 is 3 / (2-3) / 0.075, the prepared treprostinil liposomes have a high drug loading capacity (not less than 18%) and a high encapsulation efficiency (not less than 85%).

[0105] Table 4-1 Treprostinil liposome compositions with different phospholipid formulations

[0106] Note: The drug-loaded salt cation is MEG (0.30 M); the drug-loaded salt anion is HAc (0.29 M); the theoretical feed ratio is 21% (error within ±5%); the particle size range of the liposome is between 100 and 150 nm.

[0107] Table 4-2 Treprostinil liposome compositions with different phospholipid prescriptions

[0108] Note: The drug-carrying salt cation is MEG; the drug-carrying salt anion is HAc; the theoretical feed ratio is 21% (error within ±5%). The particle size range of the above liposomes is between 100 and 150 nm.

[0109] Example 5 Treprostinil liposome compositions with different internal aqueous phase pH

[0110] According to the above "general experimental procedure", the amount of acetic acid in the internal aqueous phase was adjusted (0.21-0.40 M) to prepare treprostinil liposome compositions with different internal aqueous phase pH formulations. The liposome compositions were analyzed according to the above general analytical methods.

[0111] Results: As can be seen from the table, when the acetic acid concentration in the internal aqueous phase solution is 0.21-0.40 M and the corresponding internal aqueous phase solution pH is 9.52-4.97, the prepared treprostinil liposomes have a high drug loading capacity (not less than 18%).

[0112] Table 5 Treprostinil liposome compositions with different internal aqueous phase pH

[0113] Note: The drug-loaded salt cation is MEG (0.30M); the theoretical feed ratio is 22.3% (error within ±5%); the particle size range is 100-200 nm;

[0114] Example 6 Treprostinil liposome compositions with different internal aqueous phase anions

[0115] Treprostinil liposome compositions with different internal aqueous phase anion formulations were prepared according to the aforementioned "General Experimental Procedures," and the liposome compositions were analyzed according to the aforementioned general analytical methods.

[0116] Results: As can be seen from the table, maintaining the internal aqueous phase pH at 8.5 and using acetic acid as the internal aqueous phase anion resulted in a high drug loading (not less than 18%) for the prepared treprostinil liposomes. When valeric acid and lactic acid were used as internal aqueous phase anions and the theoretical feed ratio was less than 18%, the prepared treprostinil liposomes achieved a high encapsulation efficiency (not less than 85%). Gluconic acid, malic acid, citric acid, and phosphoric acid were not suitable anions for providing a gradient in this composition.

[0117] Table 6 Treprostinil liposome compositions with different types of internal aqueous acid

[0118] Note: The drug-loaded salt cation is MEG (0.30 M); the particle size of the above liposomes ranges from 100 to 150 nm.

[0119] Example 7 Liposome pharmaceutical compositions of different weakly acidic drug types

[0120] The liposome compositions prepared according to the above “general experimental procedures” were loaded with different drugs, and the liposome compositions were analyzed according to the above general analytical methods.

[0121] Results: As can be seen from the table, the liposome compositions loaded with different drugs all have high drug loading (not less than 18%).

[0122] Table 7 Liposome pharmaceutical compositions of different weakly acidic drugs

[0123] Note: The drug-loaded salt cation is MEG (0.30 M); the drug-loaded salt anion is HAc (0.29 M); the theoretical feed ratio is 22% (error within ±5%); the particle size range of the liposome is between 100 and 150 nm.

[0124] Example 8 Treprostinil liposome pharmaceutical composition

[0125] The liposome composition prepared according to the above “General Experimental Procedure” was loaded with treprostinil sodium, and the liposome composition was analyzed according to the above general analytical method.

[0126] Results: As can be seen from the table, the liposome composition loaded with treprostinil has a high drug loading capacity and high encapsulation efficiency.

[0127] Table 8 Treprostinil liposome pharmaceutical composition

[0128] Note: The drug-loaded salt cation is MEG (0.30M); the drug-loaded salt anion is HAc (0.29M);

[0129] Example 9. Treprostinil liposome compositions with different phospholipid concentrations

[0130] According to the above "general experimental procedure", the feed ratio was kept constant and the feed was added at different phospholipid concentrations to prepare treprostinil liposome compositions. The liposome compositions were analyzed according to the above general analytical methods.

[0131] Results: As can be seen from the table, when the theoretical feed ratio is as high as 22%, the drug-loaded composition, the treprostinil liposomes prepared with a phospholipid concentration in the range of 1.4 to 4.18 mg / ml have a high drug loading capacity (not less than 18%).

[0132] Table 9 Treprostinil liposome compositions with different phospholipid concentrations

[0133] Note: The drug-loaded salt cation is MEG (0.30 M); the drug-loaded salt anion is HAc (0.29 M); the theoretical feed ratio is 22% (error within ±5%); the particle size range of the liposome is between 100 and 150 nm.

[0134] The above is an exemplary description of the implementation methods of the technical solution of the present invention. It should be understood that the scope of protection of the present invention is not limited to the above implementation methods. Any modifications, equivalent substitutions, improvements, etc. made by those skilled in the art within the spirit and principles of the present invention shall be included in the scope of protection of the claims of this application.

Claims

1. A liposome comprising a phospholipid and an internal aqueous phase; the internal aqueous phase comprises meglumine and acetic acid; and the pH of the internal aqueous phase is 4.0 to 10.

5.

2. The liposome according to claim 1, wherein The pH of the inner aqueous phase is 4.5-10.0, 6.5-10.5, such as 4.97-9.52, preferably 8.

5.

3. The liposome according to claim 1 or 2, characterized in that The concentration of meglumine in the internal aqueous phase is 0.1 to 0.8 M, for example 0.2 to 0.6 M, preferably 0.3 to 0.4 M; Preferably, the concentration of acetic acid in the inner aqueous phase is 0.1 to 0.8 M, for example 0.2 to 0.6 M; Preferably, the concentration of meglumine and acetic acid in the internal aqueous phase is 0.30-0.40M.

4. The liposome according to any one of claims 1 to 3, characterized in that The phospholipid has a bilayer lipid structure, and the bilayer lipid is composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), with a molar ratio of 3:(1-3):(0.025-0.225), for example, 3:(2-3):(0.025-0.075), 3:(1.5-2.5):(0.025-0.075); preferably 3:2:(0.025-0.075); Preferably, the concentration of the phospholipid in the liposome composition is 1 to 100 mg / mL, for example, 1 to 50 mg / mL, 1 to 20 mg / mL; Preferably, the particle size of the liposome is 50 to 500 nm, such as 100 to 200 nm, preferably 100 to 150 nm, 140 to 190 nm.

5. A liposome composition, characterized in that The liposome composition comprises the liposome according to any one of claims 1 to 4 and an external aqueous phase; the external aqueous phase is a buffer solution with a pH of 4.5 to 6.

5.

6. The liposome composition according to claim 5, characterized in that The buffer is selected from one or more of HEPES, sodium chloride, sucrose, citrate buffer, phosphate buffer, and Tris buffer, preferably citrate buffer; the concentration of the buffer is 0.05M to 0.25M, for example, 0.05M to 0.2M.

7. The method for preparing the liposome according to any one of claims 1 to 4, comprising the steps of: (1) Preparation of oil phase: Weigh hydrogenated soybean lecithin, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and cholesterol in parts by weight and dissolve them in ethanol to obtain an oil phase; (2) Preparation of aqueous phase: After dissolving meglumine in water, acetic acid was added to adjust the pH to obtain an aqueous phase, i.e., the inner aqueous phase; (3) Emulsification-extrusion: the oil phase and the aqueous phase are mixed to obtain colostrum, and the colostrum is further extruded through a polycarbonate membrane to obtain the liposome; Preferably, the preparation method further comprises the step of ultrafiltration: the liposomes obtained in step (3) are ultrafiltered with citric acid-sodium citrate buffer to replace the liposome external phase medium and remove the organic solvent to obtain the liposomes.

8. A method for preparing the liposome composition according to claim 5 or 6, comprising dispersing the liposome according to any one of claims 1 to 4 in an external aqueous phase to obtain the liposome composition.

9. Use of the liposome according to any one of claims 1 to 4 or the liposome composition according to claim 5 or 6 as a drug carrier; Preferably, the drug is a weakly acidic drug. ; Preferably, the pKa of the weakly acidic drug is between 2 and 7; Preferably, the weakly acidic drug is selected from at least one drug selected from the group consisting of prostaglandins, antipyretics and analgesics, quinoline carboxylic acid antibiotics, and interferon gene stimulator receptors.

10. The use according to claim 9, characterized in that The prostaglandin drugs such as treprostinil, beraprost, iloprost, carboprost, limaprost, epoprostenol, alprostadil, unoprostone, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); the antipyretic and analgesic drugs such as aspirin, ibuprofen, naproxen, diclofenac sodium, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); the quinoline carboxylic acid antibacterial drugs such as nalidixic acid, pyrrolopyrimic acid, fluoroquinolinic acid, sitafloxacin, ciprofloxacin, enoxacin, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); the STING drugs such as MSA-2, STING agonist-7, Vadimezan, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs).