Liposome drug delivery system and preparation method therefor, liposome formulation, use, and fat reduction drug or medical aesthetic product comprising liposome formulation

Abstract

A liposome drug delivery system and a preparation method therefor, a liposome formulation, a use, and a fat reduction drug or medical aesthetic product comprising a liposome formulation. The liposome drug delivery system comprises deoxycholic acid and phospholipid; the phospholipid comprises a first phospholipid and optionally comprises a second phospholipid; the mass ratio of the deoxycholic acid to the phospholipid is 1:2.5 to 1:35. The liposome formulation can be further prepared by using this drug delivery system, and is used for the fat reduction drug or medical aesthetic product. The liposome drug delivery system, the liposome formulation drug or medical aesthetic product have excellent fat cell selectivity, achieve the effect of well dissolving adipose tissues, are small in side effect and high in safety, and have a good market application prospect.

Description

A liposome drug delivery system and its preparation method, liposome preparation, use, fat-reducing medicine or medical beauty product containing the same

[0001] This application claims the benefit of Chinese Patent Application No. 2023112666525 filed on September 27, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to the field of medical cosmetics or medicines, and specifically to a liposome drug delivery system, and more particularly to a liposome drug delivery system and its preparation method, liposome preparation, use, and fat-reducing medicine or medical cosmetics containing the liposome drug delivery system. Background Art

[0003] Kybella, an injectable formulation containing deoxycholic acid as its active ingredient, was approved by the U.S. Food and Drug Administration (FDA) in 2015 for the treatment of moderate to severe double chin lipolysis in adults. Deoxycholic acid primarily exerts its pharmacological effects by lysing cell membranes, causing massive necrosis of fat cells. However, due to its low cell-killing specificity, deoxycholic acid not only kills fat cells but also damages other normal non-fat cells, causing inflammation at the injection site. Furthermore, Kybella's weakly alkaline pH significantly increases the incidence of side effects such as severe pain, nodules, swelling, and numbness after injection.

[0004] Another class of injectable drugs for localized fat reduction includes Essentiale, developed by A. Nattermann GmbH in Germany in the 1950s for the treatment of liver disease; Lipostabil, used for the prevention and treatment of fat embolism; and Lipobean, approved in South Korea as an adjunct treatment for hepatic coma due to cirrhosis. These injectables, consisting primarily of a mixture of polyene phosphatidylcholine and the solubilizer deoxycholic acid, have been found to reduce localized fat in clinical practice and have therefore been used off-label for localized lipolysis. However, research indicates that the active fat-reducing ingredient in these injectables is actually deoxycholic acid, and their lipolysis efficacy and side effects remain controversial. Furthermore, these injectables can also cause adverse reactions such as pain, erythema, swelling, and induration. Patent CN102970993A discloses in the background art that the disadvantages of this type of preparation for subcutaneous lipolysis mainly involve adverse reactions such as swelling, hematoma (bruises), pain and discomfort in the treatment area, such as burning and itching at the injection site after treatment, and cell necrosis.

[0005] Liposomes can be used as carriers for many drugs. Targeted liposomes can usually reduce drug toxicity. Liposome targeting generally includes two forms: passive targeting and active targeting. Passive targeting liposomes do not carry active groups. They utilize the physiological characteristics and differences of various organs in the human body to selectively enrich liposomes in certain organs or lesions, and are often used in the field of tumors. Active targeting is more difficult to achieve and often requires appropriate modification of liposomes, including antibodies, oligosaccharides, peptides, proteins, and vitamins. Research focuses on antibodies, which mainly utilize the high affinity and specificity of monoclonal antibodies to achieve targeting.

[0006] However, truly realizing the therapeutic advantages of liposomes is not easy. The following three requirements must be met: 1) the drug can be encapsulated within the liposome with high encapsulation efficiency and sufficient drug loading; 2) the liposomes are stable in vitro; and 3) the drug has good targeting. Based on current liposome technology, developing liposome preparations that meet all quality requirements and simultaneously achieve the targeting of liposome drugs has become a key and difficult research topic. How to effectively control the targeting of liposome drugs to specific cells is a technical issue that needs to be addressed when developing liposome drugs.

[0007] The industry has previously attempted to develop liposome-based drugs for localized fat reduction. Chinese patent CN1870977A discloses a deoxycholic acid liposome system containing high-purity soy phosphatidylcholine and deoxycholic acid. The liposomes have a diameter of 30 to 100 nm and are effective in treating lipomas and cellulitis. The patent also discloses that patients experienced adverse reactions during treatment, including mild tingling, tenderness, and sensitivity, as well as moderate erythema.

[0008] Regarding deoxycholic acid molecules, there are no literature reports on liposomes with adipocyte targeting capabilities. How to achieve the targeting of deoxycholic acid liposomes to adipocytes is a major difficulty, and it is also the key to achieving efficacy enhancement and toxicity reduction.

[0009] Summary of the Invention

[0010] The present invention addresses the shortcomings of the existing technology and aims to provide deoxycholate liposomes with improved encapsulation efficiency, stability, and adipocyte targeting, achieving a lipolysis effect with enhanced efficacy and reduced toxicity. As mentioned above, because this product is a localized lipolytic drug, it must be released at the injection site to ensure that the drug is effective in the desired area; otherwise, the desired therapeutic effect will not be achieved. At the same time, it must actively target adipocytes to reduce its lethality to normal cells, thereby reducing toxicity.

[0011] Through extensive research, the present inventors discovered that the deoxycholate liposomes prepared in Chinese Patent CN1870977A lack adipocyte targeting, effectively killing not only adipocytes but also non-adipocytes, resulting in significant adverse reactions. However, the deoxycholate liposomes provided in this application, while not modified with antibodies, vitamins, peptides, or sugars, still demonstrated excellent adipocyte-selective killing in both adipocyte and vascular endothelial cell cytotoxicity tests.

[0012] The deoxycholic acid liposome pharmaceutical composition provided by the present invention has good adipocyte selectivity and high safety, and also has high encapsulation efficiency, good stability and excellent quality.

[0013] In a first aspect, the present invention provides a liposome drug delivery system comprising deoxycholic acid and a phospholipid; the phospholipid comprises a first phospholipid and optionally a second phospholipid; the mass ratio of the deoxycholic acid to the phospholipid is 1:2.5 to 1:35; preferably, the mass ratio of the deoxycholic acid to the phospholipid is 1:3.4 to 1:30.4; more preferably, the mass ratio of the deoxycholic acid to the phospholipid is 1:4.4 to 1:30.4.

[0014] In some embodiments of the present invention, the mass ratio of deoxycholic acid to phospholipid is 1:4 to 1:7, preferably 1:4.3 to 1:6.5, and more preferably 1:5 to 1:6.

[0015] In the liposome drug delivery system provided by the present invention, the phospholipids include main phospholipids (also called first phospholipids) for maintaining the phospholipid bilayer, and optionally include auxiliary phospholipids (also called second phospholipids) for further modifying the surface characteristics of the liposome.

[0016] In the liposome drug delivery system provided by the present invention, the first phospholipid is selected from one or more of egg yolk phosphatidylcholine (PC), soybean phosphatidylcholine (SPC), hydrogenated soybean phosphatidylcholine (HSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE).

[0017] In some embodiments, in the liposome drug delivery system provided by the present invention, the first phospholipid is selected from soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), distearoylphosphatidylcholine (DSPC), myristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dierucoylphosphatidylcholine (DEPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); Preferred are soy lecithin (SPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC); more preferred are dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), for example, dioleoylphosphatidylcholine (DOPC).

[0018] In the liposome drug delivery system provided by the present invention, the second phospholipid is selected from one of distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylserine (DSPS), (2-dioleoylhydroxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleyl-3-dimethylamino-propane (DODMA), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dioleoylphosphatidylglycerol (DOPG) and egg yolk phosphatidylglycerol (EPG). or more; preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylglycerol (DOPG) or 1,2-dioleyl-3-dimethylamino-propane (DODMA); preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) or distearoylphosphatidylcholine (DSPG); more preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0019] In the liposome drug delivery system provided by the present invention, the mass ratio of deoxycholic acid to phospholipid is 1:3.4, 1:3.6, 1:4.4, 1:5, 1:5.4, 1:5.5, 1:6, 1:6.4 or 1:30.4.

[0020] In the liposome drug delivery system provided by the present invention, the mass ratio of the deoxycholic acid to the first phospholipid is 1:3 to 1:32, preferably 1:4 to 1:30, more preferably 1:4 to 1:6, for example 1:4, 1:5 or 1:6.

[0021] In the liposome drug delivery system provided by the present invention, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(3-32):(0-3).

[0022] In some embodiments, in the liposome drug delivery system provided by the present invention, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(4-30):(0.2-0.6); preferably, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(4-30):(0.4-0.5).

[0023] In some embodiments, in the liposome drug delivery system provided by the present invention, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(4-10):(0-0.6), preferably 1:(4-6):(0.2-0.6), and more preferably 1:(4-6):(0.4-0.5).

[0024] In some embodiments, in the liposome drug delivery system provided by the present invention, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:3:0.4, 1:4:0.4, 1:5:0.4, 1:5:0.5, 1:6:0.4 or 1:30:0.4; preferably, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:4:0.4, 1:5:0.4, 1:5:0.5, 1:6:0.4 or 1:30:0.4.

[0025] In some embodiments, in the liposome drug delivery system provided by the present invention, the mass ratio of the first phospholipid to the second phospholipid is (3-32):(0-3); preferably, the mass ratio of the first phospholipid to the second phospholipid is (4-30):(0.2-0.6), more preferably 3:0.4, 4:0.4, 5:0.4, 5:0.5, 6:0.4 or 30:0.4.

[0026] In the liposome drug delivery system provided by the present invention, the drug-lipid ratio is 1:(3-32), preferably 1:(4-30.9), for example 1:5.9, 1:3.9, 1:4.0, 1:4.9, 1:6.9, 1:5.5, 1:6 or 1:6.1.

[0027] The liposome drug delivery system provided by the present invention may further optionally contain cholesterol and / or an antioxidant; preferably, the antioxidant is selected from vitamin E and / or vitamin E acetate.

[0028] The liposome drug delivery system provided by the present invention further comprises an aqueous phase; the aqueous phase is selected from one or more of disodium succinate solution, histidine solution, physiological saline, sucrose solution and water for injection.

[0029] In some embodiments, in the liposome drug delivery system provided herein, the aqueous phase is selected from one or more of a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl, a 0.05 M disodium succinate buffer solution, a 0.025 M histidine solution, physiological saline, and an 8% sucrose solution. The 8% sucrose solution refers to an aqueous solution of sucrose having a mass concentration of 8%.

[0030] In some embodiments, in the liposome drug delivery system provided by the present invention, the aqueous phase is a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl;

[0031] Preferably, the pH value of the 0.02-0.04 M disodium succinate solution containing 0.5% NaCl is 5.0-7.0; more preferably, the pH value is 6-6.5.

[0032] In the liposome drug delivery system provided by the present invention, each unit preparation is prepared from the following components: 1-5 parts of deoxycholic acid, 15-30 parts of the first phospholipid, 0-2 parts of the second phospholipid and 100-5000 parts of the aqueous phase; preferably, the aqueous phase is 500-5000 parts.

[0033] In some embodiments, in the liposome drug delivery system provided by the present invention, each unit preparation is prepared from the following components: 1-5 parts of deoxycholic acid, 15-30 parts of the first phospholipid, 0-2 parts of the second phospholipid, 500-5000 parts of the aqueous phase, 0-2.5 parts of cholesterol and 0-0.625 parts of vitamin E acetate.

[0034] In some embodiments, in the liposome drug delivery system provided by the present invention, each unit preparation is prepared from the following components: 5 parts of deoxycholic acid, 15-30 parts of the first phospholipid, 0-2 parts of the second phospholipid, 500 parts of the aqueous phase, 0-2.5 parts of cholesterol and 0-0.625 parts of vitamin E acetate.

[0035] In some embodiments, in the liposome drug delivery system provided by the present invention, the first phospholipid is selected from one or more of soybean lecithin (SPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); preferably, the first phospholipid is selected from one or more of dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); more preferably, it is dioleoylphosphatidylcholine (DOPC).

[0036] In some embodiments, in the liposome drug delivery system provided by the present invention, the second phospholipid is selected from one or more of distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylserine (DSPS), (2-dioleoylhydroxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleyl-3-dimethylamino-propane (DODMA), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and dioleoylphosphatidylglycerol (DOPG) and egg yolk phosphatidylglycerol (EPG); preferably, the second phospholipid is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and distearoylphosphatidylcholine (DSPG); more preferably, distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

[0037] In some embodiments, in the liposome drug delivery system provided by the present invention, the aqueous phase is selected from one or more of a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl, a 0.05 M disodium succinate buffer solution, a 0.025 M histidine solution, a normal saline solution, and an 8% sucrose solution; preferably, the aqueous phase is selected from one or more of a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl, a 0.025 M histidine solution, a normal saline solution, and an 8% sucrose solution; more preferably, the aqueous phase is a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl.

[0038] More preferably, the pH value of the 0.02-0.04 M disodium succinate solution containing 0.5% NaCl is 5.0-7.0; preferably the pH value is 6-6.5.

[0039] In some embodiments, in the liposome drug delivery system provided by the present invention, the first phospholipid and the second phospholipid satisfy any one of the following combinations:

[0040] (1) soybean lecithin (SPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0041] (2) hydrogenated soy lecithin (HSPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0042] (3) distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0043] (4) Dierucylphosphatidylcholine (DEPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0044] (5) 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0045] (6) myristoylphosphatidylcholine (DMPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0046] (7) Dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0047] (8) dioleoylphosphatidylcholine (DOPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000);

[0048] (9) dioleoylphosphatidylcholine (DOPC) and distearoylphosphatidylcholine (DSPG);

[0049] (10) dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG);

[0050] (11) Dioleoylphosphatidylcholine (DOPC) and 1,2-dioleyl-3-dimethylamino-propane (DODMA).

[0051] In some embodiments, in the liposome drug delivery system provided by the present invention, the first phospholipid is dioleoylphosphatidylcholine (DOPC), the second phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the mass ratio of the first phospholipid to the second phospholipid is (4-6):(0.4-0.6); the mass ratio of the first phospholipid to the second phospholipid is preferably (4-6):(0.4-0.5), for example 5:0.4 or 5:0.5.

[0052] Alternatively, the first phospholipid is dieucoylphosphatidylcholine (DEPC), the second phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the mass ratio of the first phospholipid to the second phospholipid is (4-6):(0.4-0.6); the mass ratio of the first phospholipid to the second phospholipid is preferably (4-6):(0.4-0.5), for example 5:0.4 or 5:0.5.

[0053] Alternatively, the first phospholipid is 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the second phospholipid is distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the mass ratio of the first phospholipid to the second phospholipid is (4-6):(0.4-0.6); the mass ratio of the first phospholipid to the second phospholipid is preferably (4-6):(0.4-0.5), for example 5:0.4 or 5:0.5.

[0054] In a second aspect, the present application provides a liposome preparation comprising the above-mentioned liposome drug delivery system; the preparation is an injection solution or a lyophilized preparation.

[0055] In some embodiments, the liposome preparation provided herein, when being a lyophilized preparation, optionally further comprises a lyophilization excipient, wherein the lyophilization excipient is selected from one or more of sucrose, lactose, trehalose and mannitol; preferably sucrose.

[0056] In a third aspect, the present invention also provides a method for preparing the aforementioned liposome drug delivery system.

[0057] The preparation method of the aforementioned liposome drug delivery system provided by the present invention can be a conventional method in the art, preferably selected from the injection method, reverse evaporation method, thin film dispersion method or active drug loading method.

[0058] In some embodiments, the preparation method provided herein preferably comprises the following steps:

[0059] I. taking the deoxycholic acid, the first phospholipid, the second phospholipid, cholesterol and an antioxidant (e.g., vitamin E acetate) according to a ratio, and dissolving them in an organic solvent to prepare an organic phase;

[0060] II. removing the organic solvent from the organic phase obtained in step I, adding an aqueous phase thereto for hydration to obtain a crude suspension;

[0061] III. Homogenize the crude suspension obtained in step II to obtain a suspension;

[0062] IV. Filter the suspension obtained in step III through a 0.45 μm filter membrane or a (1.0+0.65) μm double-layer filter membrane, and then filter through a 0.22 μm microporous filter membrane to obtain liposomes.

[0063] In some embodiments, the total solid content of the organic phase in step I is 20% (w / v).

[0064] In some embodiments, the organic solvent in step I is selected from one or more of chloroform, anhydrous ethanol, and anhydrous methanol, preferably a mixed solution of chloroform and anhydrous methanol. The volume ratio of chloroform to anhydrous methanol in the mixed solution of chloroform and anhydrous methanol is preferably 1:1 to 30:1, for example, 10:1.

[0065] In some embodiments, the amount of the organic solvent used in step I is generally such that the total solid content in the formed organic phase reaches 20% (w / v).

[0066] In some embodiments, the aqueous phase in step II is selected from one or more of a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl, a 0.05 M disodium succinate buffer solution, a 0.025 M histidine solution, physiological saline, and an 8% sucrose solution; preferably, the aqueous phase is a 0.02-0.04 M disodium succinate solution containing 0.5% NaCl; more preferably, the pH value of the 0.02-0.04 M disodium succinate solution containing 0.5% NaCl is 5.0-7.0 (e.g., 6.0-6.5).

[0067] In some embodiments, the amount of the aqueous phase in step II is determined based on the amount of deoxycholic acid. The amount of the aqueous phase is preferably such that the mass concentration of deoxycholic acid in the liposome solution is 5-20 mg / mL, more preferably 10 mg / mL.

[0068] In some embodiments, the amount of the aqueous phase in step II is preferably 80%-95%, for example 90%, of the total volume of the liposome.

[0069] In some embodiments, the method for removing the organic solvent in step II is vacuum distillation, which is preferably performed at 45-60° C. The purpose of the vacuum distillation is to remove the organic solvent and form a lipid film.

[0070] In some embodiments, the hydration temperature in step II is 20-60°C, preferably 50-60°C, or 20-25°C.

[0071] In some embodiments, the hydration time in step II is 60-120 minutes.

[0072] In some embodiments, the homogenization condition in step III is 8000-12000 psi for 3-5 times.

[0073] In some embodiments, in step IV, the suspension obtained in step III is filtered through a 0.45 μm filter membrane or a (1.0+0.65) μm double-layer filter membrane to remove large particles, and then sterilized by filtration through a 0.22 μm microporous filter membrane to obtain liposomes.

[0074] On the other hand, the present invention also provides a method for preparing the liposome preparation, which is a freeze-dried preparation, including the method for preparing the liposome drug delivery system. After homogenization in step III and before filtration with a filter membrane in step IV, the suspension is mixed with a freeze-dried excipient, and then an aqueous phase (for example, a 0.025M disodium succinate solution containing 0.5% NaCl with a pH of 6 to 6.5) is added; then, the suspension is filtered with a 0.45μm filter membrane and then filtered with a 0.22μm microporous filter membrane to obtain liposomes, and the liposomes are freeze-dried.

[0075] In some embodiments, the aqueous phase is added after the lyophilized excipients are completely dissolved.

[0076] In some embodiments, after mixing with the lyophilized excipient, the aqueous phase is added so that the weight percentage of the lyophilized excipient in the solution reaches 5-8%, for example 6%.

[0077] In some embodiments, in the liposome drug delivery system or liposome preparation provided herein, the average particle size of the liposome is 50-300 nanometers, preferably 50-100 nanometers; more preferably, the average particle size of the liposome is 60-95 nanometers, most preferably 60-91.5 nanometers.

[0078] In the liposome drug delivery system or liposome preparation provided in the present application, the liposome encapsulation rate can reach more than 75%, preferably more than 85%, more preferably more than 90%, and most preferably more than 95%.

[0079] In the liposome drug delivery system or liposome preparation provided herein, the polydispersity coefficient of the liposome may be 0.1-0.4, preferably 0.1-0.35, more preferably 0.1-0.25, and most preferably 0.1-0.2.

[0080] In the liposome drug delivery system or liposome preparation provided in the present application, the Zeta potential of the liposome can reach -65mV to -5mV, preferably -45mV to -20mV, and more preferably -45mV to -35mV.

[0081] In a fourth aspect, the present application provides the use of a liposome drug delivery system or a liposome preparation in the preparation of fat-reducing drugs or medical cosmetic products; preferably, the fat-reducing drugs or medical cosmetic products are local fat-reducing drugs or local medical cosmetic products.

[0082] In some embodiments, in the uses provided herein, the fat-reducing drug or medical cosmetic product lyses the cell membrane of fat cells and / or promotes apoptosis of fat cells; preferably, the fat-reducing drug or medical cosmetic product is taken up by fat cells.

[0083] In a fifth aspect, the present application provides a fat-reducing drug or medical beauty product, comprising the above-mentioned liposome drug delivery system or liposome preparation, and acceptable excipients or auxiliary ingredients in the fat-reducing drug or medical beauty product.

[0084] In some embodiments, in the fat-reducing drugs or medical cosmetic products provided herein, the excipients or auxiliary ingredients are selected from one or more of VC, benzoic acid, and lidocaine.

[0085] In some embodiments, the fat-reducing drugs or medical cosmetic products provided herein are liposome preparations for local injection into adipose tissue.

[0086] The deoxycholic acid liposome drug delivery system provided in this application and the liposome preparations or medical beauty products containing the same have a good fat-reducing effect. By optimizing the composition of phospholipids in the liposomes, the selectivity for fat cells can be improved, achieving the effect of dissolving adipose tissue, with few side effects and high safety, and has good market application prospects.

[0087] The liposomes for local injection into adipose tissue provided by the present invention can efficiently dissolve adipose tissue, reduce fat accumulation, and at the same time reduce the killing of other non-fat cells (such as vascular cells, etc.). The therapeutic effect and adverse reactions are far superior to those of lipolysis injections on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1. Morphology of deoxycholic acid liposomes under electron microscopy;

[0089] FIG2 compares the killing ability of adipocytes and HUVEC (human umbilical vein endothelial cells);

[0090] FIG3 Comparison of the in vivo lipolysis effect and adverse reactions on rat tail adipose tissue. DETAILED DESCRIPTION

[0091] Abbreviations

[0092] PC:Egg yolk lecithin

[0093] SPC:Soybean Lecithin

[0094] DOPC: dioleoylphosphatidylcholine, also known as: dioleoylphosphatidylcholine

[0095] DSPC: Distearoylphosphatidylcholine

[0096] HSPC: Hydrogenated Soy Lecithin

[0097] DEPC: Dierucylphosphatidylcholine

[0098] DMPC: dimyristoylphosphatidylcholine

[0099] DPPC: Dipalmitoylphosphatidylcholine

[0100] POPC: 1-palmitoyl-2-oleoyl-phosphatidylcholine

[0101] DSPG: Distearoylphosphatidylglycerol

[0102] DOPG: dioleoylphosphatidylglycerol

[0103] DSPE-PEG2000: Distearoylphosphatidylethanolamine-polyethylene glycol 2000

[0104] DPPC: Dipalmitoylphosphatidylcholine

[0105] DSPE: Distearoylphosphatidylethanolamine

[0106] DOPE: dioleoylphosphatidylethanolamine

[0107] DOPS: dioleoylphosphatidylserine

[0108] DSPS: Distearoylphosphatidylserine

[0109] DOTAP: (2-dioleoylhydroxypropyl)trimethylammonium chloride

[0110] DODMA: 1,2-dioleyl-3-dimethylamino-propane

[0111] EPG: Egg yolk phosphatidylglycerol

[0112] PS: Phosphatidylserine

[0113] Definitions and terms:

[0114] Encapsulation efficiency (EE) refers to the ratio of the drug encapsulated in the liposome to the amount of drug administered during loading;

[0115] Calculation formula: EE=W inside of liposome / W total of input×100%

[0116] Drug loading (DL) refers to the ratio of the mass of the drug encapsulated in the liposome to the total mass of the liposome preparation;

[0117] Calculation formula: DL = W inside of liposome / W total × 100%

[0118] Drug:Lipid ratio (D:L) refers to the ratio of the mass of drug to total lipid components (phospholipids and cholesterol).

[0119] In this application, when described as "... solution", unless otherwise specified, it refers to an aqueous solution of a certain substance; for example, "disodium succinate solution, histidine solution, sucrose solution" refer to an aqueous solution with disodium succinate as the solute, an aqueous solution with histidine as the solute, and an aqueous solution with sucrose as the solute, respectively.

[0120] In the present application, when described as "optionally comprising...", it means that the substance may be included or not included.

[0121] Aqueous phase: The aqueous phase refers to the aqueous solution added during the hydration step, or the aqueous solution different from the organic phase during liposome preparation.

[0122] Solids content refers to the mass-to-volume ratio of solid raw materials to solvent in a solution or mixture, expressed in g / mL. For example, in Example 2 of this application, "weighing... to prepare an organic phase with a total solids content of 20% (w / v)" means that the total mass of deoxycholic acid, soy lecithin (SPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), cholesterol, and vitamin E acetate in anhydrous ethanol in the organic phase is 20% (g / mL) by volume (w / v).

[0123] The "0.02-0.04 M disodium succinate solution containing 0.5% NaCl" described in this application refers to: an aqueous solution containing 0.02-0.04 mol / L disodium succinate, wherein NaCl is added so that its mass percentage in the solution is 0.5%; the pH value of the solution can be further adjusted to 5-7 using a pH regulator, and the pH regulator that can be used includes but is not limited to 1M sodium hydroxide solution and 1M hydrochloric acid solution.

[0124] The physiological saline described in this application refers to a sodium chloride solution having an osmotic pressure substantially equal to that of animal or human plasma, for example, a sodium chloride aqueous solution with a mass concentration of 0.9%.

[0125] The "liposome drug delivery system" or "liposome" described in this application refers to a deoxycholic acid liposome solution or suspension prepared after homogenization and filtration steps, which can be further prepared into an injection or a lyophilized preparation.

[0126] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0127] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0128] Example 1 (Comparative Example)

[0129] Deoxycholic acid liposomes were prepared according to Example 1 of patent CN1870977A using soybean lecithin as the membrane material.

[0130] 12.5 g of high-purity soy lecithin (phosphatidylcholine content greater than 95%) and 6.325 g of deoxycholic acid were dissolved in 50 ml of ethanol. The resulting solution was then evaporated to dryness under reduced pressure. The resulting residue was dispersed in 250 ml of water and homogenized to an average liposome particle size of 30 nm to 100 nm. The resulting liposome system was then filtered through a 0.2 μm filter under sterile conditions, and the encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0131] Example 2

[0132] Preparation of deoxycholic acid liposomes (the first phospholipid is SPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0133] 5 g of deoxycholic acid, 25 g of soy lecithin (SPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were mixed and dissolved in anhydrous ethanol to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60°C to form a lipid film. The prepared film was then hydrated with 450 ml of normal saline (0.9% NaCl) at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi and the volume was adjusted to 500 ml with normal saline (0.9% NaCl). The resulting sample was first filtered through a 0.45 μm filter to remove large particles and then sterilized by filtration through a 0.22 μm microporous filter to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0134] Example 3

[0135] Preparation of deoxycholic acid liposomes (the first phospholipid is HSPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0136] 5 g of deoxycholic acid, 25 g of hydrogenated soy lecithin (HSPC), and 2 g of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were mixed and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 50-60° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0137] Example 4

[0138] Preparation of deoxycholic acid liposomes (the first phospholipid is DSPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0139] 5 g of deoxycholic acid, 25 g of distearoylphosphatidylcholine (DSPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 50-60° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0140] Example 5

[0141] Preparation of deoxycholic acid liposomes (the first phospholipid is DEPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0142] 5 g of deoxycholic acid, 25 g of dieucoylphosphatidylcholine (DEPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were mixed and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0143] Example 6

[0144] Preparation of deoxycholic acid liposomes (first phospholipid POPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0145] 5 g of deoxycholic acid, 25 g of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. Then, 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6 to 6.5 was added to the film prepared above for hydration. The hydration temperature was 20 to 25° C. and the hydration time was 60 to 120 min. After hydration, the film was homogenized three times under 8000 to 12000 psi homogenization conditions. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6 to 6.5. The resulting sample was first filtered through a 0.45 μm filter to remove large particles and then sterilized by filtration through a 0.22 μm microporous filter to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0146] Example 7

[0147] Preparation of deoxycholic acid liposomes (first phospholipid DMPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0148] 5 g of deoxycholic acid, 25 g of dimyristoylphosphatidylcholine (DMPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0149] Example 8

[0150] Preparation of deoxycholic acid liposomes (first phospholipid DPPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0151] 5 g of deoxycholic acid, 25 g of dipalmitoylphosphatidylcholine (DPPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0152] Example 9

[0153] Preparation of deoxycholic acid liposomes (first phospholipid DOPC); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0154] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), and 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) were weighed, and then 2.5 g of cholesterol and 0.625 g of vitamin E acetate were mixed and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0155] Example 10

[0156] Preparation of Lyophilized Deoxycholic Acid Liposomes

[0157] Sucrose was added to the sample obtained after the homogenization step in Example 9 and dissolved completely. The sample was then diluted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5, resulting in a sucrose content of 6% (w / w). Large particles were removed using a 0.45 μm filter, followed by sterilization by filtration using a 0.22 μm microporous filter to obtain liposomes. The liposomes were dispensed into 6 ml vials at a filling volume of 2 ml per vial and freeze-dried to obtain lyophilized deoxycholate liposomes. The lyophilized product was reconstituted with 2 ml of water for injection, and the encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0158] Example 11

[0159] Preparation of blank liposomes (blank liposomes with DOPC as the first phospholipid)

[0160] 25 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by reduced pressure evaporation at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain blank liposomes. The particle size, polydispersity index (PDI), and zeta potential were measured. The results are shown in Table 1.

[0161] Example 12

[0162] Preparation of deoxycholic acid liposomes; the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:3:0.4.

[0163] 5 g of deoxycholic acid, 15 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0164] Example 13

[0165] Preparation of deoxycholic acid liposomes; the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:4:0.4.

[0166] 5 g of deoxycholic acid, 20 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed, mixed, and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0167] Example 14

[0168] Preparation of deoxycholic acid liposomes; the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:6:0.4.

[0169] 5 g of deoxycholic acid, 30 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed, mixed, and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0170] Example 15

[0171] Preparation of deoxycholic acid liposomes (without DSPE-PEG2000); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.

[0172] 5 g of deoxycholic acid, dioleoylphosphatidylcholine (DOPC), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed, mixed, and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by reduced pressure evaporation at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 0.45 μm membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0173] Example 16

[0174] Preparation of deoxycholic acid liposomes; the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0175] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed, mixed, and dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M histidine solution at 20-25°C for 60-120 minutes. After hydration, the film was homogenized three times at 8,000-12,000 psi and then brought to a volume of 500 ml with a 0.025 M histidine solution. The resulting sample was first filtered through a 0.45 μm filter to remove large particles and then sterilized by filtration through a 0.22 μm microporous filter to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0176] Example 17

[0177] Preparation of deoxycholic acid liposomes; the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0178] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.05 M disodium succinate buffer solution at 20-25°C for 60-120 minutes. After hydration, the film was homogenized three times at 8,000-12,000 psi and then brought to a volume of 500 ml with 0.05 M disodium succinate buffer solution. The resulting sample was first filtered through a 0.45 μm filter to remove large particles and then sterilized by filtration through a 0.22 μm microporous filter to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0179] Example 18 Preparation of Reference Preparation (Kybella) (100 ml)

[0180] Weigh 900 mg of benzyl alcohol, 1000 mg of deoxycholic acid, 142 mg of sodium dihydrogen phosphate, 143 mg of sodium hydroxide, and 438 mg of sodium chloride and thoroughly dissolve them in 80 ml of water for injection. Adjust the pH to 8.3 with 1 M sodium hydroxide solution and finally dilute to 100 ml with water for injection. Sterilize by filtration through a 0.2 μm filter to obtain the reference preparation. The resulting deoxycholic acid solution has an active ingredient concentration of 10 mg / mL.

[0181] Example 19

[0182] Preparation of deoxycholic acid liposomes; the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:30:0.4.

[0183] Weigh 1 g of deoxycholic acid, 30 g of dioleoylphosphatidylcholine (DOPC), 0.4 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and then weigh 0.5 g of cholesterol and 0.125 g of vitamin E acetate, mix them, and dissolve them in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent is removed by reduced pressure evaporation at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 100 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 1.0 + 0.65 μm double-layer membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0184] Example 20

[0185] Preparation of deoxycholic acid liposomes (second phospholipid DSPG); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0186] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylcholine (DSPG), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by reduced pressure evaporation at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8000-12000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a (1.0 + 0.65) μm double-layer membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0187] Example 21

[0188] Preparation of deoxycholic acid liposomes (second phospholipid DSPE-PEG2000); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.5.

[0189] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), 2.5 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), 2.5 g of cholesterol, and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 1.0 + 0.65 μm double-layer membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0190] Example 22

[0191] Preparation of deoxycholic acid liposomes (without cholesterol); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0192] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 0.625 g of vitamin E acetate were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60° C. to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 1.0 + 0.65 μm double-layer membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0193] Example 23

[0194] Preparation of deoxycholic acid liposomes (without vitamin E acetate); the mass ratio of deoxycholic acid, the first phospholipid and the second phospholipid is 1:5:0.4.

[0195] 5 g of deoxycholic acid, 25 g of dioleoylphosphatidylcholine (DOPC), 2 g of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and 2.5 g of cholesterol were weighed and mixed, dissolved in a mixed organic solvent of chloroform and anhydrous methanol (v / v = 10:1) to prepare an organic phase with a total solid content of 20% (w / v). The organic solvent was removed by evaporation under reduced pressure at 45-60°C to form a lipid film. The prepared film was then hydrated by adding 450 ml of a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5 at 20-25° C. for 60-120 min. After hydration, the film was homogenized three times at 8,000-12,000 psi. The volume was then adjusted to 500 ml with a 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6-6.5. The resulting sample was first filtered through a 1.0 + 0.65 μm double-layer membrane to remove large particles and then sterilized by filtration through a 0.22 μm microporous membrane to obtain liposomes. The properties of the liposomes were observed, including encapsulation efficiency, particle size, polydispersity index (PDI), and zeta potential. The results are shown in Table 2.

[0196] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

[0197] Test Example 1 Example Sample Quality Inspection

[0198] Property testing: The samples prepared in the above examples were taken and their properties were observed. The test results are shown in Tables 1 and 2 below.

[0199] Detection of particle size and polydispersity coefficient: 25 μL of the liposome sample prepared in Example was diluted to 5 ml with 0.9% NaCl, and the particle size and polydispersity coefficient were detected. The test results are shown in Tables 1 and 2 below.

[0200] Zeta potential detection: 25 μL of the liposome sample prepared in Example was diluted to 5 ml with water for injection, and the sample was taken for zeta potential detection. The test results are shown in Tables 1 and 2 below.

[0201] Table 1 Quality test results of liposomes

[0202] *Note: Example 10 is a lyophilized formulation of deoxycholic acid liposomes, which was reconstituted with 2 ml of water for injection before the above quality assessment. Example 11 is a blank liposome in which the first phospholipid is DOPC. N / A indicates not applicable.

[0203] The results of the investigation in Table 1 above show that the liposome encapsulation efficiency of Example 1 (Comparative Example) is very low; the liposome encapsulation efficiency of Examples 2, 3, 4, 7, and 8 is less than 80%. In addition, the particle size of the liposomes prepared in Examples 3-4 and 7-8 is greater than 100 nm; the liposomes prepared by the four prescriptions of Examples 5, 6, 9, and 10 have an encapsulation efficiency greater than 90%, and the particle size is less than 100 nm, with good dispersion characteristics, the potential for uniform fat dissolution, and a higher Zeta potential and better stability.

[0204] Table 2 Quality test results of liposomes

[0205] The results of the investigation in Table 2 show that in Example 12 (the mass ratio of deoxycholic acid to the first phospholipid was 1:3), Example 9 (1:5), Example 13 (1:4), and Example 14 (1:6), when the mass ratio of deoxycholic acid to DOPC was 1:3 (Example 12), the suspension properties were poor and no opalescence was observed. At the same time, the encapsulation efficiency was less than 80%, the liposome quality was poor, and the particle size and polydispersity index (PDI) were also large, resulting in poor product dispersion characteristics. The quality differences of the liposome samples in Examples 9, 13, and 14 were relatively small, that is, when the mass ratio of deoxycholic acid to DOPC was 1:4, 1:5, and 1:6, the liposomes had higher encapsulation efficiency, smaller particle size and polydispersity index (PDI), and were more conducive to the release of the product in vivo. When the mass ratio of deoxycholic acid to DOPC was greater than 1:6, i.e., 1:30, the liposomes prepared in Example 19 exhibited a high encapsulation efficiency, a small particle size, and a small polydispersity index (PDI), indicating excellent liposome quality. Further increasing the amount of the first phospholipid, DOPC, did not significantly change the liposome encapsulation efficiency, particle size, and polydispersity index (PDI). Considering the economic cost and the safety risks posed by the excipient to patients, further increasing the phospholipid ratio is not recommended.

[0206] Further find out from the investigation result of Table 2, when not containing the second phospholipid DSPE-PEG2000 (embodiment 15) in the prescription, although the character, encapsulation efficiency, particle diameter and polydispersity index (PDI) of liposome suspension are all with no significant difference containing the second phospholipid, but Zeta potential is lower than-10mV, shows that the stability of product is poor. The liposomes obtained in embodiment 9, embodiment 13, embodiment 14, Zeta potential can be maintained at about-40mV, shows that liposome stability is better. Compared with Example 9, when the second phospholipid DSPE-PEG2000 (embodiment 21) consumption increased, the absolute value of Zeta potential increased, which is conducive to liposome keeping stable.

[0207] The results of the investigation in Table 2 further show that when the prescription composition contains the second phospholipid DSPG (Example 20), the encapsulation efficiency is higher, and the particle size and polydispersity index (PDI) are smaller, which is conducive to the dispersion stability of the product.

[0208] The results of the investigation in Table 2 also show that the liposome samples prepared using 0.025 M histidine solution (Example 16), 0.05 M disodium succinate buffer (Example 17) and 0.025 M disodium succinate solution containing 0.5% NaCl at a pH of 6 to 6.5 as the aqueous phase all had high encapsulation efficiencies, especially the liposomes prepared in Example 9, which had smaller particle size and polydispersity index (PDI), which was more conducive to maintaining good dispersibility.

[0209] It can be further seen from the examination of Table 2 that when the formulation does not contain cholesterol (Example 22) or vitamin E acetate (Example 23), there is no significant difference in the quality attributes such as encapsulation efficiency, particle size and polydispersity index (PDI) of the prepared liposomes.

[0210] Test Example 2: Investigating the ability to kill fat cells

[0211] Experimental plan: 3T3-L1 (mouse embryonic fibroblasts) were seeded into 96-well plates in advance and induced to differentiate into mature adipocytes for subsequent cell evaluation. Mature adipocytes and HUVEC (human umbilical vein endothelial cells) were added to 96-well plates at 10,000 cells per well, with a volume of 100 μL per well. The cells were cultured in a carbon dioxide incubator and cultured for 24 hours. The samples of Example 18 and Example 1-10 were diluted with complete medium with water for injection to a concentration of 1 mg / ml, 0.5 mg / ml, 0.25 mg / ml, and 0.125 mg / ml, respectively, containing deoxycholic acid. The lyophilized sample of Example 10 was first reconstituted with water for injection and then diluted with water for injection to the above-mentioned concentrations. The sample of Example 11 (blank liposome) was diluted in the same proportion with reference to the corresponding drug-loaded liposome to obtain a blank liposome solution. The drug-loaded liposomes containing different concentrations of deoxycholic acid, the reference formulation, and blank liposome solutions were added to mature adipocyte and HUVEC cell plates (n=4) at a volume of 100 μL per well. Following dosing, the cells were cultured in a CO2 incubator for 24 hours. 100 μL of supernatant was aspirated from each well, and 50 μL of cell viability assay reagent (Cell Counting-Lite 3D Luminescent Cell Viability Assay) was added to each well. The cells were shaken and mixed at room temperature for 5 minutes until the cells were completely lysed. After incubation for 30 minutes, the cells were transferred to a light-proof 96-well plate at a volume of 50 μL per well. Fluorescence intensity (FI) was measured using a fluorescence microplate reader, and cell viability was calculated according to the following formula. The cell cytotoxicity was calculated using GraphPad software, and the IC50 value was calculated. The results of the cell viability assay are shown in Figure 2, and the results of the cell cytotoxicity assay are shown in Table 3.

[0212] Cell viability (%) = (FI sample - FI blank) / (FI control - FI blank) × 100%

[0213] FI blank: indicates the average fluorescence intensity value after no cells are present and no sample treatment is added

[0214] FI control: indicates the average fluorescence intensity value after cells are present but no sample treatment is added

[0215] Table 3 Killing ability (IC50) on different cells (unit: mg / mL)

[0216] As can be seen from Table 3, after 24 hours of administration, Example 11 (blank liposomes) did not kill rat adipocytes and non-adipocyte HUVEC (human umbilical vein endothelial cells). The killing ability (IC50) of the other groups of test samples on rat adipocytes was basically the same, but the killing ability (IC50) of the liposomes of Examples 9 and 10 on non-adipocyte HUVEC (human umbilical vein endothelial cells) was significantly weaker than that of the reference preparation (Example 18) and Example 1 (Comparative Example), and also weaker than that of Examples 2-8. It can be seen that the liposomes of Examples 9 and 10 have better adipocyte selectivity.

[0217] As can be seen from the above table, the liposomes prepared in Example 9 and the lyophilized samples of the liposomes prepared in Example 10 have almost no difference in their ability to kill rat adipocytes and HUVEC cells. It can be seen that preparing deoxycholic acid liposomes into lyophilized products does not adversely affect product quality and therapeutic effects. Therefore, they can be further prepared into lyophilized preparations.

[0218] As can be seen from Figure 2, with the increase in the deoxycholic acid administration concentration, the cell survival rate of adipocytes and non-adipocyte HUVECs in each experimental group further decreased. When the deoxycholic acid administration concentration was 0.125 mg / mL and 0.25 mg / mL, the survival rate of non-adipocyte HUVECs in each experimental group was higher than that of adipocytes at the same administration concentration. When the deoxycholic acid administration concentration was 0.5 mg / mL, the survival rate of adipocytes and non-adipocyte HUVECs in the groups treated with Example 1 (Comparative Example), Example 2, and the reference preparation (Example 18) dropped below 10%, while the non-adipocyte survival rate of the groups treated with Example 5, Example 6, Example 9, and Example 10 remained above 50%, especially the non-adipocyte survival rate of the groups treated with Example 9 and Example 10 reached almost 80%. When the deoxycholic acid administration concentration was 1.0 mg / mL, Examples 5, Example 6, Example 9, and Example 10 also showed weaker non-adipocyte killing compared to Example 1 (Comparative Example), Example 2, and the reference preparation (Example 18). It can be seen that Examples 5, 6, 9 and 10 have very excellent adipocyte selectivity.

[0219] Test Example 3 Liposome morphology test

[0220] A freeze-dried sample of liposomes prepared in Example 10 was reconstituted with 2 ml of water for injection. The liposome morphology was observed by cryo-transmission electron microscopy. A precise amount of 5 μl of liposomes was placed on an R1.2 / 1.3 100-pore carbon film grid (Cu 200 mesh). Excess liposome suspension was quickly removed by aspiration, and the carbon film grid was frozen in a mixture of liquid ethane and methane. Data were acquired using a FEI Talos F200C electron microscope at an operating voltage of 200 kV and processed using SerialEM software.

[0221] Cryo-transmission electron microscopy revealed that the freeze-dried liposome samples were liposomes with complete vesicle structures, rather than lipid complexes. Furthermore, electron microscopy revealed that most of the liposomes exhibited a bicompartmental conformation, with a few exhibiting a multicompartmental conformation, as shown in FIG1 .

[0222] Experimental Example 4: Stability Study of Deoxycholic Acid Liposomes

[0223] The lyophilized liposome samples prepared in Example 10 were placed in a 2-8°C stability chamber. Samples were taken at 0, 1, 2, 3, and 6 months to test the encapsulation efficiency, particle size, and PDI, using the same testing methods as in Experiment 1. The results are shown in Table 4.

[0224] Table 4 Liposome stability investigation data

[0225] The above investigation results show that the liposome freeze-dried preparation prepared in Example 10 has an encapsulation efficiency that remains essentially unchanged (data changes are within the detection error of 2%) and no significant change in particle size after being stored for 6 months, indicating good stability and excellent quality.

[0226] Experimental Example 5: Investigating the lipolysis effect of liposomes on ZDF rat tail fat

[0227] Twelve male ZDF rats weighing 330-350 g were selected and randomly divided into three groups: normal saline group, DC-LIP (sample prepared in Example 10), and reference preparation (Example 18), with 4 rats in each group. The animals were anesthetized with isoflurane, and the tail fat volume was detected by Micro-CT before administration, and reconstruction was performed to calculate the initial tail fat volume. Subsequently, 2.5 mL of the test sample was injected into the tail fat tissue on both sides of the ZDF rats, respectively. Among them, the sample of Example 10 was a lyophilized preparation, which was reconstituted with 2 ml of water for injection, with a deoxycholic acid concentration of 10 mg / mL and a reference preparation drug concentration of 10 mg / mL. Two weeks after administration, the tail fat tissue was detected again by Micro-CT to calculate the volume after administration. After the last CT examination, the tail fat was weighed and the weights of each group were compared.

[0228] The lipolytic efficacy of the test sample was demonstrated by the fat volume change rate and fat weight reduction before and after administration. Fat volume change rate (%) = [(fat volume after administration - fat volume before administration) / fat volume before administration] * 100%. The experimental results are shown in FIG3 .

[0229] As can be seen from Figure 3, after two weeks of administration, the tail fat volume of the normal saline group increased by 13.8%, the tail fat volume of the DC-LIP group (Example 10) decreased by 9%, and the tail fat volume of the reference preparation group increased by 20.5%. Regarding tail fat weight, the tail fat weight of the ZDF rats in the DC-LIP group (Example 10) was significantly reduced compared to the normal saline and reference preparation groups. Compared to the normal saline group, the reference preparation group showed an increase in both fat volume and weight. During dissection, the adipose tissue at the injection site showed obvious tissue necrosis, as evidenced by a gray-brown coloration. This suggests that the reference preparation has exerted its pharmacological effect of lysing adipose tissue at the injection site. However, due to the reference preparation's indiscriminate cell membrane lysis, it caused a more severe inflammatory reaction at the injection site, resulting in an increase in fat volume and weight.

[0230] While evaluating the in vivo lipolysis effect, adverse reactions at the injection site were observed (see Table 5). During adverse reaction monitoring, no swelling, nodules, or ulcers were observed in any group on day 1 after administration. Seven days after administration, the DC-LIP group (Example 10) showed only very mild swelling (+), with no nodules or skin ulcers, while the reference preparation group showed swelling, nodules, and skin ulcers. Fourteen days after administration, swelling disappeared in the DC-LIP group (Example 10), while swelling, nodules, and ulcers persisted in the reference preparation group. No swelling, nodules, or skin ulcers were observed in the saline control group. This indicates that adverse reactions in the DC-LIP group were mild.

[0231] Table 5 Incidence of adverse reactions at the injection site

[0232] Note: ① Percentage incidence (%), the data in brackets are the number of adverse reactions and the total number of cases; ② The degree of swelling is semi-quantitatively described using a five-point system, such as grade 0, grade 1 (+), grade 2 (++), grade 3 (+++), and grade 4 (++++). The larger the grade, the more severe the adverse reaction; ③ 0 / 8 represents the number of swelling, nodules, or ulcers in the 8 caudal fat pads of 4 animals in each group (each animal has 2 caudal fat pads).

[0233] The above results of tail lipolysis and local adverse reactions show that the drug-loaded liposomes of Example 10 can not only exert the lipolysis effect faster, but also reduce the local adverse reactions of the reference preparation.

Claims

1. A liposome drug delivery system, characterized in that: It comprises deoxycholic acid and phospholipids; the phospholipids comprise a first phospholipid and optionally a second phospholipid; and the mass ratio of the deoxycholic acid to the phospholipids is 1:2.5-1:

35.

2. The liposome drug delivery system according to claim 1, characterized in that: The mass ratio of deoxycholic acid to phospholipid is 1:3.4-1:30.4, for example, 1:3.4, 1:3.6, 1:4.4, 1:5, 1:5.4, 1:5.5, 1:6, 1:6.4 or 1:30.4, preferably 1:4.4-1:30.

4.

3. The liposome drug delivery system according to at least one of claims 1-2, characterized in that: The first phospholipid is selected from one or more of egg yolk lecithin (PC), soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE); preferably soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), distearoylphosphatidylcholine (DSPC), myristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE) and dioleoylphosphatidylethanolamine (DOPE). Choline (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) or 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); more preferably soy lecithin (SPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) or 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); more preferably dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) or 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), for example dioleoylphosphatidylcholine (DOPC); And / or, the second phospholipid is selected from one or more of distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylserine (DSPS), (2-dioleoylhydroxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleyl-3-dimethylamino-propane (DODMA), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dioleoylphosphatidylglycerol (DOPG) and egg yolk phosphatidylglycerol (EPG); preferably distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylserine (DSPS), (2-dioleoylhydroxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleyl-3-dimethylamino-propane (DODMA), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), dioleoylphosphatidylglycerol (DOPG) and egg yolk phosphatidylglycerol (EPG). The present invention relates to an aqueous solution of at least one iodine-containing compound selected from the group consisting of 1,2-dioleylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylglycerol (DOPG) or 1,2-dioleyl-3-dimethylamino-propane (DODMA); preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) or distearoylphosphatidylcholine (DSPG); more preferably distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000).

4. The liposome drug delivery system according to at least one of claims 1 to 3, characterized in that The mass ratio of the deoxycholic acid to the first phospholipid is 1:3-1:32, preferably 1:4-1:30, more preferably 1:4-1:6, for example 1:4, 1:5 or 1:6; And / or, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(3-32):(0-3); preferably, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(4-30):(0.2-0.6); more preferably 1:3:0.4, 1:4:0.4, 1:5:0.4, 1:5:0.5, 1:6:0.4 or 1:30:0.4; And / or, the mass ratio of the first phospholipid to the second phospholipid is (3-32):(0-3); preferably, the mass ratio of the first phospholipid to the second phospholipid is (4-30):(0.2-0.6), more preferably 3:0.4, 4:0.4, 5:0.4, 5:0.5, 6:0.4 or 30:0.4; And / or, the drug-to-lipid ratio is 1:(3-32), preferably 1:(4-30.9), for example 1:5.9, 1:3.9, 1:4.0, 1:4.9, 1:6.9, 1:5.5, 1:6 or 1:6.

1.

5. The liposome drug delivery system according to at least one of claims 1 to 4, characterized in that The mass ratio of deoxycholic acid to phospholipid is 1:4-1:7, preferably 1:4.3-1:6.5; more preferably 1:(5-6); And / or, the mass ratio of deoxycholic acid to the first phospholipid and the second phospholipid is 1:(4-10):(0-0.6), preferably 1:(4-6):(0.2-0.6), and more preferably 1:(4-6):(0.4-0.5).

6. The liposome drug delivery system according to at least one of claims 1 to 5, characterized in that Optionally, cholesterol and / or an antioxidant are also included; preferably, the antioxidant is selected from vitamin E and / or vitamin E acetate.

7. The liposome drug delivery system according to at least one of claims 1 to 6, characterized in that It also comprises an aqueous phase; the aqueous phase is preferably selected from one or more of a disodium succinate solution, a histidine solution, a physiological saline solution, a sucrose solution and water for injection; Preferably, the aqueous phase is selected from one or more of a 0.02-0.04M disodium succinate solution containing 0.5% NaCl, a 0.05M disodium succinate buffer solution, a 0.025M histidine solution, a physiological saline solution, and an 8% sucrose solution; more preferably, the aqueous phase is a 0.02-0.04M disodium succinate solution containing 0.5% NaCl; More preferably, the pH value of the 0.02-0.04 M disodium succinate solution containing 0.5% NaCl is 5.0-7.0; preferably the pH value is 6-6.

5.

8. The liposome drug delivery system according to at least one of claims 1 to 7, characterized in that Each unit preparation is prepared from the following components: 1-5 parts of deoxycholic acid, 15-30 parts of the first phospholipid, 0-2 parts of the second phospholipid and 100-5000 parts of an aqueous phase; preferably, the aqueous phase is 500-5000 parts; Alternatively, each unit preparation is prepared from the following components: 1-5 parts of deoxycholic acid, 15-30 parts of the first phospholipid, 0-2 parts of the second phospholipid, 500-5000 parts of an aqueous phase, 0-2.5 parts of cholesterol, and 0-0.625 parts of vitamin E acetate; Preferably, each unit preparation is prepared from the following components: 5 parts of deoxycholic acid, 15-30 parts of the first phospholipid, 0-2 parts of the second phospholipid, 500 parts of the aqueous phase, 0-2.5 parts of cholesterol and 0-0.625 parts of vitamin E acetate.

9. The liposome drug delivery system according to at least one of claims 1 to 8, characterized in that The first phospholipid is selected from one or more of soybean lecithin (SPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); preferably, the first phospholipid is selected from one or more of dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); more preferably, dioleoylphosphatidylcholine (DOPC); The second phospholipid is selected from distearoylphosphatidylcholine (DSPG), dioleoylphosphatidylserine (DOPS), distearoylphosphatidylserine (DSPS), (2-dioleoylhydroxypropyl)trimethylammonium chloride (DOTAP), 1,2-dioleyl-3-dimethylamino-propane (DODMA), distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and dioleoylphosphatidylglycerol (DOPG) and egg yolk phosphatidylglycerol (EPG) one or more; preferably, the second phospholipid is selected from one or more of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) and distearoyl phosphatidylcholine (DSPG); more preferably, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); The aqueous phase is selected from one or more of a 0.02-0.04M disodium succinate solution containing 0.5% NaCl, a 0.05M disodium succinate buffer solution, a 0.025M histidine solution, a physiological saline solution, and an 8% sucrose solution; preferably, the aqueous phase is selected from one or more of a 0.02-0.04M disodium succinate solution containing 0.5% NaCl, a 0.025M histidine solution, a physiological saline solution, and an 8% sucrose solution; more preferably, the aqueous phase is a 0.02-0.04M disodium succinate solution containing 0.5% NaCl.

10. The liposome drug delivery system according to at least one of claims 1 to 9, characterized in that The first phospholipid and the second phospholipid satisfy any one of the following combinations: (1) soybean lecithin (SPC) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (2) hydrogenated soy lecithin (HSPC) and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (3) distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (4) Dierucylphosphatidylcholine (DEPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (5) 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (6) myristoylphosphatidylcholine (DMPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (7) dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); (8) Dioleoylphosphatidylcholine (DOPC) and distearoylphosphatidylethanolamine-polyethylene glycol PEG 2000 (DSPE-PEG2000); (9) dioleoylphosphatidylcholine (DOPC) and distearoylphosphatidylcholine (DSPG); (10) dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG); (11) Dioleoylphosphatidylcholine (DOPC) and 1,2-dioleyl-3-dimethylamino-propane (DODMA).

11. The liposome drug delivery system according to at least one of claims 1 to 10, characterized in that The first phospholipid is dioleoylphosphatidylcholine (DOPC), the second phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the mass ratio of the first phospholipid to the second phospholipid is (4-6):(0.4-0.6); the mass ratio of the first phospholipid to the second phospholipid is preferably (4-6):(0.4-0.5), for example 5:0.4 or 5:0.5; Alternatively, the first phospholipid is dieucoylphosphatidylcholine (DEPC), the second phospholipid is distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the mass ratio of the first phospholipid to the second phospholipid is (4-6):(0.4-0.6); the mass ratio of the first phospholipid to the second phospholipid is preferably (4-6):(0.4-0.5), for example 5:0.4 or 5:0.5; Alternatively, the first phospholipid is 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the second phospholipid is distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), and the mass ratio of the first phospholipid to the second phospholipid is (4-6):(0.4-0.6); the mass ratio of the first phospholipid to the second phospholipid is preferably (4-6):(0.4-0.5), for example 5:0.4 or 5:0.

5.

12. The method for preparing the liposome drug delivery system according to at least one of claims 1 to 11, characterized in that: The steps include: Ⅰ. taking the deoxycholic acid, the first phospholipid, the second phospholipid, cholesterol and the antioxidant according to the ratio, dissolving them in an organic solvent to prepare an organic phase; II. removing the organic solvent from the organic phase obtained in step I, and adding an aqueous phase thereto for hydration to obtain a crude suspension; III. Homogenize the crude suspension obtained in step II to obtain suspension a; IV. The suspension a obtained in step III was filtered through a 0.45 μm filter membrane or a (1.0 + 0.65) μm filter. The mixture is filtered through a double-layer filter membrane and then sterilized through filtration using a 0.22 μm microporous filter membrane to obtain liposomes; the antioxidant is preferably vitamin E acetate.

13. The preparation method according to claim 12, characterized in that: The total solid content of the organic phase in step I is 20% (w / v); And / or, the organic solvent in step I is selected from one or more of chloroform, anhydrous ethanol and anhydrous methanol, preferably a mixed solution of chloroform and anhydrous methanol; in the mixed solution of chloroform and anhydrous methanol, the volume ratio of chloroform to anhydrous ethanol is preferably 1:1 to 30:1, for example 10:1; And / or, the aqueous phase in step II is selected from one or more of a 0.02-0.04M disodium succinate solution containing 0.5% NaCl, a 0.05M disodium succinate buffer solution, a 0.025M histidine solution, a physiological saline solution, and an 8% sucrose solution; more preferably, the aqueous phase is a 0.02-0.04M disodium succinate solution containing 0.5% NaCl; more preferably, the pH value of the 0.02-0.05M disodium succinate solution containing 0.5% NaCl is 5.0-7.0, for example 6.0-6.5; And / or, the method for removing the organic solvent in step II is vacuum distillation; the vacuum distillation is preferably carried out at 45-60°C; And / or, the hydration temperature in step II is 20-60°C, preferably 50-60°C or 20-25°C; And / or, the hydration time in step II is 60-120 min; And / or, the homogenization condition in step III is 8000-12000 psi homogenization 3-5 times.

14. A liposome preparation, characterized in that: A liposome drug delivery system comprising any one of claims 1 to 11; the preparation is an injection solution or a lyophilized preparation.

15. The liposome preparation according to claim 14, characterized in that When the preparation is a lyophilized preparation, it may further optionally contain a lyophilization excipient, wherein the lyophilization excipient is selected from one or more of sucrose, lactose, trehalose and mannitol; preferably sucrose.

16. The liposomal formulation according to at least one of claims 14 to 15, characterized in that When the liposome preparation is a lyophilized preparation, the preparation method of the lyophilized preparation comprises the preparation method of the liposome drug delivery system according to at least one of claims 12-13, and after homogenization in step III and before filtering with a filter membrane in step IV, the suspension a is mixed with a lyophilized excipient, and then an aqueous phase is added; then filtered with a 0.45 μm filter membrane, and then filtered with a 0.22 μm microporous filter membrane to obtain liposomes, and the liposomes are lyophilized; The aqueous phase is preferably a 0.025M disodium succinate solution containing 0.5% NaCl at a pH of 6 to 6.5; The aqueous phase is preferably added to make the weight percentage of the lyophilized excipient in the solution reach 5%-8%, preferably 6%.

17. The liposome drug delivery system according to at least one of claims 1 to 11 or the liposome preparation according to at least one of claims 14 to 16, characterized in that: The average particle size of the liposome is 50-300 nanometers, preferably 50-100 nanometers; more preferably, the average particle size of the liposome is 60-95 nanometers, most preferably 60-91.5 nanometers; and / or, in the liposome system or liposome preparation, the liposome encapsulation efficiency is 75% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more; and / or, in the liposome system or liposome preparation, the polydispersity coefficient of the liposome is 0.1-0.4, preferably 0.1-0.35, more preferably 0.1-0.25, most preferably 0.1-0.2; And / or, in the liposome system or liposome preparation, the Zeta potential of the liposome is -65mV to -5mV, preferably -45mV to -20mV, more preferably -45mV to -35mV.

18. Use of the liposome drug delivery system of at least one of claims 1 to 11 or the liposome preparation of at least one of claims 14 to 16 in the preparation of fat-reducing drugs or medical cosmetics; preferably, the fat-reducing drugs or medical cosmetics are local fat-reducing drugs or local medical cosmetics.

19. A fat-reducing drug or medical beauty product, characterized in that: Containing at least one of the liposome drug delivery systems of claims 1 to 11 or at least one of the liposome preparations of claims 14 to 16, and excipients or auxiliary ingredients acceptable in fat-reducing drugs or medical beauty products; Preferably, the auxiliary material or auxiliary component is selected from one or more of ascorbic acid (VC), benzoic acid and lidocaine.

20. The fat-reducing drug or medical cosmetic product according to claim 19, characterized in that: The fat-reducing medicine or medical beauty product is a liposome preparation for local injection into adipose tissue.

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