Abiraterone acetate self-nanoemulsion liquid-filled capsule as well as preparation method and application thereof

Abstract

The invention discloses an abiraterone acetate self-nanoemulsion liquid-filled capsule as well as a preparation method and application thereof. The abiraterone acetate self-nanoemulsion liquid-filled capsule is good in self-nanoemulsion effect, controllable in preparation quality and remarkable in killing effect on prostate cancer cells, the bioavailability of the capsule is improved by 12.54 times compared with that of commercially available tablets, the influence of food on the bioavailability of drugs is reduced, the limitation of abiraterone tablets in clinical application is expected to be overcome, and the capsule is worthy of popularization and application. The research has important scientific significance and potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to abiraterone acetate self-filled nanoemulsion capsules, their preparation methods, and applications. Background Technology

[0002] Prostate cancer is the most common malignant tumor of the male genitourinary system, seriously threatening men's lives and health. Based on its progression, prostate cancer is currently clinically classified into localized prostate cancer and metastatic prostate cancer.

[0003] Treatment strategies for prostate cancer include surgery, chemotherapy, radionuclide therapy, androgen deprivation therapy, and immunotherapy. Surgery is effective for localized prostate cancer, while androgen deprivation therapy remains the first-line clinical treatment for metastatic prostate cancer. However, first-line drugs such as abiraterone acetate are mostly BCS Class IV drugs, characterized by low solubility and low permeability, resulting in extremely low bioavailability and limiting their clinical application.

[0004] Currently, the main dosage form of abiraterone acetate in clinical treatment is tablets. The main problems with this dosage form are: low solubility (0.001 mg / mL), low transmembrane absorption (leading to low bioavailability), easy hydrolysis in the gastrointestinal tract, instability, large oral dose (1000 mg / dose), high raw material cost, and a bioavailability of less than 10% in patients. Furthermore, the bioavailability of abiraterone acetate tablets is greatly affected by food effects, with a tenfold difference between fasting and postprandial bioavailability. Therefore, exploring new dosage forms to improve the bioavailability of abiraterone acetate is a key issue that needs to be addressed. Summary of the Invention

[0005] The purpose of this invention is to provide an abiraterone acetate self-filled nanoemulsion capsule.

[0006] The abiraterone acetate self-nanoemulsion filled capsule provided by the present invention includes a capsule shell and an abiraterone acetate self-nanoemulsion filled in the capsule shell;

[0007] The abiraterone acetate nanoemulsion is composed of abiraterone acetate and excipients in a mass ratio of (12.5-25):500. The excipients are made from the following raw materials in the following mass percentages: oil phase 20-40%, emulsifier 30-53%, and co-emulsifier 25-30%. The oil phase is selected from any two of glyceryl monooleate, glyceryl monolinoleate, and medium-chain triglycerides. The emulsifier is selected from one or two of polysorbate-80, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil. The co-emulsifier is selected from one or two of ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether.

[0008] When the co-emulsifier contains ethanol or is ethanol, the mass percentage of ethanol is ≤30%, preferably ≤15%.

[0009] Furthermore, the abiraterone acetate nanoemulsion is composed of abiraterone acetate and excipients in a mass ratio of 25:500. The excipients are made from raw materials with the following mass percentages: oil phase 30-40%, emulsifier 30-40%, and co-emulsifier 25-30%.

[0010] Preferably, in every 1000 capsules, the abiraterone acetate nanoemulsion is made from the following raw materials: 25g abiraterone acetate, 75g monolinoleic acid glyceride, 75g medium-chain triglycerides, 80-100g polysorbate, 100g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether.

[0011] Alternatively, in every 1000 capsules, the abiraterone acetate nanoemulsion is made from the following ingredients: 25g abiraterone acetate, 100g glyceryl monooleate, 100g medium-chain triglycerides, 80g polysorbate, 75g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether.

[0012] Alternatively, for every 1000 capsules, the abiraterone acetate nanoemulsion is made from the following ingredients: 25g abiraterone acetate, 100g glyceryl monooleate, 100g medium-chain triglycerides, 100g polysorbate 80, 75g polyoxyethylene hydrogenated castor oil, 50g ethanol, and 75g diethylene glycol monoethyl ether.

[0013] The most preferred formulation is that each 1000 capsules contains abiraterone acetate nanoemulsion made from the following ingredients: 25g abiraterone acetate, 75g monolinoleic acid glyceride, 75g medium-chain triglycerides, 100g polysorbate 80, 100g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether.

[0014] Referring to the market specification of abiraterone acetate tablets (250 mg / tablet) and combining the pharmacokinetic data of abiraterone acetate self-nanoemulsion filled capsules of the present invention in animals (see Example 5), based on the maximum drug loading of abiraterone acetate in excipients being 67 mg / g, capsule #1 was used, with each capsule containing 0.525 g of abiraterone acetate self-nanoemulsion, that is, each capsule containing 25 mg of abiraterone acetate.

[0015] The abiraterone acetate nanoemulsion filled in the capsules described above also falls within the scope of this invention.

[0016] The present invention also provides a method for preparing abiraterone acetate self-filled nanoemulsion capsules.

[0017] The method for preparing abiraterone acetate self-filled nanoemulsion capsules provided by the present invention includes the following steps:

[0018] 1) Mix the abiraterone acetate raw material in equal increments, pass it through a 100-mesh sieve, and collect the portion that passes through the sieve;

[0019] 2) Weigh the oil phase, emulsion, and co-emulsifier according to the proportion, mix them, and then mix them by ultrasonication, vortexing, and other operations until there is no obvious layering to obtain a blank self-nanoemulsion formulation;

[0020] 3) Mix the sieved abiraterone acetate raw material from step 1) with the blank self-nanoemulsion preparation, and vortex mix at 37°C for 24 hours to obtain abiraterone acetate self-nanoemulsion.

[0021] 4) Fill No. 1 opaque capsules at room temperature, filling each capsule with 0.525g of the abiraterone acetate nanoemulsion to obtain abiraterone acetate nanoemulsion filled capsules.

[0022] This invention provides a novel abiraterone acetate-loaded self-nanoemulsion drug delivery system, verifying its cytotoxic effect on prostate cancer cells and investigating its bioavailability in beagle dogs. The results show that this invention successfully established the formulation process and quality standards for abiraterone acetate self-nanoemulsion-filled capsules. This formulation is stable, with a self-nanoemulsion particle size of approximately 30 nm, an abiraterone acetate encapsulation rate of 91%, and stable and controllable formulation quality. The cytotoxic effect and apoptosis-inducing effect of the abiraterone acetate self-nanoemulsion on prostate cancer cells were verified. The abiraterone acetate self-nanoemulsion-filled capsules demonstrated significantly enhanced oral absorption and bioavailability in beagle dogs; compared to the control formulation, the pre-meal oral relative bioavailability of the abiraterone acetate self-nanoemulsion-filled capsules was increased by 12.54 times, and its bioavailability was not affected by food.

[0023] This invention successfully developed a novel abiraterone self-nanoemulsion filled capsule, which exhibits good self-nanoemulsification effect, controllable formulation quality, significant killing effect on prostate cancer cells, and 12.54 times higher bioavailability compared to commercially available tablets. It also reduces the impact of food on drug bioavailability, and is expected to overcome the limitations of abiraterone tablets in clinical application. This research has important scientific significance and potential clinical application value. Attached Figure Description

[0024] Figure 1 The solubility of abiraterone acetate in different media (37℃, n=3); (A) oil phase; (B) emulsifier; (C) co-emulsifier;

[0025] Figure 2The effect of oil phase on the pseudo-ternary phase diagram of abiraterone acetate in different solvent systems; (A) glyceryl monolinoleate; (B) glyceryl monooleate; (C) polyethylene glycol oleate; (D) medium-chain triglycerides;

[0026] Figure 3 The effect of emulsifiers in different solvent systems on the pseudo-ternary phase diagram of abiraterone acetate; (A) polysorbate-80; (B) polyoxyethylene castor oil; (C) polyoxyethylene hydrogenated castor oil;

[0027] Figure 4 The effect of co-emulsifiers in different solvent systems on the pseudo-ternary phase diagram of abiraterone acetate; (A) ethanol; (B) diethylene glycol monoethyl ether; (C) polyethylene glycol 400;

[0028] Figure 5 Image of abiraterone acetate self-filled nanoemulsion capsules;

[0029] Figure 6 The self-emulsification effect of nanoemulsions with different formulations;

[0030] Figure 7 TEM image of abiraterone acetate self-nanoemulsion;

[0031] Figure 8 The effect of self-generated nanoemulsions on the growth of Caco-2 cells;

[0032] Figure 9 To observe the uptake effect of fluorescently labeled nanoemulsions in Caco-2 cells using flow cytometry; (A) Flow cytometry images showing the uptake effect at different concentrations. (B) Quantitative uptake at different concentrations. (C) Flow cytometry showing the uptake effect of a fixed concentration of C6 (100 ng / mL C6) at different times. (D) Quantitative uptake results of a fixed concentration of C6 (100 ng / mL C6) at different times. (n=3)

[0033] Figure 10 To observe the uptake effect of C6-SNEDDS in Caco-2 cells using confocal microscopy;

[0034] Figure 11 The standard curve is C6.

[0035] Figure 12 is the apparent permeability coefficient of C6-SNEDDS in the Caco-2 monolayer membrane model;

[0036] Figure 13 The killing effect of abiraterone acetate nanoemulsion on prostate cancer cells;

[0037] Figure 14 The apoptosis-inducing effect of abiraterone acetate nanoemulsion on prostate cancer cells;

[0038] Figure 15 The daughter ion spectra of abiraterone and internal standard are shown in the following figures: (A) Internal standard (abiraterone-d4); (B) Abiraterone.

[0039] Figure 16 Multiple reaction monitoring profiles for abiraterone and internal standard: (A) Blank plasma; (B) Blank plasma + AB; (C) Blank plasma + IS;

[0040] Figure 17 A standard curve of abiraterone concentration in plasma;

[0041] Figure 18 Blood concentration-time curves of abiraterone after oral administration on an empty stomach in beagle dogs;

[0042] Figure 19 Blood concentration-time curves of abiraterone administered orally on an empty stomach and after a high-fat meal in beagles (A) tablets; (B) capsules. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0044] The materials and instruments used in the following embodiments are as follows:

[0045] 1. Experimental Materials

[0046] Abiraterone acetate raw material, Shanxi Zhendong Pharmaceutical Co., Ltd.; soybean oil (injection), Sinopharm Chemical Reagent Co., Ltd.; ethyl oleate (EO), Sinopharm Chemical Reagent Co., Ltd.; glycerol monooleate (GMO), Gaffars, France; glycerylmonolinoleate (Maisine CC), Gaffars, France; oleoyl polyoxyl-6glycerides (Labrafil M1944 CS), Gaffars, France; polyglyceryl oleate (Plurol oleique CC 497), Gaffars, France; medium chain triglyceride (MCT), Gaffars, France; lauroylmacorgolglycerides (Gelucire 44 / 14), Gaffars, France; propylene glycol monooctanoate. Capryol 90 (acid), Gaffars, France; Polysorbate 80 (Tween-80), Beijing Femo Biotechnology Co., Ltd.; Polyoxyethylene castor oil (EL-35), Beijing Huawi Ruike Chemical Co., Ltd.; Polyethylene glycol glycerol (labrasol, LAB), Gaffars, France; Ethoxylated hydrogenated castor oil (RH-40), Beijing Huawi Ruike Chemical Co., Ltd.; Polysorbate 20 (Tween-20), Beijing Femo Biotechnology Co., Ltd.; Polysorbate 85 (Tween-85), Beijing Femo Biotechnology Co., Ltd.; Polyethylene glycol 400, Beijing Tongguang Fine Chemical Co., Ltd.; Glycerin, Beijing Inokai Technology Co., Ltd.; Diethylene glycol monoethyl ether, Gaffars, France; Artificial gastric juice, Beijing Bizi Biotechnology Co., Ltd.; Artificial intestinal juice, Beijing Bizi Biotechnology Co., Ltd.

[0047] 2. Experimental Apparatus

[0048] BT-25S Precision Electronic Analytical Balance (1 / 100,000 gram), Sartorius GmbH, Germany; Magnetic Heating Stirrer, Beijing Chemical Glass Station Scientific Instruments Co., Ltd.; UV-1800 UV-Vis Spectrophotometer, Shanghai Meipuda Instrument Co., Ltd.; Nexus 470 Infrared Spectrometer, Thermo Fisher Scientific, USA; Agilent 1260 High Performance Liquid Chromatograph, Agilent Technologies, USA; ZORBAX SB-C18 Column (5μm, 150×4.6μm), Agilent Technologies; SB-5200 Ultrasonic Crusher, Ningbo Xinzhi Biotechnology Co., Ltd.; Potentiometric Cell (1mL), Beijing Haike Hongchuang Biotechnology Co., Ltd.; Microporous Filter Membrane. Membrane (0.22μm), Beijing Solarbio Biotechnology Co., Ltd.; Dissolution Tester (RC-6), Tianjin Tianguang Optical Instrument Co., Ltd.; Stability Test Chamber (SHH-150SD), Shanghai Jianheng Instrument Co., Ltd.

[0049] Example 1: Study on the formulation and process of abiraterone self-filled nanoemulsion capsules

[0050] 1 Experimental Methods

[0051] 1.1 Solubility

[0052] Weigh 2g of the oil phase, surfactant, and co-surfactant into 10mL centrifuge tubes, and add excess abiraterone acetate raw material. After thorough vortexing, sonicate at 37℃ for 4 hours to ensure complete dissolution. Equilibrate by shaking at 150rpm / min at 37℃ for 48 hours, allow to stand for 24 hours, and then centrifuge at 15000rpm / min for 15 minutes. Dilute the supernatant 1000 times with methanol, filter through a 0.22μm microporous membrane, and determine the solubility of the filtrate under the chromatographic conditions described above. Repeat this process three times until the dosage remains constant to obtain the saturated solubility of abiraterone acetate in each excipient.

[0053] 1.2 Prescription Screening

[0054] 1.2.1 Orthogonal Experiment for Screening Excipients

[0055] Based on the solubility of abiraterone acetate in various oil phases, emulsifiers, and co-emulsifiers, this paper initially selected four oil phases: glyceryl monooleate, glyceryl monolinoleate, polyethylene glycol oleate, and medium-chain triglycerides; four emulsifiers: polysorbate 80, polyoxyethylene castor oil, PEG-3-caprylate, and hydrogenated polyoxyethylene castor oil; and three co-emulsifiers: ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether for subsequent formulation screening.

[0056] An orthogonal experimental design was used for the four oil phases, four emulsifiers, and three co-emulsifiers mentioned above. Oil phase (A), emulsifier (B), and co-emulsifier (C) were selected as the three factors under investigation, and their proportions were determined according to the commonly used m-value in the literature. A :m B :m C An orthogonal experiment was designed with a ratio of 3:5:2 for subsequent prescription screening. (Based on m...) AbA :m A :m B :m C Weigh each excipient in a ratio of 0.01:3:5:2 and mix them in a beaker. Stir at a constant temperature until homogeneous. Add 50 mL of purified water at 37°C and stir magnetically at 100 rpm for 2 minutes. Observe the appearance of the emulsion, record the emulsification time, and observe its emulsification efficiency. The orthogonal experimental design factor levels are shown in Table 1.

[0057] Table 1. Orthogonal experimental design for screening formulations of abiraterone acetate self-nanoemulsions

[0058]

[0059] 1.2.2 Screening of excipients using pseudo-ternary phase diagram method

[0060] (1) Selection of oil phase

[0061] Using glyceryl monooleate, glyceryl monolinoleate, polyethylene glycol oleate, and medium-chain triglycerides as the oil phase, polysorbate-80 as the emulsifier, and ethanol as the co-emulsifier, the excipients were weighed in different proportions and mixed in a beaker. The mixture was stirred at a constant temperature until homogeneous. 50 mL of purified water at 37 °C was added, and the mixture was stirred at a constant temperature of 100 rpm. The appearance of the emulsion was observed, and its emulsification efficiency was assessed.

[0062] (2) Selection of emulsifier

[0063] Polysorbate-80, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil were used as emulsifiers, medium-chain triglycerides as the oil phase, and ethanol as a co-emulsifier. The excipients were weighed in different proportions and mixed in a beaker. The mixture was stirred at a constant temperature until homogeneous. 50 mL of purified water at 37 °C was added, and the mixture was stirred at a constant temperature of 100 rpm. The appearance of the emulsion was observed, and its emulsification efficiency was measured.

[0064] (3) Selection of emulsifiers

[0065] Ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether were used as co-emulsifiers, medium-chain triglycerides were used as the oil phase, and polysorbate-80 was used as the emulsifier. The excipients were weighed in different proportions and mixed in a beaker. The mixture was stirred at a constant temperature until homogeneous. 50 mL of purified water at 37 °C was added, and the mixture was stirred at a constant temperature of 100 rpm. The appearance of the emulsion was observed, and its emulsification efficiency was assessed.

[0066] Pseudo-ternary phase diagrams of different oil phases were drawn using Origin software. The formulations that formed a transparent emulsion with a slight bluish tint, exhibited stable thermodynamic properties, and were not prone to stratification were plotted as self-emulsifying regions.

[0067] 1.3 Prescription Process

[0068] Based on clinical trial data of abiraterone acetate tablets, and considering that the active pharmaceutical ingredient is a BCS IV drug with poor absorption and solubility, the convenience of the route of administration, and patient acceptance of the dosage form, the formulation was developed. The specifications of the nanoemulsion-filled capsules were based on the marketed specification of abiraterone acetate tablets (250 mg / tablet), and the capsule dosage was determined using in vivo pharmacokinetic data of abiraterone acetate nanoemulsion-filled capsules.

[0069] 2. Experimental Results

[0070] 2.1 Solubility

[0071] The saturated solubility of abiraterone acetate in various oil phases, emulsifiers, and co-emulsifiers was determined by high performance liquid chromatography (HPLC). The results are as follows: Figure 1As shown, in the oil phase, the solubility is Capryol 90 > Masine CC > GMO > Gelucire 44 / 14 > MCT > EO > Obleique CC497 > Labrafil M 1944CS > Soybean oil. Abiraterone acetate has the worst solubility in soybean oil, which also has poor flowability. Although abiraterone acetate has the highest solubility in Capryol 90, Capryol 90 does not have FDA-approved excipient approval. Gelucire 44 / 14 has poor flowability, and Obleique CC497 does not have FDA-approved excipient approval. Therefore, after comprehensive consideration, four oil phases with high solubility and good flowability were selected for the next step of formulation screening: glyceryl monooleate (GMO), glyceryl monolinoleate (maisine CC), polyethylene glycol oleate (labrafil M 1944CS), and medium-chain triglycerides (MCT). Among the emulsifiers, the solubility of abiraterone acetate is LAB > EL-35 > Tween-85 > Tween-80 > Tween-20 > RH-40; among them, Tween-85 has a higher HLB value and higher viscosity. Therefore, considering all factors, four emulsifiers were selected for the next stage of formulation screening: polysorbate-80, polyoxyethylene castor oil, polyethylene glycol glycerol caprylate, and polyoxyethylene hydrogenated castor oil. Among the co-emulsifiers, the solubility of abiraterone acetate is diethylene glycol monoethyl ether > ethanol > polyethylene glycol 400 > propylene glycol > glycerin, and it is almost insoluble in glycerin. Therefore, three co-emulsifiers with relatively high solubility—ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether—were selected for subsequent formulation screening.

[0072] 2.2 Prescription Screening

[0073] 2.2.1 Screening Results of Orthogonal Experiment

[0074] The orthogonal experimental design and results are shown in Table 2. Among the different oil phases, MCT had the shortest emulsification time, resulting in a more stable emulsion and the best emulsification effect, followed by the other three oil phases. Among the emulsifiers, Tween-80, EL-35, and RH-40 all showed good emulsification effects. LAB had the worst emulsification effect, failing to form stable nanoemulsions with any of the four oil phases. Among the co-emulsifiers, ethanol and diethylene glycol monoethyl ether formed a good emulsion with good fluidity, allowing for complete mixing with all components in the formulation. PEG 400, due to its inherent viscosity, easily separated into layers when mixed with other components in the formulation.

[0075] Table 2 Results of orthogonal experimental design for screening formulations of abiraterone acetate-based nanoemulsions

[0076]

[0077]

[0078] 2.2.2 Screening Results of Pseudo-Ternary Phase Diagram Tests

[0079] Using the oil phase, emulsifier, and co-emulsifier as each segment, a pseudo-ternary phase diagram was drawn using Origin software. The area of ​​the self-emulsifying region formed by different ratios in the phase diagram was used as an indicator. The results are as follows. Figure 2 , Figure 3 , Figure 4 As shown.

[0080] (1) Oil phase from Figure 2 It can be seen that the size of the self-emulsifying region formed by different oil phases is: MCT > GMO > Labrafil M1944CS > Maisine CC. Among these, the effective self-emulsifying region area formed by different oil phases varies considerably, with MCT forming the largest self-emulsifying region and Maisine CC forming the smallest. However, the solubility of different oil phases in abiraterone acetate must be considered comprehensively; therefore, the formulation can include multiple oil phases.

[0081] (2) Emulsifier is composed of Figure 3 It can be seen that the size of the self-emulsifying region formed by different emulsifiers is: Tween-80 > EL-35 > RH-40. The effective self-emulsifying region area formed by different emulsifiers does not differ significantly. However, a large amount of surfactant can irritate the gastrointestinal tract; therefore, the proportion of a single emulsifier must be strictly controlled, and one or more emulsifiers can be used.

[0082] (3) Emulsifiers are made from Figure 4 It was found that the size of the self-emulsifying region formed by different emulsifiers was: ethanol > diethylene glycol monoethyl ether > polyethylene glycol 400. Among these, the effective self-emulsifying region area formed by different emulsifiers varied considerably. Ethanol formed the largest self-emulsifying region. However, this nano-formulation needs to be loaded into capsule shells, and the alcohols in the nano-formulation can migrate into the capsule shell, leading to drug precipitation and shell softening. Therefore, the ethanol content in the formulation should be strictly controlled.

[0083] 2.3 Prescription Composition

[0084] Through orthogonal experimental design and the creation of pseudo-ternary phase diagrams, the emulsification regions were selected. One to two of the following excipients—glyceryl monooleate, glyceryl monolinoleate, and medium-chain triglycerides—were chosen as the oil phase; one to two of the following excipients—polysorbate-80, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil—were chosen as emulsifiers; and one to two of the following excipients—ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether—were chosen as co-emulsifiers. Eight formulations containing abiraterone acetate that met the requirements for excipient types and proportions were successfully screened. The specific composition of these formulations is shown in Table 3 below. In the eight formulations selected in this study, the total mass of excipients was 100%, the oil phase accounted for 20-40%, the emulsifier for 30-53%, and the co-emulsifier for 25%-30%. The surfactant dosage was within the FDA's maximum allowable dosage range, indicating good safety. The ethanol content in the eight formulations was <30%, which avoids ethanol migration into the gelatin capsule shell, preventing drug precipitation and softening of the capsule material, thus ensuring good stability.

[0085] Table 3. Formulation of Abiraterone Acetate Self-Nanoemulsion Formulation

[0086]

[0087]

[0088] 2.4 Prescription Process

[0089] Based on the experimental results (data such as the solubility of raw materials and drugs) from the early stage of the experiment, and considering the physicochemical properties, stability, absorption, convenience of administration route and patient compliance of abiraterone acetate, the formulation was developed into a self-filled nanoemulsion capsule.

[0090] Referring to the market specification of abiraterone acetate tablets (250 mg / tablet) and combining the pharmacokinetic data of abiraterone acetate in vivo from nanoemulsion-filled capsules (see Example 5), based on the maximum drug loading of abiraterone acetate in excipients of 67 mg / g, capsule #1 was used, with each capsule containing 0.525 g of drug formulation, that is, each capsule contains 25 mg of drug.

[0091] The prepared abiraterone acetate self-filled nanoemulsion capsules are as follows: Figure 5 As shown.

[0092] Based on the above results, the final preparation process is determined as follows:

[0093] (1) The raw material (abiraterone acetate) was mixed in equal increments and then passed through a 100-mesh sieve three times.

[0094] (2) After weighing various blank excipients, the blank formulation was obtained by ultrasonic and vortexing operations until there was no obvious stratification.

[0095] (3) Mix the active pharmaceutical ingredient with the blank preparation, vortex mix at 37°C for 24 hours, and fill into No. 1 opaque capsules at 25°C. Each capsule contains 0.525g of the drug preparation.

[0096] Example 2: Quality Characterization Study of Abiraterone Self-Nanoemulsion

[0097] The eight formulations selected in Example 1 were subjected to quality characterization to identify the optimal formulation, ultimately yielding abiraterone acetate self-nanoemulsion filled capsules suitable for clinical application. Following the requirements for capsules in Part IV of the 2020 edition of the Chinese Pharmacopoeia and based on the inspection items of national drug standards, quality studies were conducted on the abiraterone acetate self-nanoemulsion filled capsules, and corresponding quality standards were established.

[0098] 1 Experimental Methods

[0099] 1.1 Characterization of self-emulsification performance: Take 0.1g of the self-emulsifying nanoemulsion preparations of the 8 formulations prepared in Example 1, drop them into 50mL of purified water at 37℃, and stir magnetically at a constant speed and temperature of 100rpm to form nanoemulsions. Observe the self-emulsification efficiency of different formulations.

[0100] 1.2 Particle Size and Polydispersity Index 0.1 g of each of the eight self-contained nanoemulsion formulations prepared in Example 1 was added dropwise to 50 mL of purified water at 37°C. The mixture was magnetically stirred at a constant speed and temperature of 100 rpm to form a transparent microemulsion with a slight bluish tint. An appropriate amount of the microemulsion was taken, diluted appropriately, and the particle size and polydispersity index of the self-contained nanoemulsion were determined using a zetasizer (Malvern). The angle between the incident and scattered beams was 90°, the measurement temperature was 25°C, the sample equilibration time was 120 s, and the solvent was an aqueous system.

[0101] 1.3 Characterization of Encapsulation Efficiency and Drug Loading: Take 1.0 g of the prepared blank self-nanoemulsion formulation and add excess abiraterone acetate to achieve the saturated solubility of the formulation. Take 50 mg of the self-nanoemulsion formulation containing abiraterone acetate, accurately weigh it, and inject it into the liquid chromatography system described above to obtain the drug content W1. Take 50 mg of the self-nanoemulsion formulation containing abiraterone acetate, accurately weigh it, place it in a 5 mL brown volumetric flask, add purified water at 37 °C to dissolve and dilute to the mark, shake well, and allow the self-emulsifying concentrate to form a nanoemulsion under external dynamic conditions. Take a portion of the liquid and place it in a 5 mL centrifuge tube, centrifuge at 4500 × g and 4 °C for 20 min, accurately measure 1 mL of the supernatant, place it in a 10 mL brown volumetric flask, add an appropriate amount of methanol, sonicate (200 W, 40 kHz) for 10 min to break the emulsion, cool to room temperature, dilute to the mark with methanol, shake well, and inject it into the liquid chromatography system described above to obtain the drug content W2. Calculate the drug loading and encapsulation efficiency using the following formulas:

[0102] Drug loading (%) = W2 × 100%; Encapsulation efficiency (%) = W2 / W1 × 100%.

[0103] 1.4 Transmission Electron Microscopy Characterization The morphology of nanoparticles in the nanoemulsion was observed using a transmission electron microscope (TEM, JEM-1400, Leica, Germany). 0.1 g of the capsule contents was added to 50 mL of purified water and magnetically stirred at a constant speed of 100 rpm to form a transparent microemulsion with a slight bluish tint. An appropriate amount of the microemulsion was diluted appropriately and completely dispersed. The mixture was then filtered through a 0.22 μm microporous membrane. The resulting nanoparticle mixture, along with the staining agent uranyl acetate, was uniformly spread on a carbon film copper grid. After baking, the mixture was observed and recorded using a transmission electron microscope at 120 kV.

[0104] 2 Experimental Results

[0105] 2.1 Characterization of self-emulsifying properties: The apparent emulsifying effects of eight formulations of self-emulsifying nanoemulsions were observed, and the results are shown below. Figure 6 As shown in the figure. The results show that all eight formulations can form transparent nanoemulsions with a slight bluish tint in an aqueous environment.

[0106] 2.2 Particle Size and Polydispersity Index (PDI) To screen the optimal formulation from the eight formulations, the particle size of the nanoemulsions was evaluated using dynamic light scattering (DLS). The results of the formulation screening are shown in Table 4 below. The particle size and PDI of each formulation showed some differences, with the particle size range all within the range of 30-70 nm.

[0107] Table 4. Particle size and polydispersity index of each nanoemulsion formulation

[0108]

[0109] 2.3 Encapsulation Efficiency and Drug Loading: The drug loading and encapsulation efficiency results for different formulations are shown in Table 5 below. Overall, the encapsulation efficiency of each nanoemulsion formulation is above 85%.

[0110] Table 5. Drug loading and encapsulation efficiency of different formulations of self-made nanoemulsions

[0111]

[0112] 2.4 Transmission Electron Microscopy Characterization: Morphological observation of the self-emulsion particles was performed using transmission electron microscopy. The nanoemulsion solution (self-emulsion of Formulation 7) is shown in the electron microscope image. Figure 7 The results show that, at appropriate dilution ratios, the nanoemulsion particles are well dispersed, uniform in size, and exhibit near-spherical edges with a diameter of approximately 40 nm, which is consistent with the particle size results.

[0113] 3 Results Analysis

[0114] The key indicator for evaluating the quality of abiraterone acetate self-emulsifying nanoemulsions is the self-emulsification efficiency, which is mainly determined by the particle size after self-emulsification. Therefore, the screening of the formulation focuses on examining the particle size and dispersion coefficient of the capsule contents after emulsification. Using the contents characteristics, drug loading, particle size, and dispersion coefficient of abiraterone acetate self-emulsifying nanoemulsion-filled capsules as indicators, the optimal formulation was screened, and its formulation process was determined.

[0115] The drug loading and encapsulation efficiency of abiraterone acetate in eight formulations were determined by high-performance liquid chromatography (HPLC). Since abiraterone acetate is a hydrophobic drug (soluble in water less than 1 μg / mL), centrifugation after dilution of the abiraterone acetate from the nanoemulsion was sufficient to separate the drug from the active ingredient of the nanoemulsion. Formulation 1 showed a low drug loading of abiraterone acetate, while the other seven formulations all had drug loadings >50 mg / g. The encapsulation efficiency of all eight formulations was above 85%. The stability of the nanoemulsion dispersion systems in the eight formulations was evaluated by particle size, polydispersity index, and transmission electron microscopy. The particle size in all eight formulations was between 30-70 nm, indicating small size and uniform dispersion, suggesting relatively stable nanoemulsion particles.

[0116] The screening results showed that formulations 1, 2, 3, 4, 5, 6, 7, and 8 all had satisfactory self-emulsification efficiency. However, compared to other formulations, formulation 1 had a lower drug loading, formulations 3 and 4 had higher levels of single surfactants, which could cause significant irritation to the human gastrointestinal tract, and formulation 5 had a higher ethanol content, which might affect capsule shell storage. Among formulations 2, 5, 6, and 7, formulation 7 was clearly superior in terms of self-emulsification time, particle size, and PDI. Overall, formulation 7 can be considered ideal in terms of self-emulsification efficiency, drug loading, particle size after self-emulsification, dispersion, safety, and stability; therefore, it was selected as the final formulation.

[0117]

[0118] The abiraterone self-filled nanoemulsion capsules prepared according to the optimal formulation should produce a clear, transparent liquid with good flowability and a bluish sheen after self-emulsification. This formulation is stable, with a self-filled nanoemulsion particle size of approximately 30 nm and an abiraterone acetate encapsulation rate of 91%. Dissolution rates in three batches of samples were all above 80%. The abiraterone acetate content in six batches of samples ranged from 98% to 102%. The related substances content in three batches of samples was below 1%. These results indicate that the quality of this abiraterone acetate self-filled nanoemulsion capsule formulation is stable and controllable.

[0119] Example 3: Investigating the oral absorption performance of fluorescently labeled nanoemulsions

[0120] Using the Caco-2 cell model to simulate the human small intestine absorption model, we investigated the uptake capacity of coumarin C6 fluorescent probe-labeled self-nanoemulsion in gastrointestinal cells using flow cytometry and laser confocal microscopy.

[0121] 1 Materials and Instruments

[0122] 1.1 Experimental Materials DMEM cell culture medium (Dulbecco's modification of Eagle's medium Dulbecco), penicillin-streptomycin 100×, 0.25% trypsin, 4% paraformaldehyde, Hanks' solution (Beijing Zhongke Maichen Technology Co., Ltd.); fetal bovine serum (FBS) (Gibco, USA); cell culture plates, culture flasks, sterile centrifuge tubes, Transwell culture plates (polycarbonate membrane, 1.13 cm²). 2 12-well pore size (3.0 μm); Corning Incorporated, USA; Non-essential amino acids (NEAA); Beijing Xinxiyuan Biotechnology Co., Ltd.; Coumarin-6; Sigma-Aldrich China distributor; Caco-2 cell line; Institute of Hematology, Chinese Academy of Medical Sciences; Dimethyl sulfoxide; Beijing Keboshengchuang Biotechnology Co., Ltd.; CCK-8 cell counting kit; Beijing Hanpei Biotechnology Co., Ltd.; Hoechst; Beijing Bairddi Biotechnology Co., Ltd.; Dynasore, bafilomycin A1, and noccdazole; Shanghai Haoyuan Biomedical Technology Co., Ltd.; Cytochalasin D; Baoruyi (Beijing) Biotechnology Co., Ltd.; Monensin; Beijing Bizi Biotechnology Co., Ltd.; Brefeldin A. A) Nystatin and Verapamil from Beijing Mairuida Technology Co., Ltd.; Amiloride from Shijiazhuang Ximo Technology Co., Ltd.; Chlorpromazine and Methyl-β-cyclodextrin (M-β-CD) from Beijing Solarbio Technology Co., Ltd.

[0123] 1.2 Experimental Instruments: Electronic analytical balance (BT-25S), Sartorius, Germany; Magnetic heating stirrer, Beijing Chemical Glass Station Scientific Instrument Co., Ltd.; Flow cytometer (BD FACS Calibur), Becton Dickinson, USA; Laser scanning confocal microscope (LEICATCS-SP8), LEICA, Germany; Cell incubator, Thermo Scientific, USA; Centrifuge (1-14K), Sigma, Germany; Rotary evaporator (RE-52AA), Shanghai Yarong Biochemical Instrument Factory; Electric thermostatic water bath (ZHWY-103B), Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; Multifunctional microplate reader (FlexStation 3), Molecular microplate reader. Devices: Millicell ERS-2 resistance meter, Merck Chemical Technology (Shanghai) Co., Ltd.; BDS400 inverted fluorescence microscope, Chongqing Aote Optical Instrument Co., Ltd.; Clean bench (YT-CJ-2ND), Beijing Yatai Kelon Instrument Technology Co., Ltd.

[0124] 2 Experimental Methods

[0125] 2.1 Cell Culture Caco-2 cells were cultured in DMEM cell culture medium containing 10% fetal bovine serum, penicillin 100 U / mL and streptomycin 100 μg / mL, and non-essential amino acids. The cell culture conditions in the incubator were 37°C and 5% CO2.

[0126] 2.2 Effects on Caco-2 cell growth

[0127] 2.2.1 Solution Preparation To investigate the effect of fluorescently labeled self-nanoemulsions on the safety of Caco-2 cells, a self-nanoemulsion formulation loaded with coumarin 6 (C6) needs to be prepared.

[0128] Preparation of C6-SNEDDS: Weigh an appropriate amount of C6 and completely dissolve it in the blank formulation (the components of formulation 7 except for "abiraterone acetate"). Emulsify and dilute with Hanks solution to a nanoemulsion solution containing a C6 concentration of 200 μg / mL. Dilute with cell culture medium to 200, 100, 50, 10, 5, 1, and 0.5 μg / mL to obtain nanoemulsion solutions (C6-SNEDDS) containing different concentrations of C6.

[0129] Preparation of C6-ME: Take 0.2g of C6, dissolve it completely in methanol, and then dilute it with cell culture medium to the required concentration to obtain methanol solutions (C6-ME) containing different concentrations of C6.

[0130] 2.2.2 Effect on Caco-2 Cell Growth Caco-2 cells were cultured in an incubator at 37℃ and 5% CO2. After reaching a certain cell count, 200 μL of cell suspension was added to each well of a 96-well plate to achieve a cell concentration of 8000 cells / well. The cells were then pre-cultured in the incubator for 24 hours. After 24 hours, the culture medium was discarded, and different concentrations of C6-SNEDDS and C6-ME were added to the culture plates. After drug administration, the culture plates were incubated in an incubator for 48 hours. After 48 hours, serum-free culture medium containing 10% CCK8 was prepared, the original culture medium in the 96-well plate was discarded, and 100 μL of CCK8 solution was added to each well. The culture plates were incubated in an incubator for 1-4 hours until appropriate color development was achieved, and the absorbance of the cells at 450 nm was measured using a microplate reader. Cell viability = (A450 nm of treated cells / A450 nm of blank culture medium control cells) × 100%, where A450 nm is the absorbance at 450 nm.

[0131] 2.2.3 Uptake effect in Caco-2 cells

[0132] 2.2.3.1 Flow Cytometry Study of Uptake Effect: To investigate the uptake effect of fluorescently labeled self-nanoemulsifiers in Caco-2 cells, coumarin C6-loaded self-nanoemulsifiers were prepared using coumarin C6. 5.0 mL of the blank formulation was weighed, and 1.0 mg of C6 was added and mixed thoroughly to prepare a solution containing 200 μg / mL of coumarin. This solution was then emulsified with Hanks' solution and diluted to nanoemulsion solutions containing C6 concentrations of 500, 250, 100, 50, and 25 ng / mL, thus obtaining self-nanoemulsifiers (C6-SNEDDS) loaded with different concentrations of coumarin.

[0133] Caco-2 cells were planted at a density of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 100 μL in 12-well plates, with three replicates per group. 2 mL of culture medium was added to each well, and the plates were incubated at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and 2 mL of a self-contained nanoemulsion containing free or coumarin was added to each well of the 12-well plate. For the control group, 1 mL of culture medium was added. The plates were then incubated at 37°C with 5% CO2. After incubation for 15, 60, 90, 120, and 180 min, the solution was discarded. Cells were washed three times with pre-cooled 4°C PBS, digested with 0.25% trypsin, and collected by centrifugation. The supernatant was removed, and after washing, 1 mL of PBS was added to form a suspension. The suspension was then passed through a 400-mesh sieve into flow cytometry tubes, and the amount of drug taken up by the cells was measured using a flow cytometer (FCM). The excitation wavelength was 488 nm, and the emission wavelength was 530 nm.

[0134] The effects of different concentrations (500, 250, 100, 50, and 25 ng / mL) of C6-SNEDDS and different administration times (15, 60, 90, 120, and 180 min) on cell uptake were also investigated.

[0135] 2.3.2 Confocal Microscopy to Examine Uptake Effect: Caco-2 cells were placed at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in round-bottomed glass-bottom laser confocal microscopy culture dishes and incubated at 37°C with 5% CO2 for 24 h. After 24 h, the culture medium in the dishes was discarded, and medium containing 1 mL of free coumarin or C6-SNEDDS (C6 concentration 100 ng / mL) was added. After incubation for 2 h, the cells were washed three times with pre-cooled Hanks' solution, fixed with 4% paraformaldehyde, washed three times with Hanks' solution, stained with Hoechst solution for 10 min, the solution was discarded, and the cells were washed three times with Hanks' solution. Observation and image acquisition were performed using an inverted laser confocal microscope.

[0136] 2.4 Apparent transmembrane permeability coefficient in the Caco-2 monolayer membrane model

[0137] 2.4.1 Establishment of C6 content detection method

[0138] Weigh an appropriate amount of C6 and dissolve it in an appropriate amount of methanol. After complete dissolution, transfer the solution to a volumetric flask and dilute to volume to prepare a C6 stock solution with a concentration of 10 μg / mL. Dilute with anhydrous methanol to prepare solutions of 1, 2, 10, 50, 100, 200, 500, and 1000 ng / mL. Add these solutions to 96-well plates and measure their fluorescence intensity using a multi-mode microplate reader. The excitation light was 488 nm, and the emission light was 530 nm. Establish a standard curve by plotting fluorescence intensity against C6 concentration.

[0139] 2.4.2 Establishment of the Caco-2 monolayer model

[0140] Caco-2 cells were planted at a density of 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per Transwell plate. 0.5 mL of cell suspension was added to the upper layer of the Transwell plate, and 1.5 mL of cell-free DMEM complete medium was added to the lower chamber. The plates were incubated at 37°C in a 5% CO2 incubator. From day 2 to 7, the upper and lower layers were changed every other day. From day 8 to 17, the upper layer was changed daily, and the lower layer was changed every other day. After day 18, the upper and lower layers were changed daily, and the resistivity was monitored. After 21 days, the Caco-2 cell monolayer resistivity reached 250 Ω, indicating successful model establishment.

[0141] 2.4.3 Apparent transmembrane permeability in Caco-2 monolayer membranes

[0142] After the Caco-2 cell model was constructed, the liquid in the upper and lower layers of the Transwell plate was discarded. Then, 0.5 mL and 1.5 mL of Hanks solution were added to the upper and lower layers respectively. After slight shaking, the Hanks solution was discarded, and this process was repeated three times. After the last addition of Hanks, the plate was incubated at 37°C for half an hour. After incubation, the Hanks solution was aspirated, and the resistance value was monitored again. Hanks solution containing 0.5 mL of free coumarin or C6-SNEDDS (coumarin concentration of 1 μg / mL) was added to the upper layer, and fresh Hanks solution was added to the lower layer. The plate was then placed in a 37°C constant temperature shaker and shaken. At 30 min, 60 min, 90 min, 120 min, and 150 min, the Hanks solution was removed from the lower layer, and an equal amount of fresh Hanks solution was added.

[0143] The Hanks solution was concentrated and reconstituted with 300 μL of methanol. The mixture was vortexed for 3 min, sonicated for 15 min, and centrifuged at 5000 rpm for 5 min. The supernatant was collected and added to a 96-well plate. The fluorescence intensity of C6 was measured using a microplate reader. Based on the standard curve, the amount of C6 transported to the lower layer was calculated, and the apparent permeability coefficient P was calculated according to the formula. app Value, P app = dQ / dt × 1 / AC0. Where, P app dQ / dt is the apparent permeability coefficient (cm / s), dQ / dt is the transmembrane transport rate of C6 (ng / s), C0 is the initial concentration of C6 in the drug solution, and A is the transport area.

[0144] 3 Experimental Results

[0145] 3.1 Effect on Caco-2 Cell Growth After CCK8 staining, the results of enzyme-linked immunosorbent assay (ELISA) analysis showed that when the C6 concentration was 0.0-10.0 μg / mL (excipient concentration was 0-200 μg / mL), the C6-loaded nanoemulsion did not exhibit significant toxicity. This indicates that the C6-loaded nanoemulsion and blank excipient had good safety at the corresponding concentrations. At a concentration of 20 μg / mL, both free C6 and the blank nanoemulsion compound showed slight toxicity. Figure 8 Therefore, subsequent cell uptake and transmembrane transport experiments should be performed at excipient concentrations <200 μg / mL.

[0146] 3.2 Uptake effect in Caco-2 cells

[0147] 3.2.1 Flow cytometry to examine uptake effect

[0148] After treating Caco-2 cells with C6-SNEDDS, the fluorescence intensity of C6 was used as an indicator to characterize the changes in C6-SNEDDS cell uptake with different drug concentrations and administration times using flow cytometry. The absorption peaks of each formulation group are shown in the figure, and the relative quantitative results are shown in the figure (…). Figure 9 As shown in the figure.

[0149] Quantitative results ( Figure 9 B) indicates that the average fluorescence intensity of intracellular C6 increases with increasing drug concentration. However, within the drug concentration range of 100-500 ng / mL, the fluorescence intensity did not increase proportionally with the drug concentration. These results suggest that C6-SNEDDS uptake in Caco-2 cells is not a simple passive diffusion process. The increased average fluorescence intensity of intracellular C6 with prolonged drug administration time indicates that C6-SNEDDS uptake in Caco-2 cells is a time-dependent process. Subsequent C6-SNEDDS cellular uptake experiments were conducted under the same conditions: 100 ng / mL for 120 min.

[0150] 3.2.2 Confocal microscopy to examine the uptake effect

[0151] The uptake effect of C6-SNEDDS was further verified using STED inverted confocal microscopy. Fluorescent spots displayed under a 50 μm scale laser confocal microscope visually demonstrated the difference in uptake between free C6 and C6-loaded nanoemulsions. The confocal results showed that the fluorescence intensity of the C6-loaded nanoemulsion was stronger than that of the free C6.

[0152] 3.3 Transmembrane transport permeability coefficient in the Caco-2 monolayer membrane model

[0153] 3.3.1 Establishment of C6 content detection method: The fluorescence intensity of C6 standard solutions of different concentrations was measured using a multifunctional microplate reader, and the standard curve results are shown in the figure. Figure 10 The standard curve equation is y = 11.54x - 71.16, R0 2 =0.9985.

[0154] 3.3.2 Permeability coefficient for transmembrane transport in the Caco-2 monolayer membrane model

[0155] A Caco-2 monolayer cell model was established to simulate small intestinal epithelial cells to study the transmembrane transport capacity of C6-SNEDDS. Caco-2 monolayer cells were treated with free C6 (1 μg / mL) and C6-SNEDDS (1 μg / mL) respectively to investigate the difference in transmembrane transport efficiency between the two formulations. Quantitative results were obtained based on the formula for calculating the apparent permeability coefficient, as shown in the figure. Figure 11 The apparent permeability coefficient of C6-SNEDDS nanoemulsion is 2.21 × 10⁻⁶. -7 cm / s, compared to the apparent permeability of free C6 (1.32×10 cm / s). -7 The significant increase in the permeability (cm / s) indicates that the self-nanoemulsion enhances the permeability of drug transmembrane transport.

[0156] 4 Results Analysis

[0157] Abiraterone acetate, a BCS Class IV drug, suffers from poor solubility and permeability, severely hindering its absorption in the gastrointestinal tract and consequently affecting its bioavailability. This invention utilizes a self-nanoemulsion drug delivery system as a carrier, significantly improving drug solubility. However, after the self-nanoemulsion delivery carrier forms a nanoemulsion under spontaneous peristalsis in the gastrointestinal tract, its transmembrane transport requires traversing the intestine to the epithelial cells and from the intracellular space to the basolateral side of the cell membrane, finally entering the bloodstream. Therefore, the intestinal epithelial monolayer is a crucial barrier to the oral absorption of nanoemulsions. The Caco-2 cell monolayer model is a feasible human small intestinal absorption model, possessing a well-differentiated brush-edge surface and expressing typical small intestinal-associated hydrolases and various cytochrome subtypes, which can be used to simulate the drug absorption process. In this embodiment, Caco-2 cells were used as a model, and coumarin 6, a fluorescent compound similar in properties to abiraterone acetate, was used as a fluorescent probe. Flow cytometry and fluorescence microscopy were employed to study the cellular uptake process of the self-nanoemulsion prepared previously. Furthermore, the apparent permeability coefficient was used to investigate the transmembrane transport efficiency of the self-nanoemulsion carrier after oral administration.

[0158] All excipients used in the preparation of the nanoemulsion carrier in this invention are FDA-approved for use in oral drug delivery systems, exhibit low toxicity, and demonstrate excellent biocompatibility. Experimental results show that the carrier at concentrations <200 μg / mL has virtually no impact on the integrity of Caco-2 cells and exhibits no cytotoxicity.

[0159] After spontaneous peristalsis in the gastrointestinal tract, nanoemulsions form and then undergo uptake and membrane-diffuse transport within gastrointestinal cells. Flow cytometry and inverted microscopy were used to quantitatively and qualitatively study the uptake of coumarin C6-labeled nanoemulsions. The results demonstrated that the uptake of fluorescently labeled nanoemulsions was affected by drug concentration and time. Qualitative analysis using confocal inverted microscopy showed that the uptake intensity of fluorescently labeled nanoemulsions was stronger than that of the free C6 group.

[0160] The permeation capacity of fluorescently labeled nanoemulsions was studied using a monolayer cell model differentiated from Caco-2 cells. The results showed that the nanoemulsions prepared in this invention significantly enhanced drug transmembrane transport efficiency, demonstrating their advantage in enhancing the oral absorption of abiraterone acetate.

[0161] Example 4: Killing effect of abiraterone self-generated nanoemulsion on prostate cancer cells

[0162] This study aimed to investigate the cytotoxic and apoptosis-inducing effects of abiraterone acetate nanoemulsion on prostate cancer cells. Cytotoxicity assays and flow cytometry were used to examine its cytotoxic and apoptosis-inducing effects on prostate cancer cells, exploring its therapeutic efficacy against prostate cancer.

[0163] 1 Materials and Instruments

[0164] 1.1 Experimental Materials F12K cell culture medium, penicillin-streptomycin 100×, 0.25% trypsin, Hanks' solution (Beijing Zhongke Maichen Technology Co., Ltd.); fetal bovine serum (FBS) (Gibco, USA); PC-3 cell line (Institute of Hematology, Chinese Academy of Medical Sciences); CCK-8 cell counting kit (Beijing Hanpei Biotechnology Co., Ltd.); Annexin V / PI apoptosis kit (Meilunyun (Beijing) Cell Biotechnology Co., Ltd.).

[0165] 1.2 Experimental Instruments: Electronic analytical balance (BT-25S), Sartorius GmbH, Germany; magnetic heating stirrer, Beijing Chemical Glass Station Scientific Instruments Co., Ltd.; flow cytometer (BD FACS Calibur), Becton Dickinson, USA; cell incubator, Thermo Scientific, USA; centrifuge (1-14K), Sigma, Germany; clean bench (YT-CJ-2ND), Beijing Yatai Kelon Instrument Technology Co., Ltd.

[0166] 2 Experimental Methods

[0167] 2.1 Cell culture of PC-3 cells: F12K cells were cultured in F12K cell culture medium containing 10% fetal bovine serum and double antibiotics (penicillin 100 U / mL and streptomycin 100 μg / mL) at an incubator temperature of 37℃ and 5% CO2.

[0168] 2.2 Killing effect on prostate cancer cells

[0169] Preparation of AbA-SNEDDS: Weigh abiraterone acetate and add it to the blank preparation to prepare a self-nanoemulsion preparation (Formula 7). Emulsify with Hanks solution and dilute to a nanoemulsion solution containing an AbA concentration of 20 mg / mL. Dilute with cell culture medium to 50, 25, 10, 5, 2.5, and 1 μg / mL to obtain self-nanoemulsion solutions containing different concentrations of AbA.

[0170] Preparation of AbA-ME: Accurately weigh free abiraterone acetate powder and dissolve it in methanol to a concentration of 20 mg / mL. Dilute with cell culture medium to concentrations of 50, 25, 10, 5, 2.5, and 1 μg / mL to obtain methanol solutions containing different concentrations of AbA. Culture PC-3 cells in an incubator at 37°C and 5% CO2 until a certain cell density is reached. Add 200 μL of cell suspension to each well of a 96-well plate to achieve a cell concentration of 8000 cells / well. Pre-culture the plate in a cell culture incubator for 24 hours. After 24 hours, discard the culture medium and add AbASNEDDS and AbA-ME containing different concentrations of abiraterone acetate to the plate. After drug administration, incubate the plate in a cell culture incubator for 72 hours. After 72 hours, prepare serum-free culture medium solution containing 10% CCK8, discard the original culture medium in the 96-well plate, and add 100 μL of CCK8 solution to each well. Incubate the culture plate in an incubator for 1-4 hours until appropriate color development occurs, then measure the absorbance at 450 nm using a microplate reader. Cell viability = (A450 nm of treated cells / A450 nm of blank culture medium control cells) × 100%, where A450 nm is the absorbance at 450 nm.

[0171] 2.3 Effect on inducing apoptosis in prostate cancer cells

[0172] Flow cytometry was used to investigate apoptosis in cells, and the specific binding characteristics of Annexin V antibody to phosphatidylserine (PS) were used to detect apoptosis. Prostate cancer PC-3 cells were first cultured in an incubator at 37°C and 5% CO2, and then divided into 6-well plates at a density of 1 × 10⁶ cells per well. 4 Cells were cultured at high density in 6-well plates. Three experimental groups (blank cell group, AbA-ME group, and AbA-SNEDDS group) were designed and added to 6-well plates at appropriate concentrations. Cells were incubated at 37°C with 5% CO2 for 24 hours to induce apoptosis. The 10× binding buffer from the apoptosis kit was diluted to 1× with distilled water and incubated on ice. After incubation, cells were washed three times with pre-cooled PBS. The precipitate was collected by centrifugation. In both the control and experimental groups, fluorescein isothiocyanate (FITC) was added first, and adherent cells were digested with trypsin without EDTA and stained in the dark for 10 minutes. After thorough mixing, cells were stained with propidium iodide (PI) fluorescent staining solution in the dark for 5 minutes. After mixing, the cells were analyzed by flow cytometry, and the results were statistically analyzed.

[0173] 3 Experimental Results

[0174] 3.1 Killing effect on prostate cancer cells

[0175] This experiment investigated the cytotoxic effects of different concentrations of free AbA and AbA self-nanoemulsion on prostate cancer cells PC-3. The results showed that both free AbA and AbA self-nanoemulsion had a cytotoxic effect on PC-3 cells in a dose-dependent manner. Figure 13 When the AbA concentration was 1-50 μg / mL, both free AbA and AbA-loaded nanoemulsions exhibited cytotoxic effects on PC-3 cells. The IC50 value of the AbA-loaded nanoemulsion against PC-3 cells was calculated to be 3.97 μg / mL. The IC50 value of free abiraterone acetate against PC-3 cells was 6.14 μg / mL. These results indicate that AbA-loaded nanoemulsions have a significant cytotoxic effect on PC-3 cells.

[0176] 3.2 Effect on inducing apoptosis in prostate cancer cells

[0177] To investigate the apoptosis effect, PC-3 cells were treated with free AbA and AbA-based nanoemulsions, respectively. The apoptosis rate was measured by flow cytometry after 24 hours. The results showed that after a period of drug treatment, the apoptosis rate of the AbA-based nanoemulsion group was 18.77±0.60%, while the apoptosis rate of PC-3 cells treated with free AbA was 16.17±1.86%. The apoptosis rate of PC-3 cells treated with AbA-based nanoemulsions was higher than that of free AbA, by a factor of 1.17. Figure 14 )

[0178] Example 5: Bioavailability Study of Abiraterone Self-filled Nanoemulsion Capsules

[0179] The aim was to evaluate the bioavailability of the prepared abiraterone acetate self-filled nanoemulsion capsule formulation (prepared using the optimal formulation) in beagle dogs through pharmacokinetic studies, and to select the optimal dose and administration time based on pharmacokinetic data, providing a basis for clinical dosing regimens.

[0180] 1 Materials and Instruments

[0181] 1.1 Experimental Materials: Abiraterone (Shanxi Zhendong Pharmaceutical Co., Ltd.); Abiraterone acetate tablets (Shanxi Zhendong Pharmaceutical Co., Ltd.); Capsule shells (Shanxi Zhendong Pharmaceutical Co., Ltd.); Heparin (Beijing Solarbio Science & Technology Co., Ltd.); Abiraterone-D... 4 Shandong Wodesen Biotechnology Co., Ltd.; Acetonitrile (chromatographic grade), FEPURE; Methanol (gradient grade), FEPURE; Ultrapure water.

[0182] 1.2 Experimental Instruments: Electronic analytical balance (MSA36S-CE), Sartorius, Germany; Pure water system (Milli-Q, Millipore); Centrifuge (Allegra X-30R Centrifuge), BECKMAN COULTER; Centrifugal concentrator and dryer (LNG-T83A), Taicang Hualida Experimental Equipment Co., Ltd.; Adjustable mixer (MX-S), Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd.; Nitrogen blower (DC-12), Shanghai Anpu Experimental Technology Co., Ltd.; Triple quadrupole liquid chromatography-mass spectrometry (degasser, SRD-3600; high performance liquid chromatograph, HPG-3400RS; autosampler, WPS-3000TRS; column oven, TCC-3000RS; mass spectrometer, TSQ Endura), Thermo Fisher Scientific (China) Co., Ltd.

[0183] 1.3 Experimental animals: Beagles (adult male Beagles, weighing 10±2 kg) were purchased from the Experimental Animal Center of Peking University School of Medicine and housed there. During the experiment, the ambient temperature was controlled and standardized sterilization procedures were followed. All feeding and handling were conducted under clean conditions and in accordance with international guidelines for animal experimentation (Animal Experimentation Ethics Approval No.: LA2022535).

[0184] 2 Experimental Methods

[0185] 2.1 Establishment of LC-MS / MS determination method

[0186] 2.1.1 Chromatographic conditions: The chromatographic column used was octadecylsilane-bonded silica gel. (ACQUITY) BEHC18 column (2.1×50mm, 1.7μm); methanol-water as the mobile phase, flow rate 0.35mL / min, temperature 25℃, injection plate temperature 4℃, injection volume 5μL. The gradient elution program is shown in Table 6 below.

[0187] Table 6 Gradient elution program

[0188]

[0189] 2.1.2 Mass spectrometry conditions: Heated electrospray ionization (H-ESI) was used for positive ion detection. The detection mode was Selected Reaction Detection Scan (SRM); the positive ion spray voltage was 4000 V; the ion transfer tube temperature was 300 °C; the vaporizer temperature was 260 °C; the collision gas (CID) was 1.5 mTorr; the sheath gas was 20 Arb; the auxiliary gas was 10 Arb; AB (abiraterone) and the internal standard AB-d4 (abiraterone-d4) were used for analysis, as shown in Table 7 below.

[0190] Table 7 Mass Spectrometry Separation Conditions

[0191]

[0192] 2.1.3 Plasma Sample Processing Methods

[0193] Quality control samples: Take 100 μL of blank plasma, 100 μL of abiraterone (AB) quality control working solution (1 ng / mL, 50 ng / mL, 500 ng / mL, 1500 ng / mL), and 100 μL of abiraterone-d 4 (AB-d 4 Working solution (40 ng / mL), 150 μL acetonitrile in

[0194] Mix the sample in a 1.5 mL EP tube by vortexing and centrifuge at 4°C and 13000 rpm for 15 min. Collect the supernatant for analysis. The entire procedure should be performed on ice.

[0195] Plasma sample: Take 100 μL of sample, 100 μL of acetonitrile, and 100 μL of AB-d. 4 The working solution (40 ng / mL) and 150 μL of acetonitrile were added to a 1.5 mL EP tube, vortexed, and centrifuged at 4 °C and 13000 rpm for 15 min. The supernatant was then injected for analysis. The entire procedure was performed on ice.

[0196] 2.1.4 Specificity was assessed by injecting 100 μL of blank plasma, 100 μL of blank plasma with 100 μL of abiraterone (AB, 0.5 ng / mL), and 100 μL of blank plasma with 100 μL of abiraterone (AB, 40 ng / mL) working solution, respectively, after processing the samples according to the sample processing method described in 2.1.3 above.

[0197] 2.1.5 Standard Curve: Take 100 μL of blank plasma, 100 μL of abiraterone (AB) standard series solution, and 100 μL of abiraterone-d... 4 (AB-d 4 Working solution (40 ng / mL) and 150 μL of acetonitrile were added to 1.5 mL EP tubes. After treating the plasma sample with acetonitrile, solutions with concentrations of 0.5, 1, 2, 10, 50, 100, 200, 500, 1000, and 2000 ng / mL were prepared. Analysis was performed using the LC-MS / MS method established above. The response ratio of AB and internal standard was plotted on the ordinate, and the concentration on the x-axis, 1 / x. 2 We use the weights to construct a linear regression curve.

[0198] 2.2.2 Bioavailability study in Beagle dogs

[0199] 2.2.2.1 Animal Grouping: Ten healthy adult male beagles, weighing 8±2 kg, were randomly divided into two groups of five each. Two experimental cycles were conducted: the first cycle was a fasting test, and the second cycle was a postprandial test, with a 7-day washout period between cycles. Before the experiment, the beagles were placed in individual cages and allowed to acclimatize for one week under standard temperature (25-28℃), humidity (50-60%), and light (12h light / 12h darkness) conditions, with free access to food and water. The experiment was approved by the Laboratory Animal Welfare and Ethics Committee of the Biomedical Ethics Committee of Peking University (LA2022535), and the animal experiments were conducted in accordance with relevant national and international guidelines for animal experiments.

[0200] 2.2.2.2 The animal drug administration experiment design was divided into fasting and postprandial experiments. For the fasting experiment, animals needed to fast for 12 hours before the experiment, administer the drug on an empty stomach, and be fed 4 hours after administration. For the postprandial experiment, animals needed to fast for 10 hours before the experiment, administer the drug after a high-fat meal (feeding and administration were completed within 30 minutes). The experimental control was abiraterone acetate tablets (provided by Zhendong Research Institute), with a dosage of 250 mg / animal. The experimental capsules were abiraterone acetate self-filled nanoemulsion capsules prepared according to the optimal formulation of this invention, with a dosage of 125 mg / animal.

[0201] 2.2.2.3 Sampling and Plasma Sample Processing Following drug administration, 1 mL of blood was collected from the vein of a beagle dog at 12 sampling points at 0h, 0.25h, 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 6h, 8h, 12h, and 24h, and placed in heparinized EP tubes. The samples were immediately placed on ice after collection. Within 30 minutes, the samples were centrifuged at 13000 rpm for 15 minutes at 4℃, and the supernatant plasma sample was collected and stored at -80℃ for later analysis.

[0202] 2.3 Experimental Results

[0203] 2.3.1 Methodological Investigation Results

[0204] 2.3.1.1 Spectra of Analyte and Internal Standard The daughter ion spectra of abiraterone (AB) and the internal standard (IS, AB-d4) are as follows: Figure 15 As shown. The quantitative ion pairs for abiraterone (AB) are m / z 350.2→m / z 156.1, and the quantitative ion pairs for the internal standard (IS,AB-d4) are m / z 354.3→m / z 160.1.

[0205] 2.3.1.2 Mass spectrometry of specific blank beagle plasma and beagle plasma supplemented with abiraterone (AB) and internal standard (IS, AB-d4) is shown below. Figure 16 As shown in the figure. The results show that AB and IS did not have any interfering peaks in different blank plasma samples.

[0206] 2.3.1.3 The standard curve is plotted with the response ratio of abiraterone (AB) and the internal standard (IS, AB-d4) on the ordinate and concentration on the abscissa, 1 / x 2 Assign weights to construct a linear regression curve, such as... Figure 17 As shown. The R-value of the standard curve in the experiment. 2 The value of 0.9991 indicates that abiraterone (AB) exhibits good linearity in beagle plasma within the range of 0.5–2000 ng / mL. The standard curve is y = 0.035x - 0.0011.

[0207] 2.3.2 Bioavailability in Beagle Dogs The plasma concentrations of abiraterone (AB) in beagle dogs were measured using the LC-MS / MS method established in the methodology. The pharmacokinetic parameters are shown in Tables 8, 9, 10, and 11. The plasma concentration-time curves for abiraterone acetate tablets and abiraterone acetate capsules filled with nanoemulsion after oral administration to beagle dogs on an empty stomach are shown below. Figure 18 As shown. Following oral administration of abiraterone acetate via nanoemulsion-filled capsules and tablets to beagle dogs on an empty stomach, the plasma C-levels of abiraterone were... max The concentrations were (587.4±141.6) and (64.88±42.27) ng / mL, respectively; AUC 0-t The concentrations were (1134.0±243.70) h·ng / mL and (180.8±153.7) h·ng / mL, respectively; t 1 / 2 The relative bioavailability of abiraterone acetate self-filled nanoemulsion capsules to beagle dogs via oral administration on an empty stomach was 1254.07% compared to abiraterone acetate tablets.

[0208] The following are blood concentration-time curves comparing abiraterone acetate tablets and capsules administered orally to beagle dogs on an empty stomach and after a meal: Figure 19As shown, there was no significant difference in the time to peak concentration (TTP) of abiraterone acetate self-nanoemulsion-filled capsules between fasting and high-fat meals in beagles. The postprandial oral bioavailability was 1.26 compared to the fasting level, while the postprandial oral bioavailability was 17.5 compared to the fasting level when abiraterone acetate tablets were administered after both fasting and high-fat meals. This indicates that abiraterone acetate self-nanoemulsion-filled capsules can reduce the pre- and postprandial differences in drug administration.

[0209] Table 8. Pharmacokinetic parameters of beagle dogs taken orally on an empty stomach.

[0210]

[0211] Table 9. Pharmacokinetic parameters of abiraterone acetate self-nanoemulsion filled capsules administered orally to beagle dogs on an empty stomach.

[0212]

[0213] Table 10 Pharmacokinetic parameters of abiraterone acetate tablets administered orally after meals to beagles

[0214]

[0215] Table 11 Pharmacokinetic parameters of abiraterone acetate self-emulsifying capsules administered orally after meals to beagles

[0216]

[0217] In summary, the abiraterone acetate self-filled nanoemulsion capsules prepared in this study significantly improved the bioavailability of the control formulation, abiraterone acetate tablets, in beagle dogs, with a 12.54-fold increase in relative bioavailability after pre-meal oral administration. Furthermore, the abiraterone acetate self-filled nanoemulsion capsules significantly reduced the difference in bioavailability between pre- and post-meal oral administration.

Claims

1. An abiraterone acetate self-nanoemulsion filled capsule, comprising a capsule shell and an abiraterone acetate self-nanoemulsion filled within the capsule shell; in, The abiraterone acetate nanoemulsion is composed of abiraterone acetate and excipients in a mass ratio of (12.5-25):

500. The excipients are made from the following raw materials in the following mass percentages: oil phase 20-40%, emulsifier 30-53%, and co-emulsifier 25%-30%. The oil phase is selected from any two of glyceryl monooleate, glyceryl monolinoleate, and medium-chain triglycerides. The emulsifier is selected from one or two of polysorbate-80, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil. The co-emulsifier is selected from one or two of ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether.

2. The abiraterone acetate self-filled nanoemulsion capsule according to claim 1, characterized in that: The abiraterone acetate nanoemulsion is composed of abiraterone acetate and excipients in a mass ratio of 25:

500. The excipients are made from the following raw materials in the following mass percentages: oil phase 30-40%, emulsifier 30-40%, and co-emulsifier 25-30%.

3. The abiraterone acetate self-filled nanoemulsion capsule according to claim 1 or 2, characterized in that: Each 1000 capsules contains abiraterone acetate nanoemulsion made from the following ingredients: 25g abiraterone acetate, 75g monolinoleic acid glyceride, 75g medium-chain triglycerides, 80g polysorbate, 100g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether. Alternatively, in every 1000 capsules, the abiraterone acetate nanoemulsion is made from the following ingredients: 25g abiraterone acetate, 100g glyceryl monooleate, 100g medium-chain triglycerides, 80g polysorbate, 75g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether. Alternatively, for every 1000 capsules, the abiraterone acetate nanoemulsion is made from the following ingredients: 25g abiraterone acetate, 100g glyceryl monooleate, 100g medium-chain triglycerides, 100g polysorbate 80, 75g polyoxyethylene hydrogenated castor oil, 50g ethanol, and 75g diethylene glycol monoethyl ether. The most preferred formulation is that each 1000 capsules contains abiraterone acetate nanoemulsion made from the following ingredients: 25g abiraterone acetate, 75g monolinoleic acid glyceride, 75g medium-chain triglycerides, 100g polysorbate 80, 100g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether.

4. The abiraterone acetate self-filled nanoemulsion capsule according to any one of claims 1-3, characterized in that: The capsule shell is made of No. 1 capsule, and each capsule contains 0.525g of the abiraterone acetate nanoemulsion, that is, each capsule contains 25mg of abiraterone acetate.

5. An abiraterone acetate nanoemulsion, comprising abiraterone acetate and excipients in a mass ratio of (12.5-25):500, wherein the excipients are made from raw materials in the following mass percentages: oil phase 20-40%, emulsifier 30-53%, and co-emulsifier 25%-30%; wherein the oil phase is selected from any two of glyceryl monooleate, glyceryl monolinoleate, and medium-chain triglycerides; the emulsifier is selected from one or two of polysorbate-80, polyoxyethylene castor oil, and polyoxyethylene hydrogenated castor oil; and the co-emulsifier is selected from one or two of ethanol, polyethylene glycol 400, and diethylene glycol monoethyl ether.

6. The abiraterone acetate self-nanoemulsion according to claim 5, characterized in that: The abiraterone acetate nanoemulsion is composed of abiraterone acetate and excipients in a mass ratio of 25:

500. The excipients are made from the following raw materials in the following mass percentages: oil phase 30-40%, emulsifier 30-40%, and co-emulsifier 25-30%.

7. The abiraterone acetate self-filled nanoemulsion capsule according to claim 5 or 6, characterized in that: The abiraterone acetate nanoemulsion is made from the following raw materials: 25g abiraterone acetate, 75g monolinoleic acid glyceride, 75g medium-chain triglycerides, 80-100g polysorbate, 100g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether. Alternatively, the abiraterone acetate nanoemulsion is made from the following raw materials: 25g abiraterone acetate, 100g glyceryl monooleate, 100g medium-chain triglycerides, 80g polysorbate, 75g polyoxyethylene castor oil, 75g ethanol, and 75g diethylene glycol monoethyl ether. Alternatively, the abiraterone acetate nanoemulsion is made from the following raw materials: 25g abiraterone acetate, 100g glyceryl monooleate, 100g medium-chain triglycerides, 100g polysorbate 80, 75g polyoxyethylene hydrogenated castor oil, 50g ethanol, and 75g diethylene glycol monoethyl ether.

8. A method for preparing abiraterone acetate self-filled nanoemulsion capsules according to any one of claims 1-4, comprising the following steps: 1) Mix the abiraterone acetate raw material in equal increments, pass it through a 100-mesh sieve, and collect the portion that passes through the sieve; 2) Weigh the oil phase, emulsion, and co-emulsifier according to the proportion, mix them, and then mix them by ultrasonication, vortexing, and other operations until there is no obvious layering to obtain a blank self-nanoemulsion formulation; 3) Mix the sieved abiraterone acetate raw material from step 1) with the blank self-nanoemulsion preparation, and vortex mix at 37°C for 24 hours to obtain abiraterone acetate self-nanoemulsion. 4) Fill No. 1 opaque capsules at room temperature, filling each capsule with 0.525g of the abiraterone acetate nanoemulsion to obtain abiraterone acetate nanoemulsion filled capsules.

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