Liposome pharmaceutical composition 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 medicine lipidosome, namely the treprostinil lipidosome prepared from the lipidosome has a relatively good curative effect on treating peripheral artery occlusion diseases or pulmonary arterial hypertension.

Description

A liposome pharmaceutical composition 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 202311236761.2, filed with the State Intellectual Property Office of China on September 22, 2023, entitled “A liposomal pharmaceutical composition and its application”;

[0004] Priority to the prior application, patent application number 202311260280.5, filed with the State Intellectual Property Office of China on September 26, 2023, entitled “A liposomal pharmaceutical composition, its preparation method and application”;

[0005] The entire contents of said prior application are incorporated into the present application by reference. Technical Field

[0006] The present invention belongs to the field of medicine, and in particular relates to a liposome pharmaceutical composition, a preparation method and an application thereof. Background Art

[0007] 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.

[0008] 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.

[0009] In recent years, many researchers have effectively loaded weakly basic drugs into the liposome aqueous phase by remote loading technology (for example, pH gradient method or ammonium sulfate gradient method), and improved the encapsulation efficiency of their drugs. However, the research on weakly acidic drug liposomes is relatively less. The development of weakly acidic drug liposome preparations still has many unresolved problems, such as the loading capacity of weakly acidic drugs is not high or the encapsulation efficiency is not good, and the technology of the present invention has further improved the drug loading capacity, so that in clinical application, patient compliance and therapeutic effect can be improved.

[0010] Summary of the Invention

[0011] In order to improve the above technical problems, the present invention is implemented through the following technical solutions:

[0012] The present invention provides a liposome pharmaceutical composition comprising a weakly acidic drug and liposomes; the liposomes are one of the following:

[0013] (1) The liposome comprises phospholipids and an internal aqueous phase; or

[0014] (2) The liposome comprises phospholipids, an inner aqueous phase and an outer aqueous phase;

[0015] wherein the internal aqueous phase comprises meglumine and a weak acid;

[0016] The external aqueous phase suspends the liposomes.

[0017] 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.

[0018] According to an embodiment of the present invention, the pKa of the weakly acidic drug is between 2 and 7.

[0019] According to an embodiment of the present invention, the drug loading of the weakly acidic drug is not less than 18%, for example, 18% to 40%, and can also be 19%-30%, or 20-25%, such as 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.3%, 23.5% or 35%.

[0020] According to an embodiment of the present invention, the encapsulation efficiency of the drug liposome is not less than 85%, preferably not less than 90%. According to an embodiment of the present invention, when the drug loading of the weakly acidic drug is less than 18%, the encapsulation efficiency of the drug liposome is preferably not less than 85%, preferably not less than 90%. More preferably, the drug loading of the drug liposome is not less than 18%, and the encapsulation efficiency of the drug liposome is not less than 85%, preferably not less than 90%.

[0021] 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;

[0022] According to an embodiment of the present invention, the prostaglandin drugs are 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 are such as aspirin, ibuprofen, naproxen, diclofenac sodium, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); the quinoline carboxylic acid antibacterial drugs are 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 are such as MSA-2, STING agonist-7, Vadimezan, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs).

[0023] According to an embodiment of the present invention, the pH of the internal aqueous phase is 4.0 to 10.5, such as 4.5 to 10.0, 6.5 to 10.5, preferably 4.97 to 9.52;

[0024] 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.

[0025] 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 weak acid provides the anionic portion of the internal aqueous phase.

[0026] According to an embodiment of the present invention, the weak acid is selected from "carboxylic acids", for example, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, lactic acid or a combination thereof. Preferably, formic acid, acetic acid, propionic acid or a combination thereof.

[0027] Different from the contents disclosed in the prior art, the internal aqueous phase in the embodiment of the present invention does not contain carbonate or bicarbonate, but the internal aqueous phase system is realized by meglumine and weak acid.

[0028] 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;

[0029] According to an embodiment of the present invention, the concentration of the weak 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;

[0030] According to an embodiment of the present invention, the meglumine and the weak acid are meglumine and acetic acid; preferably, the concentration of the meglumine and acetic acid in the internal aqueous phase is 0.30-0.40M.

[0031] 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).

[0032] 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).

[0033] 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 liposome pharmaceutical composition of the present invention, the concentration of the phospholipid used 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.

[0034] 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.

[0035] 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.

[0036] 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, 0.08M to 0.2M, and preferably 0.09M to 0.12M.

[0037] 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, wherein the pH value of the outer aqueous phase is higher than the pKa of the weak acid drug.

[0038] The present invention also provides a treprostinil liposome based on the liposome pharmaceutical composition, wherein the weakly acidic drug in the liposome pharmaceutical composition is treprostinil or a derivative thereof, the treprostinil or the derivative thereof is encapsulated in the inner aqueous phase of the liposome, and the inner aqueous phase contains meglumine;

[0039] According to an embodiment of the present invention, the derivative of treprostinil is selected from hydrates, solvates or complexes of treprostinil; or is selected from pharmaceutically acceptable salts, esters or prodrugs thereof of treprostinil, such as treprostinil sodium salt, treprostinil potassium salt, treprostinil diethanolamine salt, treprostinil methyl ester, treprostinil ethyl ester, and treprostinil fumaryl diketopiperidine prodrug.

[0040] According to an embodiment of the present invention, the drug loading of treprostinil or its derivative is not less than 18%. When it is lower than the maximum drug loading (18%), the encapsulation efficiency is not less than 85%, preferably not less than 90%.

[0041] The molecular structure of treprostinil is as follows:

[0042] The present invention also provides the use of the liposome of the treprostinil or its derivatives in preparing medicines.

[0043] According to an embodiment of the present invention, the drug is a drug for treating pulmonary hypertension, pulmonary hypertension, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease, asthma, ischemic disease, heart failure, arteriosclerosis, postoperative anticoagulation, central retinal vein occlusion, thrombotic microangiopathy, peripheral vascular disease, heart and lung transplantation and the like.

[0044] According to an embodiment of the present invention, the drug is a drug for treating peripheral arterial occlusive disease or pulmonary hypertension.

[0045] The present invention also provides a method for treating peripheral arterial occlusive disease or pulmonary hypertension using the liposomes of treprostinil or its derivatives, comprising administering a therapeutically effective amount of the liposomes of treprostinil or its derivatives to a patient. The present invention also provides liposomes of treprostinil or its derivatives for treating peripheral arterial occlusive disease or pulmonary hypertension.

[0046] The present invention also provides a method for preparing a liposome pharmaceutical composition, the method comprising: loading liposomes with at least one weakly acidic drug, preferably, the loading amount of the weakly acidic drug is not less than 18%;

[0047] According to an embodiment of the present invention, the method for preparing the liposome comprises the following steps:

[0048] (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;

[0049] (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;

[0050] (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.

[0051] According to an embodiment of the present invention, the molar ratio of hydrogenated soy lecithin (HSPC), cholesterol (CHOL) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000) is 3:2:(0.025-0.225), for example 3:2:(0.025-0.15), preferably 3:2:(0.025-0.075).

[0052] According to an embodiment of the present invention, the method for preparing liposomes 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.

[0053] According to an embodiment of the present invention, when the liposomes include an external aqueous phase, the method for preparing the liposomes further comprises dispersing the liposomes in the external aqueous phase to obtain liposomes containing the external aqueous phase;

[0054] 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;

[0055] 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.

[0056] According to an embodiment of the present invention, the preparation method of the liposome pharmaceutical composition comprises the following steps: dissolving a weakly acidic drug in an external aqueous phase and mixing the mixture with the liposome solution to obtain the liposome pharmaceutical composition; wherein the concentration of the liposome is 1.0 to 20 mg / ml, preferably 1.0 to 5.0 mg / ml (calculated as phospholipid concentration).

[0057] According to an embodiment of the present invention, the method for preparing the liposome pharmaceutical composition comprises the following steps:

[0058] (1) dissolving the weakly acidic drug in a sodium citrate aqueous solution to obtain a drug solution;

[0059] (2) mixing the liposome solution with a citrate buffer solution to obtain a carrier solution;

[0060] (3) adding the drug solution into the carrier solution for loading to obtain the liposome drug composition. Beneficial effects

[0061] The drug liposomes provided by the present invention have a high drug loading capacity or a high encapsulation efficiency. Specifically, the drug liposomes have a high drug loading capacity (maximum drug loading capacity of not less than 18%); when the drug liposomes are below the maximum drug loading capacity (18%), the encapsulation efficiency is not less than 85%, preferably not less than 90%. Preferably, the drug liposomes of the present invention also have the effects of a high drug loading capacity (e.g., not less than 18%) and a high encapsulation efficiency (e.g., not less than 85%, preferably not less than 90%). DETAILED DESCRIPTION

[0062] 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.

[0063] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0064] 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:

[0065] Encapsulation efficiency EE%=(1–W f / W t )×100% (Formula 1)

[0066] 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.

[0067] 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)

[0068] 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.

[0069] 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);

[0070] 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.

[0071] General experimental procedures

[0072] 1. Preparation of Treprostinil Liposome Composition

[0073] Example 1: Preparation of Treprostinil Liposomes

[0074] (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 the internal aqueous phase (such as meglumine solution or sodium bicarbonate solution, taking 0.3M, pH = 8.5 sodium bicarbonate solution as the internal aqueous phase as an example, 86.7g of sodium bicarbonate solution is injected), and stirred and hydrated at 60-65°C for 20 minutes; after the emulsification is completed, the colostrum is extruded through a small-scale liposome extruder at 60-65°C, and extruded through a 200nm and / or 100nm polycarbonate membrane at a pressure of 0.6-1.0MPa multiple times to make the sample particle size 100-200nm and PDI less than 0.2. Then, the sample is cooled to room temperature, and the liposome external phase medium is replaced with 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.

[0075] (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.

[0076] General analytical methods

[0077] 1. Quantitative Characterization of Treprostinil Liposomal Composition

[0078] a. Preparation of reference solution:

[0079] 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.

[0080] b. Free and total treprostinil concentrations:

[0081] 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.

[0082] 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.

[0083] The specific HPLC chromatographic conditions are as follows:

[0084] c. Encapsulation efficiency (EE) and drug loading (LE):

[0085] 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:

[0086] Encapsulation efficiency

[0087] 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.

[0088] The concentration of the liposomal drug composition was calculated using the following formula 3:

[0089] 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.

[0090] d. Average particle size and polydispersity index (PdI):

[0091] 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).

[0092] 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.

[0093] Example 1. Treprostinil liposome compositions with different drug-loaded salt types and different feed ratios

[0094] 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.

[0095] 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".

[0096] 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%.

[0097] 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.

[0098] Table 1 Treprostinil liposome compositions prepared with different drug-loaded salts 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.

[0099] Example 2. Treprostinil liposome compositions containing meglumine-acetic acid drug-loaded salts of different particle sizes

[0100] 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.

[0101] 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.

[0102] Table 2 Treprostinil compositions of different particle sizes 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%);

[0103] Example 3 Treprostinil liposome compositions with different internal aqueous phase concentrations

[0104] 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.

[0105] 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%).

[0106] Table 3-1 High theoretical feed ratio - Treprostinil liposome composition with different internal aqueous phase (meglumine) concentrations 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;

[0107] Table 3-2 Low theoretical feed ratio - Treprostinil liposome composition with different internal aqueous phase (meglumine) concentrations 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;

[0108] Example 4 Treprostinil liposome compositions with different phospholipid prescriptions

[0109] 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.

[0110] 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%).

[0111] Table 4-1 Treprostinil liposome compositions with different phospholipid formulations 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.

[0112] Table 4-2 Treprostinil liposome compositions with different phospholipid prescriptions 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.

[0113] Example 5 Treprostinil liposome compositions with different internal aqueous phase pH

[0114] 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.

[0115] 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%).

[0116] Table 5 Treprostinil liposome compositions with different internal aqueous phase pH 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;

[0117] Example 6 Treprostinil liposome compositions with different internal aqueous phase anions

[0118] 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.

[0119] 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.

[0120] Table 6 Treprostinil liposome compositions with different types of internal aqueous acid Note: The drug-loaded salt cation is MEG (0.30 M); the particle size of the above liposomes ranges from 100 to 150 nm.

[0121] Example 7 Liposome pharmaceutical compositions of different weakly acidic drug types

[0122] 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.

[0123] Results: As can be seen from the table, the liposome compositions loaded with different drugs all have high drug loading (not less than 18%).

[0124] Table 7 Liposome pharmaceutical compositions of different weakly acidic drugs 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.

[0125] Example 8 Treprostinil liposome pharmaceutical composition

[0126] 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.

[0127] Results: As can be seen from the table, the liposome composition loaded with treprostinil has a high drug loading capacity and high encapsulation efficiency.

[0128] Table 8 Treprostinil liposome pharmaceutical composition Note: The cation of the drug-loaded salt is MEG (0.30 M); the anion of the drug-loaded salt is HAc (0.29 M);

[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 22%, the drug-loaded composition with a phospholipid concentration in the range of 1.4 to 4.18 mg / ml has a high drug loading capacity (not less than 18%).

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

[0133] 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 pharmaceutical composition comprising a weakly acidic drug and liposomes; The liposome is one of the following: (1) The liposome comprises phospholipids and an inner aqueous phase; (2) The liposome comprises phospholipids, an inner aqueous phase and an outer aqueous phase; in, The internal aqueous phase comprises meglumine and a weak acid, and the pH of the internal aqueous phase is 4.0 to 10.5, for example, 4.5 to 10.0, 6.5 to 10.5, preferably 4.97 to 9.52; The external aqueous phase suspends the liposomes; 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.

2. The liposome pharmaceutical composition according to claim 1, wherein The pKa of the weakly acidic drug is between 2 and 7; Preferably, the weakly acidic drug is selected from the group consisting of: prostaglandins, antipyretics, quinoline carboxylic acid antibiotics, interferon gene stimulator (STING) receptors, or at least one drug thereof (such as pharmaceutically acceptable salts, esters, or prodrugs); Preferably, the prostaglandin drug is selected from treprostinil, beraprost, iloprost, carboprost, limaprost, epoprostenol, alprostadil, unoprostone, or derivatives thereof; the antipyretic and analgesic drug is selected from aspirin, ibuprofen, naproxen, diclofenac sodium, or derivatives thereof; the quinoline carboxylic acid antibacterial drug is selected from nalidixic acid, pyroximic acid, fluoroquinolinic acid, sitafloxacin, ciprofloxacin, enoxacin, or derivatives thereof; the STING drug is selected from MSA-2, STING agonist-7, Vadimezan, or derivatives thereof; Preferably, the drug loading of the drug liposome is less than 18%, and the encapsulation efficiency is not less than 85%, preferably not less than 90%; Preferably, the drug loading of the weakly acidic drug is not less than 18% and not more than 40%.

3. The liposome pharmaceutical composition according to claim 1 or 2, characterized in that The weak acid is selected from carboxylic acids, for example, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, lactic acid or a combination thereof; preferably formic acid, acetic acid, propionic acid or a combination thereof; Preferably, 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; Preferably, the concentration of the weak acid in the inner aqueous phase is 0.1-0.8M, such as 0.2-0.6M.

4. The liposome pharmaceutical composition according to any one of claims 1 to 3, characterized in that The meglumine and the weak acid are meglumine and acetic acid; preferably, the concentration of the meglumine and acetic acid in the internal aqueous phase is 0.30-0.40M.

5. The liposome pharmaceutical composition according to any one of claims 1 to 4, characterized in that The phospholipid has a bilayer lipid structure, and the bilayer lipid is composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), with a molar ratio of 3:(1-3):(0.025-0.225), preferably 3:2:(0.025-0.150); Preferably, the concentration of the phospholipid in the liposome composition is 1-100 mg / mL, such as 1-50 mg / mL, 1-20 mg / mL.

6. The liposome pharmaceutical composition according to any one of claims 1 to 5, characterized in that The particle size of the liposome is 50 to 500 nm, for example, 100 to 200 nm; Preferably, 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.

7. The liposome pharmaceutical composition according to any one of claims 1 to 6, characterized in that The external aqueous phase is a buffer solution with a pH of 4.5 to 6.5; Preferably, 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, 0.08M to 0.2M, preferably 0.09 to 0.12M.

8. A treprostinil liposome based on the liposome pharmaceutical composition according to any one of claims 1 to 7, wherein: The weakly acidic drug in the liposome pharmaceutical composition is treprostinil or a derivative thereof, and the treprostinil or a derivative thereof is encapsulated in the inner aqueous phase of the liposome; Preferably, the derivative of treprostinil is selected from hydrates, solvates or complexes of treprostinil, or pharmaceutically acceptable salts, esters or prodrugs thereof of treprostinil, such as treprostinil sodium salt, treprostinil potassium salt, treprostinil diethanolamine salt, treprostinil methyl ester, treprostinil ethyl ester, and treprostinil fumaryl diketopiperidine prodrug.

9. Use of the treprostinil liposome or its derivatives according to claim 8 in the preparation of medicines; Preferably, the drug is a drug for treating pulmonary hypertension, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease, asthma, ischemic disease, heart failure, arteriosclerosis, postoperative anticoagulation, central retinal vein occlusion, thrombotic microangiopathy, peripheral vascular disease, heart and lung transplantation, and the like; Preferably, the drug is a drug for treating peripheral arterial occlusive disease or pulmonary hypertension.

10. A method for preparing the liposome pharmaceutical composition according to any one of claims 1 to 7, comprising loading the liposomes with at least one weakly acidic drug; Preferably, the drug loading of the weakly acidic drug is not less than 18%; Preferably, the method for preparing the liposome pharmaceutical composition comprises the following steps: dissolving a weakly acidic drug in the external aqueous phase and mixing the mixture with the liposome solution to obtain the liposome pharmaceutical composition; The concentration of the liposome is 1.0 to 20 mg / ml, preferably 1.0 to 5.0 mg / ml (calculated as phospholipid concentration); Preferably, the method for preparing the liposome pharmaceutical composition comprises the following steps: (1) dissolving the weakly acidic drug in a sodium citrate aqueous solution to obtain a drug solution; (2) mixing the liposome solution with a citrate buffer solution to obtain a carrier solution; (3) adding the drug solution into the carrier solution for loading to obtain the liposome drug composition.