Preparation method of pharmaceutical composition for treating atopic dermatitis

Abstract

The invention provides a preparation method of a pharmaceutical composition for treating atopic dermatitis, and relates to the field of pharmacy, and the preparation method comprises the following steps: S1, preparing upatinib enteric-coated microspheres; step S2, preparing asiaticoside nanoparticles; s3, preparation of the pharmaceutical composition: firstly mixing the upatinib enteric-coated microspheres with xanthan gum, adding a phosphate buffer solution, and adjusting the pH value with sodium bicarbonate to obtain a microsphere suspension; adding the asiaticoside nanoparticles and carbomer 940 into a buffering agent, uniformly stirring, and adding glutamine to obtain a gel solution; and finally, slowly mixing the microsphere suspension and the gel solution under magnetic stirring, sequentially adding a flavoring agent and a bacteriostatic agent, adjusting the pH value, and freeze-drying to obtain the pharmaceutical composition. According to the method, the pharmaceutical composition of asiaticoside and upatinib is prepared, so that the use dosage of upatinib can be effectively reduced, and the pharmaceutical composition takes effect quickly and is high in stability and low in adverse reaction occurrence rate.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceuticals, and in particular to a method for preparing a pharmaceutical composition for treating atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD) is a chronic, recurrent, inflammatory skin disease; because patients often have other atopic diseases such as allergic rhinitis and asthma, it is considered a systemic disease. Atopic dermatitis patients often have severe itching, which seriously affects their daily life. The treatment of atopic dermatitis cannot be delayed.

[0003] Studies have shown that the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway plays an important role in the pathogenesis of atopic dermatitis. Therefore, JAK inhibitors are an emerging therapy for the treatment of atopic dermatitis. Upadacitinib is a highly selective Janus kinase 1 (JAK1) inhibitor that inhibits the expression of cytokines (interleukin-4 (IL-4), IL-13, IL-31, γ-interferon, etc.) involved in the pathogenesis of atopic dermatitis through the Janus kinase-signal transducer and activator of transcription pathway, thereby ultimately blocking the occurrence and development of atopic dermatitis. The launch of upadacitinib sustained-release tablets provides more options for the treatment of patients with atopic dermatitis; however, during the use of upadacitinib, adverse reactions such as oropharyngeal pain, adverse cardiovascular events, abdominal pain and diarrhea, and hypersensitivity are prone to occur. Therefore, oral upadacitinib requires strict control of the dosage of the patient, which greatly reduces the efficacy of upadacitinib.

[0004] Centella asiatica (Aisaticoside, CAS No.: 16830-15-2) is a natural triterpenoid saponin extracted from Centella asiatica, which has multiple biological effects such as angiogenesis, anti-inflammation, wound healing and osteogenic differentiation. Anti-inflammatory compound preparations containing plant extracts such as Centella asiatica can effectively reduce inflammatory responses, protect vascular endothelial cells, and thus play an anti-atherosclerotic role; at the same time, the research results of Xie Shengyang et al. show that the porous microspheres of Centella asiatica can repair skin damage, thereby accelerating the regeneration of damaged skin. Studies in recent years have shown that Centella asiatica exhibits a wide range of biological activities in in vitro cell culture and in vivo animal models, mainly in terms of anti-oxidation, anti-inflammation and protection against DNA damage. Modern pharmacological studies have shown that Centella asiatica has strong pharmacological activity, can promote wound healing, stimulate biosynthesis, improve skin problems and anti-inflammatory; however, due to the surface activity of saponins in Centella asiatica, which has a stimulating effect on gastric mucosa and saponins can accelerate intestinal peristalsis, oral administration of Centella asiatica has potential risks such as nausea, diarrhea, and stomach discomfort. Summary of the invention

[0005] In view of the problems existing in the above prior art, the object of the present invention is to provide a method for preparing a pharmaceutical composition for treating atopic dermatitis, which can effectively reduce the dosage of upadacitinib by preparing a pharmaceutical composition of asiaticoside and upadacitinib; and through the synergistic combination of the pharmaceutical composition, it is used for the prevention and treatment of atopic dermatitis, with rapid onset of effect, high stability and low incidence of adverse reactions.

[0006] The purpose of the present invention is achieved through the following technical solutions: A method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: first, adding upadacitinib and polylactic acid-co-glycolic acid (PLGA) into ethyl acetate, and ultrasonically dispersing them to form an organic phase; under stirring, slowly dripping the organic phase into a water phase of polyvinyl alcohol (PVA) to form colostrum; vacuum drying and washing the colostrum to obtain microspheres; and fluidizing and coating the microspheres with an ethanol solution of hydroxypropyl methylcellulose acetate succinate (HPMCAS) to obtain the microspheres; Step S2, preparing asiaticaoside nanoparticles: firstly, including asiaticaoside and β-cyclodextrin in warm water, adding chitosan solution, and stirring evenly; then slowly dropping sodium tripolyphosphate solution for cross-linking, and adding hyaluronic acid solution after cross-linking to form asiaticaoside nanoparticles; Step S3, preparing a pharmaceutical composition: firstly, mixing upadacitinib enteric-coated microspheres with xanthan gum, and adding phosphate buffer, and after ultrasonic dispersion, adding sodium bicarbonate to adjust the pH to 5.5 to obtain a microsphere suspension for standby use; then, adding asiatica glycoside nanoparticles and carbomer 940 to the buffer, stirring evenly, and then adding glutamine to obtain a gel solution for standby use; finally, slowly mixing the microsphere suspension and the gel solution under magnetic stirring, and sequentially adding a flavoring agent and an antibacterial agent, adjusting the pH to 5.5, and freeze-drying to obtain a pharmaceutical composition.

[0007] Based on further optimization of the above scheme, the molecular weight of the polylactic acid-glycolic acid copolymer is 10 kDa; the mass volume ratio of upadacitinib, polylactic acid-glycolic acid copolymer and ethyl acetate is 1 mg: 9-10 mg: 0.1-0.2 mL; the aqueous phase concentration of polyvinyl alcohol is 0.2-0.4% w / v, and the aqueous phase volume of polyvinyl alcohol is 10-20 times the volume of the organic phase.

[0008] Based on further optimization of the above scheme, the method for preparing the aqueous phase of polyvinyl alcohol is specifically as follows: dissolve 0.2-0.4 g of polyvinyl alcohol in 100 mL of ultrapure water, heat to 75-80° C., stir to dissolve, cool and filter, and the filtrate is the aqueous phase of polyvinyl alcohol.

[0009] Based on further optimization of the above scheme, in step S1, the stirring rate of the colostrum formation process is 600-800 rpm, and the stirring time is 45-60 min; the vacuum drying temperature is 32-36°C, the pressure is 45-50 mbar, and the drying time is 12-16 h; the washing step includes ultrapure water washing, centrifugal separation and freeze drying.

[0010] Based on further optimization of the above scheme, the particle size of the microspheres obtained in step S1 is 20 to 30 μm.

[0011] Based on further optimization of the above scheme, the concentration of the ethanol solution of hydroxypropyl methylcellulose acetate succinate is 7.5-8.5% (w / v); after fluidized bed coating, the microspheres are qualified when the weight gain is 14%-16%.

[0012] Based on further optimization of the above scheme, the mass volume ratio of Centella asiatica, β-cyclodextrin and chitosan solution in step S2 is 20 mg: 40-60 mg: 2-3 mL; the concentration of chitosan solution is 1% w / v (i.e., 1 g of chitosan is dissolved in 100 mL of deionized water); during the inclusion process, the amount of warm water used is 8-12 mL, the temperature is 45-55° C., and ultrasonic treatment is performed for 28-32 min during the inclusion process.

[0013] Based on further optimization of the above scheme, the mass ratio of sodium tripolyphosphate to chitosan is 1:10-15, the concentration of the sodium tripolyphosphate solution is 0.2-0.4% w / v (i.e., 0.2-0.4 g of sodium tripolyphosphate is dissolved in 100 mL of deionized water); the concentration of the hyaluronic acid solution is 0.5% w / v (i.e., 0.5 g of hyaluronic acid is dissolved in 100 mL of deionized water), and the mass ratio of hyaluronic acid to sodium tripolyphosphate is 5-10:1-2.

[0014] Based on further optimization of the above scheme, in step S2, the stirring time for adding the chitosan solution is 28 to 32 minutes, and the stirring speed is 450 to 550 rpm; the cross-linking time is 18 to 22 minutes, and the stirring rate is 800 to 1000 rpm during the cross-linking process; after adding the hyaluronic acid solution, the stirring speed is 200 to 400 rpm and the stirring is 0.9 to 1.1 hours.

[0015] Based on further optimization of the above scheme, in step S3, the mass ratio of upadacitinib to asiaticoside in the upadacitinib enteric-coated microspheres and asiaticoside nanoparticles is 2.5-3.5:38-42; the mass ratio of xanthan gum to upadacitinib enteric-coated microspheres is 1:2; and the mass ratio of carbomer 940 to xanthan gum is 1:3-5.

[0016] Based on further optimization of the above scheme, the phosphate buffer adopts any one of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer, and the pH of the phosphate buffer is 5.5; the volume mass ratio of phosphate buffer to upadacitinib enteric-coated microspheres is 1-2 mL:3 mg.

[0017] Based on further optimization of the above scheme, the buffer adopts citric acid solution or sodium dihydrogen phosphate solution with a pH of 6.0, and the volume mass ratio of the buffer to Centella asiatica nanoparticles is 1-2 mL:20 mg; the mass ratio of glutamine to Centella asiatica nanoparticles is 0.8-1:2.

[0018] Based on further optimization of the above scheme, the flavoring agent is one or more of sucrose, sucralose, and acesulfame potassium, the antibacterial agent is one or more of sorbic acid, methylparaben, benzalkonium chloride, and sodium benzoate, and the mass ratio of the flavoring agent, the antibacterial agent and upadacitinib enteric-coated microspheres is 5-10:0.1-0.5:1.

[0019] The following are the technical effects of the solution of the present invention: The present invention prepares upadacitinib enteric-coated microspheres and asiaticoside nanoparticles, and then uses the optimized suspension of upadacitinib enteric-coated microspheres to compound with the asiaticoside nanoparticle solution to obtain a pharmaceutical composition containing asiaticoside and upadacitinib, which effectively reduces the dosage of upadacitinib and improves the therapeutic effect of atopic dermatitis; the upadacitinib enteric-coated microspheres in the pharmaceutical composition prepared by the present invention are released in a weakly acidic environment (i.e., pH>5.5), thereby avoiding release in a strong acidic environment in the stomach after oral administration, thereby effectively protecting the gastric mucosa, relieving gastric discomfort, and avoiding irritation and damage to the stomach; at the same time, the asiaticoside nanoparticles in the pharmaceutical composition adhere to the intestinal mucus layer, thereby reducing the direct contact between the drug and the intestinal epithelial tissue, and playing a role in mucosal protection. Through the synergistic effect between upadacitinib enteric-coated microspheres and Centella asiatica nanoparticles, the more frequent and more severe gastrointestinal symptoms caused by the interaction of Centella asiatica and upadacitinib are avoided (i.e., the saponins of Centella asiatica stimulate the gastric mucosa and accelerate intestinal peristalsis, combined with the adverse symptoms of oral upadacitinib, leading to more severe abdominal pain, diarrhea, nausea and vomiting), thereby reducing irritation to the gastrointestinal tract and promoting intestinal mucosal repair.

[0020] In addition, the pharmaceutical composition prepared by the present invention has high solubility, can avoid the problems of poor water solubility of Centella asiatica glycoside and low solubility of upadacitinib under acidic conditions, and has good oral compliance, good solution stability, and rapid onset of action; at the same time, the pharmaceutical composition can effectively reduce the sedimentation rate of enteric-coated microspheres and nanoparticles, prolong the action time of the drug in the gastrointestinal tract, and improve the efficacy of the drug. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be clearly and completely described below in the form of specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, that is, the protection scope of the present invention is not limited by the specific implementation methods.

[0022] Embodiment 1: A method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: First, upadacitinib and polylactic acid-co-glycolic acid (PLGA) with a molecular weight of 10 kDa were added to ethyl acetate and ultrasonically dispersed (conventional ultrasonic dispersion process can be used, which is not specifically limited in this embodiment), and the mass volume ratio of upadacitinib, polylactic acid-co-glycolic acid and ethyl acetate was 1 mg:9 mg:0.1 mL to form an organic phase; Under stirring conditions, the organic phase is slowly dripped into the aqueous phase of polyvinyl alcohol (PVA) to form colostrum, wherein the aqueous phase concentration of polyvinyl alcohol is 0.2% w / v, and the aqueous phase volume of polyvinyl alcohol is 10 times the volume of the organic phase (for example, if the volume of the organic phase is 1 mL, 10 mL of aqueous phase is used), the stirring rate is 600 rpm, and the stirring time is 60 min (stirring is carried out at room temperature of about 25°C to avoid excessive evaporation of the solvent and agglomeration of emulsified microspheres). The specific method for preparing the aqueous phase of polyvinyl alcohol is as follows: 0.2 g of polyvinyl alcohol is dissolved in 100 mL of ultrapure water, heated to 75°C, stirred to dissolve, cooled and filtered (i.e., cooled to room temperature), and the filtrate is the aqueous phase of polyvinyl alcohol.

[0023] The colostrum is vacuum dried and washed to obtain microspheres with a particle size of 20 to 30 μm; wherein: the vacuum drying temperature is 32°C, the pressure is 45 mbar, and the drying time is 16 hours; the washing steps include ultrapure water washing (i.e., ultrapure water is added to the solid powder obtained after vacuum drying, and the amount of ultrapure water is determined according to the actual amount of powder, generally completely immersed), centrifugal separation (centrifugal speed is 14000 rpm, and the centrifugal time is 15 minutes) and freeze drying (conventional freeze drying process can be used); The microspheres were fluidized bed coated with an ethanol solution of hydroxypropyl methylcellulose acetate succinate (HPMCAS) (coating can be performed using a common fluidized bed), the concentration of the ethanol solution of hydroxypropyl methylcellulose acetate succinate was 7.5% (w / v), and after fluidized bed coating, the microspheres gained 14% weight, thereby obtaining upadacitinib enteric-coated microspheres.

[0024] Step S2, preparing asiaticaside nanoparticles: First, asiatica glycoside and β-cyclodextrin were included in warm water, chitosan solution was added, and stirred evenly (stirring rate was 450rpm, stirring time was 32min); wherein, the mass volume ratio of asiatica glycoside, β-cyclodextrin and chitosan solution was 20mg:40mg:2mL; the concentration of chitosan solution was 1% w / v (i.e., 1g chitosan was dissolved in 100mL deionized water); during the inclusion process, the amount of warm water (using deionized water) was 8mL, the temperature was 45°C, and ultrasonic treatment was performed for 28min during the inclusion process (to ensure uniform dispersion); Then slowly add sodium tripolyphosphate solution for cross-linking, the mass ratio of sodium tripolyphosphate to chitosan is 1:10, the concentration of sodium tripolyphosphate solution is 0.2% w / v (i.e., 0.2 g sodium tripolyphosphate is dissolved in 100 mL of deionized water), the cross-linking time is 18 min, and the stirring speed is 800 rpm during the cross-linking process; After cross-linking, hyaluronic acid solution was added and stirred at a speed of 200 rpm for 0.9 h. The concentration of the hyaluronic acid solution was 0.5% w / v (i.e., 0.5 g hyaluronic acid was dissolved in 100 mL of deionized water), and the mass ratio of hyaluronic acid to sodium tripolyphosphate was 5:1. After stirring, free matter was removed by centrifugation (14000 rpm, 28 min), and then freeze-drying was performed (conventional freeze-drying process was used) to form Centella asiatica nanoparticles.

[0025] Step S3, preparing a pharmaceutical composition: First, upadacitinib enteric-coated microspheres are mixed with xanthan gum, the mass ratio of xanthan gum to upadacitinib enteric-coated microspheres is 1:2, and a phosphate buffer is added, the phosphate buffer adopts sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and the pH of the phosphate buffer is 5.5 (the pH value is regulated by regulating the ratio between sodium dihydrogen phosphate and disodium hydrogen phosphate. In this embodiment, the volume ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate is about 51:1), the volume mass ratio of phosphate buffer to upadacitinib enteric-coated microspheres is 1 mL:3 mg, and sodium bicarbonate is added after ultrasonic dispersion to adjust the pH to 5.5 to obtain a microsphere suspension for standby use; Then, add asiaticaside nanoparticles and carbomer 940 to the buffer, wherein the mass ratio of upadacitinib to asiaticaside in upadacitinib enteric-coated microspheres and asiaticaside nanoparticles is 2.5:38, the mass ratio of carbomer 940 to xanthan gum is 1:3, the buffer is a citric acid solution with a pH of 6.0, the volume mass ratio of the buffer to asiaticaside nanoparticles is 1 mL:20 mg, stir evenly (stirring speed is 400-500 rpm), then add glutamine, the mass ratio of glutamine to asiaticaside nanoparticles is 0.8:2, and obtain a gel solution for standby use; Finally, the microsphere suspension and the gel solution are slowly mixed under magnetic stirring, and flavoring agents and antibacterial agents are added in sequence. Sucrose is used as the flavoring agent, and sorbic acid is used as the antibacterial agent. The mass ratio of the flavoring agent, the antibacterial agent and upadacitinib enteric-coated microspheres is 5:0.1:1. The pH is adjusted to 5.5 (sodium bicarbonate or sodium hydroxide solution can be used to adjust the pH value), and freeze-dried (conventional freeze-drying process can be used) to obtain a pharmaceutical composition.

[0026] Embodiment 2: A method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: First, upadacitinib and poly(lactic acid-co-glycolic acid) (PLGA) with a molecular weight of 10 kDa were added to ethyl acetate and ultrasonically dispersed (conventional ultrasonic dispersion process can be used, which is not specifically limited in this embodiment), and the mass volume ratio of upadacitinib, poly(lactic acid-co-glycolic acid) and ethyl acetate was 1 mg:9.5 mg:0.15 mL to form an organic phase; Under stirring conditions, the organic phase is slowly dripped into the aqueous phase of polyvinyl alcohol (PVA) to form colostrum, wherein the aqueous phase concentration of polyvinyl alcohol is 0.3% w / v, and the aqueous phase volume of polyvinyl alcohol is 15 times the volume of the organic phase (for example, if the volume of the organic phase is 1 mL, 15 mL of aqueous phase is used), the stirring rate is 700 rpm, and the stirring time is 52 min (stirring is carried out at room temperature of about 25°C to avoid excessive evaporation of the solvent and agglomeration of emulsified microspheres). The specific method for preparing the aqueous phase of polyvinyl alcohol is as follows: 0.3 g of polyvinyl alcohol is dissolved in 100 mL of ultrapure water, heated to 77°C, stirred to dissolve, and filtered after cooling (i.e., cooled to room temperature), and the filtrate is the aqueous phase of polyvinyl alcohol.

[0027] The colostrum is vacuum dried and washed to obtain microspheres with a particle size of 20 to 30 μm; wherein: the vacuum drying temperature is 34°C, the pressure is 47 mbar, and the drying time is 14 hours; the washing steps include ultrapure water washing (i.e., ultrapure water is added to the solid powder obtained after vacuum drying, and the amount of ultrapure water is determined according to the actual amount of powder, generally completely immersed), centrifugal separation (centrifugal speed is 15000 rpm, and the centrifugal time is 12 minutes) and freeze drying (conventional freeze drying process can be used); The microspheres are fluidized bed coated with an ethanol solution of hydroxypropyl methylcellulose acetate succinate (HPMCAS) (coating can be performed using a common fluidized bed), the concentration of the ethanol solution of hydroxypropyl methylcellulose acetate succinate is 8% (w / v), and after fluidized bed coating, the weight of the microspheres increases by 15%, thereby obtaining upadacitinib enteric-coated microspheres.

[0028] Step S2, preparing asiaticaside nanoparticles: First, asiatica glycoside and β-cyclodextrin were included in warm water, chitosan solution was added, and stirred evenly (stirring rate was 500rpm, stirring time was 30min); wherein, the mass volume ratio of asiatica glycoside, β-cyclodextrin and chitosan solution was 20mg:50mg:2.5mL; the concentration of chitosan solution was 1% w / v (i.e., 1g chitosan was dissolved in 100mL deionized water); during the inclusion process, the amount of warm water (deionized water) was 10mL, the temperature was 50℃, and ultrasonic treatment was performed for 30min during the inclusion process (to ensure uniform dispersion); Then slowly add sodium tripolyphosphate solution for cross-linking, the mass ratio of sodium tripolyphosphate to chitosan is 1:12, the concentration of sodium tripolyphosphate solution is 0.3% w / v (i.e., 0.3 g sodium tripolyphosphate is dissolved in 100 mL of deionized water), the cross-linking time is 20 min, and the stirring speed is 900 rpm during the cross-linking process; After cross-linking, hyaluronic acid solution was added and stirred at 300 rpm for 1 hour. The concentration of the hyaluronic acid solution was 0.5% w / v (i.e., 0.5 g hyaluronic acid was dissolved in 100 mL of deionized water), and the mass ratio of hyaluronic acid to sodium tripolyphosphate was 7:1.5. After stirring, free matter was removed by centrifugation (15000 rpm, 30 min), and then freeze-drying was performed (conventional freeze-drying process was used) to form Centella asiatica nanoparticles.

[0029] Step S3, preparing a pharmaceutical composition: First, upadacitinib enteric-coated microspheres are mixed with xanthan gum, the mass ratio of xanthan gum to upadacitinib enteric-coated microspheres is 1:2, and phosphate buffer is added, the phosphate buffer adopts sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and the pH of the phosphate buffer is 5.5 (the pH value is regulated by regulating the ratio between sodium dihydrogen phosphate and disodium hydrogen phosphate. In this embodiment, the volume ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate is about 51:1), the volume mass ratio of phosphate buffer to upadacitinib enteric-coated microspheres is 1.5 mL:3 mg, and sodium bicarbonate is added after ultrasonic dispersion to adjust the pH to 5.5 to obtain a microsphere suspension for standby use; Then, add the asiaticoside nanoparticles and carbomer 940 into the buffer, wherein the mass ratio of upadacitinib to asiaticoside in the upadacitinib enteric-coated microspheres and the asiaticoside nanoparticles is 3:40, the mass ratio of carbomer 940 to xanthan gum is 1:4, the buffer is a citric acid solution with a pH of 6.0, the volume mass ratio of the buffer to the asiaticoside nanoparticles is 1.5 mL:20 mg, stir evenly (stirring speed is 450 rpm), then add glutamine, the mass ratio of glutamine to asiaticoside nanoparticles is 0.9:2, and obtain a gel solution for standby use; Finally, the microsphere suspension and the gel solution are slowly mixed under magnetic stirring, and flavoring agents and antibacterial agents are added in sequence. The flavoring agent is sucrose, and the antibacterial agent is sodium benzoate. The mass ratio of the flavoring agent, the antibacterial agent and upadacitinib enteric-coated microspheres is 7.5:0.3:1. The pH is adjusted to 5.5 (sodium bicarbonate or sodium hydroxide solution can be used to adjust the pH value), and freeze-dried (conventional freeze-drying process can be used) to obtain a pharmaceutical composition.

[0030] Embodiment 3: A method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: First, upadacitinib and polylactic acid-co-glycolic acid (PLGA) with a molecular weight of 10 kDa were added to ethyl acetate and ultrasonically dispersed (conventional ultrasonic dispersion process can be used, which is not specifically limited in this embodiment), and the mass volume ratio of upadacitinib, polylactic acid-co-glycolic acid and ethyl acetate was 1 mg: 10 mg: 0.2 mL to form an organic phase; Under stirring conditions, the organic phase is slowly dripped into the aqueous phase of polyvinyl alcohol (PVA) to form colostrum, wherein the aqueous phase concentration of polyvinyl alcohol is 0.4% w / v, and the aqueous phase volume of polyvinyl alcohol is 20 times the volume of the organic phase (for example, if the volume of the organic phase is 1 mL, 20 mL of aqueous phase is used), the stirring rate is 800 rpm, and the stirring time is 45 min (stirring is carried out at room temperature of about 25°C to avoid excessive evaporation of the solvent and agglomeration of emulsified microspheres). The specific method for preparing the aqueous phase of polyvinyl alcohol is as follows: 0.4 g of polyvinyl alcohol is dissolved in 100 mL of ultrapure water, heated to 80°C, stirred to dissolve, and filtered after cooling (i.e., cooled to room temperature), and the filtrate is the aqueous phase of polyvinyl alcohol.

[0031] The colostrum is vacuum dried and washed to obtain microspheres with a particle size of 20 to 30 μm; wherein: the vacuum drying temperature is 36°C, the pressure is 50 mbar, and the drying time is 12 hours; the washing steps include ultrapure water washing (i.e., ultrapure water is added to the solid powder obtained after vacuum drying, and the amount of ultrapure water is determined according to the actual amount of powder, generally completely immersed), centrifugal separation (centrifugal speed is 16000 rpm, and the centrifugal time is 15 minutes) and freeze drying (conventional freeze drying process can be used); The microspheres were fluidized bed coated with an ethanol solution of hydroxypropyl methylcellulose acetate succinate (HPMCAS) (coating can be performed using a common fluidized bed), the concentration of the ethanol solution of hydroxypropyl methylcellulose acetate succinate was 8.5% (w / v), and after fluidized bed coating, the microspheres gained 16% in weight, thereby obtaining upadacitinib enteric-coated microspheres.

[0032] Step S2, preparing asiaticaside nanoparticles: First, asiatica glycoside and β-cyclodextrin were included in warm water, chitosan solution was added, and stirred evenly (stirring rate was 550rpm, stirring time was 28min); wherein, the mass volume ratio of asiatica glycoside, β-cyclodextrin and chitosan solution was 20mg:60mg:3mL; the concentration of chitosan solution was 1% w / v (i.e., 1g chitosan was dissolved in 100mL deionized water); during the inclusion process, the amount of warm water (deionized water) was 12mL, the temperature was 55℃, and ultrasonic treatment was performed for 32min during the inclusion process (to ensure uniform dispersion); Then slowly drop sodium tripolyphosphate solution for cross-linking, the mass ratio of sodium tripolyphosphate to chitosan is 1:15, the concentration of sodium tripolyphosphate solution is 0.4% w / v (i.e., 0.4 g sodium tripolyphosphate is dissolved in 100 mL of deionized water), the cross-linking time is 22 min, and the stirring speed is 1000 rpm during the cross-linking process; After cross-linking, hyaluronic acid solution was added and stirred at 400 rpm for 1.1 h. The concentration of the hyaluronic acid solution was 0.5% w / v (i.e., 0.5 g hyaluronic acid was dissolved in 100 mL of deionized water), and the mass ratio of hyaluronic acid to sodium tripolyphosphate was 10:1.8. After stirring, free matter was removed by centrifugation (16000 rpm, 32 min), and then freeze-drying was performed (conventional freeze-drying process was used) to form Centella asiatica nanoparticles.

[0033] Step S3, preparing a pharmaceutical composition: First, upadacitinib enteric-coated microspheres are mixed with xanthan gum, the mass ratio of xanthan gum to upadacitinib enteric-coated microspheres is 1:2, and phosphate buffer is added, the phosphate buffer adopts potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer and the pH of the phosphate buffer is 5.5 (the pH value is regulated by regulating the ratio between potassium dihydrogen phosphate and dipotassium hydrogen phosphate. In this embodiment, the volume ratio of potassium dihydrogen phosphate and dipotassium hydrogen phosphate is about 51:1), the volume mass ratio of phosphate buffer to upadacitinib enteric-coated microspheres is 2 mL:3 mg, and sodium bicarbonate is added after ultrasonic dispersion to adjust the pH to 5.5 to obtain a microsphere suspension for standby use; Then, add asiaticaside nanoparticles and carbomer 940 to the buffer, wherein the mass ratio of upadacitinib to asiaticaside in upadacitinib enteric-coated microspheres and asiaticaside nanoparticles is 3.5:42, the mass ratio of carbomer 940 to xanthan gum is 1:5, the buffer is a sodium dihydrogen phosphate solution with a pH of 6.0, the volume mass ratio of the buffer to asiaticaside nanoparticles is 2 mL:20 mg, stir evenly (stirring speed is 500 rpm), then add glutamine, the mass ratio of glutamine to asiaticaside nanoparticles is 1:2, and obtain a gel solution for standby use; Finally, the microsphere suspension and the gel solution are slowly mixed under magnetic stirring, and a flavoring agent and an antibacterial agent are added in sequence. The flavoring agent is sucralose, and the antibacterial agent is methylparaben. The mass ratio of the flavoring agent, the antibacterial agent and upadacitinib enteric-coated microspheres is 10:0.5:1. The pH is adjusted to 5.5 (sodium bicarbonate or sodium hydroxide solution can be used to adjust the pH value), and freeze-dried (conventional freeze-drying process can be used) to obtain a pharmaceutical composition.

[0034] Embodiment 4: An oral solvent for treating atopic dermatitis, a pharmaceutical composition finally prepared using any one of Examples 1 to 3, characterized in that the pharmaceutical composition is redissolved with purified water, fixed to volume, and stirred evenly, and finally the prepared solution is subjected to secondary filtration using PES 0.45μm and 0.22μm filter membranes, and the filtrate is encapsulated, and the filtrate is the oral solution.

[0035] Comparative Example 1: A method for preparing a pharmaceutical composition, comprising: Step S1, placing anhydrous citric acid in 80% of the prescribed amount of purified water, stirring for 5 minutes until it is completely dissolved, to obtain a citric acid solution with a pH of 6.0; Step S2, adding upadacitinib and asiaticain in sequence, the mass ratio of upadacitinib to asiaticain being 3:40, and the mass volume ratio of asiaticain to citric acid solution being 20 mg:1.5 mL, and stirring for 30 min until completely dissolved; Step S3, adding sodium citrate, sodium benzoate and sucrose in sequence, the mass ratio of sodium citrate, sodium benzoate, sucrose antibacterial agent to upadacitinib is 0.35:0.3:7.5:1, stirring for 10 minutes until completely dissolved; finally freeze-drying to obtain a pharmaceutical composition.

[0036] Comparative Example 2: A method for preparing a pharmaceutical composition, comprising: Step S1, using conventional upadacitinib powder.

[0037] Step S2, preparing asiaticaside nanoparticles: the same as the preparation steps in Example 2.

[0038] Step S3, preparing a pharmaceutical composition: First, upadacitinib powder and xanthan gum are mixed, the mass ratio of xanthan gum to upadacitinib powder is 1:2, and phosphate buffer is added, the phosphate buffer adopts sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and the pH of the phosphate buffer is 5.5, the volume mass ratio of phosphate buffer to upadacitinib powder is 1.5mL:3mg, and after ultrasonic dispersion, sodium bicarbonate is added to adjust the pH to 5.5 to obtain a suspension for standby use; Then, add asiaticaside nanoparticles and carbomer 940 to a buffer, wherein the mass ratio of upadacitinib to asiaticaside in asiaticaside nanoparticles is 3:40, the mass ratio of carbomer 940 to xanthan gum is 1:4, the buffer is a citric acid solution with a pH of 6.0, the volume mass ratio of the buffer to asiaticaside nanoparticles is 1.5 mL:20 mg, stir evenly (stirring speed is 450 rpm), then add glutamine, the mass ratio of glutamine to asiaticaside nanoparticles is 0.9:2, and obtain a gel solution for standby use; Finally, the suspension and the gel solution are slowly mixed under magnetic stirring, and a flavoring agent and an antibacterial agent are added in sequence. The flavoring agent is sucrose, and the antibacterial agent is sodium benzoate. The mass ratio of the flavoring agent, the antibacterial agent and upadacitinib powder is 7.5:0.3:1. The pH is adjusted to 5.5 (sodium bicarbonate or sodium hydroxide solution can be used to adjust the pH value), and freeze-dried (conventional freeze-drying process can be used) to obtain a pharmaceutical composition.

[0039] Comparative Example 3: A method for preparing a pharmaceutical composition, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: the same as the preparation steps in Example 2.

[0040] Step S2: using conventional Centella asiatica powder.

[0041] Step S3, preparing a pharmaceutical composition: First, upadacitinib enteric-coated microspheres are mixed with xanthan gum, the mass ratio of xanthan gum to upadacitinib enteric-coated microspheres is 1:2, and phosphate buffer is added, the phosphate buffer adopts sodium dihydrogen phosphate-disodium hydrogen phosphate buffer and the pH of the phosphate buffer is 5.5 (the pH value is regulated by regulating the ratio between sodium dihydrogen phosphate and disodium hydrogen phosphate. In this embodiment, the volume ratio of sodium dihydrogen phosphate and disodium hydrogen phosphate is about 51:1), the volume mass ratio of phosphate buffer to upadacitinib enteric-coated microspheres is 1.5 mL:3 mg, and sodium bicarbonate is added after ultrasonic dispersion to adjust the pH to 5.5 to obtain a microsphere suspension for standby use; Then, sodium asiaticaside and carbomer 940 are added to the buffer, wherein the mass ratio of upadacitinib to asiaticaside in the upadacitinib enteric-coated microspheres is 3:40, the mass ratio of carbomer 940 to xanthan gum is 1:4, the buffer is a citric acid solution with a pH of 6.0, the volume mass ratio of the buffer to asiaticaside powder is 1.5 mL:20 mg, and the mixture is stirred evenly (stirring speed is 450 rpm), and then glutamine is added, and the mass ratio of glutamine to asiaticaside powder is 0.9:2, to obtain a gel solution for standby use; Finally, the microsphere suspension and the gel solution are slowly mixed under magnetic stirring, and flavoring agents and antibacterial agents are added in sequence. The flavoring agent is sucrose, and the antibacterial agent is sodium benzoate. The mass ratio of the flavoring agent, the antibacterial agent and upadacitinib enteric-coated microspheres is 7.5:0.3:1. The pH is adjusted to 5.5 (sodium bicarbonate or sodium hydroxide solution can be used to adjust the pH value), and freeze-dried (conventional freeze-drying process can be used) to obtain a pharmaceutical composition.

[0042] Comparative Example 4: A method for preparing a pharmaceutical composition, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: the same as the preparation steps in Example 2.

[0043] Step S2, preparing asiaticaside nanoparticles: the same as the preparation steps in Example 2.

[0044] Step S3, preparing a pharmaceutical composition: the preparation steps are basically the same as those in Example 2, except that sodium carboxymethyl cellulose (CMC-Na) is used instead of xanthan gum.

[0045] Comparative Example 5: A method for preparing a pharmaceutical composition, comprising: Step S1, preparing upadacitinib enteric-coated microspheres: the same as the preparation steps in Example 2.

[0046] Step S2, preparing asiaticaside nanoparticles: the same as the preparation steps in Example 2.

[0047] Step S3, preparing a pharmaceutical composition: the preparation steps are basically the same as those in Example 2, except that Carbomer 934P is used instead of Carbomer 940.

[0048] The pharmaceutical compositions prepared in Examples 1 to 3 and Comparative Examples 1 to 5 were respectively used to prepare oral solvents by the method described in Example 4, and the long-term stability of the products was investigated according to the stability test of Part II of the 2015 edition of the Chinese Pharmacopoeia. The test conditions were: temperature 25±2°C, humidity: RH60%±10%, and the inspection time was 0, 3, 6, 9, 12, 18, and 24 months. The test results are as follows:

[0049] 160 SD rats were selected and divided into 8 groups, each with 20 rats, and the 8 groups were gavaged with the oral solvent prepared by the pharmaceutical composition in Examples 1 to 3 and Comparative Examples 1 to 5, respectively, and a single dose was administered. The incidence of diarrhea in the rats was observed 6 hours after administration, and the intestinal IL-8 level of the rats was tested (at the same time, a blank group was set, that is, 20 SD rats were randomly selected, no medication was given, and the incidence of diarrhea and intestinal IL-8 level were tested). The results are as follows:

[0050] It is obvious from the above table that the pharmaceutical composition prepared by the scheme of the present invention, i.e., the embodiment, can significantly reduce the side effects of upadacitinib and asiaticoside on the gastrointestinal tract, and the incidence of diarrhea after administration is low, and the intestinal IL-8 level is not significantly different from that of the blank control group (i.e., the control group not administered with the drug); while directly mixing upadacitinib with asiaticoside, the incidence of diarrhea is high and the intestinal IL-8 level is significantly increased, which proves that the direct alternating use of upadacitinib and asiaticoside will cause frequent and severe gastrointestinal symptoms.

Claims

1. A method for preparing a pharmaceutical composition for treating atopic dermatitis, characterized in that: include: Step S1, preparing upadacitinib enteric-coated microspheres: first, adding upadacitinib and polylactic acid-co-glycolic acid copolymer into ethyl acetate, and ultrasonically dispersing them to form an organic phase; under stirring, slowly dripping the organic phase into an aqueous phase of polyvinyl alcohol to form colostrum; vacuum drying and washing the colostrum to obtain microspheres; and fluidizing and coating the microspheres with an ethanol solution of hydroxypropyl methylcellulose acetate succinate to obtain microspheres; Step S2, preparing asiaticaoside nanoparticles: firstly, including asiaticaoside and β-cyclodextrin in warm water, adding chitosan solution, and stirring evenly; then slowly dropping sodium tripolyphosphate solution for cross-linking, and adding hyaluronic acid solution after cross-linking to form asiaticaoside nanoparticles; Step S3, preparing a pharmaceutical composition: firstly, mixing upadacitinib enteric-coated microspheres with xanthan gum, and adding phosphate buffer, and after ultrasonic dispersion, adding sodium bicarbonate to adjust the pH to 5.5 to obtain a microsphere suspension for standby use; then, adding asiatica glycoside nanoparticles and carbomer 940 to the buffer, stirring evenly, and then adding glutamine to obtain a gel solution for standby use; finally, slowly mixing the microsphere suspension and the gel solution under magnetic stirring, and sequentially adding a flavoring agent and an antibacterial agent, adjusting the pH to 5.5, and freeze-drying to obtain a pharmaceutical composition.

2. A method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 1, characterized in that: The molecular weight of the polylactic acid-glycolic acid copolymer is 10 kDa; the mass volume ratio of upadacitinib, polylactic acid-glycolic acid copolymer and ethyl acetate is 1 mg: 9-10 mg: 0.1-0.2 mL; the aqueous phase concentration of polyvinyl alcohol is 0.2-0.4% w / v, and the aqueous phase volume of polyvinyl alcohol is 10-20 times the volume of the organic phase.

3. A method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 1 or 2, characterized in that: In step S1, the stirring rate of the colostrum formation process is 600-800 rpm, and the stirring time is 45-60 min; the vacuum drying temperature is 32-36° C., the pressure is 45-50 mbar, and the drying time is 12-16 h; and the washing step includes ultrapure water washing, centrifugal separation, and freeze drying.

4. A method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 1 or 3, characterized in that: The particle size of the microspheres obtained in step S1 is 20 to 30 μm.

5. A method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 1 or 3, characterized in that: In step S2, the mass volume ratio of asiaticoside, β-cyclodextrin and chitosan solution is 20 mg: 40-60 mg: 2-3 mL; the concentration of chitosan solution is 1% w / v; during the inclusion process, the amount of warm water used is 8-12 mL, the temperature is 45-55° C., and the inclusion process is ultrasonically treated for 28-32 min.

6. The method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 5, characterized in that: The mass ratio of sodium tripolyphosphate to chitosan is 1:10-15, the concentration of the sodium tripolyphosphate solution is 0.2-0.4% w / v; the concentration of the hyaluronic acid solution is 0.5% w / v, and the mass ratio of hyaluronic acid to sodium tripolyphosphate is 5-10:1-2.

7. The method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 5, characterized in that: In the step S2, the stirring time of adding the chitosan solution is 28 to 32 minutes, and the stirring speed is 450 to 550 rpm; the cross-linking time is 18 to 22 minutes, and the stirring rate is 800 to 1000 rpm during the cross-linking process; after adding the hyaluronic acid solution, the stirring speed is 200 to 400 rpm and the stirring is 0.9 to 1.1 hours.

8. The method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 1, characterized in that: In the step S3, the mass ratio of upadacitinib to asiaticoside in the upadacitin enteric-coated microspheres and asiaticoside nanoparticles is 2.5-3.5:38-42; the mass ratio of xanthan gum to upadacitinib enteric-coated microspheres is 1:2; and the mass ratio of carbomer 940 to xanthan gum is 1:3-5.

9. The method for preparing a pharmaceutical composition for treating atopic dermatitis according to claim 1, characterized in that: The flavoring agent is one or more of sucrose, sucralose, and acesulfame potassium, and the antibacterial agent is one or more of sorbic acid, methylparaben, benzalkonium chloride, and sodium benzoate. The mass ratio of the flavoring agent, the antibacterial agent, and upadacitinib enteric-coated microspheres is 5-10:0.1-0.5:1.

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