TW202415382A - Liposome drug composition, preparation method and use thereof - Google Patents

Abstract

The present disclosure provides a liposome and its composition, when used as a drug carrier, through a specific drug loading condition process can obtain (1) a high drug loading amount (the maximum drug loading amount is not less than 18%); (2) when it is below the maximum drug loading (18%), the encapsulation rate shall not be less than 85%. The present disclosure also provides a preparation method for the liposome and its composition, as well as its use as a drug carrier. The treprostinil liposome prepared from the present disclosure have good therapeutic effects on treating peripheral arterial occlusive diseases or pulmonary hypertension.

Description

Liposome drug composition and preparation method and use thereof

The present invention claims: The priority of the prior application filed with the National Intellectual Property Administration of China on September 30, 2022, with patent application number 202211216387.5, entitled "A liposome, drug composition, preparation method and application thereof"; The priority of the prior application filed with the National Intellectual Property Administration of China on September 22, 2023, with patent application number 202311236761.2, entitled "A liposome drug composition and application thereof"; The priority of the prior application filed with the National Intellectual Property Administration of China on September 26, 2023, with patent application number 202311260280.5, entitled "A liposome drug composition, preparation method and application thereof"; The full text of the prior application is incorporated into the present invention by reference.

The present invention belongs to the field of medicine, and specifically relates to a liposome drug composition and a preparation method and use thereof.

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 enhanced drug circulation time in the blood, reduced cytotoxicity, sustained release of drugs, and delivery of specific drugs to selected tissues. When using liposomes for the delivery of therapeutic drugs, high drug encapsulation efficiency and drug content are required to reduce the potential toxicity of phospholipid excipients.

The prior art of US8932627B2 and US20190022004A1 uses acetate or bicarbonate to load drugs in molecular form into liposomes. The drugs are stored in the inner aqueous phase in ionic form and the release rate of the drugs is controlled to treat respiratory diseases. However, the drug loading capacity of liposomes obtained by using prescriptions loaded with acetate or bicarbonate is limited. 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.

In recent years, many researchers have used remote loading techniques (e.g., pH gradient method or ammonium sulfate gradient method) to effectively load weak alkaline drugs into the aqueous phase of liposomes and improve their drug encapsulation efficiency. However, there are relatively few studies on weakly acidic drug liposomes. There are still many unresolved issues in the development of weakly acidic drug liposome preparations, such as low loading amount of weakly acidic drugs or poor encapsulation efficiency. The technology of the present invention further increases the drug loading amount, in order to improve patient compliance and therapeutic effects in clinical applications.

In order to improve the above technical problems, the present invention is implemented by the following technical solutions:

The present invention provides a liposome drug composition, comprising a weak acid drug and a liposome; the liposome is one of the following: (1) the liposome comprises phospholipids and an internal aqueous phase; or (2) the liposome comprises phospholipids, an internal aqueous phase and an external aqueous phase; wherein the internal aqueous phase comprises meglumine and a weak acid; the external aqueous phase suspends the liposome.

According to the implementation scheme 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.

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

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%.

According to the embodiment of the present invention, the encapsulation rate of the drug liposome is not less than 85%, preferably not less than 90%. According to the embodiment of the present invention, when the drug loading of the weakly acidic drug is less than 18%, the encapsulation rate 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 rate of the drug liposome is not less than 85%, preferably not less than 90%.

According to the implementation scheme of the present invention, the weakly acidic drug can be selected from: at least one drug in the group consisting of prostaglandins, antipyretics, analgesics, quinoline carboxylic acid antibiotics, and stimulator of interferon genes (STING) receptors; According to the 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, pyroximate, 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).

According to the embodiment of the present invention, the pH of the inner aqueous phase is 4.0-10.5, for example, 4.5-10.0, 6.5-10.5, preferably 4.97-9.52;

According to an embodiment of the present invention, the pH of the inner 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.

According to an embodiment of the present invention, the meglumine in the inner aqueous phase provides the cationic portion of the inner aqueous phase, and the weak acid provides the anionic portion of the inner aqueous phase.

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, it is formic acid, acetic acid, propionic acid or a combination thereof.

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

According to an embodiment of the present invention, the concentration of meglumine in the inner aqueous phase is 0.1-0.8 M, for example 0.2-0.6 M, preferably 0.3-0.4 M, such as 0.3 M, 0.35 M, 0.4 M;

According to an embodiment of the present invention, the concentration of the weak acid in the inner aqueous phase is 0.1-0.8 M, for example 0.2-0.6 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;

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 the acetic acid in the inner aqueous phase is 0.30-0.40 M.

According to an embodiment of the present invention, the phospholipid has a bilayer lipid structure, and the bilayer lipid is composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), and the molar ratio is 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).

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

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

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, 140-190 nm.

According to the implementation scheme of the present invention, the external aqueous phase is a buffer solution with a pH of 4.5 to 6.5.

According to the embodiment of the present invention, the buffer for achieving the corresponding pH value of the external water phase is well known in the art and is not particularly limited. The commonly used buffer in the art is selected from one or more of HEPES, sodium chloride, sucrose, citrate buffer, phosphate buffer, and Tris buffer. According to the preferred embodiment of the present invention, the buffer can be selected from citrate buffer; the concentration of the buffer is 0.05M~0.25M, for example, 0.08M~0.2M, preferably 0.09~0.12M.

According to an embodiment of the present invention, the liposome has a pH gradient property 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.

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

According to the 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 of treprostinil, such as treprostinil sodium salt, treprostinil potassium salt, treprostinil diethanolamine salt, treprostinil methyl ester, treprostinil ethyl ester, and treprostinil fumarate diketopyridine prodrug.

According to the implementation scheme of the present invention, the drug loading of treprostinil or its derivative is not less than 18%. When it is less than the maximum drug loading (18%), the encapsulation efficiency is not less than 85%, and preferably not less than 90%.

The molecular structure of Treprostinil is as follows:

The present invention also provides the use of the liposomes of treprostinil or its derivatives in the preparation of drugs.

According to the implementation scheme of the present invention, the drug is a drug for treating diseases such as pulmonary arterial 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, and heart and lung transplantation.

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

The present invention also provides a method for treating peripheral arterial occlusive disease or pulmonary artery 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 artery hypertension.

The present invention also provides a method for preparing a liposome drug composition, which comprises loading liposomes with at least one weakly acidic drug, preferably, the loading amount of the weakly acidic drug is not less than 18%.

According to the embodiment of the present invention, the method for preparing liposomes comprises the following steps: (1) Preparation of oil phase: weighing hydrogenated soybean lecithin, distearyl phosphatidylethanolamine-polyethylene glycol 2000 and cholesterol by weight and dissolving them in ethanol to obtain an oil phase; (2) Preparation of water phase: dissolving meglumine in water, adding acetic acid to adjust the pH, and obtaining a water phase, i.e., an inner water phase; (3) Emulsification-extrusion: mixing the oil phase and the water phase to obtain colostrum, and further extruding the colostrum through a polycarbonate membrane to obtain the liposomes.

According to an embodiment of the present invention, the molar ratio of hydrogenated soy lecithin (HSPC), cholesterol (CHOL) and distearyl 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).

According to the embodiment of the present invention, the method for preparing liposomes further comprises an ultrafiltration step: 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.

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;

According to the implementation scheme of the present invention, the external aqueous phase is a buffer solution with a pH of 4.5 to 6.5;

According to the embodiment of the present invention, the buffer for achieving the corresponding pH value of the external water phase is well known in the art and is not particularly limited. The commonly used buffer in the art 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 0.05 M to 0.25 M, for example, 0.08 M to 0.2 M.

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

According to the embodiment of the present invention, the preparation method of the liposome drug composition comprises the following steps: (1) dissolving the weak acid drug in a sodium citrate aqueous solution to obtain a drug solution; (2) mixing the liposome solution with a citrate buffer to obtain a carrier solution; (3) adding the drug solution to the carrier solution for loading to obtain the liposome drug composition.

Beneficial Effects

The drug liposomes provided by the present invention have a high drug loading or a high encapsulation rate. Among them, the drug liposomes have a high drug loading (the maximum drug loading is not less than 18%); when the drug liposomes are less than the maximum drug loading (18%), the encapsulation rate is not less than 85%, preferably not less than 90%. Preferably, the drug liposomes of the present invention also have the effects of high drug loading (for example, not less than 18%) and high encapsulation rate (for example, not less than 85%, preferably not less than 90%).

The following will further explain the technical solution of the present invention in combination with specific embodiments. It should be understood that the following embodiments are only for illustrative purposes to illustrate and explain the present invention, and should not be interpreted as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection intended by the present invention.

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

The drug loading (LE) of the present invention is expressed as a weight percentage. The definitions and explanations of encapsulation efficiency (EE) and drug loading (LE) are as follows: Encapsulation efficiency EE% = (1 – _Wf / _Wt ) × 100% (Formula 1) Wherein _Wf represents the amount of free drug in the external medium; _Wt represents the total amount of drug in the liposome suspension.

The drug loading of the liposome drug composition is calculated using the following formula 2: LE% = [W _e / W _m ] × 100% = [(W _t × EE%) / (W _l + W _t × EE%) ] × 100% (Formula 2) Where LE represents the percentage of drug loading in the liposome; W _e represents the amount of drug encapsulated in the liposome; W _m represents the total weight of the drug-loaded liposome (including the amount of liposome carrier and encapsulated drug). Among them, W _t is the total amount of drug in the liposome suspension; EE% is the encapsulation rate; W _l is the total lipid content of the liposome carrier.

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); nanolaser microscopy (malvern, ZS90); tangential flow ultrafiltration equipment (Millipore Pellicon);

Experimental materials: treprostinil sodium (Porton); hydrogenated soybean phosphatidylcholine (HSPC) (Lipoid); distearyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000) (Lipoid); cholesterol (CHOL) (Nippon Seika); sodium bicarbonate (NaHCO ₃ ), sodium acetate (NaAc), meglumine (MEG) (merck); other materials are commonly used injection-grade excipients.

General Experimental Procedure

1. Preparation of Treprostinil Liposomal Composition

Example 1: Preparation of Treprostinil Liposomes

(1) Preparation of blank liposomes: First, weigh 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)), add them into a clean and dry container, add 11.3 g of anhydrous ethanol, seal the container, and dissolve it 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 sodium bicarbonate solution with pH=8.5 as the internal aqueous phase as an example, 86.7 g of sodium bicarbonate solution is injected), and stirred and hydrated at 60~65°C for 20 min; after the emulsification is completed, the colostrum is extruded through a small-scale liposome extruder at 60~65°C, and extruded multiple times through a 200 nm and/or 100 nm polycarbonate membrane at a pressure of 0.6~1.0 MPa, so that the sample particle size is 100~200 nm and the PDI is less than 0.2. Then, the sample was cooled to room temperature, and the liposome external phase medium was replaced with 0.05 M citric acid-sodium citrate buffer at pH 5.5 using a tangential flow TFF filtration device. The blank liposomes after ultrafiltration were stored at 2-8°C for future use.

(2) Preparation of Treprostinil Liposomes: Dissolve Treprostinil sodium in 0.05 M sodium citrate solution, then add the Treprostinil sodium solution to the blank liposomes according to a certain theoretical dosage (for example, a theoretical drug loading of 22% (w/w)), add an appropriate amount of citric acid-sodium citrate buffer to adjust the concentration of citric acid-sodium citrate in the liposome external phase to 0.09-0.12 M, and incubate at 35-45°C for 30-60 min to obtain a Treprostinil liposome composition.

General analytical methods

1. Quantitative Characterization of Treprostinil Liposomal Composition

a. Preparation of reference solution:

Accurately weigh about 10 mg of treprostinil sodium bulk drug into a 50 ml volumetric flask, add 2.5 ml of purified water to dissolve, and dilute to the mark with methanol, shake well, and use this as the treprostinil sodium reference solution.

b. Free and total treprostinil concentration:

Accurately measure 1 ml of the liposome drug composition solution and place it in a 20 ml volumetric flask, add about 3 ml of methanol and shake gently, remove the bubbles generated, dilute to the mark with methanol, shake evenly, and obtain the solution. Take a sample for HPLC analysis to determine the total content of treprostinil in the composition.

Determination of free treprostinil: 1 ml of treprostinil liposomes was placed in an ultracentrifuge tube and centrifuged in an ultracentrifuge with the following parameters (temperature: 4°C, speed: 100,000 rpm, time: 30 min). After 30 min, the sample was taken out and the supernatant was analyzed by HPLC.

The specific HPLC column analysis conditions are as follows: Name String analysis conditions Column chromatography column Kromasil 10-5-C ₁₈ (250×4.6，5 μm) Column temperature 35℃ Wavelength 271 nm Flow rate 1.0 ml/min Injection volume 20 μl Injection chamber temperature 20℃ Mobile phase A 0.1% trifluoroacetic acid aqueous solution Mobile phase B 0.1% trifluoroacetic acid in acetonitrile Isocratic elution Time(minutes) Mobile phase A(%) Mobile phase B(%) 0 45 55 15 45 55

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

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:

Encapsulation efficiency EE% = (1 – C _f /C _t ) × 100% = (Formula 1-1) In the formula, _Cf represents the amount of free drug in the external medium; _Ct represents the total amount of drug in the liposome suspension; _Ac is the peak area of the total drug content; _Ab is the peak area of the free drug; a is the dilution multiple of the total drug content, 20.

The concentration of the liposomal drug composition was calculated using the following formula 3: (Formula 3) C is the concentration of the liposome composition, mg/ml; W _std is the sample weight of the reference substance, mg; A _std is the average peak area of the peak areas of five injections of the reference substance solution; T is the content of the main drug in the reference substance, %; D is the dilution volume of the reference substance solution; A _sample is the peak area of the liposome total drug content solution; a is the dilution volume of the liposome total drug content solution.

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

Take 30 µl of the treprostinil liposome composition, dilute it to 2 ml with purified water, mix it, and use the dynamic light scattering principle to detect the particle size distribution and polydispersity coefficient of the composition using a Marvin nanolaser microscopy instrument (parameters: temperature 25°C, refractive index 1.33, equilibrium time 120s, and 3 cycles).

The cations and anions described in the embodiments represent the material concentrations that provide the corresponding ions and are not the ion concentrations after the material is ionized.

Example 1. Treprostinil liposome compositions with different drug-carrying salt types and different feed ratios

In order to study the effect of different types of drug loading salts on the drug loading of treprostinil liposomes, treprostinil liposome compositions comprising different internal aqueous phases (sodium bicarbonate (NaHCO ₃ ), 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 section entitled “General Experimental Procedures”, which have specific treprostinil liposomes as shown in Table 1.

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 section entitled "General Analytical Methods".

From the results in the table below, it can be seen that due to the limited water volume in liposomes, as the feed amount increases, the drug loading increases first (from 8.22% to 19.72%), and then remains basically unchanged. Therefore, when the feed amount is 21%, the encapsulation rate is 87.55% and the drug loading is 19.72%. However, if the feed amount is increased again, the drug loading remains basically unchanged, which will lead to a significant decrease in the encapsulation rate. Secondly, at high feed rates, the maximum drug loading of the treprostinil liposomes of the meglumine prescription was 19.72%, while other prescriptions (such as sodium bicarbonate or sodium acetate, etc.) could not exceed 18%; at low feed rates, the encapsulation rate of the meglumine prescription could be no less than 85%, or even no less than 90%. For example, in Example C001, the drug loading was only 8.22%, but the encapsulation rate could reach 97.86%. Even at a drug loading of 17.39%, the encapsulation rates of other prescriptions were only 75.65%, and the encapsulation rates in most of the comparison examples were significantly lower than 60%.

It can also be seen from the table below that, compared with the comparative examples A001 or B001, both have the same feed ratio of 21%, but the encapsulation rate of the meglumine prescription is 87.55%, which is higher than 75.65% or 79.59% of sodium bicarbonate or sodium acetate, and the drug loading is 19.72%, which is also significantly higher than 17.39% or 17.84%. Similarly, under other conditions of the same feed ratio, the meglumine prescription of the present invention is also superior to the sodium bicarbonate or sodium acetate prescription.

Table 1 Treprostinil liposome compositions prepared with different drug loading salts No. Drug-loaded salts Theoretical feed ratio* (%) EE (%) Drug loading(%) Cation Anions Comparison Example Comparison Example A001 Na+ (0.4M) HCO3 – (0.4M) twenty one 75.65 17.39 Comparison Example A002 27 53.82 16.66 Comparison Example A003 32 37.49 14.75 Comparison Example A004 36 32.70 15.53 Comparison Example B001 Na+ (0.3M) Ac- (0.3M) twenty one 79.59 17.84 Comparison Example B002 27 57.02 17.52 Comparison Example B003 32 45.37 17.53 Comparison Example B004 36 36.41 16.87 Embodiment Embodiment C001 MEG (0.3M) HAc (0.29M) 8 97.86 8.22 Embodiment C002 15 96.54 14.79 Embodiment C003 twenty one 87.55 19.72 Embodiment C004 27 64.30 18.84 Embodiment C005 32 52.26 19.14 Embodiment C006 36 42.99 18.83 Note: *Theoretical feed ratio indicates the theoretical drug loading (within ±5%). The particle size range of liposomes is between 100 and 150 nm.

Example 2. Treprostinil liposome compositions containing meglumine-acetic acid salt with different particle sizes

Treprostinil liposome compositions containing different particle sizes (100-150 nm, 150-200 nm) were prepared according to the above-mentioned "General Experimental Procedure". The liposome compositions were analyzed according to the above-mentioned general analytical method.

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 140-190 nm, the encapsulation efficiency and drug loading of the treprostinil liposomes are comparable.

Table 2 Treprostinil compositions with different particle sizes No. Size (nm) EE (%) Drug loading(%) Embodiment Embodiment C007 127.5 79.58 18.28 Embodiment C008 135.0 84.36 18.44 Embodiment C003 141.9 87.55 19.72 Embodiment C009 149.1 86.69 19.25 Embodiment C010 189.4 86.69 19.76 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); the theoretical feed ratio is 21% (within ±5%).

Example 3 Treprostinil liposome compositions with different internal aqueous phase concentrations

Treprostinil liposome compositions containing different concentrations of meglumine-acetic acid (meglumine concentration was 0.20 ~ 0.60 M, pH was 8.5) were prepared according to the above-mentioned "General Experimental Procedure". The liposome compositions were analyzed according to the above-mentioned general analytical method.

Results: It can be seen from the table that when the theoretical feed ratio is as high as 21%, the treprostinil liposomes prepared when the aqueous phase concentration is 0.30~0.40 M have a high drug loading (not less than 18%); when the theoretical feed ratio is lower than 18%, the treprostinil liposomes prepared when the aqueous phase concentration is between 0.30~0.40 M can obtain a high encapsulation efficiency (encapsulation efficiency is not less than 90%).

Table 3-1 High theoretical feed ratio - Treprostinil liposome compositions with different internal aqueous phase (meglumine) concentrations No. Drug-loaded salt cation MEG concentration (M) EE (%) Drug loading(%) Embodiment Embodiment C003 0.30 87.55 19.72 Example C011 0.35 82.49 18.92 Embodiment C012 0.40 78.37 18.32 Comparison Example Comparison Example C013 0.20 52.76 13.04 Comparative Example C014 0.25 72.87 17.13 Note: The drug-carrying salt anion is HAc; the theoretical feed ratio is 21% (error within ±5%); the particle size range of liposomes is between 100 and 150 nm.

Table 3-2 Low theoretical feed ratio - Treprostinil liposome composition with different internal aqueous phase (meglumine) concentrations No. Drug loading salt cation concentration (M) EE (%) Drug loading(%) Embodiment Example C015 0.30 96.54 14.79 Embodiment C016 0.35 95.82 15.28 Embodiment C017 0.40 95.69 15.05 Note: The drug-carrying salt anion is HAc; the theoretical feed ratio is 15% (error within ±5%); the particle size range of liposomes is between 100~150 nm.

Example 4 Treprostinil liposome compositions with different phospholipid prescriptions

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

Results: It can be seen from the table that 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 (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 (not less than 18%) and a high encapsulation efficiency (not less than 85%).

Table 4-1 Treprostinil liposome compositions with different phospholipid prescriptions No. Phospholipid formula and composition molar ratio (HSPC/CHOL/DSPE-mPEG2000) EE (%) Drug loading(%) Embodiment Embodiment C018 3 / 2 / 0.025 79.44 19.02 Embodiment C019 3 / 2 / 0.050 82.68 19.45 Embodiment C003 3 / 2 / 0.075 87.55 19.72 Embodiment C020 3 / 2 / 0.150 81.71 18.32 Comparison Example Comparison Example C021 3 / 2 / 0.225 82.41 16.94 Notes: The drug-carrying salt cation is MEG (0.30 M); the drug-carrying salt anion is HAc (0.29 M); the theoretical feed ratio is 21% (error within ±5%); the particle size range of liposomes is between 100 and 150 nm.

Table 4-2 Treprostinil liposome compositions with different phospholipid prescriptions No. Phospholipid formula and composition molar ratio (HSPC/CHOL/DSPE-mPEG2000) EE (%) Drug loading(%) Comparison Example Comparative Example C022 3 / 1 / 0.075 63.26 16.29 Comparative Example C023 3 / 1.5 / 0.075 58.46 14.93 Embodiment Embodiment C003 3 / 2 / 0.075 87.55 19.72 Example C024 3 / 2.5 / 0.075 87.96 18.51 Example C025 3 / 3 / 0.075 88.72 18.50 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.

Example 5 Treprostinil liposome compositions with different internal aqueous phase pH

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 prescriptions. The liposome compositions were analyzed according to the above general analytical method.

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

Table 5 Treprostinil liposome compositions with different internal aqueous phase pH No. Anions and pH EE (%) Drug loading(%) Acetic acid(M) pH Embodiment Example C026 0.40 4.97 81.17 19.87 Example C027 0.35 5.26 80.23 19.63 Example C028 0.30 6.50 81.40 19.52 Example C029 0.29 8.55 86.91 19.36 Example C030 0.27 9.01 85.22 19.19 Example C031 0.21 9.52 85.42 19.08 Note: The drug-carrying salt cation is MEG (0.30 M); the theoretical feed ratio is 22.3% (error within ±5%); the particle size range is 100~200 nm.

Example 6 Treprostinil liposome compositions with different inner aqueous phase anions

Treprostinil liposome compositions with different inner aqueous phase anion formulations were prepared according to the above-mentioned "General Experimental Procedures". The liposome compositions were analyzed according to the above-mentioned general analytical methods.

Results: As can be seen from the table, when the pH of the inner aqueous phase is kept at 8.5 and acetic acid is used as the inner aqueous phase anion, the prepared treprostinil liposome has a high drug loading (not less than 18%); when valeric acid and lactic acid are used as the inner aqueous phase anions, the prepared treprostinil liposome can obtain a high encapsulation rate (encapsulation rate not less than 85%) when the theoretical feed ratio is lower than 18%. Gluconic acid, malic acid, citric acid, and phosphoric acid are not suitable as anions for providing gradients in this composition.

Table 6 Treprostinil liposome compositions with different types of aqueous acid No. Anions Theoretical feed ratio (%) EE (%) Drug loading(%) Embodiment Embodiment C003 Acetic acid 21.9 87.55 19.72 Example C032 Valeric acid 13.9 94.06 13.18 Example C033 Lactic acid 14.2 87.25 12.63 Comparison Example Comparative Example C034 Gluconic acid 1.1 14.91 0.17 Comparative Example C035 Apple acid 1.1 25.22 0.29 Comparative Example C036 Citric Acid 1.1 20.99 0.23 Comparative Example C037 Phosphoric acid 5.3 41.39 2.28 Note: The drug-carrying salt cation is MEG (0.30 M); the particle size of the above liposomes ranges from 100 to 150 nm.

Example 7 Liposomal drug compositions of different weakly acidic drug types

The liposome compositions prepared according to the above-mentioned "General Experimental Procedure" were loaded with different drugs, and the liposome compositions were analyzed according to the above-mentioned general analytical method.

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

Table 7 Liposomal drug compositions of different weakly acidic drugs No. Drugs EE (%) Drug loading(%) Embodiment Embodiment C003 Treprostinil sodium 87.55 19.72 Example C038 Beraprost Sodium 81.42 18.72 Notes: The drug-carrying salt cation is MEG (0.30 M); the drug-carrying salt anion is HAc (0.29 M); the theoretical feed ratio is 22% (error within ±5%); the particle size range of liposomes is between 100 and 150 nm.

Example 8 Treprostinil liposomal drug composition

The liposome composition prepared according to the above-mentioned "General Experimental Procedure" was loaded with treprostinil sodium, and the liposome composition was analyzed according to the above-mentioned general analytical method.

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

Table 8 Treprostinil liposomal drug composition No. Theoretical feed ratio (%) Particle size (nm) EE (%) Drug loading(%) Embodiment Embodiment C003 twenty one 141.9 87.55 19.72 Embodiment C039 twenty two 136.3 91.13 20.37 Example C040 29 182.8 72.63 23.32 Example C041 20 138.6 90.50 18.00 Note: The cation of the drug loading salt is MEG (0.30 M); the anion of the drug loading salt is HAc (0.29 M).

Example 9 Treprostinil liposome compositions with different phospholipid concentrations

According to the above-mentioned "general experimental procedure", the feed ratio was kept consistent, and the treprostinil liposome composition was prepared according to different phospholipid concentrations. The liposome composition was analyzed according to the above-mentioned general analytical method.

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

Table 9 Treprostinil liposome compositions with different phospholipid concentrations No. Feed phospholipids (mg/ml) EE (%) Drug loading(%) Embodiment Example C042 4.18 76.57 18.05 Embodiment C003 2.09 87.55 19.72 Example C043 1.40 82.32 18.88 Comparison Example Comparative Example C044 8.35 59.04 14.38 Comparative Example C045 6.26 66.12 15.51 Comparative Example C046 1.04 76.71 17.66 Notes: The drug-carrying salt cation is MEG (0.30 M); the drug-carrying salt anion is HAc (0.29 M); the theoretical feed ratio is 22% (error within ±5%); the particle size range of liposomes is between 100 and 150 nm.

The above is an exemplary description of the implementation of the technical solution of the present invention. It should be understood that the protection scope of the present invention is not limited to the above implementation. Any modification, equivalent replacement, improvement, etc. made by a person with ordinary knowledge in the technical field to which the present invention belongs within the spirit and principles of the present invention shall be included in the protection scope of the patent application of the present invention.

without

Claims (10)

A liposome drug composition, comprising a weak acid drug and liposomes; The liposomes are one of the following: (1) The liposomes comprise phospholipids and an internal aqueous phase; (2) The liposomes comprise phospholipids, an internal aqueous phase and an external aqueous phase; Wherein, the internal aqueous phase comprises meglumine and a weak acid, and 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; The external aqueous phase suspends the liposomes; The weak acid drug is encapsulated in the internal aqueous phase of the liposomes, and the liposomes encapsulating the weak acid drug are suspended in the external aqueous phase. The liposome drug composition as described in claim 1, wherein the pKa of the weak acid drug is between 2 and 7; Preferably, the weak acid drug is selected from: at least one drug selected from: prostaglandins, antipyretics, quinoline carboxylic acid antibiotics, interferon gene stimulator (STING) receptors, or their derivatives (such as pharmaceutically acceptable salts, esters or prodrugs); Preferably, the prostaglandin drugs are selected from treprostinil, beraprost, iloprost, carboprost, limaprost, epoprostenol, alprostadil, unoprostone, or their derivatives; the antipyretic and analgesic drugs are selected from aspirin, ibuprofen, naproxen, diclofenac sodium, or their derivatives; the quinoline carboxylic acid antibacterial drugs are selected from nalidixic acid, pyroximate, fluoroquinolinic acid, sitafloxacin, ciprofloxacin, enoxacin, or their derivatives; the STING drugs are selected from MSA-2, STING agonist-7, Vadimezan, or their derivatives; Preferably, the drug loading of the drug liposome is less than 18%, and its encapsulation rate 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%. The liposome drug composition as described in claim 1 or 2, wherein the weak acid is selected from carboxylic acids, such as 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 inner aqueous phase is 0.1~0.8 M, such as 0.2~0.6 M, preferably 0.3~0.4 M; Preferably, the concentration of the weak acid in the inner aqueous phase is 0.1~0.8 M, such as 0.2~0.6 M. The liposome drug composition according to any one of claims 1 to 3, wherein the meglumine and the weak acid are meglumine and acetic acid; preferably, the concentration of the meglumine and the acetic acid in the inner aqueous phase is 0.30-0.40 M. A liposome drug composition as described in any one of claims 1 to 4, wherein the phospholipid has a bilayer lipid structure, and the bilayer lipid is composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol (CHOL) and distearyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), and the molar ratio is 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. A liposome drug composition as described in any one of claims 1 to 5, wherein the particle size of the liposome is 50-500 nm, for example, 100-200 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. The liposome drug composition as described in any one of claims 1 to 6, wherein the external aqueous phase is a buffer having 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 0.05 M to 0.25 M, for example, 0.08 M to 0.2 M, preferably 0.09 to 0.12 M. A treprostinil liposome based on the liposome drug composition as described in any one of claims 1 to 7, wherein the weakly acidic drug in the liposome drug composition is treprostinil or its derivative, and the treprostinil or its derivative is encapsulated in the inner aqueous phase of the liposome; Preferably, the treprostinil derivative is selected from hydrates, solvates or complexes of treprostinil, or pharmaceutically acceptable salts, esters or prodrugs of treprostinil, such as treprostinil sodium salt, treprostinil potassium salt, treprostinil diethanolamine salt, treprostinil methyl ester, treprostinil ethyl ester, and treprostinil fumaric diketopyridine prodrug. A use of the treprostinil liposome or its derivative as described in claim 8 in the preparation of a drug; Preferably, the drug is a drug for treating pulmonary arterial hypertension, pulmonary hypertension, pulmonary fibrosis, interstitial lung disease, chronic obstructive pulmonary disease, asthma, ischemic disease, heart failure, arteriosclerosis, postoperative anticoagulation, central retinal vein obstruction, thrombotic microangiopathy, peripheral vascular disease, heart and lung transplantation and other diseases; Preferably, the drug is a drug for treating peripheral arterial occlusive disease or pulmonary arterial hypertension. A method for preparing a liposome drug composition as claimed in any one of claims 1 to 7, the method comprising loading liposomes with at least one weak acidic drug; Preferably, the drug loading amount of the weak acidic drug is not less than 18%; Preferably, the method for preparing the liposome drug composition comprises the following steps: dissolving the weak acidic drug in an external aqueous phase and mixing it with a solution of the liposomes to obtain the liposome drug composition; wherein the concentration of the liposomes is 1.0 to 20 mg/ml, preferably 1.0 to 5.0 mg/ml (based on phospholipid concentration); Preferably, the method for preparing the liposome drug composition comprises the following steps: (1) dissolving the weak acidic drug in an aqueous sodium citrate solution to obtain a drug solution; (2) Mixing the liposome solution with citrate buffer to obtain a carrier solution; (3) Adding the drug solution to the carrier solution for loading to obtain the liposome drug composition.