WO2024119375A1

WO2024119375A1 - Jak inhibitor long-acting microspheres, preparation method therefor, and use thereof

Info

Publication number: WO2024119375A1
Application number: PCT/CN2022/136978
Authority: WO
Inventors: 曹青日; 吴欣虹
Original assignee: 苏州大学
Priority date: 2022-12-06
Filing date: 2022-12-06
Publication date: 2024-06-13

Abstract

JAK inhibitor long-acting sustained-release microspheres, a preparation method therefor, and use thereof. A JAK inhibitor and a biocompatible polymer (containing a surfactant) are separately dissolved in an organic solvent, and then mixed to serve as a dispersed phase, an aqueous solution of polyvinyl alcohol serves as a continuous phase, and the dispersed phase is injected into the continuous phase under stirring to form an oil-in-water emulsion; then the organic solvent is stirred to be volatilized, the oil-in-water emulsion is then injected into the aqueous solution of polyvinyl alcohol for curing, finally washing is carried out using distilled water, and microspheres are collected and freeze-dried to obtain the JAK inhibitor long-acting microspheres. The microspheres stably encapsulating high-content JAK inhibitors can be effectively prepared, achieving industrialization; the prepared JAK inhibitor microspheres can ensure the stable and effective drug concentration in vivo, and prolong the action time of drugs, thereby meeting the requirements of clinical application.

Description

A JAK inhibitor long-acting microsphere and its preparation method and application

Technical Field

The present invention relates to the technical field of pharmaceutical preparations, and in particular to a method for preparing long-acting microspheres of a JAK inhibitor.

Background technique

JAK is a non-receptor tyrosine protein kinase that mediates the signals produced by cytokines and transmits them through the JAK-STAT signaling pathway. There are four subtypes of JAK, namely JAK1, JAK2, JAK3, and TYK2, and there are overlapping binding targets between the subtypes. Currently, JAK1 has become a target for diseases such as inflammation, cancer, and immunity; JAK2 is a target for blood system-related diseases; and JAK3 is mainly a hot target for autoimmune diseases. At present, five JAK inhibitors have been approved for marketing in China, namely Novartis's ruxolitinib, Eli Lilly's baricitinib, Pfizer's tofacitinib, AbbVie's upadacitinib, and Pfizer's abrocitinib. The indications mainly cover rheumatoid arthritis, myelofibrosis, atopic dermatitis, etc.

Tofacitinib, ruxolitinib, baricitinib, upadacitinib and abrocitinib are JAK inhibitors for the treatment of rheumatoid diseases. They can effectively inhibit the activity of JAK1 and JKA3, thereby blocking the signal transduction of multiple inflammatory cytokines and effectively inhibiting the occurrence of inflammation. Compared with traditional anti-rheumatic drugs, JAK inhibitors can not only alleviate symptoms, but can even stop the damage of the disease to the body. They can be used for the treatment of organ transplantation, rheumatoid arthritis, ulcerative colitis, psoriasis and other inflammatory-related diseases.

The dosage form of existing JAK inhibitors is mainly tablets. For example, tofacitinib citrate tablets have been approved in the United States. Each tablet contains 8 mg tofacitinib citrate, twice a day, for the treatment of rheumatoid arthritis, etc. Because of its long course of treatment, patients have poor compliance with medication, and considering that it has certain toxicity to the liver, it is of great significance to develop long-acting microspheres of JAK inhibitors. At present, the dosage form of JAK inhibitors listed in China is mainly tablets, and the development of sustained-release preparations is relatively small. Among them, ethyl cellulose is used as a sustained-release coating material in CN113384544A, and a variety of excipients such as penetration enhancers, suspending agents, adhesives and lubricants are added to make tofacitinib sustained-release tablets. The sustained-release effect can only reach 8 hours. Because a variety of excipients are added during its preparation process, the content of tofacitinib is 6% to 12%, and the drug content is low. Therefore, the development of a new type of sustained-release preparation with a longer sustained-release time and high drug content-long-acting microspheres of JAK inhibitors has a wider range of application scenarios. Moreover, long-acting microsphere preparations of JAK inhibitors have not been developed in the market. At the same time, microspheres, as a new type of drug carrier, have great development advantages. Therefore, in order to further prepare drugs that can more effectively inhibit non-receptor tyrosine protein kinases, it is necessary to develop a longer-acting sustained-release preparation.

technical problem

The purpose of the present invention is to provide a method for preparing long-acting sustained-release microspheres of JAK inhibitors, which is simple, efficient, has a high encapsulation rate, and can effectively solve the problems of instability of free drugs and short sustained-release time of traditional sustained-release preparations.

Technical Solutions

To achieve the above object, the present invention provides the following technical solutions.

A method for preparing long-acting microspheres of a JAK inhibitor comprises the following steps: mixing a JAK inhibitor solution with a solution containing a surfactant and a biocompatible polymer to obtain an oil phase; then dropping the oil phase into a first aqueous phase, stirring and pouring the mixture into a second aqueous phase, and then stirring, washing, and drying to obtain the long-acting microspheres of the JAK inhibitor.

In the present invention, in the oil phase, the organic solvent is one or more of benzyl alcohol, dichloromethane, ethyl acetate, dimethyl sulfoxide, tetrahydrofuran or methanol, preferably, the organic solvent is benzyl alcohol, dichloromethane and ethyl acetate; the biocompatible polymer is one or more of polylactic acid-co-glycolic acid (PLGA), polylactic acid (PLA), polylactic acid-polyethylene glycol (PLA-PEG) and polycaprolactone (PCL); preferably, the biocompatible polymer is polylactic acid-co-glycolic acid, and its relative molecular mass is 12000-150000 g/mol, wherein the molar ratio of lactide to glycolide is 85:15-50:50. Preferably, the molar ratio of PLGA lactide to glycolide is 75:25-50:50, more preferably 75:25. The surfactant is one or more of poloxamer, polyethylene glycol, polyoxyethylene sorbitan monooleate, castor oil polyoxyethylene ether or sodium dodecylbenzene sulfonate.

In the present invention, the weight ratio of the JAK inhibitor, the biocompatible polymer and the surfactant is 1: (1-50): (0.05-5), preferably 1: (2-5): (0.1-1); the concentration of the JAK inhibitor solution is 1%-20% (w/v), preferably 1%-10%, and the solvent is one or more of ethyl acetate, dichloromethane or benzyl alcohol; in the solution containing the surfactant and the biocompatible polymer, the concentration of the biocompatible polymer is 1%-50% (w/v), preferably 1%-25% (w/v), and the solvent is one or more of ethyl acetate, dichloromethane or benzyl alcohol.

In the present invention, the volume ratio of the oil phase to the first aqueous phase is 1:1-50, preferably 1:2-15, and more preferably 1:3-10; the volume ratio of the second aqueous phase to the first aqueous phase is 2-20:1, preferably 2-15:1, and more preferably 3-10:1; preferably, the aqueous phase is a polyvinyl alcohol aqueous solution.

The present invention drips the oil phase into the first aqueous phase, stirs conventionally for 0.5 to 2 hours, then pours into the second aqueous phase, continues stirring for 0.5 to 1 hour, then washes and freeze-dries for 20 to 100 hours to obtain JAK inhibitor long-acting microspheres. Preferably, the present invention drips the oil phase into the first aqueous phase, stirs for 0.5 to 1 hour, then pours into the second aqueous phase, then stirs for 0.5 to 1 hour, then washes and freeze-dries for 30 to 60 hours to obtain JAK inhibitor long-acting microspheres.

The invention discloses the application of the JAK inhibitor long-acting microspheres in the preparation of sustained-release drugs.

The present invention forms an O/W emulsion by dissolving a JAK inhibitor and a biocompatible polymer in an organic solvent as a dispersant and a polyvinyl alcohol aqueous solution as a continuous phase, and then injects the organic solvent into the polyvinyl alcohol aqueous solution for a certain period of time to obtain a JAK inhibitor long-acting microsphere. The JAK inhibitor and the biocompatible polymer are co-dissolved in an organic solvent, which can reduce drug loss and increase drug loading and encapsulation efficiency; the microspheres can be controlled to have a suitable particle size by adopting a reasonable process; in addition, further treatment with a polyvinyl alcohol aqueous solution can make the microspheres harder, avoid deformation during the collection process of the microspheres, and the microspheres have a good morphology.

Beneficial Effects

The present invention has the following advantages.

(1) The present invention uses JAK inhibitors, biocompatible polymers, and surfactants as raw materials to prepare drug-containing polymer solutions, adjusts the release rate of JAK inhibitor long-acting microspheres, and achieves long-acting release of JAK inhibitor microspheres.

(2) The present invention selects the raw material ratio and preparation process, and the prepared JAK inhibitor long-acting microspheres have a high encapsulation rate, a suitable particle size, a round shape, a 4h release rate of 8%, no burst release phenomenon, and a subsequent long-term release, which can be slowly released for two weeks to one month, effectively improving patient compliance.

(3) The preparation method of the present invention is simple, efficient and suitable for industrial production.

The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a chemical structural formula of a conventional JAK inhibitor.

FIG2 is a scanning electron micrograph of the JAK inhibitor long-acting microspheres prepared in Example 2 (400×).

FIG3 is an in vitro release curve of the JAK inhibitor long-acting microspheres of Example 2 and Comparative Examples 1-3 at 37° C. (0.02% TW20-PBS solution).

FIG4 shows the release curves of the JAK inhibitor long-acting microspheres of Example 2 and Comparative Examples 1-3 at 37° C. (0.02% TW20-PBS solution) in the first three days.

Embodiments of the present invention

Microspheres are skeleton-type solid spheres formed by dissolving or dispersing drugs in biocompatible polymers, and their particle size ranges from 1 to 250 μm. After the drugs are made into microspheres, they have the following characteristics: masking the bad smell of the drugs, improving the stability of the drugs, reducing irritation to the stomach or reducing the inactivation of the drugs in the stomach, solidifying the liquid drugs for easy storage or re-formation into other dosage forms, controlling the drug release rate, etc. The method for preparing JAK inhibitor long-acting microspheres of the present invention is as follows.

(1) A JAK inhibitor is mixed with a biocompatible polymer (including a surfactant) and an organic solvent as a dispersed phase, and a polyvinyl alcohol aqueous solution is used as a continuous phase. The dispersed phase is added dropwise into the continuous phase under conventional magnetic stirring to form an oil-in-water emulsion.

(2) Stirring to evaporate part of the organic solvent in the emulsion, and injecting the resulting dispersed system into the polyvinyl alcohol aqueous solution.

(3) Rinse with distilled water and collect the microspheres, and freeze-dry (freeze dryer model: FDU-2110) to obtain JAK inhibitor long-acting microspheres.

JAK inhibitors are tofacitinib, ruxolitinib, baricitinib, upadacitinib, and abrocitinib, and the chemical structure is shown in Figure 1. The specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and examples. The raw materials used in the present invention are existing products, and the specific preparation operations and tests are conventional techniques, such as stirring is a conventional technique, stirring the volatile organic solvent, and the oil phase is continuously dripped into the polyvinyl alcohol aqueous solution, and filtered using a 0.22 μm filter membrane. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. In the present invention, in the solution concentration, w/v represents the mass of the solute contained in every 100 ml of solution (expressed in g), for example, 40 mg tofacitinib is dissolved in 1.5 ml of benzyl alcohol, and the concentration of the solution is 2.7%.

Example 1.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 86 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 2.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 20 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 3.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 4.

Weigh 40 mg of baricitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 5.

Weigh 40 mg of ruxolitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse them, and use them as the oil phase. Then, drop the oil phase into 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 6.

40 mg of upadacitinib was weighed and dissolved in 1.5 ml of benzyl alcohol to obtain a drug solution; 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of poloxamer 407 were dissolved in 2.5 ml of ethyl acetate to obtain a polymer solution; the drug solution and the polymer solution were mixed and vortexed to disperse as the oil phase. The oil phase was then added dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stirred for 40 min, and then the dispersed system was poured into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stirred for 2 h, filtered, washed with water 3 times, pre-frozen in a -80℃ refrigerator for 8 h, and freeze-dried for 48 h to obtain microsphere powder.

Example 7.

Weigh 40 mg of abrocitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then, drop the oil phase into 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 8.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 20 mg of poloxamer 188 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 25 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 0.5 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 9.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 15 mg of poloxamer 338 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 30 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 45 minutes, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Example 10.

Weigh 50 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of ester-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 30 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, and then pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 1 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Comparative example 1.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 12 kDa) in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, pre-freeze in a -80℃ refrigerator for 8 h, and freeze-dry for 48 h to obtain microsphere powder.

Comparative Example 2.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Comparative example 3.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%, containing 1mg/ml sodium chloride), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Comparative Example 4.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%, add sodium hydroxide to adjust the pH to 10), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Comparative Example 5.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLA (carboxyl-terminated, Mw=90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution, vortex disperse them, and use them as the oil phase. Then, drop the oil phase into 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, pre-freeze in a -80℃ refrigerator for 8 h, and freeze-dry for 48 h to obtain microsphere powder.

Comparative Example 6.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PCL (carboxyl-terminated, Mw=90 kDa) and 10 mg of poloxamer 407 in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution, vortex disperse them, and use them as the oil phase. Then, drop the oil phase into 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Comparative Example 7.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; dissolve 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of oxyethylene (80) sorbitan monooleate in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse, and use it as the oil phase. Then, drop the oil phase into 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Comparative Example 8.

Weigh 40 mg of tofacitinib and dissolve it in 1.5 ml of benzyl alcohol to obtain a drug solution; take 200 mg of PLGA (molar ratio of carboxyl-terminated lactide to glycolide = 75:25, Mw = 90 kDa) and 10 mg of castor oil polyoxyethylene 35 ether and dissolve them in 2.5 ml of ethyl acetate to obtain a polymer solution; mix the drug solution and the polymer solution with each other, vortex disperse them, and use them as the oil phase. Then, add the oil phase dropwise to 20 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 40 min, pour the dispersed system into 100 ml of polyvinyl alcohol aqueous solution (1wt%), stir for 2 h, filter, wash with water 3 times, put it in a -80℃ refrigerator for pre-freezing for 8 h, and freeze-dry it for 48 h to obtain microsphere powder.

Test example.

Microsphere morphology. The microspheres prepared in Example 2 were taken and the microsphere morphology was measured by scanning electron microscopy. The results are shown in Figure 2. It can be seen that the tofacitinib long-acting microspheres have an appropriate particle size, are round, and have a good morphology.

Microsphere particle size: Take the microspheres prepared above, suspend them in water, and measure the particle size of the microspheres with a laser particle size distribution analyzer. The results are shown in Table 1. The obtained microsphere particle size (specifically the median diameter) is less than 100 μm, which is suitable for injection. Microsphere drug loading and encapsulation efficiency: Weigh 10 mg of the microspheres prepared above, add acetonitrile to dissolve the microspheres, add pH=4.2, 0.02% PBS-TW20 to 50 ml, take the supernatant, centrifuge at 12500rpm, take 700μl for HPLC analysis, and measure the total drug concentration. Weigh 10 mg of microspheres, add pH=4.2, 0.02% PBS-TW20 to 50 ml, take the supernatant, perform HPLC analysis, and measure the free drug concentration. The total drug amount minus the free drug amount is the encapsulated drug amount. The results are shown in Table 1. The calculation formulas for drug loading and encapsulation efficiency are as follows.

.

The present invention prepares microspheres by adding an appropriate amount of surfactant to the oil phase, and the encapsulation rate of the prepared tofacitinib microspheres is higher than that of the method of adding sodium hydroxide or sodium chloride to the water phase to form pores, which is greater than 85%. It shows that the surfactant can not only effectively regulate the release of microspheres by forming pores, but also ensure a high encapsulation rate of microspheres. In addition, compared with PLA and PCL, the encapsulation rate of the microspheres prepared by PLGA is higher, which is greater than 85%. It shows that the encapsulation rate of the microspheres prepared by using PLGA macromolecular polymer is higher. In summary, the microspheres prepared by the present invention have a high encapsulation rate, and can also effectively regulate the release of tofacitinib microspheres, so that tofacitinib can be slowly released within one month.

In vitro release profiles of microspheres.

6.8 g of potassium dihydrogen phosphate and 0.79 g of sodium hydroxide were dissolved in 1000 mL of water, and 0.5% (w/v) Tween 20 was added to obtain a release medium PBS-Tween 20 solution.

6.8 g of potassium dihydrogen phosphate and 0.79 g of sodium hydroxide were dissolved in 1000 mL of water to obtain a release medium PBS solution.

Weigh 20 mg of tofacitinib sustained-release microspheres prepared in Example 2 and Comparative Example 1-2 respectively in a 50 mL stoppered conical flask, add 50 ml of 0.02% TW20-PBS solution, put into a constant temperature 37 ° C shaker (50 rpm), sample 1 ml at different time points such as 4 h, 1 d, 3 d, 7 d, and 14 d, centrifuge, take 0.7 ml of supernatant for HPLC analysis, resuspend the remaining microspheres with fresh medium and continue to sample, calculate the cumulative release percentage, and draw a cumulative release percentage-time release curve. The results are shown in Figures 3 and 4. It can be seen that tofacitinib is slowly released from the microspheres, the release curve is gentle, and the release can be sustained for one month. Among them, Example 2 adjusts the release rate by adding a surfactant to the high molecular weight PLGA (90 kDa), and the drug can be slowly released within one month. If an appropriate amount of surfactant is not added to the oil phase, the tofacitinib microspheres prepared by using small molecular weight PLGA (12 kDa) to form pores release 24.78% on the first day (large burst release), while the tofacitinib microspheres prepared by using large molecular weight PLGA (90 kDa) basically do not release in the first two weeks. It can also be seen that the preparation of microspheres by adding an appropriate amount of surfactant to the oil phase can not only improve the encapsulation rate but also significantly improve the release of tofacitinib long-acting microspheres compared to the method of adding sodium chloride to the solid phase to form pores in the microspheres. That is, the phenomenon of large burst release or basically no release of tofacitinib long-acting microspheres in the early stage is significantly improved, meeting the clinical drug needs.

The present invention adopts a method of combining PLGA macromolecular polymer with adding surfactant in the oil phase, and the prepared microspheres have a high encapsulation rate and the drug can be slowly released within one month, and can also ensure that the prepared tofacitinib microspheres have a high encapsulation rate.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and these improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

A method for preparing long-acting microspheres of a JAK inhibitor, characterized in that it comprises the following steps: mixing a JAK inhibitor solution with a solution containing a surfactant and a biocompatible polymer to obtain an oil phase; then dropping the oil phase into a first aqueous phase, stirring and pouring the second aqueous phase into the first aqueous phase, and then stirring, washing, and drying to obtain the long-acting microspheres of the JAK inhibitor.
The method for preparing long-acting JAK inhibitor microspheres according to claim 1, characterized in that, in the oil phase, the organic solvent is one or more of benzyl alcohol, dichloromethane, ethyl acetate, dimethyl sulfoxide, tetrahydrofuran or methanol; the biocompatible polymer is one or more of polylactic acid-co-glycolic acid, polylactic acid, polylactic acid-polyethylene glycol, and polycaprolactone; the surfactant is one or more of poloxamer, polyethylene glycol, oxyethylene sorbitan monooleate, castor oil polyoxyethylene ether or sodium dodecylbenzene sulfonate.
The method for preparing the JAK inhibitor long-acting microspheres according to claim 1, characterized in that the weight ratio of the JAK inhibitor, the biocompatible polymer, and the surfactant is 1: (1-50): (0.05-5).
The method for preparing the JAK inhibitor long-acting microspheres according to claim 1 is characterized in that the concentration of the JAK inhibitor solution is 1% to 20%; in the solution containing a surfactant and a biocompatible polymer, the concentration of the biocompatible polymer is 1% to 50%.
The method for preparing long-acting JAK inhibitor microspheres according to claim 1, characterized in that the volume ratio of the oil phase to the first aqueous phase is 1:1 to 50; the volume ratio of the second aqueous phase to the first aqueous phase is 2 to 20:1.
The method for preparing long-acting JAK inhibitor microspheres according to claim 1, characterized in that the aqueous phase is a polyvinyl alcohol aqueous solution.
The method for preparing long-acting JAK inhibitor microspheres as claimed in claim 1 is characterized in that the oil phase is added dropwise to the first aqueous phase, stirred for 0.5 to 2 hours, then poured into the second aqueous phase, then stirred for 0.5 to 1 hour, then washed and dried to obtain long-acting JAK inhibitor microspheres.
The method for preparing the long-acting JAK inhibitor microspheres according to claim 1, characterized in that the drying is freeze-drying.
JAK inhibitor long-acting microspheres prepared by the method for preparing JAK inhibitor long-acting microspheres as claimed in claim 1.
Use of the JAK inhibitor long-acting microspheres described in claim 9 in the preparation of sustained-release drugs.