Fatty emulsion injection containing deoxycholic acid, and preparation method and use thereof

Abstract

The application provides a deoxycholic acid-containing fat emulsion injection, which is an O / W type submicroemulsion and comprises deoxycholic acid, soybean oil for injection, lecithin for injection, glycerol, anhydrous disodium hydrogen phosphate, alpha-tocopherol and water for injection. The application further discloses a preparation method of the deoxycholic acid-containing fat emulsion injection, which comprises the steps of oil phase preparation, water phase preparation, initial emulsion preparation, drug loading, high-pressure homogenization, sterilization and filling and sealing. The deoxycholic acid-containing fat emulsion injection prepared by the method has the advantages of low irritation, high tolerance, targeted slow release, high fat-dissolving efficiency, excellent stability and high safety, and can be used for preparing a medicine for improving local subcutaneous fat accumulation of adults and has important clinical value.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical formulation technology, specifically relating to a fat emulsion injection containing deoxycholic acid, its preparation method and uses, especially its application in local fat reduction and improvement of subcutaneous fat accumulation. Background Technology

[0002] Deoxycholic acid injection (trade name: Kybella) is a pharmaceutical agent developed by the American biopharmaceutical company Kythera Biopharmaceuticals for improving moderate to severe submental fat (double chin) in adults. In April 2015, the U.S. FDA officially approved Kybella for marketing, making it the world's first approved injectable lipolysis product.

[0003] Deoxycholic acid injection, its active ingredient is deoxycholic acid (DCA), chemically named 3α,12α-dihydroxy-5β-cholanic acid, with the molecular formula C24H40O4 and a molecular weight of 392.57 g / mol. The chemical structure of deoxycholic acid is as follows:

[0004] Deoxycholic acid is a naturally occurring secondary bile acid in human bile. It has surface activity and can destroy the adipocyte membrane and induce adipocyte apoptosis, thereby achieving local fat reduction. Currently available deoxycholic acid injections are aqueous solutions, which have the following drawbacks: (1) strong irritation, high ionic strength, and a high incidence of injection pain, redness, swelling, and induration; (2) short half-life and rapid diffusion: it is highly water-soluble and easily diffuses to non-target tissues, resulting in poor fat-dissolving targeting and the need for multiple injections.

[0005] Fat emulsion injections are O / W submicroemulsions with vegetable oils such as soybean oil as the oil phase, lecithin as the emulsifier, and glycerol as the isotonic regulator. They have a particle size of approximately 100-500 nm and offer advantages such as sustained release, targeted delivery, low irritation, and good biocompatibility. Loading deoxycholic acid into fat emulsions can significantly reduce free drug concentration, decrease irritation, prolong local retention time, and improve lipolysis targeting; however, no related reports have been found to date. Summary of the Invention

[0006] This invention provides a deoxycholic acid fat emulsion injection that is low in irritation, highly stable, targeted and sustained-release, and has high safety, as well as its preparation method and uses, solving the problems of high irritation and poor targeting of existing aqueous injections.

[0007] The technical solution of this invention is achieved through the following means: In a first aspect, the present invention provides a fat emulsion injection containing deoxycholic acid, wherein the injection is an O / W type submicroemulsion, comprising deoxycholic acid, soybean oil for injection, lecithin for injection, glycerol, anhydrous disodium hydrogen phosphate, 0.01~0.05g of α-tocopherol and water for injection.

[0008] In one or more embodiments, each 100 mL of the injection comprises: 0.5-2.0 g of deoxycholic acid, 10-20 g of soybean oil for injection, 1.0-1.5 g of lecithin for injection, 2.0-2.5 g of glycerol, 0.25-0.30 g of anhydrous disodium hydrogen phosphate, 0.01-0.05 g of α-tocopherol, and water for injection to a final volume of 100 mL.

[0009] In one or more embodiments, each 100 mL of the injection comprises: 1.0 g of deoxycholic acid, 15.0 g of soybean oil for injection, 1.2 g of lecithin for injection, 2.2 g of glycerol, 0.268 g of anhydrous disodium hydrogen phosphate, 0.02 g of α-tocopherol, and water for injection to a final volume of 100 mL.

[0010] In this invention, the injectable lecithin is not limited. Those skilled in the art can select different injectable lecithin according to actual needs, such as hydrogenated soybean lecithin or egg yolk lecithin.

[0011] In one or more embodiments, the injectable has a pH of 7.5 to 8.0 and an average particle size of 150 to 200 nm.

[0012] In one or more embodiments, the injectable has a pH of 7.8 and an average particle size of 180 nm.

[0013] In a second aspect, the present invention provides a method for preparing the fat emulsion injection containing deoxycholic acid as described in any one of the claims, comprising the following steps: (1) Preparation of oil phase: Take soybean oil for injection, add α-tocopherol, heat and stir to dissolve, and obtain oil phase; (2) Preparation of aqueous phase: Take water for injection, heat it, add lecithin, glycerol and anhydrous disodium hydrogen phosphate, stir until completely dissolved to obtain aqueous phase; (3) Preparation of colostrum: The oil phase is slowly added to the aqueous phase and sheared at high speed for 8-12 min to obtain the colostrum; (4) Drug loading: Dissolve deoxycholic acid in a small amount of water for injection, adjust the pH to 7.5-8.0, slowly add it to the colostrum, and continue shearing for 4-6 minutes; (5) High-pressure homogenization: Cool the primary emulsion to room temperature and homogenize it under high pressure, controlling the average particle size to be 150~300nm; (6) Sterilization and filling: After filtration with a microporous membrane, the product is filled under aseptic conditions to obtain a fat emulsion injection containing deoxycholic acid.

[0014] In one or more embodiments, the heating temperature in step (1) is 60~70°C. In one or more embodiments, the heating temperature in step (2) is 60~70°C and the stirring speed is 8000 rpm.

[0015] In one or more embodiments, the temperature of the high-speed shearing in step (3) is 60~70°C and the shearing speed is 12000 rpm.

[0016] In one or more embodiments, the high-pressure homogenization conditions in step (5) are: 800 bar, 3 cycles.

[0017] In one or more embodiments, the microporous filter membrane in step (6) has a pore size of 0.22 μm, and the filling is performed by filling a vial with nitrogen gas.

[0018] Thirdly, the present invention provides the use of the fat emulsion injection containing deoxycholic acid as described in any one of the claims, or the fat emulsion injection containing deoxycholic acid obtained by any one of the preparation methods, in the preparation of a drug for improving local subcutaneous fat accumulation in adults.

[0019] Compared with related technologies, the present invention has the following advantages: (1) Low irritation and high tolerance: The pH of the injection of the present invention is close to the physiological pH and the concentration of free drug is low, which can significantly reduce the risk of injection pain, redness and swelling and induration; (2) Targeted sustained release and efficient fat dissolving: The fat emulsion in the injection of the present invention has a particle size of 150~300nm, which can be selectively taken up by adipose tissue, and the local retention time is extended by 2~3 times, reducing the number of injections; (3) Excellent stability: The injection of the present invention can be stored at 4°C in the dark for 12 months without significant changes in particle size, pH and content; (4) High safety: The injection of the present invention is free of benzyl alcohol (to avoid hemolysis / sensitization), free of preservatives, and has good biocompatibility.

[0020] To provide a clearer understanding of this application, the specific embodiments of this application will be described below in conjunction with the accompanying drawings. Attached Figure Description

[0021] Figure 1 To detect the animal stimulation results in the experimental group of Example 3 24 hours later.

[0022] Figure 2 To detect the animal stimulation results in the experimental group of Example 3 48 hours later.

[0023] Figure 3To detect the animal stimulation results in the control group of Example 3 24 hours later.

[0024] Figure 4 To detect the animal stimulation results in the control group of Example 3 48 hours later. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0026] It should be understood that the described embodiments are merely some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of the embodiments of this application.

[0027] In the following description, when referring to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. In the description of this application, it should be understood that the terms "first," "second," "third," etc., are used only to distinguish similar objects and are not necessarily used to describe a specific order or sequence, nor should they be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances. The singular forms "a," "the," and "the" used in this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise. The word "if" as used herein can be interpreted as "when," "when," or "in response to determination."

[0028] Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0029] Unless otherwise specified, the experimental methods used in the following examples and comparative examples were conducted under conventional conditions or as recommended by the raw material supplier; all raw material packages used were commercially available.

[0030] Example 1: Fat emulsion injection A containing deoxycholic acid and its preparation method A fat emulsion injection A containing deoxycholic acid is an O / W type submicron emulsion. Each 100 mL of the injection contains: 1.0 g of deoxycholic acid, 15.0 g of soybean oil for injection, 1.2 g of hydrogenated soybean lecithin, 2.2 g of glycerol, 0.268 g of anhydrous disodium hydrogen phosphate, 0.02 g of α-tocopherol, and water for injection to a final volume of 100 mL.

[0031] The preparation method of the fat emulsion injection A containing deoxycholic acid includes the following steps: (1) Preparation of oil phase: Take soybean oil for injection, add α-tocopherol, stir and dissolve at 65°C to obtain oil phase; (2) Preparation of aqueous phase: Take water for injection, heat it to 65°C, add hydrogenated soybean lecithin, glycerol and anhydrous disodium hydrogen phosphate, stir at 8000 rpm until completely dissolved to obtain aqueous phase; (3) Preparation of colostrum: The oil phase is slowly added to the aqueous phase and sheared at high speed for 10 min to obtain the colostrum; the shearing temperature is 65℃ and the shearing speed is 12000 rpm; (4) Drug loading: Dissolve deoxycholic acid in a small amount of water for injection, adjust the pH to 7.8, slowly add it to the colostrum, and continue shearing for 5 minutes; (5) High-pressure homogenization: Cool the colostrum to room temperature and homogenize it under high pressure (800 bar, 3 cycles) to control the average particle size to 200 nm; (6) Sterilization and filling: After filtration with a 0.22μm microporous membrane, the solution is filled into vials under aseptic conditions with nitrogen gas to obtain fat emulsion injection A containing deoxycholic acid.

[0032] Example 2: Fat emulsion injection B containing deoxycholic acid and its preparation method A fat emulsion injection B containing deoxycholic acid is an O / W type submicron emulsion. Each 100 mL of the injection contains: 0.5 g of deoxycholic acid, 10 g of soybean oil for injection, 1.0 g of egg yolk lecithin, 2.0 g of glycerol, 0.25 g of anhydrous disodium hydrogen phosphate, 0.01 g of α-tocopherol, and water for injection to a final volume of 100 mL.

[0033] The preparation method of the fat emulsion injection B containing deoxycholic acid includes the following steps: (1) Preparation of oil phase: Take soybean oil for injection, add α-tocopherol, stir and dissolve at 60°C to obtain oil phase; (2) Preparation of aqueous phase: Take water for injection, heat it to 70°C, add egg yolk lecithin, glycerol, and anhydrous disodium hydrogen phosphate, and stir at 8000 rpm until completely dissolved to obtain the aqueous phase; (3) Preparation of colostrum: The oil phase is slowly added to the aqueous phase and sheared at high speed for 8 min to obtain the colostrum; the shearing temperature is 70℃ and the shearing speed is 12000 rpm; (4) Drug loading: Dissolve deoxycholic acid in a small amount of water for injection, adjust the pH to 7.5, slowly add it to the colostrum, and continue shearing for 6 minutes; (5) High-pressure homogenization: Cool the colostrum to room temperature and homogenize it under high pressure (800 bar, 3 cycles) to control the average particle size to 150 nm; (6) Sterilization and filling: After filtration with a 0.22μm microporous membrane, the solution is filled into vials under aseptic conditions with nitrogen gas to obtain fat emulsion injection B containing deoxycholic acid.

[0034] Example 3: Fat emulsion injection C containing deoxycholic acid and its preparation method A fat emulsion injection C containing deoxycholic acid, which is an O / W type submicron emulsion, comprises, per 100 mL: 2.0 g of deoxycholic acid, 20 g of soybean oil for injection, 1.5 g of hydrogenated soybean lecithin, 2.5 g of glycerol, 0.30 g of anhydrous disodium hydrogen phosphate, 0.05 g of α-tocopherol, and water for injection to a final volume of 100 mL.

[0035] The preparation method of the fat emulsion injection C containing deoxycholic acid includes the following steps: (1) Preparation of oil phase: Take soybean oil for injection, add α-tocopherol, stir and dissolve at 60~70℃ to obtain oil phase; (2) Preparation of aqueous phase: Take water for injection, heat it to 70°C, add hydrogenated soybean lecithin, glycerol and anhydrous disodium hydrogen phosphate, stir at 8000 rpm until completely dissolved to obtain aqueous phase; (3) Preparation of colostrum: The oil phase is slowly added to the aqueous phase and sheared at high speed for 12 min to obtain the colostrum; the shearing temperature is 60℃ and the shearing speed is 12000 rpm. (4) Drug loading: Dissolve deoxycholic acid in a small amount of water for injection, adjust the pH to 8.0, slowly add it to the colostrum, and continue shearing for 4 min; (5) High-pressure homogenization: Cool the colostrum to room temperature and homogenize it under high pressure (800 bar, 3 cycles) to control the average particle size to 180 nm; (6) Sterilization and filling: After filtration with a 0.22μm microporous membrane, the solution is filled into vials under aseptic conditions with nitrogen to obtain fat emulsion injection C containing deoxycholic acid.

[0036] Comparative Example 1: Fat emulsion injection D containing deoxycholic acid and its preparation method A fat emulsion injection D containing deoxycholic acid has the same composition and preparation method as in Example 1, except that BHT (butylated hydroxytoluene) is used instead of α-tocopherol in Example 1.

[0037] Test Example 1: Accelerated Stability Test The deoxycholic acid-containing fat emulsion injections (AD) prepared in the examples and comparative examples were subjected to accelerated stability tests (stored for 6 months at 40℃±2℃ and RH 75%±5%). Changes in key indicators, peroxide value (POV), and acid value (AV) were measured, and the results are shown in Table 1. Table 1 Results of accelerated stability tests

[0038] Peroxide value reflects the initial degree of oxidation of oils and fats; the smaller the increase, the stronger the antioxidant capacity. After the experiment, the peroxide value increase of injection AC was only about 50%, while that of injection D was 212%, indicating that α-tocopherol, as a natural antioxidant, exhibited a superior ability to inhibit free radical generation and effectively slow down the oxidation process in this invention.

[0039] The increase in acid value originates from the hydrolysis of glycerides to produce free fatty acids, and its change is related to the stability of the emulsion system and the synergistic effect of antioxidant activity. After the experiment, the acid value of injectable AC increased by only about 25%, while that of injectable D increased by 133%, indicating that in this invention, α-tocopherol may indirectly inhibit hydrolysis side reactions and maintain system stability by slowing down the oxidative chain reaction.

[0040] After the experiment, neither injection AC nor injection C showed any stratification or precipitation, indicating that the emulsion system had good physical stability. However, injection C showed emulsification and a slightly yellow color in the fourth month, suggesting that the accumulation of oxidation products led to the destruction of the interfacial film and the aggregation of emulsion droplets.

[0041] Based on the above three indicators, the fat emulsion injections containing deoxycholic acid prepared in Examples 1-3 are significantly better than those in Comparative Example 1 in terms of both chemical and physical stability, indicating that the antioxidant effect of α-tocopherol in this emulsion system is significantly better than that of BHT.

[0042] Test Example 2: Long-term rate stability test The fat emulsion injection A containing deoxycholic acid prepared in Example 1 was subjected to a long-term stability test. After being placed at 4°C in the dark for 12 months, its appearance, particle size, pH and deoxycholic acid content were tested.

[0043] The test results are as follows: Appearance: It remains a white, uniform emulsion, without layering or sedimentation; Particle size: 195nm→202nm (change <5%); pH: 7.8 → 7.7; Content: 99.5% → 98.2% (degradation <2%).

[0044] The above results indicate that the fat emulsion injection A containing deoxycholic acid prepared in Example 1 has excellent stability and can be used for industrial production.

[0045] Test Example 3: Animal Stimulation Test Experimental materials: Guinea pigs were used in the experiment and were sourced from Qingdao Kangda Aibo Biotechnology Co., Ltd.

[0046] Commercially available deoxycholic acid injection (licensed manufacturer: Allergan Pharmaceuticals International Ltd, specification: 10mg / ml, trade name: Kybella) The experimental group was prepared by subcutaneous injection of fat emulsion A containing deoxycholic acid, as described in Example 1, into the back of guinea pigs. The control group was prepared by injection of commercially available deoxycholic acid aqueous solution (10 mg / mL). The results are as follows: Experimental group: slight redness and swelling at 24 hours, completely subsided at 48 hours, with no induration; Control group: Significant redness, swelling and pain were observed at 24 hours, and induration was still present at 48 hours.

[0047] The above results indicate that the fat emulsion injection A containing deoxycholic acid prepared in Example 1 has low irritation and higher safety.

[0048] In summary, the deoxycholic acid-containing fat emulsion injection of the present invention has the advantages of low irritation, high tolerance, targeted sustained release, high efficiency in fat dissolution, excellent stability, and high safety. It can be used to prepare drugs to improve local subcutaneous fat accumulation in adults and has important clinical value.

[0049] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A fat emulsion injection containing deoxycholic acid, characterized in that, The injection is an O / W type submicroemulsion, which contains deoxycholic acid, soybean oil for injection, lecithin for injection, glycerol, anhydrous disodium hydrogen phosphate, 0.01~0.05g of α-tocopherol and water for injection.

2. The fat emulsion injection containing deoxycholic acid according to claim 1, characterized in that, Each 100 mL of the injection comprises: 0.5-2.0 g of deoxycholic acid, 10-20 g of soybean oil for injection, 1.0-1.5 g of lecithin for injection, 2.0-2.5 g of glycerol, 0.25-0.30 g of anhydrous disodium hydrogen phosphate, 0.01-0.05 g of α-tocopherol, and water for injection to a final volume of 100 mL.

3. The fat emulsion injection containing deoxycholic acid according to claim 2, characterized in that, Each 100 mL of the injection contains: 1.0 g of deoxycholic acid, 15.0 g of soybean oil for injection, 1.2 g of lecithin for injection, 2.2 g of glycerol, 0.268 g of anhydrous disodium hydrogen phosphate, 0.02 g of α-tocopherol, and water for injection to a final volume of 100 mL.

4. The fat emulsion injection containing deoxycholic acid according to any one of claims 1, characterized in that, The injection has a pH of 7.5 to 8.0 and an average particle size of 150 to 200 nm.

5. The fat emulsion injection containing deoxycholic acid according to claim 4, characterized in that, The injection has a pH of 7.8 and an average particle size of 180 nm.

6. The method for preparing the fat emulsion injection containing deoxycholic acid according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of oil phase: Take soybean oil for injection, add α-tocopherol, heat and stir to dissolve, and obtain oil phase; (2) Preparation of aqueous phase: Take water for injection, heat it, add lecithin, glycerol and anhydrous disodium hydrogen phosphate, stir until completely dissolved to obtain aqueous phase; (3) Preparation of colostrum: The oil phase is slowly added to the aqueous phase and sheared at high speed for 8-12 min to obtain the colostrum; (4) Drug loading: Dissolve deoxycholic acid in a small amount of water for injection, adjust the pH to 7.5-8.0, slowly add it to the colostrum, and continue shearing for 4-6 minutes; (5) High-pressure homogenization: Cool the primary emulsion to room temperature and homogenize it under high pressure, controlling the average particle size to be 150~300nm; (6) Sterilization and filling: After filtration with a microporous membrane, the product is filled under aseptic conditions to obtain a fat emulsion injection containing deoxycholic acid.

7. The preparation method according to claim 6, characterized in that, The heating temperature in step (1) is 60~70℃; the heating temperature in step (2) is 60~70℃, and the stirring speed is 8000rpm.

8. The preparation method according to claim 6, characterized in that, The temperature of the high-speed shearing in step (3) is 60~70℃, and the shearing speed is 12000rpm.

9. The preparation method according to claim 6, characterized in that, The high-pressure homogenization conditions in step (5) are: 800 bar, 3 cycles; the pore size of the microporous filter membrane in step (6) is 0.22 μm, and the filling is nitrogen-filled into vials.

10. The use of the fat emulsion injection containing deoxycholic acid according to any one of claims 1-6 or the fat emulsion injection containing deoxycholic acid obtained by the preparation method according to any one of claims 7-9 in the preparation of a drug for improving local subcutaneous fat accumulation in adults.

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