Abiraterone acetate enteric microcapsule and preparation method thereof

Abstract

The present application relates to the technical field of pharmaceutical preparation, and in particular to an abiraterone acetate enteric microcapsule and a preparation method thereof. The present application uses an emulsion diffusion method to prepare abiraterone acetate nanocapsules, then uses an enteric material to load the abiraterone acetate nanocapsules, and finally uses a spray drying technology to prepare abiraterone acetate oral enteric microcapsules. Through pharmacokinetic tests of a rat chylomicron blocking model, it is verified that the abiraterone acetate enteric microcapsules prepared by the present application are absorbed by the lymphatic pathway in the small intestine, so it is proved that the present application can enhance the permeability and solubility of abiraterone acetate in the body, improve the oral bioavailability thereof in the body, the preparation method is simple and controllable, has good repeatability, the particle size of the prepared nanocapsules is 182.3±5.7 nm, the Zeta potential is-15.0±1.1 mV, and the encapsulation rate is 92.4±1.7%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to an enteric microcapsule containing abiraterone acetate. BACKGROUND

[0002] Abiraterone acetate (AA) is an inhibitor of androgen synthesis enzyme CYP17A1, which is clinically used for the treatment of metastatic castration-resistant prostate cancer (mCRPC), and belongs to a liposoluble drug. Abiraterone acetate belongs to the fourth class of drugs in the biopharmaceutics classification system (BCS), and has the characteristics of low permeability and low solubility. The bioavailability of abiraterone acetate is extremely low when orally absorbed, and the food effect is significant. The recommended clinical dose is as high as 1000 mg / d, which must be taken on an empty stomach, and a large part of the drug is excreted in the form of a prototype or a precipitate after taking, so that a lot of expensive drugs are excreted in the form of crystals, precipitates or prototypes without exerting their pharmacological effects. Therefore, it is of great significance to improve the solubility and permeability of abiraterone acetate, enhance its oral bioavailability in vivo, and avoid the food effect.

[0003] Patent CN 108785256 B proposes a solid dispersion of abiraterone acetate, which can improve the solubility and eliminate the food effect to some extent, but lacks specific data on bioavailability and efficacy demonstrated by animal experiments. And abiraterone acetate is released in the stomach, which may cause loss of the drug. Patent CN 113134007 A also proposes a solid dispersion of abiraterone acetate, but does not position to release in the intestine. CN 113546037 A proposes an abiraterone acetate suppository, and the drug is absorbed through the rectum, but it is inconvenient for patients to take the drug. CN 105616364 A proposes an abiraterone acetate tablet, which has good stability, but the bioavailability is lower than that of the present application.

[0004] An enteric drug delivery system is a drug delivery technology that allows the drug to be released in the small intestine and large intestine, so that the drug can exert local or systemic therapeutic effect in the human intestinal tract, and can avoid the stimulation of the drug on the stomach or degradation in the stomach, thereby improving the efficacy of the drug. However, not all drugs are absorbed through the intestine. SUMMARY

[0005] The present application aims to promote the absorption of abiraterone acetate in the gastrointestinal tract, achieve targeted release in the intestine, minimize the food effect, improve the bioavailability, and reduce the cost of the preparation. The present application provides an abiraterone acetate enteric microcapsule and a preparation method thereof.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0007] An abiraterone acetate enteric microcapsule, the abiraterone acetate enteric microcapsule is obtained by wrapping abiraterone acetate nanoparticles outside with an enteric material; wherein the mass ratio of abiraterone acetate nanoparticles to enteric material is 11-19:10.

[0008] The enteric material is Eudragit L100-55 and hydroxypropyl methyl cellulose, wherein the mass ratio of Eudragit L100-55 to hydroxypropyl methyl cellulose is 3:4.

[0009] The abiraterone acetate nanoparticles are composed of components with the following mass percentages:

[0010] Abiraterone acetate 5-11%

[0011] Polymer material 22-28%

[0012] Oil core 64-70%.

[0013] The polymer material is PLGA 4000.

[0014] The oil core is an oil core prepared from 5 parts by mass of caprylocaproyl glycerides and 1 part by mass of oleic acid polyethylene glycol glycerides.

[0015] A method for preparing abiraterone acetate enteric microcapsules:

[0016] 1) Mix abiraterone acetate, polymer material, and oil phase in the above proportions to obtain a mixed solution 1, mix the mixed solution 1 and an organic phase solvent in a mass ratio of 1:16-21 to obtain a mixed solution 2, and wait for use;

[0017] 2) Add the mixed solution 2 to the aqueous phase, wherein the mass ratio of the mixed solution 2 to the aqueous phase is 1:3-5, and use a cell ultrasonic crusher at 300W for 3min to obtain a primary emulsion;

[0018] 3) Pour the primary emulsion into water, the mass ratio of water to the primary emulsion is 1:3-5, stir and diffuse for 2.5-3.5h, and finally remove the organic solvent under reduced pressure and rotary evaporation at a temperature of 45-55℃ and concentrate to between one sixth and one ninth of the original volume to obtain an abiraterone acetate nanoparticle suspension;

[0019] 4) Dissolve Eudragit L100-55 and hydroxypropyl methyl cellulose in a sodium phosphate buffer solution (pH=6.5, 5mM) respectively to obtain a solution, mix the hydroxypropyl methyl cellulose solution and the Eudragit L100-55 solution in a volume ratio of 1:2 to obtain an enteric material solution;

[0020] 5) mixing the above abiraterone acetate nanoparticle suspension with the enteric material solution in a volume ratio of 2:3, ultrasonic dispersion, and then preparing abiraterone acetate enteric microcapsules by spray drying.

[0021] The mass ratio of Eudragit L100-55 to hydroxypropyl methyl cellulose in the enteric capsule enteric material is 3:4.

[0022] The organic phase solvent is ethyl acetate.

[0023] The water phase is a 2-3% poloxamer 188 solution.

[0024] In step 4), Eudragit L100-55 and hydroxypropyl methyl cellulose are weighed and dissolved in a sodium phosphate buffer to obtain a 3.75 mg / ml Eudragit L100-55 solution and a 10 mg / ml hydroxypropyl methyl cellulose solution.

[0025] The application of the abiraterone acetate enteric microcapsules, wherein the abiraterone acetate enteric microcapsules are used in the treatment of prostate cancer.

[0026] The beneficial effects of the present application are:

[0027] The abiraterone acetate enteric microcapsules of the present application are prepared in the form of nanoparticles coated with enteric microcapsules, which increases the solubility of abiraterone acetate. The pharmacokinetic test of the rat chylomicron blocking model confirms that the preparation is absorbed by the lymphatic pathway in the small intestine, and further proves that positioning abiraterone acetate in the intestinal tract for release is reasonable and effective, thereby solving the problems of precipitation in the stomach and reduced bioavailability, further saving the raw drug, reducing the cost, and maximizing the avoidance of food effect. The in vivo pharmacokinetic study of the obtained abiraterone acetate enteric microcapsules shows that the oral bioavailability is 12.030%, which is higher than that of ordinary abiraterone acetate nanoparticles and abiraterone acetate oral suspension.

[0028] Meanwhile, the present application evaluates the efficacy in a castration-resistant prostate cancer nude mouse model, and the anti-cancer effect is significant. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The solubility effect diagram of abiraterone acetate in various oils provided in Example 2 of the present application.

[0030] Figure 2 The in vitro release experiment result diagram of abiraterone acetate enteric microcapsules provided in Example 6 of the present application.

[0031] Figure 3 The transmission electron microscope diagram of abiraterone acetate nanocapsules provided in Example 10 of the present application.

[0032] Figure 4 The scanning electron microscope image of the abiraterone acetate enteric microcapsule provided in the embodiment 10 of the present application.

[0033] Figure 5 The pharmacokinetic effect diagram of the abiraterone acetate suspension, the abiraterone acetate nanocapsule and the abiraterone acetate enteric microcapsule provided in the embodiment 11 of the present application.

[0034] Figure 6 The blood concentration-time curve diagram of the abiraterone acetate enteric microcapsule provided in the embodiment 12 of the present application under the condition of with or without injection of candicidin.

[0035] Figure 7 The dissection tumor effect diagram of the nude mice in each group (the abiraterone acetate suspension, the abiraterone acetate nanocapsule and the abiraterone acetate enteric microcapsule) provided in the embodiment 13 of the present application after the experiment.

[0036] Figure 8 The tumor weight effect diagram of the nude mice in each group (the abiraterone acetate suspension, the abiraterone acetate nanocapsule and the abiraterone acetate enteric microcapsule) provided in the embodiment 13 of the present application.

[0037] Figure 9 The weight effect diagram of the nude mice in each group provided in the embodiment of the present application during the experiment.

[0038] Figure 10 The serum CYP17A level effect diagram of each group (the abiraterone acetate suspension, the abiraterone acetate nanocapsule and the abiraterone acetate enteric microcapsule) provided in the embodiment of the present application.

[0039] Figure 11 The serum PSA level effect diagram of each group (the abiraterone acetate suspension, the abiraterone acetate nanocapsule and the abiraterone acetate enteric microcapsule) provided in the embodiment of the present application. DETAILED DESCRIPTION

[0040] The specific embodiments of the present application are further described in conjunction with examples below, and it should be pointed out that the specific embodiments described herein are only for the purpose of illustration and explanation of the present application, and are not limited to the present application.

[0041] The present application verifies the absorption of the preparation in the small intestine through the lymphatic pathway by the pharmacokinetic test of the rat chylomicron blocking model, therefore, the present application can greatly improve the bioavailability, improve the drug efficacy, save the raw material drug, and due to the intestinal positioning drug of the present application, the food effect can also be avoided to the greatest extent.

[0042] Example 1 Preparation of the abiraterone acetate enteric microcapsule

[0043] Weigh 20 mg of abiraterone acetate, 75 mg of PLGA4000, 150 mg of capric and caprylic acid glyceride, 30 mg of polyethylene glycol oleate into 5 mL of ethyl acetate, after fully stirring and dissolving, pour into 20 mL of 2.5% poloxamer aqueous phase, ultrasonic cell crusher at 200W for 3min, pour the primary emulsion into 100 mL of water, stir and diffuse for 3h, finally rotary evaporation at 50℃ to remove organic solvent and concentrate to 16.7ml to obtain abiraterone acetate nanoparticle suspension (see Figure 3 ) Repeat 6 times.

[0044] Dissolve 375 mg of Eudragit L100-55 in 100 mL of sodium phosphate buffer (pH = 6.5, 5 mM), mix with 50 mL of 1% HPMC solution to obtain an enteric material solution.

[0045] Pour 100 mL of abiraterone acetate nanoparticle suspension into 150 mL of enteric material solution, after ultrasonic dispersion, spray dry and collect the microcapsule powder to obtain abiraterone acetate enteric microcapsules (see Figure 4 ).

[0046] It can be seen from Figure 3 that the nanocapsules are round and have obvious core-shell structure. It can be seen from Figure 4 that the surface of abiraterone acetate enteric microcapsule powder is smooth and the particle size is uniform.

[0047] Example 2 Selection of oil core

[0048] The oil core of the nanocapsule is composed of oil and W / O emulsifier. Safe and stable auxiliary materials with high solubility of abiraterone acetate are selected to construct nanocapsules with high encapsulation efficiency:

[0049] Specifically:

[0050] Weigh 0.2 g of AA drug powder into a 5 mL EP tube, add 2 g of capric and caprylic acid glyceride, soybean oil, ethyl oleate, isopropyl myristate, Labrasol (caprylic / capric acid polyethylene glycol glyceride), Labrafil M1944CS (oleic acid polyethylene glycol glyceride) Labrafac Lipophile 1349 (medium chain triglyceride) Labrafil M2125 CS (linoleic acid polyethylene glycol glyceride), parallel 3 times, and place in a 37℃ constant temperature water bath shaker, shake and balance for 48h. After 48h, centrifuge at 5000rpm for 10min, take 100mg of supernatant, dissolve with acetonitrile, and determine the solubility of AA in various oil phases and emulsifiers by HPLC. The HPLC parameters are shown in Example 8.

[0051] Determination of solubility of different oil phases and W / O emulsifiers, seeFigure 1 From the figure, it can be seen that the solubility of oleic acid polyethylene glycol glycerol ester and ethyl oleate is the highest, which is 48.02 mg / mL and 45.63 mg / mL respectively. However, ethyl oleate has certain toxicity and tissue irritation, so caprylocaproyl glycerides is selected as the oil of the nanocapsule. Therefore, caprylocaproyl glycerides and oleic acid polyethylene glycol glycerol ester are finally determined as the oil phase and emulsifier of the oil core of the nanocapsule.

[0052] Example 3 Selection of polymer concentration

[0053] AA nanocapsules are prepared according to the basic process of Example 1, other factors are kept unchanged, and the changes of particle size, encapsulation rate, appearance and stability of each prescription are investigated when the concentration of polymer PLGA4000 is 0.5wt%, 1%, 1.5%, and 2% respectively. The results are as follows:

[0054]

[0055] With the increase of polymer concentration, the particle size of the nanocapsule increases, and the stability decreases. The encapsulation rate shows a trend of first rising and then being stable. Because the polymer material has a certain solubility to the drug, it can improve the encapsulation rate of the nanocapsule to a certain extent. By comprehensively considering the above indexes, 1% (w / v) is selected as the concentration of the polymer.

[0056] Example 4 Selection of the ratio of oil core to polymer

[0057] AA nanocapsules are prepared according to the basic prescription of Example 1 and the concentration of polymer recorded in Example 3 is 0.5%, other factors are kept unchanged, and the changes of particle size, encapsulation rate, appearance and stability of different prescriptions are determined when the mass ratio of different oil phases and polymers is 8:1, 5:1, 3:1, and 2:1. The results are as follows:

[0058]

[0059] When the polymer concentration is fixed, the viscosity of the organic phase increases when the proportion of oil in the organic phase is large, which causes the particle size of the nanocapsule to increase. However, the increase of the oil phase improves the solubility of the organic phase to AA and improves the encapsulation rate of the nanocapsule, so 3:1 can be selected as the ratio between the oil core and the polymer.

[0060] Example 5 Mass fraction of emulsifier in aqueous phase

[0061] Abiraterone acetate nanocapsules are prepared according to the method of Example 1, wherein the concentration of polymer is 0.5%, the mass ratio of oil to polymer is 3:1, other conditions are kept unchanged, and the changes of particle size, encapsulation rate, appearance and stability of different prescriptions under different concentrations of water (1wt%, 1.25%, 1.5%, 2%) are determined. The results are as follows:

[0062]

[0063] The greater the concentration of emulsifier in the aqueous phase, the smaller the particle size of the primary emulsion, and the final particle size of the nanocapsule formed by solvent diffusion is also smaller, which helps to enhance the stability of the system. The prepared nanocapsules are later loaded into enteric materials by spray drying technology. Too high emulsifier concentration will reduce the drug loading capacity, so 1.25% is selected as the optimal emulsifier concentration in the aqueous phase.

[0064] Selection of oil-water volume ratio in the preparation process of Example 6

[0065] Abiraterone acetate nanoparticles were prepared according to the method of Example 7, wherein the polymer concentration was 0.5%, the mass ratio of oil to polymer was 3:1, and the mass fraction of emulsifier in the aqueous phase was 1.25%. The particle size, encapsulation efficiency, appearance and stability of the obtained prescription were changed under different water-oil two-phase ratios (volume ratio was 5:1, 4:1, 2:1, 1:1). The results are as follows:

[0066]

[0067] Preparation of abiraterone acetate enteric microcapsules in Example 7

[0068] 20 mg of AA, 0.125 g of caprylocaproyl glycerides, 0.025 g of oleic acid polyethylene glycol glycerides and 0.05 g of PLGA were dissolved in 5 mL of ethyl acetate under magnetic stirring, and an organic phase was obtained after complete dissolution. Pour into 20 mL of aqueous phase containing 1.25% (w / v) poloxamer 188, under ice bath condition, emulsify by probe ultrasonic at 300 W power for 3 min, then pour into 100 mL of diffusion phase, i.e. purified water, under magnetic stirring at a speed of 180 rpm, diffuse for 3 h, rotary evaporate the organic solvent ethyl acetate, and concentrate to 16.7 ml to obtain abiraterone acetate nanoparticle suspension. Repeat 6 times.

[0069] 375 mg of Eudragit L100-55 was dissolved in 100 mL of sodium phosphate buffer (pH = 6.5, 5 mM), and mixed with 50 mL of 1% HPMC solution to obtain an enteric material solution.

[0070] 100 mL of abiraterone acetate nanoparticle suspension was poured into 150 mL of enteric material solution, and after ultrasonic dispersion, the microcapsule powder was collected by spray drying to obtain abiraterone acetate enteric microcapsules (see Figure 4 ), with a yield of 33.4%.

[0071] Determination of the encapsulation efficiency and drug loading capacity of abiraterone acetate enteric microcapsules in Example 8

[0072] Determination of the encapsulation efficiency of abiraterone acetate enteric microcapsules: The ultrafiltration centrifugation method was used to determine the encapsulation efficiency of abiraterone acetate enteric microcapsules. 500ul of abiraterone acetate enteric microcapsule suspension prepared according to Example 7 was taken and centrifuged at 5000rpm for 15min in an ultrafiltration centrifuge tube (0.5mL, 100kD). The filtrate was collected, diluted to 5mL with acetonitrile, and filtered through a 0.45μm microporous membrane before being determined by HPLC according to the following chromatographic conditions:

[0073] Chromatographic column: Promosil C18 (4.6mm x 250mm, 5μm)

[0074] Mobile phase: Phosphate buffer (0.05M, pH = 3): acetonitrile = 22:78

[0075] Flow rate: 1mL / min

[0076] Detection wavelength: 254nm

[0077] Injection volume: 10μL

[0078] Column temperature: 40℃

[0079] Determination of the concentration C of abiraterone acetate free , and the regression equation was y = 13167x + 4514.1 (R2= 0.9996). An equal volume (i.e. 500μL) of abiraterone acetate oral nanocapsule suspension without ultrafiltration was used, and after being broken with acetonitrile, diluted to 5mL, filtered through a 0.45μm microporous membrane, and determined by HPLC according to the above chromatographic conditions to determine the concentration C of the broken abiraterone acetate nanocapsule suspension total , and the experiment was repeated three times. The ratio of the two was the result of the encapsulation efficiency (EE%), and the specific calculation formula was

[0080] Determination of the drug loading of abiraterone acetate enteric microcapsules: A certain mass W (10mg) of abiraterone acetate enteric microcapsule powder (prepared according to the ratio of Example 7) was weighed, dissolved in DMSO, diluted to 5mL, filtered through a 0.45μm microporous membrane, and the concentration C of AA was determined by HPLC. The calculation formula of the drug loading (DL%) was as follows:

[0081] The final encapsulation efficiency of abiraterone acetate enteric microcapsules was 92.4±1.7%, and the drug loading was 2.74±0.12%.

[0082] Example 9 In vitro release experiment of abiraterone acetate enteric microcapsules

[0083] Free abiraterone acetate (abiraterone acetate molecules released from nanocapsules) and total abiraterone acetate (free abiraterone acetate and abiraterone acetate encapsulated in nanocapsules) released from abiraterone acetate enteric microcapsules in pH 1.2 and pH 6.8 media were determined according to the following method:

[0084] 500 μL of the abiraterone acetate enteric microcapsule suspension prepared according to Example 7 (abiraterone acetate: 4 mg, meeting the sink condition) was added to a centrifuge tube containing 40 mL of HC1 / KC1 buffer (0.2 M, pH 1.2, containing 0.5% Tween 80) to simulate the gastric environment, the tube was sealed and shaken in a 37°C water bath shaker for 2 hours. After 2 hours of incubation, the centrifuge tube was centrifuged at 3500 rpm for 5 minutes, 4 mL of supernatant was collected while the remaining acidic release medium was discarded. Then, 40 mL of pH 6.8 phosphate buffer (0.2 M, pH 6.8, containing 0.5% Tween 80) was added to the tube to simulate the small intestine environment. At the following time points (0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h), the tube was centrifuged at 3500 rpm for 5 minutes, 4 mL of supernatant was collected and replaced with an equal volume of fresh release medium (pH 6.8 phosphate buffer (0.2 M, pH 6.8, containing 0.5% Tween 80)).

[0085] All samples collected at different time points were divided into two parts, one part was separated to obtain free AA molecules by the method of encapsulation rate determination, the filtrate was analyzed by HPLC to determine the amount of free abiraterone acetate; the other part was broken with DMSO to 5 mL, filtered by 0.45 μm microporous membrane, and then the total amount of abiraterone acetate was determined by HPLC method. According to the amount of free abiraterone acetate and total abiraterone acetate at each time point, the in vitro release curve of abiraterone acetate enteric microcapsules was drawn (see Figure 2 ).

[0086] pH 1.2 and pH 6.8 buffers containing 0.5% Tween 80 were used to simulate the gastrointestinal environment, and the addition of Tween 80 can improve the solubility of abiraterone acetate in different pH release media to meet the sink condition.

[0087] As shown in Figure 2 , free AA reached a steady state after 6 hours, the final cumulative release of free abiraterone acetate was less than 12%, and the cumulative release amount was low. With the increase of the pH of the release medium to 6.8, the release rate of total abiraterone acetate increased significantly, in which the microcapsule surface material composed of enteric polymer Eudragit L100-55 and hydroxypropyl methyl cellulose was dissolved. Therefore, the encapsulated abiraterone acetate enteric microcapsules were released in large quantities and caused the curve to rise. As shown in Figure 1As shown, after 8h, the curve tends to plateau, and the final cumulative release of total abiraterone acetate reaches 87.1±2.2%. It can be calculated that the proportion of abiraterone acetate released from the microcapsules is about 75%. Since the encapsulation of drugs into nanoparticles can enhance the biofilm penetration of BCS IV drugs, the oral bioavailability of abiraterone acetate can be improved.

[0088] Example 10 Morphological investigation

[0089] The morphology of abiraterone acetate nanoparticles was visualized by transmission electron microscopy. Specifically, the suspension of abiraterone acetate nanoparticles prepared according to the method of Example 7 was diluted 20-fold with water until the blue opalescence was essentially invisible, and a drop was deposited on a carbon-coated copper grid. The liquid was absorbed along the edges with filter paper, and the sample was negatively stained with a 2% phosphotungstic acid solution for 3 min. The liquid was then absorbed with filter paper, and the grid was air-dried at room temperature. The results are shown in Figure 3 . As shown, the transmission electron micrograph of abiraterone acetate nanoparticles shows that the nanoparticles are relatively round and have a clear core-shell structure. Figure 3

[0090] The surface of abiraterone acetate enteric microcapsules was morphologically evaluated by scanning electron microscopy. The abiraterone acetate enteric microcapsule powder was uniformly coated on the conductive glue of the sample holder, and the excess powder was blown off. The powder on the sample holder was then sputtered with gold, and then observed under a scanning electron microscope. The results are shown in Figure 4 .

[0091] As can be seen from Figure 4 , the surface of the abiraterone acetate enteric microcapsule powder is smooth, and the particle size is uniform.

[0092] Example 11 Pharmacokinetic investigation

[0093] ​30 SD rats (250±20g, male) were randomly divided into 5 groups, 6 rats in each group. After feeding for one week, the experiment was carried out, and the rats were fasted for 12 hours before administration and allowed to drink water freely. Three groups of rats were orally administered with abiraterone acetate suspension, abiraterone acetate nanoparticles prepared according to Example 7, and abiraterone acetate enteric microcapsules 20 mg / kg, respectively, prepared according to the method of Example 7. The rats were taken blood from the orbital venous plexus at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours. 500 μL of whole blood was taken into a 1.5 mL EP tube containing heparin, centrifuged at 10,000 rpm for 10 min, and the supernatant was taken out and stored at -20℃ for later use. The fourth group of rats was injected with abiraterone acetate solution (i.e., abiraterone acetate was dissolved in solvent to prepare a solution with a concentration of 1 mg / mL; the solvent was a mixture of 10% anhydrous ethanol, 20% Cremophor EL, and 70% normal saline) 5 mg / kg through the tail vein, and blood was taken from the orbital venous plexus of the rats at 0.167, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 hours, and the processing steps were the same as above. 100 μL of SD rat drug-containing plasma was accurately pipetted into a 1.5 mL EP tube, 50 μL of internal standard solution (carbamazepine acetonitrile solution, 2 μg / mL) was added, 50 μL of acetonitrile was added, and the mixture was vortexed for 30 s; 300 μL of precipitant acetonitrile was added, vortexed for 90 s, centrifuged at 10,000 rpm for 10 min; 450 μL of supernatant was taken, filtered through a 0.22 μm microporous filter, and then transferred to a sample vial, 5 μL of which was analyzed according to the above chromatographic and mass spectrometric conditions to process the plasma sample. The blood plasma samples were detected for abiraterone acetate and abiraterone by UPLC-MS / MS method, the pharmacokinetic data of each group were calculated by DAS2.0 software, and the blood drug concentration-time curve was drawn.

[0094] Chromatographic conditions:

[0095] Chromatographic column: waters ACQUITY UPLC BEH C18 (2.1*150mm, 1.7 μm)

[0096] Mobile phase: acetonitrile: 0.1% formic acid water = 90:10 (v / v)

[0097] Column temperature: 25℃

[0098] Injection volume: 5 μL

[0099] Collection time: 3 min

[0100] Detection mode: positive ion

[0101] Ionization mode: electrospray ionization (ESI)

[0102] Ion detection mode: multiple reaction monitoring (MRM)

[0103] Capillary voltage: 3.5kV

[0104] Extraction cone voltage: 4V

[0105] Source temperature: 110℃

[0106] Desolvation temperature: 400℃

[0107] Other parameters: see the table below:

[0108]

[0109] The regression equations for the plasma standard curves of abiraterone and abiraterone acetate were y = 0.0150148x + 0.0251556 (R = 0.996191, R0). 2 =0.992396), y=0.142051x+0.283414 (R=0.990088, R 2 =0.980274)

[0110] Since abiraterone acetate (AA) is rapidly converted to abiraterone (ART) after entering the body, and the blood concentration of AA is almost undetectable in vivo, the blood concentration-time curve of ART is used to reflect the in vivo absorption of the formulation and to calculate relevant pharmacokinetic parameters.

[0111] Following oral administration, the plasma concentration-time curves for abiraterone acetate suspension, abiraterone acetate nanoparticles, abiraterone acetate enteric-coated microcapsules, and abiraterone acetate injected with AA are shown in the figure. Figure 5 .

[0112] Depend on Figure 5 It is known that the blood concentration of abiraterone acetate suspension is extremely low, with almost no absorption. The Tmax of abiraterone acetate enteric-coated microcapsules is later than that of abiraterone acetate nanoparticles. At the same dosage, the blood concentration of abiraterone acetate is higher than that of abiraterone acetate nanoparticles, indicating that oral enteric-coated microcapsules of abiraterone acetate have a certain sustained-release effect, and the oral bioavailability is higher than that of oral abiraterone acetate nanocapsules.

[0113] Example 12 Study on absorption mechanism

[0114] In Experiment 11, rats in the fifth group were intraperitoneally injected with 3 mg / mL actinomyceteone saline solution 1.5 h before drug administration (dose was 3 mg / kg) to establish a rat model of lymphatic absorption blockade. 1.5 h after intraperitoneal injection of the actinomyceteone solution, rats were administered 20 mg / kg of abiraterone acetate enteric-coated microcapsules prepared in the same manner as in Example 7 via gavage. Blood samples were collected from the orbital venous plexus of rats at 0, 0.5, 1, 2, 3, 4, 6, and 8 h. The plasma concentrations of abiraterone acetate and abiraterone were detected by UPLC-MS / MS, and plasma concentration-time curves were plotted.

[0115] Cycloheximide, chemical name cycloheximide, is a protein synthesis inhibitor, which can inhibit the secretion of chylomicron in small intestinal epithelial cells and induce apoptosis of mesenteric lymph nodes. In this experiment, cycloheximide (3 mg / kg) was injected intraperitoneally to construct a chylomicron metabolic blocking model rat. Although this blockage is irreversible, it does not affect other absorption pathways. The blood concentration-time curves of oral enteric microcapsules in the cycloheximide injection group and the non-cycloheximide injection group are shown in Figure 6 .

[0116] As can be seen from the figure, the blood concentration of abiraterone in the cycloheximide injection group was below the minimum detection limit for the first 4 hours, and the blood concentration of abiraterone could be detected after 6 hours, but was below 20 ng / mL. The blockage of chylomicron metabolism greatly reduces the absorption of the drug, and thus the above results show that the main absorption pathway of abiraterone acetate enteric microcapsules is lymphatic absorption in the gastrointestinal tract similar to chylomicron metabolism, thereby enhancing the oral bioavailability of abiraterone acetate, which has poor solubility and permeability.

[0117] Example 13 Pharmacodynamic experiment

[0118] LNCap-C4-2 cells were cultured in a 37℃, humidity-saturated 5% CO2 incubator with RPMI-1640 culture medium containing 10% FBS. When the cells covered the culture bottle, they were digested and passaged with 0.25% trypsin, and were passaged every 3-4 days.

[0119] Male BALB / c-nu nude mice were raised in the SPF animal experimental center of Shenyang Pharmaceutical University, with temperature and humidity controlled at 20-25℃ and 40%-60%, respectively. The animals were free to eat and drink during the experiment, and the experimental protocol was approved by the Animal Ethics Committee of Shenyang Pharmaceutical University. After the nude mice were adaptively raised in the SPF animal experimental center for 1 week, they were castrated and the testes of the male mice were removed, and they were continued to be raised for a week for recovery. The logarithmic growth phase C4-2 cells were collected, resuspended with cold serum-free culture medium, and the cell density was adjusted to 2.5×10 8 cells / mL, and each nude mouse was inoculated with 5×10 7 cells / 200μL. Inoculation was performed in the axillary and blood-rich parts. During the inoculation process, the cell suspension was stored at low temperature and did not directly contact the ice box. Before inoculation, the cell suspension was gently mixed to keep the number of cells inoculated in each nude mouse approximately consistent.

[0120] When the average tumor volume reached 100mm 3 The tumor volume was removed when it was below 100mm 3The remaining nude mice were randomly divided into three groups according to the tumor volume: ① model group (negative control group): given blank solvent (equal volume of normal saline); ② abiraterone acetate suspension group (positive control group), the dosage was 196 mg / kg (0.5% CMC-Na-AA suspension, 5 mg / mL); ③ abiraterone acetate enteric microcapsules prepared by the method of Example 7, the dosage was 196 mg / kg (suspended in water after weighing, 0.25 g / mL). The administration was performed by oral gavage, 5 days of continuous administration per week, 2 days of rest, once a day, 7 days as a cycle, a total of 3 weeks.

[0121] Conventional index detection:

[0122] 1. General condition of nude mice: the mental state of nude mice was observed every day, the body weight of each mouse was weighed and recorded every 3 days, and the average body weight of each group was used to draw the body weight change curve of nude mice;

[0123] 2. Change of nude mouse transplanted tumor volume: the long diameter and short diameter of the tumor were measured every 3 days by vernier caliper, the transplanted tumor volume was calculated according to the tumor volume formula: V = (a*b 2 ) / 2 (a, b are the long diameter and short diameter of the tumor, respectively), and the average change of the transplanted tumor volume of each group was used to draw the tumor growth curve;

[0124] 3. After the end of the experiment, the body weight was weighed, the blood was taken by enucleation, and after the death, the tumor mass was dissected and peeled off, and the Figure 7 tumor weight was weighed, and the tumor weight distribution graph of each group was drawn;

[0125] 4. The peeled tumor tissue was photographed and recorded by digital camera.

[0126] Serum index detection:

[0127] After the end of the experiment, the blood was taken by enucleation. The CYP17A1 enzyme activity and PSA level in the serum of tumor-bearing nude mice after three weeks of administration were measured by enzyme-linked immunoassay (ELISA) using mouse CYP17A1 kit and mouse PSA kit. The specific operation is as follows:

[0128] 1. Blood sampling by enucleation of mouse eye: the left thumb and index finger of the left hand were used to grasp the mouse's both ears and the skin behind the neck, the little finger was used to fix the tail; the middle finger was used to press the left forelimb of the mouse at the heart of the sternum, the thumb was twisted, the eye skin of the blood sampling side was pressed, the eye was congested and protruded; the eye was clamped with a curved forceps, so that the blood flowed vertically into the centrifuge tube to avoid contamination; after the blood flow was completed, the mouse was killed by breaking the neck.

[0129] 2. Detection of PSA level and CYP17A1 enzyme activity in serum

[0130] The determination of serum indexes was carried out according to the steps of the kit, and the specific steps were as follows:

[0131] (1) Serum---Center 1000×g for 10 minutes to remove particles and polymers.

[0132] (2) Storage------ Store it at -80℃ and avoid repeated freezing.

[0133] (3) Mix all reagents thoroughly before use.

[0134] (4) Perform 3 replicates for each standard and blank well. Dilute the specimen 1:4 with specimen diluent and add 50 μL to the reaction well.

[0135] (5) Add 50 μL of the diluted standard to the reaction well and 50 μL of the sample to be tested to the reaction well. Cover the membrane plate, gently shake to mix, and incubate at 37°C for 30 min.

[0136] (6) Shake off the liquid in the well, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 5 times.

[0137] (7) Add 50 μL of avidin-HRP to each well except for the blank wells, gently shake to mix, and incubate at 37°C for 30 minutes.

[0138] (8) Shake off the liquid in the holes, fill each hole with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 5 times.

[0139] (9) Add 50 μL each of colorimetric reagent A and B to each well, gently shake to mix, and incubate at 37°C for 10 minutes. Avoid light exposure.

[0140] (10) Remove the microplate and quickly add 50 μL of stop solution. The results should be measured immediately after adding the stop solution. Measure the OD value of each well at a wavelength of 450 nm.

[0141] All experimental results were derived from three independent repeated trials and are expressed as mean ± SD. One-way ANOVA was performed using SPSS 22.0 statistical software. p < 0.05 was considered statistically significant.

[0142] Result: As Figure 8 As shown, in nude mice, the average tumor weight of the model control group was 0.35±0.14g; the average tumor weight of the positive control group and the preparation group was significantly smaller than that of the model control group, at 0.12±0.020g and 0.036±0.0071g, respectively, with tumor inhibition rates as high as 65.7% and 89.7%, respectively.

[0143] During the experiment, the body weight of tumor-bearing mice was weighed and recorded every 3 days. In the CRPC model of nude mice, the body weight of tumor-bearing mice in the preparation group had no obvious change at the initial stage of administration, but with the prolongation of treatment time, the body weight of tumor-bearing mice increased slightly, and there was no significant difference compared with the positive control group at the end of the experiment Figure 9 ). In addition, after the administration of each group was completed, we measured the enzyme activity of androgen synthesis enzyme CYP17A1 and the level of prostate specific antigen PSA in the serum of nude mice in each group by enzyme-linked immunoassay. The PSA level in the serum is used to reflect the growth of C4-2 cell transplanted tumor in nude mice. CYP17A1 is the target of AA treatment of castration-resistant prostate cancer, and AA is a CYP17A1 inhibitor, which can reduce the level of androgen in the body, especially in tumor tissue, by inhibiting the activity of CYP17A1, thereby inhibiting the proliferation and growth of cancer cells. As shown in Figure 10 and Figure 11 , the enzyme activity of CYP17A1 and the level of PSA in the serum of the model control group were 100%, and the level of PSA in the positive control group was 62.97±0.52%, and the enzyme activity of CYP17A1 was 42.28±2.28%; while the level of PSA and the enzyme activity of CYP17A1 in the preparation group were 48.93±0.69% and 19.98±0.88%, respectively. The two serum indicators, both P values were less than 0.01 compared with the model group, indicating that the difference between the two groups had very significant statistical significance. Compared with the positive control, P values were less than 0.05, indicating that the difference between the two groups had statistical significance. It is shown that both groups effectively reduce the PSA level in the CRPC model mice, inhibit the activity of CYP17A1 in the mice, and it can also be seen that the efficacy of the abiraterone acetate enteric microcapsule in the preparation group is more significant than that of the positive control drug abiraterone acetate suspension.

[0144] The above results show that the present application can effectively inhibit the proliferation of prostate cancer cells and has no obvious toxic side effects.

Claims

1. An enteric-coated microcapsule of abiraterone acetate, characterized in that: Abiraterone acetate enteric-coated microcapsules are obtained by encapsulating abiraterone acetate nanoparticles with an enteric-coating material; wherein the mass ratio of abiraterone acetate nanoparticles to enteric-coating material is 11-19:

10. The abiraterone acetate nanoparticles are composed of the following components by mass percentage: Abiraterone acetate 5-11% Polymer materials 22-28% Oil content 64-70%; The mass ratio of oil core to polymer material is 3:1; The polymer material is 1% w / v PLGA 4000; The oil core is formulated with 5 parts by weight of caprylic / capric triglyceride and 1 part by weight of polyethylene glycol oleate. The enteric material is Eudragit L100-55 and hydroxypropyl methylcellulose, wherein the mass ratio of Eudragit L100-55 to hydroxypropyl methylcellulose is 3:4; The method for preparing the abiraterone acetate enteric-coated microcapsules is as follows: 1) Mix abiraterone acetate, polymer material, and oil core in the above proportions to obtain mixture 1. Mix mixture 1 and organic solvent at a mass ratio of 1:16-21 to obtain mixture 2, which is ready for use. 2) Add the above mixture 2 to the aqueous phase, wherein the mass ratio of mixture 2 to the aqueous phase is 1:4, and sonicate the cells at 300 W for 3 minutes to obtain colostrum; 3) Pour the above colostrum into water, with a water-to-colostrum mass ratio of 1:3-5, stir and diffuse for 2.5-3.5 h, and finally remove the organic solvent by rotary evaporation under reduced pressure at 45-55℃ and concentrate to between one-sixth and one-ninth of the original volume to obtain abiraterone acetate nanoparticle suspension. 4) Weigh Eudragit L100-55 and hydroxypropyl methylcellulose at a mass ratio of 3:4, and then dissolve them separately in pH 6.5 and 5 mM sodium phosphate buffer solutions. Mix the hydroxypropyl methylcellulose solution and the Eudragit L100-55 solution at a volume ratio of 1:2 to obtain the enteric material solution. 5) The above abiraterone acetate nanoparticle suspension and enteric-coated material solution were mixed at a volume ratio of 2:3, ultrasonically dispersed, and then spray-dried to prepare abiraterone acetate enteric-coated microcapsules; The aqueous phase is a 1.25% poloxamer 188 solution.

2. The method for preparing abiraterone acetate enteric-coated microcapsules according to claim 1, characterized in that: 1) Mix abiraterone acetate, polymer material, and oil core in the above proportions to obtain mixture 1. Mix mixture 1 and organic solvent at a mass ratio of 1:16-21 to obtain mixture 2, which is ready for use. 2) Add the above mixture 2 to the aqueous phase, wherein the mass ratio of mixture 2 to the aqueous phase is 1:4, and sonicate the cells at 300 W for 3 minutes to obtain colostrum; 3) Pour the above colostrum into water, with a water-to-colostrum mass ratio of 1:3-5, stir and diffuse for 2.5-3.5 h, and finally remove the organic solvent by rotary evaporation under reduced pressure at 45-55℃ and concentrate to between one-sixth and one-ninth of the original volume to obtain abiraterone acetate nanoparticle suspension. 4) Weigh Eudragit L100-55 and hydroxypropyl methylcellulose at a mass ratio of 3:4, and then dissolve them separately in pH 6.5 and 5 mM sodium phosphate buffer solutions. Mix the hydroxypropyl methylcellulose solution and the Eudragit L100-55 solution at a volume ratio of 1:2 to obtain the enteric material solution. 5) The above abiraterone acetate nanoparticle suspension and enteric-coated material solution were mixed at a volume ratio of 2:3, ultrasonically dispersed, and then spray-dried to prepare abiraterone acetate enteric-coated microcapsules; The aqueous phase is a 1.25% poloxamer 188 solution.

3. The method for preparing abiraterone acetate enteric-coated microcapsules according to claim 2, characterized in that: The organic solvent is ethyl acetate.

4. The method for preparing abiraterone acetate enteric-coated microcapsules according to claim 2, characterized in that: In step 4), Eudragit L100-55 and hydroxypropyl methylcellulose were weighed and dissolved in sodium phosphate buffer to obtain Eudragit L100-55 solution with a concentration of 3.75 mg / ml and hydroxypropyl methylcellulose solution with a concentration of 10 mg / ml.

5. The application of abiraterone acetate enteric-coated microcapsules according to claim 1, characterized in that: The application of the abiraterone acetate enteric-coated microcapsules in the preparation of drugs for treating prostate cancer.

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