A rifabutin microsphere thermosensitive gel and its preparation method

Abstract

This invention relates to the field of pharmaceutical technology and proposes a rifapentine microsphere thermosensitive gel and its preparation method. The rifapentine microsphere thermosensitive gel includes rifapentine microspheres and a thermosensitive gel matrix. The rifapentine microspheres comprise a continuous aqueous phase and an oil phase dispersed in the aqueous phase. The oil phase includes rifapentine, an emulsifier, and vegetable oil. The aqueous phase includes glycerol and water. The emulsifier consists of poloxamer and lecithin in a mass ratio of 5:4~6. The rifapentine microsphere thermosensitive gel provided by this invention has good encapsulation efficiency and drug loading, solving the problems of insufficient encapsulation efficiency and drug loading in existing rifapentine microsphere thermosensitive gels.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a rifapentine microsphere thermosensitive gel and its preparation method. Background Technology

[0002] Rifapentine, a semi-synthetic broad-spectrum bactericidal agent of the rifamycin class, is a key drug in current clinical anti-tuberculosis treatment. Its anti-tuberculosis activity is approximately 3-4 times that of rifampin, with advantages such as a long half-life, mild adverse reactions, and the need for only 1-2 doses per week. It can be integrated with short-course chemotherapy regimens and plays an irreplaceable role in the initial treatment and retreatment of tuberculosis, as well as in the treatment of non-tuberculous mycobacterial infections. However, for acute and critically ill tuberculosis patients with rapidly progressing disease and concentrated infection foci, there is an urgent need for a novel formulation that can act quickly, target and enrich the affected area, and reduce systemic toxicity, in order to achieve rapid infection control and improve patient prognosis.

[0003] Thermosensitive gels are a type of temperature-responsive smart drug delivery carrier. At low temperatures, they are fluid solutions, facilitating drug administration. Once inside the body, they rapidly undergo a sol-gel transition triggered by body temperature, achieving in-situ retention and sustained release of the drug. Lipospheres, as a novel drug delivery system, can effectively encapsulate hydrophobic drugs, improving solubility, and simultaneously enabling passive targeted enrichment at inflamed sites. Rifapentine possesses strong lipid solubility and extremely low water solubility, making it an ideal candidate for liposphere drug delivery systems. Combining thermosensitive gels with lipospheres to construct a drug delivery system can fully leverage the synergistic drug delivery advantages of both, compensating for the shortcomings of single drug delivery systems.

[0004] Currently, rifapentine formulations used clinically are mainly oral capsules and dispersible tablets. These formulations suffer from drawbacks such as low bioavailability, poor photothermal stability, inability to target tuberculosis infection sites, and the risk of liver and kidney damage with long-term administration. For critically ill patients, oral formulations have slow onset of action and insufficient drug concentration at the lesion site, making it difficult to quickly control the condition. Existing rifapentine microsphere thermosensitive gels also have significant technical limitations: particle size is approximately greater than 400 nm, encapsulation efficiency is approximately 55%, drug freeness is high, and targeting is poor. Large particles tend to deposit in the upper respiratory tract, preventing deep delivery to target organs, and the low encapsulation efficiency results in insufficient effective drug utilization, failing to meet treatment needs. Therefore, developing a method for preparing rifapentine microsphere thermosensitive gels with suitable particle size, high encapsulation efficiency, and stable drug loading has become an urgent direction in current formulation research. Summary of the Invention

[0005] To address the above technical problems, this invention provides a rifapentine microsphere thermosensitive gel and its preparation method. The rifapentine microsphere thermosensitive gel provided by this invention incorporates poloxamer and lecithin as emulsifiers, improving the encapsulation efficiency and drug loading capacity of the rifapentine microsphere thermosensitive gel, thus solving the problem of insufficient encapsulation efficiency and drug loading capacity in existing technologies.

[0006] The specific technical solution of the present invention is as follows: According to one aspect of the present invention, a rifapentine microsphere thermosensitive gel is provided, comprising rifapentine microspheres and a thermosensitive gel matrix; The rifabutin microspheres comprise a continuous aqueous phase and an oil phase dispersed in the aqueous phase; The oil phase includes rifapentine, emulsifiers, and vegetable oils; The aqueous phase comprises glycerol and water; The emulsifier is composed of poloxamer and lecithin in a mass ratio of 5:4 to 6.

[0007] In the above technical solution, the volume ratio of the rifabutin microspheres to the thermosensitive gel matrix is ​​4:1~3.

[0008] In the above technical solution, the rifapentine content in the rifapentine microspheres is 3.8 mg / mL to 4.2 mg / mL.

[0009] In the above technical solution, the thermosensitive polymer in the thermosensitive gel matrix is ​​a PLGA-PEG-PLGA triblock copolymer.

[0010] In the above technical solution, the mass ratio of the emulsifier to the volume of the rifapentine microspheres is 1~7g:100mL.

[0011] In the above technical solution, the vegetable oil includes at least one of soybean oil, corn oil, rapeseed oil, and olive oil.

[0012] In the above technical solution, the oil phase further includes a co-emulsifier, which includes at least one selected from oleic acid, stearic acid, or lauric acid; and / or The oil phase also includes an antioxidant, which includes at least one of vitamin E, ascorbyl palmitate, or propylparaben.

[0013] In the above technical solution, the preparation method of the rifabutin microspheres includes the following steps: S1. Mix rifapentine with the remaining raw materials in the oil phase and add it to anhydrous ethanol. Mix until completely dissolved, then evaporate the anhydrous ethanol to obtain the oil phase. S2. Add water to glycerol and mix well to obtain the aqueous phase; S3. Add the aqueous phase dropwise to the oil phase, emulsify and homogenize to obtain homogenized rifabutin microspheres.

[0014] In the above technical solution, in step S3, during emulsification, the rotation speed is 6500~7500 r / min and the time is 7~10 min; the homogenization pressure is 650~750 bar; and the homogenization is performed 3~4 times.

[0015] According to another aspect of the present invention, the present invention also provides a method for preparing the above-mentioned rifapentine microsphere thermosensitive gel, comprising the following steps: Rifapentine microspheres were mixed with a thermosensitive gel matrix to obtain the rifapentine microsphere thermosensitive gel.

[0016] Compared with existing technologies, the rifapentine microsphere thermosensitive gel of the present invention has higher encapsulation efficiency and drug loading. The present invention uses poloxamer and lecithin in a mass ratio of 5:4~6 as emulsifiers, which improves the encapsulation efficiency and drug loading of the rifapentine microsphere thermosensitive gel. Poloxamer effectively reduces the interfacial tension between the oil and aqueous phases, and its own thermosensitive properties synergistically enhance the thermosensitive response performance of the formulation. Lecithin forms a lipid film structure on the surface of the microspheres. Effectively encapsulating rifapentine molecules reduces leakage and improves its lipophilicity, promoting uniform dispersion within the microspheres. When combined in a 5:4 to 6 ratio, the two components produce a synergistic effect. This not only reduces the interfacial tension between the oil and aqueous phases, further enhancing emulsification and forming a microsphere system with uniform particle size and good dispersibility, thus increasing the encapsulation rate and drug loading of rifapentine within the microspheres, but also synergistically improves the solubility and stability of rifapentine, preventing degradation and precipitation during storage and use. Attached Figure Description

[0017] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0018] Figure 1 The in vitro cumulative release curves of rifapentine free drug, rifapentine microspheres, and rifapentine microsphere thermosensitive gel drug obtained in Example 4 of the present invention are shown. Figure 2 The above are the blood drug concentration-time curves for the ORC group, ORLTH group, and PRTH group of this invention. Figure 3 The tissue distribution results for ORC, ORLTH and PRTH groups at 4h, 8h, 12h, 24h, 72h, 120h, 192h and 288h are shown. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention more apparent, the invention is described in detail below. It should be understood that the invention is not limited to the description herein.

[0020] Rifapentine Rifapentine, a highly effective and long-acting rifamycin antibiotic, is encapsulated in lipid microspheres, which reduces drug degradation and loss during delivery. Combined with the local in-situ gelation properties of thermosensitive gel, it can prolong the drug's residence time at the lesion site, improve bioavailability, reduce the frequency of administration, and thus enhance the therapeutic effect on infectious diseases such as tuberculosis.

[0021] Polosham The poloxamer used in this invention can reduce the interfacial tension between the oil phase and the aqueous phase, effectively promote the dispersion and stability of rifapentine in the oil phase, and thus promote the formation and stability of rifapentine ester microspheres, thereby effectively improving the encapsulation efficiency and drug loading of rifapentine, solving the problem of insufficient encapsulation efficiency and drug loading in the prior art.

[0022] Lecithin The lecithin used in this invention can form a lipid membrane structure on the surface of rifapentine microspheres, effectively encapsulating rifapentine molecules, reducing their leakage, and improving the lipid solubility of rifapentine, promoting its uniform dispersion in the microspheres, thereby increasing the encapsulation efficiency and drug loading of rifapentine.

[0023] vegetable oil The vegetable oil used in this invention is a known vegetable oil in the art that can be used in liposphere thermosensitive gels, and this invention is not limited to the vegetable oils listed below. As examples, the vegetable oil can be soybean oil, corn oil, rapeseed oil, olive oil, or castor oil. In the rifapentine liposphere thermosensitive gel of this invention, the role of the vegetable oil is to dissolve and carry the lipid-soluble drug rifapentine, ensuring its uniform dispersion within the lipospheres; simultaneously, it works with emulsifiers to maintain the uniform and stable structure of the microspheres, reducing drug aggregation, and can delay drug release after administration, improving the biocompatibility of the formulation.

[0024] co-emulsifier The co-emulsifier used in this invention is a known co-emulsifier in the art that can be used in liposphere thermosensitive gels, and this invention is not limited to the co-emulsifiers listed below. For example, the co-emulsifier can be oleic acid, stearic acid, lauric acid, myristic acid, or palmitic acid. In the rifapentine microsphere thermosensitive gel of this invention, the co-emulsifier functions synergistically with the emulsifier to further reduce the interfacial tension between the oil and aqueous phases, improve the stability of the emulsion system, and reduce microsphere aggregation or stratification; simultaneously, it helps to form smaller, more uniformly distributed lipospheres during the emulsification and homogenization process, improving the overall stability of the formulation and ensuring the uniformity and stability of the system after subsequent mixing with the thermosensitive gel matrix.

[0025] antioxidants The antioxidants used in this invention are known in the art and can be used in liposphere thermosensitive gels, and this invention is not limited to the antioxidants listed below. For example, antioxidants may be vitamin E, ascorbate palmitate, propylparaben, or propyl gallate. In the rifapentine microsphere thermosensitive gel of this invention, the antioxidants inhibit or delay the oxidative degradation of rifapentine and vegetable oils in the oil phase, preventing the active drug from becoming ineffective due to oxidation and improving the chemical stability of the rifapentine microsphere system; simultaneously, they can delay the oxidative deterioration of the formulation during storage and use, thereby ensuring the overall quality and efficacy stability of the thermosensitive gel formulation.

[0026] glycerin The glycerol used in this invention is the type of glycerol known in the art that can be used in liposphere thermosensitive gels. In the rifapentine liposphere thermosensitive gel of this invention, the role of glycerol is to regulate the osmotic pressure of the liposphere system, thereby improving the stability of the liposphere system and reducing the aggregation and rupture of lipospheres during storage and homogenization.

[0027] Thermosensitive polymer The thermosensitive polymer used in this invention is a thermosensitive polymer known in the art that can be used in liposphere thermosensitive gels, and this invention is not limited to the thermosensitive polymers listed below. As examples, the thermosensitive polymer can be a PLGA-PEG-PLGA triblock copolymer, a PEG-PLGA diblock copolymer, or a PCL-PEG-PCL triblock copolymer. In the rifapentine microsphere thermosensitive gel of this invention, the thermosensitive polymer functions as follows: it is liquid at room temperature, facilitating uniform mixing and administration with rifapentine microspheres; after injection into the body, it rapidly undergoes a phase transition at body temperature, forming a semi-solid or solid gel, encapsulating and fixing the rifapentine microspheres to the lesion site; through the physical barrier effect of the gel skeleton and the biodegradability of the polymer, the slow and continuous release of rifapentine from the lipospheres is controlled, achieving long-acting sustained release and reducing the frequency of administration.

[0028] Rifapentine microsphere thermosensitive gel The rifapentine microsphere thermosensitive gel of the present invention comprises rifapentine microspheres and a thermosensitive gel matrix; Rifafenib microspheres consist of a continuous aqueous phase and an oil phase dispersed in the aqueous phase; The oil phase includes rifapentine, emulsifiers, and vegetable oils; The aqueous phase includes glycerol and water; The emulsifier consists of poloxamer and lecithin in a mass ratio of 5:4~6.

[0029] In the rifapentine microsphere thermosensitive gel, the mass ratio of poloxamer to lecithin is 5:4~6, preferably 5:5~6.

[0030] In the rifapentine microsphere thermosensitive gel, the volume ratio of rifapentine microspheres to the thermosensitive gel matrix is ​​4:1~3, preferably 4:2~3.

[0031] In the rifapentine microsphere thermosensitive gel, the rifapentine content in the rifapentine microspheres is 3.8 mg / mL to 4.2 mg / mL, preferably 3.8 mg / mL to 4.0 mg / mL, and more preferably 3.8 mg / mL to 3.9 mg / mL.

[0032] In this invention, the rifapentine content is 3.8 mg / mL to 4.2 mg / mL. By scientifically controlling the drug loading ratio, it avoids stability problems such as rifapentine supersaturation precipitation and formulation stratification that may be caused by high concentration of drug loading, while ensuring the full dissolution of rifapentine.

[0033] In the rifapentine microsphere thermosensitive gel, the mass ratio of the emulsifier to the volume of the rifapentine microsphere is 1~7g:100mL, preferably 3~6g:100mL, and more preferably 4~5g:100mL.

[0034] In the rifapentine microsphere thermosensitive gel, the mass ratio of vegetable oil in the rifapentine microspheres to the volume ratio of the rifapentine microspheres is 8~12g:100mL, preferably 8~10g:100mL, and more preferably 9~10g:100mL.

[0035] In the rifapentine microsphere thermosensitive gel, the mass ratio of the co-emulsifier to the volume of the rifapentine microsphere is 0.3~0.8g:100mL, preferably 0.4~0.7g:100mL, and more preferably 0.5~0.6g:100mL.

[0036] In the rifapentine microsphere thermosensitive gel, the mass ratio of the antioxidant to the volume of the rifapentine microsphere is 0.15~0.35g:100mL, preferably 0.2~0.35g:100mL, and more preferably 0.2~0.3g:100mL.

[0037] In the rifapentine microsphere thermosensitive gel, the mass ratio of glycerol to the volume of rifapentine microspheres is 2~3g:100mL, preferably 2~2.8g:100mL, and more preferably 2.5~2.8g:100mL.

[0038] In the rifapentine microsphere thermosensitive gel, the thermosensitive polymer in the thermosensitive gel matrix is ​​a PLGA-PEG-PLGA triblock copolymer.

[0039] Preparation method of rifabutin microspheres The present invention also provides a method for preparing rifabutin microspheres as described above, comprising: S1. Mix rifapentine with the remaining raw materials in the oil phase and add it to anhydrous ethanol. Mix until completely dissolved, then evaporate the anhydrous ethanol to obtain the oil phase. S2. Add water to glycerol and mix well to obtain the aqueous phase; S3. Add the aqueous phase dropwise to the oil phase, emulsify and homogenize to obtain homogenized rifabutin microspheres.

[0040] In the preparation method of rifabutin microspheres, during emulsification, the rotation speed is 6500~7500 r / min and the time is 7~10 min; the homogenization pressure is 650~750 bar; and the number of homogenizations is 3~4.

[0041] The rifapentine microsphere thermosensitive gel of this invention, by limiting the rotation speed during emulsification to 6500~7500 r / min, the homogenization pressure to 650~750 bar, and the number of homogenization cycles to 3~4, controls the average particle size of the lipospheres to within 200 nm and ensures uniform particle size distribution (PDI≤0.2). The lipospheres prepared in this way not only avoid the deposition of large-diameter particles in the upper respiratory tract, but also enable drug delivery to deep target organs, improve the targeted aggregation effect, and thus significantly improve drug utilization.

[0042] Preparation method of thermosensitive gel matrix The present invention also provides a method for preparing the thermosensitive gel matrix as described above, comprising: adding water to a PLGA-PEG-PLGA triblock copolymer to obtain a PLGA-PEG-PLGA aqueous solution, swelling at 4°C until completely dissolved, to obtain a thermosensitive gel matrix.

[0043] In the preparation method of the thermosensitive gel matrix, the mass ratio of PLGA-PEG-PLGA triblock copolymer to water is 15~25g:80mL.

[0044] Preparation method of rifapentine microsphere thermosensitive gel The present invention also provides a method for preparing rifapentine microsphere thermosensitive gel as described above, comprising: mixing rifapentine microspheres with a thermosensitive gel matrix to obtain rifapentine microsphere thermosensitive gel.

[0045] In the preparation method of rifapentine microsphere thermosensitive gel, the volume ratio of rifapentine microspheres to PLGA-PEG-PLGA gel matrix is ​​4:1~3.

[0046] The rifapentine microsphere thermosensitive gel of this invention, with the targeting properties of lipospheres and the in-situ retention advantage of thermosensitive gel, allows the drug to be rapidly enriched in the target organs of tuberculosis infection, greatly increasing the local drug concentration. Compared with traditional oral preparations and existing dosage forms, the drug concentration in the target organs is significantly increased, and an effective bactericidal concentration can be achieved in a short time after administration.

[0047] To further illustrate the present invention, the following examples will provide a detailed description. The raw materials used in the following examples and comparative examples of the present invention are all commercially available products. The molecular weight distribution of the PLGA-PEG-PLGA triblock copolymer is PLGA (1500~2000)-PEG (1000~1500)-PLGA (1500~2000), the LA:GA ratio is 5:25, and the phase transition temperature is 35±2℃.

[0048] Example 1 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.038g rifapentine, 0.8g corn oil, 0.1g emulsifier, 0.03g oleic acid, 0.015g vitamin E, 0.2g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, emulsifier, oleic acid, and vitamin E are weighed according to the formula and added to 2 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 55°C until completely dissolved. After evaporating and removing the anhydrous ethanol, an oil phase is obtained, wherein the emulsifier consists of poloxamer and lecithin in a mass ratio of 5:4. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 55°C to obtain an aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 10 min at 6500 r / min using a high-speed shear emulsifier to obtain a primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain a refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, sealed with nitrogen, and stored away from light to obtain rifapentine microspheres. 15 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a uniform, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:1 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0049] Example 2 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.6g emulsifier, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, emulsifier, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, an oil phase is obtained, wherein the emulsifier consists of poloxamer and lecithin in a mass ratio of 5:4. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain an aqueous phase. At 60°C, the aqueous phase is added dropwise to the oil phase, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain a primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain a refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0050] Example 3 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.042g rifapentine, 1.2g corn oil, 0.7g emulsifier, 0.08g oleic acid, 0.035g vitamin E and 0.3g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, emulsifier, oleic acid, and vitamin E are weighed according to the formula and added to 4 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 65°C until completely dissolved. After evaporating to remove the anhydrous ethanol, an oil phase is obtained, wherein the emulsifier consists of poloxamer and lecithin in a mass ratio of 5:4. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 65°C to obtain an aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 7 minutes at 7500 r / min using a high-speed shear emulsifier to obtain a primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain a refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 25 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a uniform, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:3 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0051] Example 4 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.6g emulsifier, 0.05g oleic acid, 0.025g vitamin E, 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, emulsifier, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, an oil phase is obtained, wherein the emulsifier consists of poloxamer and lecithin in a mass ratio of 5:5. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain an aqueous phase. At 60°C, the aqueous phase is added dropwise to the oil phase, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain a primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain a refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0052] Example 5 Except for replacing the emulsifier composed of poloxamer and lecithin in a mass ratio of 5:4 with an emulsifier composed of poloxamer and lecithin in a mass ratio of 5:6, everything else is the same as in Example 2.

[0053] Example 6 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum three times at 300 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0054] Example 7 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum three times at 500 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0055] Example 8 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum three times at 600 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0056] Example 9 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum three times at 800 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0057] Example 10 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum twice at 700 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0058] Example 11 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum four times at 700 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0059] Example 12 Except for replacing the method of homogenizing the colostrum three times at 700 bar to obtain the refined emulsion with homogenizing the colostrum five times at 700 bar to obtain the refined emulsion, everything else is the same as in Example 4.

[0060] Comparative Example 1 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.6g lecithin, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, lecithin, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, the oil phase is obtained. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain the aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain the primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain the refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0061] Comparative Example 2 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.1g lecithin, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, lecithin, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, the oil phase is obtained. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain the aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain the primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain the refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0062] Comparative Example 3 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.5g lecithin, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, lecithin, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, the oil phase is obtained. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain the aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain the primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain the refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0063] Comparative Example 4 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.6g poloxamer, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, poloxamer, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, the oil phase is obtained. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain the aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain the primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain the refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0064] Comparative Example 5 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.25g poloxamer, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, poloxamer, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, the oil phase is obtained. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain the aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain the primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain the refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0065] Comparative Example 6 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.4g poloxamer, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, poloxamer, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, the oil phase is obtained. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain the aqueous phase. The aqueous phase is added dropwise to the oil phase at 60°C, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain the primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain the refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0066] Comparative Example 7 Taking 10 mL of rifabutic acid microspheres as an example, the prescription is as follows: 0.04g rifapentine, 1.0g corn oil, 0.22g emulsifier, 0.05g oleic acid, 0.025g vitamin E and 0.25g glycerin, and purified water to a final volume of 10mL; The preparation process of rifapentine microspheres is as follows: Rifapentine, corn oil, emulsifier, oleic acid, and vitamin E are weighed according to the formula and added to 3 mL of anhydrous ethanol. The mixture is placed in a heated, constant-temperature magnetic stirring bath and stirred at 60°C until completely dissolved. After evaporating to remove the anhydrous ethanol, an oil phase is obtained, wherein the emulsifier consists of poloxamer and lecithin in a mass ratio of 1:10. Glycerin is weighed according to the formula and added to purified water. The mixture is stirred and dissolved at 60°C to obtain an aqueous phase. At 60°C, the aqueous phase is added dropwise to the oil phase, and the mixture is sheared for 8 minutes at 7000 r / min using a high-speed shear emulsifier to obtain a primary emulsion. The primary emulsion is placed in a high-pressure homogenizer and homogenized three times at 700 bar to obtain a refined emulsion. The refined emulsion is rapidly cooled to room temperature in an ice bath, brought to a final volume of 10 mL, sealed under nitrogen, and stored away from light to obtain rifapentine microspheres. 20 g of rifapentine microspheres are weighed... PLGA-PEG-PLGA triblock copolymer was added to 80 mL of purified water to obtain PLGA-PEG-PLGA aqueous solution. The solution was swelled and completely dissolved at 4 °C to form a homogeneous, particle-free thermosensitive gel matrix, which was then stored away from light. Rifapentine microspheres were mixed with the PLGA-PEG-PLGA gel matrix at a volume ratio of 4:2 and stirred at low speed under ice bath conditions until homogeneous. The mixture was kept away from light and bubble generation was avoided throughout the process to obtain rifapentine microsphere thermosensitive gel.

[0067] Experimental Example 1 The rifapentine microspheres and rifapentine microsphere thermosensitive gels prepared in Examples 1-12 and Comparative Examples 1-7 were tested according to the following methods: I. Particle size, polydispersity index (PDI), and zeta potential of rifapentine microspheres: An appropriate amount of rifapentine microsphere sample was taken, diluted 10 times with purified water, and ultrasonically dispersed in an ice bath for 3 min. The diluted solution was added to the sample cell of a Malvern nanolaser particle size analyzer. The detection parameters were set as follows: detection temperature 25℃, scattering angle 90°, equilibration time 60s. Each sample was measured in triplicate, and the average hydrated particle size, polydispersity index (PDI), and zeta potential of the lipid microspheres were recorded. The average value of the results was taken. The test results are shown in Table 1 below. II. Encapsulation efficiency and drug loading of rifabutin microspheres: 1. Stability: The rifapentine microspheres obtained in Example 4 showed no stratification or oil separation after being placed at room temperature for 24 hours, and no drug content retention rate, encapsulation rate, and drug loading rate after being placed under low temperature and light protection conditions for 10 days. The core drug loading indicators showed no significant decrease. 2. Standard curve preparation: Weigh 3.8 mg, 4.0 mg, and 4.2 mg of rifapentine, dilute with methanol to prepare three standard solutions with concentrations of 3.8 mg / mL, 4.0 mg / mL, and 4.2 mg / mL. HPLC was used to determine the peak area, and a standard curve was plotted. The regression equation was y = 14.59212x + 2.13086 (r = 0.9998). 3. Encapsulation efficiency determination: Take 1 mL of rifapentine microsphere sample, centrifuge at 12000 r / min for 30 min, collect the supernatant, and determine the free drug content by HPLC; take another 1 mL of lipid microsphere sample, add 0.4 mL of methanol, sonicate to fully demulsify, add 8 mL of methanol, and determine the total drug content by HPLC. Encapsulation efficiency (%) = (total drug content - free drug content) / total drug content × 100%; 4. Drug loading determination: Take the free drug content and the total drug content measured after demulsification, and calculate the actual drug loading. Actual drug loading = total drug content measured after demulsification - free drug content; Drug loading (%) = actual drug loading / total mass of rifapentine microspheres × 100%; The test results are shown in Table 1 below.

[0068] Table 1 Performance test results of Examples 1-12 and Comparative Examples 1-7

[0069] The encapsulation efficiency and drug loading of Examples 1-5 were higher than those of Comparative Examples 1-6, indicating that the present invention, by adding poloxamer and lecithin, not only improved the encapsulation efficiency and drug loading of rifapentine microspheres, but also made the particle size less than 200 nm, with a dispersion index (PDI) ≤ 0.2, good dispersibility, and a zeta potential absolute value > 16.0 mV, indicating good system stability.

[0070] The encapsulation efficiency and drug loading of Example 2 were higher than those of Comparative Example 7, indicating that the present invention further improved the encapsulation efficiency and drug loading of rifapentine microspheres by adding poloxamer and lecithin in a ratio of 5:4~6.

[0071] The encapsulation efficiency and drug loading of Example 4 were higher than those of Examples 6-12, indicating that the present invention improved the encapsulation efficiency and drug loading of rifabutin microspheres by limiting the homogenization pressure to 700 bar and the homogenization times to 3.

[0072] III. Rifafenibose microsphere thermosensitive gel: 1. Gelation characteristics: Measured by rheometer, gelation temperature 33~34℃, gelation time ≤25s, viscosity 500~510cP at 25℃, viscosity ≥2000cP at 35℃; 2. Dosage compatibility: The formulation is a low-viscosity, clear, orange liquid at room temperature, with good needle penetration properties; 3. Stability: The rifapentine microsphere thermosensitive gel formulation obtained in Example 4 has good uniformity, no particulate matter, no free oil droplets, and a drug retention rate of ≥90% and an encapsulation rate of ≥71% after 7 days, showing excellent drug loading stability. 4. Freeze-thaw stability: The rifapentine microsphere thermosensitive gel obtained in Example 4 was frozen at -20℃ for 24 hours, and then placed in a constant temperature water bath at 25℃ until it was completely thawed and in a homogeneous state (no ice crystals, no precipitation, no stratification). Immediately after that, the next freeze-thaw cycle was carried out. After 3 freeze-thaw cycles, there were no significant changes in the appearance, particle size, and gelation characteristics of the formulation. The drug content retention rate was 89.12%, the encapsulation rate was ≥68%, the drug loading was ≥3.0%, there was no risk of accumulation, and the biocompatibility was excellent. It is suitable for different transportation and storage conditions.

[0073] Experimental Example 2 I. In vitro drug release experiment 1. Dialysis bag pretreatment: Cut the dialysis bag into 8cm segments. Boil the dialysis bag in an aqueous solution containing 2g / 100mL sodium bicarbonate and 1mmol / L EDTA·2Na (pH=8.0) for 10 minutes. Rinse the dialysis bag with distilled water, then boil it again in an aqueous solution containing 1mmol / L EDTA·2Na (pH=8.0) for 10 minutes. After cooling, place it in distilled water and store it in a refrigerator at 4°C for later use. Ensure that the dialysis bag is always submerged in the solution and wear gloves when handling it.

[0074] 2. In vitro drug release test: Take equal amounts of rifapentine free drug, rifapentine microspheres, and rifapentine microsphere thermosensitive gel (rifapentine free drug, rifapentine microspheres, and rifapentine microsphere thermosensitive gel are all rifapentine microspheres and rifapentine microsphere thermosensitive gel obtained in Example 4), and set up 3 parallel samples for each group. Transfer them into pretreated dialysis bags and seal both ends of the dialysis bags. Then immerse the dialysis bags in test tubes containing release medium (release medium is pH 7.4 PBS buffer containing 0.5% Tween-80), and control the total volume of the system inside and outside the dialysis bag to be 20 mL. Place the test tube in a constant temperature water bath shaker at (37±0.1)℃ and shake at 100 r / min. At the preset sampling time point, take 1 mL of the test solution from the external release medium of the test tube and immediately replenish with an equal volume of fresh release medium. Determine the drug concentration using high performance liquid chromatography (HPLC), calculate the cumulative release amount Q of RPT according to the corresponding formula, and finally plot the in vitro cumulative release curve of the drug (as shown below). Figure 1As shown (in the figure), the preset sampling time points are 0 h, 2 h, 3 h, 5 h, 7 h, 10 h, 13 h, 16 h, 19 h, 25 d, and 30 d respectively; the drug cumulative release rates (%) of rifapentine free drug, rifapentine lipid microspheres, and rifapentine lipid microsphere thermosensitive gel at these times are shown in Table 1 below.

[0075] Table 1 Drug cumulative release rates of rifapentine free drug, rifapentine lipid microspheres, and rifapentine lipid microsphere thermosensitive gel

[0076] The cumulative release amount Q = [CnVn+(Cn - 1Vn - 1 + Cn - 2Vn - 2 + …… + C1V1)] / m×100%, where Cn refers to the rifapentine concentration obtained at the nth sampling, V refers to the total volume of the release medium; Vn - 1 refers to the volume of the medium taken out at the (n - 1)th sampling, and m represents the weight of the un-released rifapentine; Fitting the in vitro release kinetic model of rifapentine lipid microsphere thermosensitive gel, the results show that its in vitro release conforms to the Riger - Peppas kinetic model, with a high model fitting degree (R² = 0.993) and a release exponent n = 0.673, that is, the in vitro drug release of rifapentine in the lipid microsphere thermosensitive gel is the synergistic effect of drug free diffusion and gel surface erosion; According to Figure 1 It can be seen that the cumulative release rate of free rifapentine exceeds 90% at 36 h, and the release rate is too fast; the cumulative release rate of rifapentine lipid microsphere thermosensitive gel reaches 82.35% at 30 d, without an obvious burst release effect, and the drug concentration at each time point is higher than the minimum inhibitory concentration of rifapentine.

[0077] II. In vivo pharmacokinetic experiment 1. Experimental animals SPF - grade female SD rats with a body weight of (180 ± 30) g were selected, purchased from Beijing Huafukang Biotechnology Co., Ltd., and the animal production license number: SCXK (Beijing) 2024 - 0003; all rats were raised in an environment with a temperature of (22 ± 2) °C, a relative humidity of 50% - 60%, and a 12 - h light - dark cycle, with free access to food and water. After 7 d of adaptive feeding, the experiment was carried out. This experiment was reviewed and approved by the Experimental Animal Ethics Committee of the Hebei Institute for Drug and Medical Device Control (approval number: LL2025 - 02), and all operations followed the "Guidelines for Laboratory Animal Ethics" to minimize animal suffering to the greatest extent.

[0078] 2. Experimental methods Solution preparation Weigh an appropriate amount of rifapentine reference standard, add acetonitrile, sonicate to dissolve and dilute to volume to prepare a 1 μg / mL rifapentine stock solution, and serially dilute to prepare a series of working solutions of reference standard from 5 to 10000 ng / mL; separately weigh rifampicin internal standard, prepare a 1 μg / mL internal standard stock solution using the same method, dilute to a 50 ng / mL internal standard working solution, and store at 4℃ protected from light for later use.

[0079] Animal grouping and dosing regimen SD rats were randomly divided into 3 groups of 24 rats each. The grouping and administration methods are as follows: ORC group (traditional oral administration group, administered via gavage instead of oral administration): Rifapentine raw material suspension, administered via gavage at a dose of 13.5 mg / kg (calculated as rifapentine). The preparation method of rifapentine raw material suspension is as follows: Weigh 1.35 g of rifapentine, add 0.2 mL of Tween-80 and grind to moisten. Use an aqueous solution containing 0.5 g of CMC-Na as the dispersion medium, grind and disperse evenly, add water to make up to 10 mL, and sonicate to obtain rifapentine raw material suspension. ORLTH group (oral gel group): The rifapentine microsphere thermosensitive gel obtained in Example 4 was administered by gavage at the same dosage as before. PRTH group (lung administration group): Rifapentine microsphere thermosensitive gel (obtained in Example 4) was administered via endotracheal intubation, simulating local lung administration via clinical fiberoptic bronchoscopy, with the same dosage as before.

[0080] Biological sample collection and pretreatment Plasma samples: Blood samples of 0.3-0.5 mL were collected from the inner canthus of the eyes of rats in each group at 15 min, 30 min, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h after drug administration. The plasma was separated by centrifugation at 4℃ and 4500 r / min for 15 min. 50 μL of plasma was taken, and 150 μL of acetonitrile containing internal standard was added to precipitate the protein. After vortexing for 2 min, the mixture was centrifuged at 12000 r / min for 15 min. The supernatant was collected and concentrated by nitrogen blowing. The mixture was then reconstituted with the mobile phase and ready for analysis. Tissue samples: Rats in each group were anesthetized and sacrificed at 4h, 8h, 12h, 24h, 72h, 120h, 192h, and 288h after drug administration. Heart, liver, spleen, lung, and kidney tissues were quickly separated, rinsed with physiological saline, and dried with filter paper. PBS buffer was added at a ratio of 1:3 (w / v), homogenized in an ice bath, and centrifuged at 12000 rpm for 15 min. The supernatant was collected, and the drug was extracted according to the plasma sample pretreatment method for tissue distribution detection.

[0081] LC-MS / MS detection conditions Chromatographic conditions: An Agilent Poroshell 120 EC-C18 column (2.7 μm, 4.5 × 50 mm) was used; mobile phase A was 0.1% formic acid aqueous solution (containing 5 mM ammonium formate), and mobile phase B was 0.1% formic acid acetonitrile solution, with gradient elution; flow rate was 0.5 mL / min, column temperature was 35 ℃, injection volume was 2 μL, and run time was 5 min.

[0082] Mass spectrometry conditions: Electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM) mode; rifapentine monitoring ion pair m / z 877.0→174.0, collision energy 38 eV, declustering voltage 120 V; rifampin internal standard monitoring ion pair m / z 823.3→723.3, collision energy 35 eV, declustering voltage 120 V; drying temperature 450℃.

[0083] 2.1 The pharmacokinetic results are shown in Table 2 below. Figure 2 As shown, each experiment was repeated 3 times, and the data in the table are the mean ± standard deviation of the 3 results; Table 2. Pharmacokinetic parameters of RPT in ORC, ORLTH and PRTH groups

[0084] Table 1 shows that compared with the ORC group, the pharmacokinetic parameters of both the ORLTH and PRTH groups were significantly optimized, and the differences were statistically significant (P<0.05). The area under the curve (0-t) of the PRTH group and ORLTH group were 307.68±19.85 mg·h / L and 305.42±27.16 mg·h / L, respectively, which were significantly higher than those of the ORC group (298.76±22.36 mg·h / L), indicating a significant improvement in bioavailability.

[0085] The elimination half-lives of the drugs in the PRTH and ORLTH groups were prolonged to 52.41±3.68h and 45.78±3.59h, respectively, which were much longer than the 22.36±2.14h in the ORC group, indicating a significant prolongation of the drug's duration of action in vivo. The time to peak plasma concentration was delayed to 11.62±4.35h and 8.76±1.35h in the PRTH group and ORLTH group, respectively. The average residence time of drug molecules in the body was significantly prolonged, the drug release was more stable, and there was no obvious sudden increase in plasma concentration. according to Figure 3 The plasma concentration curves of the PRTH and ORLTH groups did not show the peak shape of "rapid release + rapid decline" as in the ORC group. Instead, they showed a flat curve of "slow rise, delayed peak, long plateau period, and slow elimination", which has obvious sustained-release characteristics.

[0086] 2.2 Organizational distribution patterns are as follows Figure 3 As shown according to Figure 3 It can be seen that all three groups of drugs follow the kinetic law of absorption-distribution-elimination in rats, with significant differences in tissue distribution.

[0087] The drug concentration in lung tissue of the PRTH group was significantly higher than that of the ORC and ORLTH groups at all time points (P<0.05). The effective bactericidal concentration was maintained even 288 hours after administration, achieving efficient enrichment of the target organ.

[0088] In the early stages of administration, the amount of drug accumulation in the liver tissue of the ORLTH and PRTH groups was significantly lower than that of the ORC group, and the peak value of drug accumulation in the liver tissue of the ORLTH and PRTH groups was lower than that of the ORC group, thus reducing the risk of liver toxicity. The drug was evenly distributed in the heart, spleen, and kidney tissues, with no abnormal accumulation, indicating good safety.

[0089] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rifabutin microsphere thermosensitive gel, characterized in that, Including rifabutin microspheres and a temperature-sensitive gel matrix; The rifabutin microspheres comprise a continuous aqueous phase and an oil phase dispersed in the aqueous phase; The oil phase includes rifapentine, emulsifiers, and vegetable oils; The aqueous phase comprises glycerol and water; The emulsifier is composed of poloxamer and lecithin in a mass ratio of 5:4 to 6.

2. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The volume ratio of the rifapentine microspheres to the thermosensitive gel matrix is ​​4:1~3.

3. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The rifapentine microspheres contain 3.8 mg / mL to 4.2 mg / mL of rifapentine.

4. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The thermosensitive polymer in the thermosensitive gel matrix is ​​a PLGA-PEG-PLGA triblock copolymer.

5. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The mass ratio of the emulsifier to the volume of the rifapentine microspheres is 1~7g:100mL.

6. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The vegetable oil includes at least one of soybean oil, corn oil, rapeseed oil, and olive oil.

7. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The oil phase further includes a co-emulsifier, which includes at least one selected from oleic acid, stearic acid, or lauric acid; and / or The oil phase also includes an antioxidant, which includes at least one of vitamin E, ascorbyl palmitate, or propylparaben.

8. The rifabutin microsphere thermosensitive gel according to claim 1, characterized in that, The method for preparing the rifabutin microspheres includes the following steps: S1. Mix rifapentine with the remaining raw materials in the oil phase and add it to anhydrous ethanol. Mix until completely dissolved, then evaporate the anhydrous ethanol to obtain the oil phase. S2. Add water to glycerol and mix well to obtain the aqueous phase; S3. Add the aqueous phase dropwise to the oil phase, emulsify and homogenize to obtain homogenized rifabutin microspheres.

9. The rifabutin microsphere thermosensitive gel according to claim 8, characterized in that, In step S3, during emulsification, the rotation speed is 6500~7500 r / min and the time is 7~10 min; the homogenization pressure is 650~750 bar; and the homogenization is performed 3~4 times.

10. A method for preparing a rifabutin microsphere thermosensitive gel, characterized in that, The preparation of the rifapentine microsphere thermosensitive gel according to any one of claims 1 to 9 comprises the following steps: Rifapentine microspheres were mixed with a thermosensitive gel matrix to obtain the rifapentine microsphere thermosensitive gel.

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