A pharmaceutical composition, a pharmaceutical preparation and use thereof

Abstract

This invention belongs to the field of pharmaceutical technology and relates to a pharmaceutical composition, pharmaceutical preparation, and application. Specifically, it discloses a furanone derivative, 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone. It involves obtaining five crystal forms (I, II, III, IV, and amorphous) using different crystallization methods. Characterization and stability studies of the different crystal forms are conducted. The pharmaceutical composition containing a therapeutically effective dose of the polymorphs of this compound exhibits anti-inflammatory and antitumor pharmacological activities and can be used for the treatment of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the application of polymorphs of furanone compounds. Background Technology

[0002] Polymorphism refers to a solid substance exhibiting two or more different spatial arrangements, resulting in different physical and chemical properties. Small organic molecule compounds commonly exhibit polymorphism, including different crystal forms, solvates, and amorphous crystals. Different crystal forms of the same drug can have different melting points, solubilities, dissolution characteristics, and stability, leading to variations in bioavailability and thus affecting clinical application and efficacy. Statistics show that over 50% of chemical drugs have polymorphism issues, and approximately 80% of clinical drugs are administered in solid form. Therefore, the polymorphism of solid drugs directly impacts their clinical application. Furthermore, different crystal forms of polymorphic drugs often exhibit stability issues, changing during raw material synthesis, purification, formulation processing, and storage, thus affecting their efficacy. Therefore, conducting research on the crystal stability of polymorphic drugs is directly related to the selection of drug crystal forms, drug synthesis and purification processes, formulation processes, and drug packaging and storage conditions.

[0003] FONE is a derivative obtained by esterification of 4-(hydroxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(hydroxy)-5-[(hydroxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone with acetic anhydride. 1 H-NMR and 13 The C-NMR structure was confirmed as 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone. Studies have found that this compound possesses anti-inflammatory and antitumor pharmacological activities, but research on its polymorphism has not yet been reported. Therefore, polymorphism studies of FONE were conducted to provide a basis for the development and application of this compound as a candidate drug. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a pharmaceutical composition, a pharmaceutical preparation, and its application, wherein the pharmaceutical composition uses multiple crystal forms of FONE as raw materials.

[0005] The technical solution of this invention is implemented as follows:

[0006] On one hand, the present invention provides a crystalline form of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone, wherein the crystalline form is crystalline form I, and the characteristic peak with a relative peak height (Height%) ≥ 5% is determined by X-ray powder diffraction. The specific diffraction peak positions, diffraction angle 2θ (°), and relative peak height (Height%) are as follows:

[0007]

[0008] Crystal form I was determined using a melting point apparatus at a heating rate of 1 °C / min, with a melting range of 125.0–127.8 °C. Crystal form I was also determined using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) at a heating rate of 10 °C / min. Its TGA curve showed no significant weight loss in the range of 40–160 °C; its DSC curve exhibited characteristic endothermic peaks at 120.2 °C and 130.0 °C.

[0009] The preparation method for crystal form I employs atomization rapid evaporation. The procedure involves: dissolving the compound in a solvent with stirring; then rapidly evaporating the solution via atomization to obtain a solid powder; wherein the solvent is ethanol; and the operating parameters for atomization rapid evaporation are: atomization pressure 0.15 MPa, solution flow rate 5 mL / min, nitrogen gas as the evaporation medium, and a flow rate of 0.4 m³ / min. 3 / min, inlet temperature 100℃.

[0010] Secondly, the present invention provides a crystalline form of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone, wherein the crystalline form is crystalline form II, and the characteristic peak with a relative peak height (Height%) ≥ 5% is determined by X-ray powder diffraction. The specific diffraction peak positions, diffraction angle 2θ (°), and relative peak height (Height%) are as follows:

[0011]

[0012] Crystal form II was determined using a melting point apparatus at a heating rate of 1 °C / min, and its melting range was 125.8–127.8 °C. Crystal form II was also determined using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) at a heating rate of 10 °C / min. Its TGA curve showed first-order weight loss at 105–120 °C, with a weight loss rate of 5.715%. Its DSC curve showed characteristic endothermic peaks at 105.6 °C and 130.2 °C.

[0013] The preparation methods for crystal form II are cooling crystallization and dissolution crystallization.

[0014] The operation steps of the cooling crystallization method are as follows: the compound is stirred and dissolved in a hot solvent, the solution is cooled to 4°C to precipitate crystals, which are then collected by filtration and dried at 40°C to obtain the product; wherein the solvent is tetrahydrofuran.

[0015] The operation steps of the dissolution-precipitation crystallization method are as follows: the compound is dissolved in the main solvent by stirring, the above solution is added to the stirring precipitation solvent, the solid is precipitated, filtered and collected, and dried at 40°C to obtain the product; wherein the main solvent is tetrahydrofuran, the precipitation solvent is one of n-hexane and purified water, more preferably purified water, and the volume ratio of the main solvent to the precipitation solvent is 1:1 to 1:10, more preferably 1:4 to 1:6.

[0016] Thirdly, the present invention provides a crystalline form of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone, wherein the crystalline form is crystalline form III, and the characteristic peak with a relative peak height (Height%) ≥ 5% is determined by X-ray powder diffraction. The specific diffraction peak positions, diffraction angle 2θ (°), and relative peak height (Height%) are as follows:

[0017]

[0018] Crystal form III was determined using a melting point apparatus at a heating rate of 1 °C / min, with a melting range of 128.0–129.8 °C. Crystal form III was also determined using thermogravimetric analysis (TGA) and differential thermal imaging (DTI) at a heating rate of 10 °C / min. Its TGA curve showed no significant weight loss in the range of 40–160 °C; its DTI curve exhibited a characteristic endothermic peak at 130.8 °C.

[0019] The methods for preparing crystal form III include cooling crystallization, dissolution crystallization, and slow solvent evaporation crystallization.

[0020] The cooling crystallization method involves dissolving the compound in a hot solvent with stirring, cooling the solution to 4°C to precipitate crystals, collecting the crystals by filtration, and drying them at 40°C to obtain the final product. The solvent is selected from one of the following: hexane, diethyl ether, dichloromethane, n-butanol, ethanol, ethyl acetate, isopropanol, dioxane, acetonitrile, methanol, and dimethyl sulfoxide, with ethanol being a more preferred option.

[0021] The operation steps of the dissolution-precipitation crystallization method are as follows: the compound is dissolved in the main solvent by stirring, the above solution is added to the stirring precipitation solvent, the solid is precipitated, filtered and collected, and dried at 40°C to obtain the product; wherein the main solvent is selected from one of p-xylene, dichloromethane, ethanol, ethyl acetate, dioxane, acetonitrile, methanol, and dimethyl sulfoxide, and is more preferably ethanol; the precipitation solvent is selected from one of n-hexane and purified water, and is more preferably purified water.

[0022] The operation steps of the slow evaporation crystallization method are as follows: the compound is stirred and dissolved in a solvent, left at room temperature, the solvent is slowly evaporated to precipitate a solid, which is collected and dried at 40°C to obtain the product; wherein the solvent is selected from one of dichloromethane, ethanol, and acetonitrile.

[0023] Fourthly, the present invention provides a crystalline form of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone, wherein the crystalline form is crystalline form IV, and the characteristic peak with a relative peak height (Height%) ≥ 5% is determined by X-ray powder diffraction. The specific diffraction peak positions, diffraction angle 2θ (°), and relative peak height (Height%) are as follows:

[0024]

[0025] Crystal form IV was determined using a melting point apparatus at a heating rate of 1 °C / min, with a melting range of 127.8–129.4 °C. Crystal form IV was also determined using thermogravimetric analysis (TGA) and differential thermal analysis (DTA) at a heating rate of 10 °C / min. Its TGA curve showed no significant weight loss in the range of 40–160 °C; its DTA curve exhibited a characteristic endothermic peak at 132.8 °C.

[0026] The preparation method for crystal form IV adopts the cooling crystallization method. The operation steps are as follows: the compound is stirred and dissolved in a hot solvent, the solution is cooled to 4°C to precipitate crystals, which are then collected by filtration and dried at 40°C to obtain the crystals; wherein the solvent is acetone.

[0027] Fifthly, the present invention provides a crystalline form of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone, wherein the crystalline form is amorphous. X-ray powder diffraction analysis shows a diffuse, irregularly raised diffraction pattern without sharp peaks. The amorphous form is measured using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Its TGA curve shows no significant weight loss before 160°C; its DSC curve shows no characteristic endothermic peaks.

[0028] The amorphous preparation method is the melt-cold crystallization method. The operation steps are as follows: the compound is melted at 140℃ into a molten liquid, and then placed at -20℃ for rapid cooling and solidification to obtain the product.

[0029] In a sixth aspect, the present invention provides a pharmaceutical composition comprising one or more crystal forms selected from crystal form I, crystal form II, crystal form III, crystal form IV, and amorphous form.

[0030] Polymorphic drugs possess multiple crystalline structures, and different crystalline forms often exhibit crystalline stability issues. When a drug's crystalline form is unstable, it can transform under the influence of environmental, process, and storage conditions (such as temperature, humidity, light, and pressure), directly leading to changes in the drug's physicochemical properties and ultimately affecting its efficacy and clinical application. Therefore, conducting crystalline stability studies on different drug crystalline forms is directly related to the selection of crystalline forms, synthesis and purification processes, formulation processes, and drug packaging and storage conditions.

[0031] Considering that crystal form II is a tetrahydrofuran solvate, and tetrahydrofuran is a Class II solvent with biosafety concerns, this crystal form is not suitable for pharmaceutical applications. Therefore, only crystal form I, III, IV, and V (amorphous) of FONE were investigated for crystal form stability. Specific investigation conditions included: influencing factor tests (high temperature, high humidity, and strong light), pressure tests, grinding tests, stirring tests in water, and high temperature tests. X-ray powder diffraction was performed on samples under different investigation conditions, and the results were compared with the X-ray powder diffraction results of the original sample crystal form to evaluate its crystal form stability.

[0032] The results showed that crystal form III was the most thermodynamically stable crystal form, maintaining its original crystal form under all experimental conditions; crystal form I transformed into crystal form III under high temperature environment and stirring in water (60℃); crystal form IV maintained its crystal form stability under high humidity and light influence conditions, but transformed into crystal form III under other experimental conditions; crystal form V / amorphous maintained its crystal form stability under high humidity, light influence and pressure conditions, transformed into crystal form I under grinding and high temperature (60℃) conditions, and transformed into crystal form III under stirring in water and high temperature (130℃) conditions.

[0033] Mixed crystals: The FONE mixed crystals involved in this invention are solid mixtures obtained by mixing different drug crystal forms (crystal form I, crystal form II, crystal form III, crystal form IV and amorphous respectively) obtained by the previous method in any non-zero proportion, or solid mixtures of different crystal forms formed during the crystal form transformation process of non-stable crystal form (crystal form I, crystal form II, crystal form IV and amorphous respectively) solid drugs under the influence of external environmental factors.

[0034] Dosage, formulation and application of pharmaceutical compositions containing crystalline components

[0035] The pharmaceutical composition involved in this invention is a pharmaceutical crystal form and a pharmaceutically acceptable excipient or carrier, wherein the excipient or carrier includes solubilizers, suspending agents, emulsifiers, solvents, wetting agents, diluents, disintegrants, adsorbents, binders, lubricants, water-soluble matrices, oil-soluble matrices, emulsion matrices, water-soluble carriers, hydrophobic carriers, inclusion materials, coating materials, etc.

[0036] The pharmaceutical composition involved in this invention has a daily dosage in the range of 20-1200 mg.

[0037] The pharmaceutical compositions involved in this invention are characterized by the presence of a drug and a pharmaceutically acceptable excipient or carrier, formulated into solid dosage forms, liquid dosage forms, and semi-solid dosage forms, or first formulated into solid dispersions, inclusion complexes, microspheres, nanoparticles, etc., and then further formulated into the aforementioned solid dosage forms, liquid dosage forms, and semi-solid dosage forms. Dosage forms include tablets, capsules, dry suspensions, granules, powders, films, microcapsules, droplets, powder inhalers, aerosols, sprays, solutions, suspensions, emulsions, injections, ointments, suppositories, gels, patches, etc., and routes of administration include oral, pulmonary inhalation, injection, external application, and topical application.

[0038] The pharmaceutical compositions involved in this invention can be used as anti-inflammatory and anti-tumor drugs for the treatment of related diseases, including acute lung injury, chronic obstructive pulmonary disease, pulmonary fibrosis, osteoarthritis, colitis, tumors, etc.

[0039] The present invention has the following beneficial effects:

[0040] (1) Using different crystallization techniques (including cooling crystallization, antisolvent crystallization, slow evaporation crystallization, rotary evaporation crystallization, melt quenching crystallization, and spray drying crystallization), five crystal forms of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone were prepared for the first time, namely crystal form I, crystal form II, crystal form III, crystal form IV and crystal form V (amorphous). The solid state of the drug in the above different crystal forms was characterized to obtain the characteristic parameters of the drug in different crystal forms.

[0041] (2) The stability of different crystal forms of drugs was investigated, including influencing factor tests (high temperature, high humidity and strong light), pressure condition tests, grinding condition tests, stirring tests in water and high temperature tests. It was found that crystal form III is the stable crystal form of the drug, while other crystal forms (crystal form I, crystal form IV and amorphous) will transform into crystal form III under different conditions.

[0042] (3) The drug composition formed by the drug and conventional excipients or carriers in the field can be made into different dosage forms such as solid dosage form, liquid dosage form and semi-solid dosage form to meet the drug use needs of different routes of administration and can be used for the treatment of inflammation and tumor-related diseases. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 Optical microscope images of the drugs with crystal form I obtained in Example 1, crystal form II obtained in Example 2, crystal form III obtained in Example 4, crystal form IV obtained in Example 7, and crystal form V (amorphous) obtained in Example 8.

[0045] Figure 2 The X-ray powder diffraction patterns are those of the crystal form I obtained in Example 1, the crystal form II obtained in Example 2, the crystal form III obtained in Example 4, the crystal form IV obtained in Example 7, and the crystal form V (amorphous) obtained in Example 8.

[0046] Figure 3 The TG-DSC spectra of the drugs with crystal form I obtained in Example 1, crystal form II obtained in Example 2, crystal form III obtained in Example 4, crystal form IV obtained in Example 7, and crystal form V (amorphous) obtained in Example 8 are shown.

[0047] Figure 4 The image shows the DSC spectrum of the crystal form V (amorphous) drug obtained in Example 8, obtained through a heating-cooling-reheating process.

[0048] Figure 5 The results of the crystal stability study of the drugs with crystal form I obtained in Example 1, crystal form II obtained in Example 2, crystal form III obtained in Example 4, crystal form IV obtained in Example 7, and crystal form V (amorphous) obtained in Example 8 under the influence of various factors.

[0049] Figure 6 The results of the crystal stability tests of the drugs with crystal form I obtained in Example 1, crystal form II obtained in Example 2, crystal form III obtained in Example 4, crystal form IV obtained in Example 7, and crystal form V (amorphous) obtained in Example 8 were obtained under pressure conditions, grinding conditions, stirring in water, and high temperature.

[0050] Figure 7This figure shows the effect of oral administration of the drug on blood immune cells in a mouse model of acute lung injury induced by Poly(I:C) endotracheal instillation. In the figure, # indicates P < 0.05, ## indicates P < 0.01, and ### indicates P < 0.001 compared with the Con group; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 compared with the Mod group. n=8.

[0051] Figure 8 The effect of oral administration of the drug on lung injury in a mouse model of acute lung injury induced by Poly(I:C) endotracheal instillation.

[0052] Figure 9 The figure shows the effect of drug administration via gavage on inflammatory factors in bronchoalveolar lavage fluid in a mouse model of acute lung injury induced by Poly(I:C) tracheal instillation. In the figure, ### indicates P < 0.001 compared with the Con group; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 compared with the Mod group. n=8.

[0053] Figure 10 This figure shows the effect of nasal administration of the drug inclusion complex on lung injury in a mouse model of acute lung injury induced by LPS nasal instillation. In the figure, ## indicates P < 0.01 compared with the Con group; * indicates P < 0.05, and ** indicates P < 0.01 compared with the Mod group, n=6.

[0054] Figure 11 The figure shows the effect of nasal administration of the drug inclusion complex on inflammatory factors in bronchoalveolar lavage fluid and lung tissue in a mouse model of acute lung injury induced by LPS nasal instillation. In the figure, ### indicates P < 0.001 compared with the Con group; ** indicates P < 0.01 and *** indicates P < 0.001 compared with the Mod group, n=6.

[0055] Figure 12 The graph shows the effect of oral administration of the drug solid dispersion on lung function in mice with chronic obstructive pulmonary disease induced by tracheal instillation of LPS combined with cigarette smoke. In the graph, ## indicates P < 0.01, ### indicates P < 0.001 compared with the Con group; * indicates P < 0.05, ** indicates P < 0.01 compared with the Mod group; ^ indicates P < 0.05 compared with DEX; && indicates P < 0.01 compared with the original drug group, n=5.

[0056] Figure 13 The figure shows the effect of oral administration of solid drug dispersion on lung injury in mice with chronic obstructive pulmonary disease induced by endotracheal instillation of LPS combined with cigarette smoke. In the figure, ### indicates P < 0.001 compared with the Con group; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001 compared with the Mod group; ^ indicates P < 0.05 compared with the DEX group; & indicates P < 0.05 compared with the original drug group, n=5.

[0057] Figure 14 The figure shows the effect of oral administration of solid drug dispersion on inflammatory factors in bronchoalveolar lavage fluid of mice with chronic obstructive pulmonary disease induced by tracheal instillation of LPS combined with cigarette smoke. In the figure, ### indicates P < 0.001 compared with the Con group; * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 compared with the Mod group. n=5.

[0058] Figure 15 Micro-CT images showing the effect of intra-articular injection of drug microspheres on the knee joint of rats with anterior cruciate ligament transection combined with medial meniscectomy to establish an osteoarthritis model. ** indicates P < 0.01, *** indicates P < 0.001 compared to the sham-operated group; # indicates P < 0.05, ## indicates P < 0.01, ### indicates P < 0.001 compared to the saline group; n = 3; scale bar: 5 mm.

[0059] Figure 16 Diagram illustrating the effect of intra-articular injection of drug microspheres on cartilage tissue in rats with anterior cruciate ligament transection combined with medial meniscectomy to establish an osteoarthritis model. In the figure, *** indicates P < 0.001 compared to the sham-operated group; # indicates P < 0.05; ## indicates P < 0.01 compared to the saline group; n = 3; scale bar: 500 μm.

[0060] Figure 17 This figure illustrates the effects of intra-articular injection of drug microspheres on chondrocyte extracellular matrix degradation and inflammatory factor secretion in rats with anterior cruciate ligament transection combined with medial meniscectomy to establish an osteoarthritis model. In the figure, *** indicates P < 0.001 compared to the sham-operated group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to the saline group; n = 3; scale bar: 100 μm. Detailed Implementation

[0061] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0063] Example 1

[0064] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form I.

[0065] The specific steps are as follows: Weigh 5.0g of the drug into a beaker, add 250mL of ethanol, and stir to dissolve; rapidly evaporate the above solution via atomization to obtain a solid powder, thus obtaining the drug; the operating parameters are: atomization pressure 0.15 MPa, solution flow rate 5 mL / min, evaporation medium is nitrogen, and flow rate is 0.4 m³ / min. 3 / min, inlet temperature 100℃.

[0066] X-ray powder diffraction was used to determine, such as Figure 2 As shown, the characteristic peaks with a relative peak height (Height%) ≥ 5% have the following characteristics: peak position, diffraction angle 2θ (°), and relative peak height (Height%). Table 1 below shows the details:

[0067] Table 1:

[0068]

[0069] Crystal form I was determined using a melting point apparatus at a heating rate of 1 °C / min, with a melting range of 125.0–127.8 °C. Crystal form I was also determined using thermogravimetric analysis and differential thermal imaging. Figure 3 As shown, with a heating rate of 10 °C / min, its thermogravimetric curve shows no significant weight loss in the range of 40–160 °C; its differential thermal scanning curve shows characteristic endothermic peaks at 120.2 °C and 130.0 °C.

[0070] Example 2

[0071] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form II.

[0072] The specific steps are as follows: Weigh 5.0 g of the drug into a beaker, add 5 mL of tetrahydrofuran, and stir to dissolve under a 50 ℃ water bath; cool the above solution to room temperature, and then place it in a refrigerator at 4 ℃ for 24 h to precipitate the solid, filter and collect it, and dry it under vacuum at 40 ℃ to obtain the final product.

[0073] X-ray powder diffraction was used to determine, such as Figure 2As shown in Table 2, the characteristic peaks with a relative peak height (Height%) ≥ 5% have the following characteristics: peak position, diffraction angle 2θ (°), and relative peak height (Height%).

[0074] Table 2:

[0075]

[0076] Crystal form II was determined using a melting point apparatus at a heating rate of 1 °C / min, and its melting range was 125.8–127.8 °C. Crystal form II was also determined using thermogravimetric analysis and differential thermal imaging. Figure 3 As shown, with a heating rate of 10 °C / min, its thermogravimetric curve exhibits first-order weight loss at 105–120 °C, with a weight loss rate of 5.715%; its differential thermal scanning curve shows characteristic endothermic peaks at 105.6 °C and 130.2 °C.

[0077] Example 3

[0078] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form II.

[0079] The specific steps are as follows: Weigh 5.0 g of the drug into a beaker, add 15 mL of tetrahydrofuran, and stir to dissolve; add the above solution to 75 mL of n-hexane while stirring, precipitate the solid, filter and collect, and dry under vacuum at 40 °C to obtain the final product.

[0080] Example 4

[0081] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form III.

[0082] The specific steps are as follows: Weigh 5.0 g of the drug into a beaker, add 50 mL of ethanol, and stir to dissolve under a 75°C water bath; cool the above solution to room temperature, and then place it in a refrigerator at 4°C for 24 h to precipitate the solid, filter and collect it, and dry it under vacuum at 40°C to obtain the final product.

[0083] X-ray powder diffraction was used to determine, such as Figure 2 As shown, the characteristic peaks with a relative peak height (Height%) ≥ 5% have the following specific diffraction peak positions, diffraction angle 2θ (°), and relative peak height (Height%):

[0084] Table 3:

[0085]

[0086] Crystal form III was determined using a melting point apparatus at a heating rate of 1 °C / min, and its melting range was 128.0–129.8 °C. Crystal form III was also determined using thermogravimetric analysis and differential thermal imaging. Figure 3 As shown, with a heating rate of 10 ℃ / min, its thermogravimetric curve shows no significant weight loss in the range of 40~160℃; its differential thermal scanning curve has a characteristic endothermic peak at 130.8 ℃.

[0087] Example 5

[0088] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form III.

[0089] The specific steps are as follows: Weigh 5.0g of the drug into a beaker, add 20 mL of acetone, and stir to dissolve; add the above solution to 100 mL of purified water while stirring, precipitate the solid, filter and collect, and dry under vacuum at 40 ℃ to obtain the final product.

[0090] Example 6

[0091] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form III.

[0092] The specific steps are as follows: Weigh 5.0g of the drug into a beaker, add 10 mL of dichloromethane, and stir to dissolve; transfer the above drug solution into a vial, cover the mouth of the vial with filter paper, place it at room temperature, the solvent slowly evaporates, the solid precipitates, filter and collect, and vacuum dry at 40 ℃ to obtain the drug.

[0093] Example 7

[0094] This embodiment provides a method for preparing 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone crystal form IV.

[0095] The specific steps are as follows: Weigh 5.0 g of the drug into a beaker, add 5.0 mL of acetone, and stir to dissolve under water-soluble conditions at 40 ℃; cool the above solution to room temperature, and then place it in a refrigerator at 4 ℃ for 24 h to precipitate the solid, filter and collect it, and dry it under vacuum at 40 ℃ to obtain the final product.

[0096] Example 8

[0097] This embodiment provides a method for preparing the amorphous form of 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone.

[0098] The specific steps are as follows: Weigh 5.0 g of the drug and place it in a beaker. Melt the drug in an oil bath at 140°C until it reaches a molten state. Transfer the beaker to a freezer at -20°C and cool it rapidly for 24 hours to obtain the final product.

[0099] X-ray powder diffraction was used to determine, such as Figure 2 As shown, the diffraction pattern consists of diffuse, irregular ridges without sharp diffraction peaks. Amorphous diffraction patterns were determined using thermogravimetric analysis (TGA) and differential thermal scanning calorimetry (DTC). Figure 3 Its thermogravimetric curve shows no significant weight loss before 160℃; its differential thermal scanning curve shows no characteristic endothermic peak.

[0100] Application Example 1

[0101] Preparation of drug dry suspension

[0102] Weigh 10 g of crystal form III drug and 5 g of soybean phospholipids, add them to 250 mL of purified water, then add 1 kg of 0.3 mm zirconia balls, stir and grind in a medium at 700 rpm for 3 h. Then pass the mixture through a 120-mesh sieve to collect the drug suspension. Weigh 25 g of lactose, add it to the above drug suspension, stir to dissolve, and then spray dry (process parameters: injection flow rate 3 mL / min, atomization pressure 0.1 MPa, drying medium: air, air flow rate 0.4 m³ / min). 3 (At a flow rate of 100°C / min, the inlet temperature is 100°C) to obtain solid powder; take 20 g of solid powder, add 0.5 g of sodium carboxymethyl cellulose, stir and mix well to obtain the final product.

[0103] The above-mentioned dry suspension, when evenly dispersed in water, forms a drug suspension that remains in suspension for 30 minutes without sedimentation. The above-mentioned drug suspension can be administered orally or via nebulization.

[0104] Application Example 2

[0105] Preparation of pharmaceutical solid dispersion formulations

[0106] Weigh 15 g of crystal form III drug and 75 g of polyethylene glycol 4000, add them to 450 mL of ethanol, and stir to dissolve to obtain a drug solution; subject the above solution to spray drying (process parameters: injection flow rate 2 mL / min, atomization pressure 0.1 MPa, drying medium: nitrogen, nitrogen flow rate 0.4 m³ / min). 3 (At a flow rate of 60°C / min and an inlet temperature of 60°C), solid powder is collected to obtain a drug solid dispersion.

[0107] Take 10 g of the above solid dispersion, add 0.3 g of micronized silica gel, mix well, and dispense into No. 0 hollow gelatin capsules to obtain capsules.

[0108] Take 10 g of the above solid dispersion, add 0.3 g of sodium carboxymethyl starch and 0.3 g of micronized silica gel, mix evenly, and compress into tablets using a 10 mm shallow arc die to obtain tablets.

[0109] The disintegration time of the above-mentioned capsules and tablets is within 15 minutes, and the drug dissolution percentage is above 80% after 20 minutes; the above-mentioned drug solid dispersion formulations can be administered orally.

[0110] Application Example 3

[0111] Preparation of liquid formulations of drug inclusion complexes

[0112] 2.5 g of crystal form III drug was dissolved in 20 mL of anhydrous ethanol to obtain a drug solution. 33.07 g of HP-γ-CD was dissolved in 50 mL of purified water to obtain an inclusion material solution. Under a 50 ℃ water bath, the above drug solution was added to the inclusion material solution in a thin stream while stirring at a speed of 300 r / min. After the drug solution was added, stirring was continued for 2 h. Finally, the drug inclusion complex was obtained by freeze drying.

[0113] Weigh 15.3 g of the above drug inclusion complex and add it to 100 mL of purified water and stir to dissolve. Then weigh 0.1 g of potassium sorbate and 0.01 g of EDTA-2Na and add them to the above solution. Stir to dissolve and adjust the pH of the solution to 5.5-6.5 with 0.1 mol / L hydrochloric acid. Autoclave at 121℃ for 20 min and aseptically dispense into plastic bottles, each containing 10 mL.

[0114] The above-mentioned liquid preparations can be administered via nasal drops, oral administration, or nebulized inhalation.

[0115] Application Example 4

[0116] Preparation of drug microsphere formulations

[0117] Weigh 0.12 g of crystal form III drug and 1.2 g of PLGA (molecular weight 30 KD, lactic acid to glycolic acid ratio 75:25), add to 10 mL of dichloromethane, sonicate to dissolve, add the above solution dropwise to 40 mL of 1% (w / v) polyvinyl alcohol (PVA1788) aqueous solution while stirring at 500 r / min, then transfer to 200 mL of purified water, stir at 400 r / min for 3 h at room temperature, evaporate the organic solvent, centrifuge and wash with water 3 times, freeze dry to obtain drug microspheres.

[0118] The above-mentioned drug microspheres can be evenly dispersed in sterile water for injection and administered via intra-articular injection.

[0119] Application Example 5

[0120] Preparation of pharmaceutical hydrogel formulations

[0121] Weigh 2.0 g of carbomer and add it to 60 mL of purified water. Stir to disperse and allow to swell overnight. Weigh 1.2 g of triethanolamine and dissolve it in 10 mL of water to obtain a triethanolamine solution. Add this solution to the above carbomer solution and stir to dissolve and swell to obtain a blank gel. Weigh 0.3 g of crystal form III drug, add 15 g of propylene glycol, 10 g of glycerin, 1 g of borneol and 0.1 g of ethylparaben, stir well, add this solution to the above blank gel, and stir to mix well to obtain the drug hydrogel preparation.

[0122] The above-mentioned hydrogel preparations can be applied topically to the affected area for administration.

[0123] Example 1: Characterization of drug polymorphism

[0124] The following tests were performed on the drugs of crystal form I obtained in Example 1, crystal form II obtained in Example 2, crystal form III obtained in Example 4, crystal form IV obtained in Example 7, and crystal form V (amorphous) obtained in Example 8:

[0125] (1) Optical microscopy imaging

[0126] Appropriate amounts of drug samples of crystal form I, crystal form II, crystal form III, crystal form IV, and crystal form V (amorphous) were taken and placed on glass slides. Their morphology was observed and photographed using an optical microscope. Figure 1 .

[0127] Different drug crystal forms have different crystal habits. Crystal form I is a powdery crystal with a particle size of less than 100 μm; crystal form II is a blocky crystal with a length of about 1000 μm, a width of about 600 μm, and a height of about 700 μm; crystal form III is a needle-like crystal with a length of about 700 μm and a width and height of about 30 μm; crystal form IV is a rod-like crystal with a length of about 500 μm and a width and height of about 80 μm; crystal form V (amorphous) is a transparent block, and its size is related to the degree of breakage of the material during sampling.

[0128] (2) X-ray powder diffraction (PXRD) detection

[0129] X-ray powder diffraction was used for detection and analysis. The test parameters were as follows: test temperature 4℃, Cu-Kα target, tube voltage 40 kV, tube current 40 mA, 2 Theta range of 5~50°, and scan step size of 0.05°.

[0130] The powder diffraction pattern of the crystal form I drug is shown in [reference needed]. Figure 2 The characteristic peaks with a relative intensity peak height (Height%) ≥ 5% are shown in Table 1 for their diffraction peak positions, diffraction angle 2θ (°), and relative intensity peak height (Height%).

[0131] The powder diffraction pattern of drug crystal form II is shown below. Figure 2 The characteristic peaks with a relative intensity peak height (Height%) ≥ 5% are shown in Table 2 for their diffraction peak positions, diffraction angle 2θ (°), and relative intensity peak height (Height%).

[0132] The powder diffraction pattern of drug crystal form III is shown in [reference needed]. Figure 2 The characteristic peaks with a relative intensity peak height (Height%) ≥ 5% are shown in Table 3 for their diffraction peak positions, diffraction angle 2θ (°), and relative intensity peak height (Height%).

[0133] The powder diffraction pattern of drug crystal form IV is shown below. Figure 2 The characteristic peaks with a relative intensity peak height (Height%) ≥ 5% are shown in Table 4 for their diffraction peak positions, diffraction angle 2θ (°), and relative intensity peak height (Height%).

[0134] Table 4:

[0135] .

[0136] The powder diffraction pattern of drug crystal form V (amorphous) is shown in [reference needed]. Figure 2 The diffraction pattern shows irregular bulges without obvious sharp diffraction peaks.

[0137] (3) Drug melting point detection

[0138] Appropriate amounts of drug samples of crystal form I, crystal form II, crystal form III, and crystal form IV were placed in capillary tubes, and their melting points were determined using an automatic melting point apparatus. The results are shown in Table 5. The parameters were set as follows: initial temperature 100℃, heating rate 1℃ / min.

[0139] Table 5. Melting point determination results of different crystal forms of the drug

[0140]

[0141] (4) Drug thermogravimetric and differential thermal scanning detection

[0142] 2–3 mg of drug samples of crystal form I, crystal form II, crystal form III, crystal form IV, and crystal form V (amorphous) were weighed into aluminum crucibles. The lids were sealed to the crucibles using a crucible capping device. An empty reference crucible was prepared using the same method. Differential scanning calorimetry was used for determination. The results are shown in the figure. Figure 3 The detection parameters are as follows: measurement temperature: RT~500℃, heating rate: 10℃ / min, nitrogen atmosphere: 50 mL / min.

[0143] The thermogravimetric curve of crystal form I showed no significant weight loss before 160℃; its differential thermal scanning curve showed endothermic peaks at 120.2℃ and 130.0℃. Based on the melting point determination and crystal form stability study results, it was determined that it began to transform into crystal form III when heated to around 120℃.

[0144] The thermogravimetric curve of crystal form II shows a weight loss of 5.7% in the range of 105–120 °C, indicating that this crystal form is a solvate and will lose solvent molecules at 105 °C. Its differential thermal scanning curve shows endothermic peaks at 105.6 °C and 130.2 °C, respectively. Combining the thermogravimetric curve and the results of the crystal form stability study, it is determined that after losing solvent molecules, it transforms into crystal form III, and an endothermic peak consistent with that of crystal form III appears at 130.2 °C.

[0145] The thermogravimetric curve of crystal form III showed no significant weight loss before 160℃; its differential thermal scanning curve showed a characteristic melting endothermic peak at 130.8℃, indicating that crystal form III would not undergo crystal transformation during heating.

[0146] The thermogravimetric curve of crystal form IV showed no significant weight loss before 160℃; its differential thermal scanning curve showed a characteristic endothermic peak at 132.8℃.

[0147] The thermogravimetric analysis (TGA) curve for crystal form V (amorphous) showed no significant weight loss before 160 °C. Its differential thermal analysis (DTA) curve exhibited characteristic endothermic peaks at 120.0 and 131.9 °C. The appearance of these characteristic endothermic peaks may be related to the instability of the amorphous crystal state, and the fact that the sample preparation and crushing process caused the compound to transform from an amorphous state to other crystal forms. Further DTA measurements were performed on the compound sample using a cyclic operation of heating to 160 °C, cooling to 40 °C, and then heating back to 160 °C. The heating-cooling process simulated the melting method to obtain the amorphous state of the compound. No characteristic endothermic peaks appeared in the DTA curve after the reheating. The results are shown in [Figure number missing]. Figure 4 .

[0148] Example 2: Evaluation of the crystal stability of drug polymorphs

[0149] Polymorphic drugs often exhibit polymorphic stability issues, transforming into each other during raw material synthesis, purification, formulation processing, and storage, thus affecting their efficacy. Studying the polymorphic stability of drugs allows us to understand the stability of different polymorphs and the conditions for transformation between them, providing a basis for screening stable polymorphs, optimizing raw material and formulation processes, and selecting storage conditions. Considering that polymorph II is a tetrahydrofuran solvate, and tetrahydrofuran is a Class II solvent with safety concerns, the polymorphic stability evaluation of drugs only examines polymorphs I, III, IV, and V (amorphous) drugs. Specific content includes:

[0150] (1) Influencing Factors Experiment

[0151] ① High temperature test

[0152] Appropriate amounts of drug samples of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) were placed in open-mouthed glass bottles with stoppers, spread into a thin layer of 1 mm thickness, and placed at 60℃ and 75% relative humidity for 10 days before sampling and analysis using X-ray powder diffraction.

[0153] ② High humidity test

[0154] Appropriate amounts of drug samples of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) were placed in open-mouthed glass bottles with stoppers, spread into a thin layer of 1 mm thickness, and placed at 25℃ and 90% relative humidity for 10 days before sampling and analysis using X-ray powder diffraction.

[0155] ③ Illumination test

[0156] Appropriate amounts of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) drug samples were placed in stoppered glass bottles, spread into a thin layer of 1 mm thickness, and placed open in a light box. After 10 days under an illuminance of 4500 Lx ± 500 Lx, the samples were taken and analyzed using an X-ray powder diffractometer.

[0157] (2) Pressure condition test

[0158] Approximately 300 mg of drug samples of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) were taken and tableted at pressures of 2 tons and 6 tons respectively. After 1 minute, the tablets were crushed, sampled, and analyzed using X-ray powder diffraction.

[0159] (3) Grinding conditions test

[0160] Approximately 300 mg of drug samples of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) were ground in an agate mortar for 1 min, 3 min and 5 min respectively, and samples were taken and analyzed using X-ray powder diffraction.

[0161] (4) Stirring test in water

[0162] Approximately 200 mg of each of the following drug forms (I, III, IV, and V – amorphous) were placed in a 50 mL single-necked flask, and 20 mL of purified water was added. The samples were stirred at 40 °C and 60 °C for 24 h, respectively. After filtration, the samples were dried under vacuum at 40 °C. The samples were then analyzed using X-ray powder diffraction.

[0163] (5) High temperature test

[0164] Approximately 200 mg of drug samples of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) were taken respectively, heated to 130℃ at a heating rate of 10℃ / min, held at the temperature for 5 min, and then sampled and analyzed using X-ray powder diffraction.

[0165] The results of stability studies on drug samples of crystal form I, crystal form III, crystal form IV and crystal form V (amorphous) are shown in Table 6.

[0166] Table 6. Results of crystal stability tests for different crystalline drug forms under different experimental conditions.

[0167]

[0168] The experimental results of influencing factors show that crystal form I ( Figure 5 a) and crystal type III ( Figure 5 b) The drug sample did not undergo any crystal form change under high temperature, high humidity, and light conditions, indicating good stability of both crystal forms; the results of the influencing factor test showed that ( Figure 5c, d), Crystal form IV ( Figure 5 c) The drug sample did not undergo crystal form change under high humidity and light conditions, but was unstable under high temperature conditions and transformed into crystal form III; crystal form V / amorphous ( Figure 5 d) The drug sample did not undergo crystal form change under high humidity and light conditions, but it was unstable under high temperature conditions and transformed into crystal form I.

[0169] High temperature test results show that ( Figure 6 a) The crystal form of the drug sample of crystal form I changes, and its diffraction pattern tends to be that of crystal form III, thus transforming into crystal form III; the crystal structure of the drug sample of crystal form III remains unchanged, and its stability is good; the crystal form of the drug sample of crystal form IV changes, and its diffraction pattern tends to be that of crystal form III, thus transforming into crystal form III; the crystal form of the drug sample of crystal form V / amorphous changes, and its diffraction pattern tends to be that of crystal form III, thus transforming into crystal form III.

[0170] The results of the grinding factor test show that ( Figure 6 (b) The crystal form of drug samples of crystal form I and crystal form III is stable and has not changed; the crystal form of drug sample of crystal form IV changes, and its diffraction pattern tends to be crystal form III, thus transforming into crystal form III; the crystal form of drug sample of crystal form V / amorphous changes, and its diffraction pattern tends to be crystal form I, thus transforming into crystal form I.

[0171] The results of the pressure condition test show that ( Figure 6 c) The crystal form stability of crystal form I, crystal form III, and crystal form V / amorphous drug samples is good and has not changed; the crystal form of crystal form IV drug samples changes, and their diffraction patterns tend to be crystal form III, transforming into crystal form III.

[0172] The results of the stirring test in water show that ( Figure 6 d) Crystal form I drug samples are stable at 40℃, but at 60℃, their diffraction patterns tend to be crystal form III, transforming into crystal form III; crystal form III drug samples have good crystal form stability and do not change; crystal form IV and crystal form V / amorphous drug samples undergo crystal form transformation, and their diffraction patterns tend to be crystal form III, transforming into crystal form III.

[0173] Based on the above results, the stability of different crystal forms of the drug and the transformation relationship between crystal forms are as follows:

[0174] Crystal form III is the most thermodynamically stable crystal form and retains its original crystal form under all experimental conditions; crystal form I transforms into crystal form III under high temperature environment and stirring in water (60℃); crystal form IV remains stable under high humidity and light influence conditions, but will transform into crystal form III under other experimental conditions; crystal form V / amorphous remains stable under high humidity, light influence and pressure conditions, transforms into crystal form I under grinding and high temperature (60℃) conditions, and transforms into crystal form III under stirring in water and high temperature (130℃) conditions.

[0175] Example 3: Investigation of drug inhibition of human lung cancer A549 cells and human breast cancer MCF-7 cells

[0176] Objective: To evaluate the antitumor activity of a drug through in vitro cell assays.

[0177] Cells: The human breast cancer cell line MCF-7 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the human lung cancer cells were donated by the Academy of Sciences of the Chinese People's Liberation Army.

[0178] Medicine: Self-made, purity over 99%.

[0179] Method: Take 8×10 4 Single-cell suspensions were seeded at a density of 100 μL per well in 96-well plates and cultured until cell adhesion reached approximately 60%. Drug treatment was then initiated. Six concentrations were established for A549 cells: 0, 4 μM, 8.5 μM, 10 μM, 13.5 μM, and 18 μM. Six concentrations were also established for MCF-7 cells: 0, 2 μM, 4 μM, 6 μM, 8.5 μM, and 10 μM. After 24 hours of treatment, the inhibitory effect of the drug on tumor cells was measured using the MTT assay, and the IC50 was calculated. 50 value.

[0180] Table 7. Results of the MTT assay on the inhibitory rate of the drug on MCF-7 and A549 cells (mean±sd, n=3)

[0181]

[0182] Results: Table 7 shows the inhibition rates of different drug concentrations on tumor cells after 24 hours of drug treatment. The IC50 of the drug on A549 cells was also analyzed. 50 The concentration was (9.65±1.50) μM, and the IC50 concentration for MCF-7 cells was [missing value]. 50 The concentration was (6.4±3.29) μM, indicating certain anti-breast cancer and anti-lung cancer activity.

[0183] Example 4: Efficacy evaluation of drug administration via gavage in treating a Poly(I:C) endotracheal drip-induced acute lung injury mouse model.

[0184] Animals: 78 healthy male SPF-grade C57BL / 6 mice, 6–8 weeks old, weighing 19–21 g.

[0185] Medicine: Self-made, purity over 99%.

[0186] Grouping: Animals were randomly divided into 6 groups according to body weight: control group (Con), model group (Mod), prednisolone acetate group (3 mg / kg, PA), and low, medium and high dose drug groups (50, 100 and 200 mg / kg).

[0187] Protocol: On day 1, mice in the model group and treatment group were administered Poly(I:C) solution (20 mg / kg) via intratracheal infusion, while mice in the control group were administered the corresponding volume of physiological saline via intratracheal infusion. Starting from day 2, the treatment group was administered the drug once daily by gavage for 7 consecutive days, while mice in the control group and model group were administered the corresponding volume of 0.5% CMC-Na solution by gavage. Within 24 hours after the last administration, blood was collected from the eyeballs of mice. After anticoagulation, the blood immune cells, including neutrophils, monocytes, lymphocytes, eosinophils, and basophils, were measured using a BC-5000 VET animal blood cell analyzer. The animals were euthanized and dissected. Mouse lung tissue was collected, fixed in 4% paraformaldehyde for 24 hours, dehydrated using standard methods, embedded in paraffin, sectioned (5 μm), stained with hematoxylin and eosin (HE), and mounted to prepare paraffin sections of mouse lung tissue. The pathological changes in mouse lung tissue were observed and photographed. Bronchoalveolar lavage fluid (BALF) was collected from mice for the detection of inflammatory factors.

[0188] Results: Blood immune cell assays showed that, compared with Con, the Mod group mice had significantly increased numbers of neutrophils, monocytes, lymphocytes, eosinophils, and basophils (P < 0.001, < 0.01, < 0.05, < 0.01, < 0.01, respectively). The PA group significantly reduced the levels of all cell types except monocytes. The high-dose drug group inhibited the growth of all the above cell types (see...). Figure 7 H&E staining results showed that, compared with the Con group, the Mod group mice exhibited significant inflammatory infiltration and pulmonary edema in their lung tissue, along with hyaline membrane formation, airway wall thickening, and luminal narrowing. The PA group and all drug administration groups (50, 100, and 200 mg / kg) effectively alleviated lung inflammation, with the high-dose drug showing comparable efficacy to the positive control drug (see [link to relevant documentation]). Figure 8 The results of BALF inflammatory factor detection showed that, compared with the Con group, the levels of TNF-α, IL-1β, IL-6, IL-17A, and IFN-γ in the BALF of the Mod group mice were significantly increased (P < 0.001). After administration of PA and medium and high dose drug groups (100, 200 mg / kg), the levels of each index were significantly decreased (see...). Figure 9 The medium and high doses of the drug are comparable to the positive control drug.

[0189] Example 5: Efficacy evaluation of nasal administration of drug inclusion complex liquid formulation for treating LPS-induced acute lung injury model in mice.

[0190] Animals: 60 healthy male SPF-grade C57BL / 6 mice, 6-8 weeks old, weighing 19-21 g.

[0191] Drug: The liquid formulation of the drug inclusion complex obtained in Example 11 was used, and the administration method was nasal drops.

[0192] Grouping: Animals were randomly divided into 5 groups according to body weight: control group (Con), model group (Mod), budesonide group (0.5 mg / kg, Bud), low-dose drug group and high-dose drug group (Drug-CD-ND-L at 5 mg / kg and Drug-CD-ND-H at 10 mg / kg).

[0193] Protocol: On day 1, mice in the model group and the treatment group were intranasally instilled with LPS solution (10 mg / kg), while mice in the control group were intranasally instilled with the corresponding volume of physiological saline. Starting from day 2, the treatment group received intranasal administration twice daily (morning and afternoon) for 7 consecutive days, while mice in the control and model groups were administered the corresponding volume of physiological saline by gavage. After the last administration, the animals were sacrificed and dissected within 24 hours. Lung tissue was collected and its wet and dry weights were calculated. The lung tissue was fixed in 4% paraformaldehyde for 24 hours, dehydrated using standard methods, embedded in paraffin, sectioned (5 μm), stained with hematoxylin and eosin (HE), and mounted. Paraffin sections of mouse lung tissue were prepared, and the pathological changes in the mouse lung tissue were observed and photographed. Bronchoalveolar lavage fluid (BALF) and lung tissue homogenate were collected for the detection of inflammatory factors.

[0194] Results: Lung wet-dry weight results showed that, compared with the Con group, the lung wet-dry weight level in the Mod group was significantly increased, and all treatment groups effectively reduced lung wet-dry weight (see...). Figure 8 H&E staining results showed that, compared with the Con group, the Mod group had severe alveolar structure destruction, alveolar structure disappearance, large-area inflammatory cell infiltration, and significant tissue inflammation; compared with the Mod group, the Bud group, low-dose and high-dose administration groups could alleviate lung injury to varying degrees, restore normal alveolar structure, and reduce inflammatory cell infiltration (see...). Figure 8 The results of BALF and lung tissue inflammatory factor detection showed that, compared with the Con group, the levels of inflammatory factors in BALF and lung tissue were significantly increased in the Mod group (P < 0.001). Compared with the Mod group, the levels of TNF-α, IL-6, and IL-1β in BALF and lung tissue of the Bud group, low-dose group, and high-dose group were significantly decreased (P < 0.001). The high-dose drug was comparable to the positive control drug (see...). Figure 11 ).

[0195] Example 6: Pharmacodynamic evaluation of a mouse model of chronic obstructive pulmonary disease induced by intratracheal instillation of LPS combined with cigarette smoke administration via gavage.

[0196] Animals: KM mice, male, SPF grade, 6-8 weeks old, 78 in total.

[0197] Drug: The solid dispersion of the drug obtained in Example 10 was used, and the administration method was gavage.

[0198] Grouping: Animals were randomly divided into 6 groups according to body weight: control group (Con), model group (Mod), dexamethasone group (1 mg / kg, DEX), drug solid dispersion (Drug-SD) low and high dose groups (100, 200 mg / kg), and original drug group (200 mg / kg).

[0199] Protocol: A mouse model of chronic obstructive pulmonary disease (COPD) was established by intratracheal instillation of LPS combined with cigarette smoke induction. Mice were instilled with LPS solution (1.25 mg / kg) via intratracheal instillation on days 1 and 15. From days 2 to 14 and days 16 to 28, mice were placed in a smoke exposure chamber with a total particulate smoke concentration controlled at 300 mg / m³. 3 The mice were administered medication by gavage for 1 hour each time, twice a day, 6 days a week for 4 weeks. Starting on day 29, each treatment group was given the medication by gavage for 7 days, while the model group was given 0.5% CMC-Na, with a gavage volume of 0.1 mL / 10 g, once a day. After the administration, the lung function of each group of mice was tested using a PFT pulmonary function testing instrument. Then, the animals were sacrificed and dissected, and the wet and dry weights of the lung tissue were calculated. The lung tissue was fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, sectioned (5 μm), stained with HE and Masson staining, and mounted. Paraffin sections of mouse lung tissue were prepared, and the pathological changes of mouse lung tissue were observed and photographed. Morphological quantitative analysis was performed on HE and Masson pathological sections, including inflammation score, mean alveolar septum (mLI), mean alveolar number (MAN), and collagen deposition. The bronchoalveolar lavage fluid (BALF) of mice was used to detect inflammatory factors.

[0200] Results: Lung function tests in mice showed that, compared with the Con group, the FEV1 of the Mod group mice was significantly lower. 0.1 Forced vital capacity (FVC), forced vital capacity (FVC), and dynamic lung compliance (Cydn) were significantly decreased, while total lung capacity (TLC), functional residual capacity (FRC), and airway resistance (RI) were significantly increased. This indicates that airway obstruction in mice leads to airflow limitation, increased airway resistance, decreased compliance, and deterioration of lung function, consistent with the pathological characteristics of a COPD model. Low and high doses of the drug solid dispersion significantly increased FEV1 in mice. 0.1 / FVC, the high-dose group also significantly increased Cydn and FVC, and significantly reduced TLC, FRC and RI, while the original drug group only significantly improved FEV1. 0.1 / FVC and TLC, DEX only significantly improves FEV 0.1 / FVC, FVC, and TLC, the solid dispersion of the drug showed better improvement in lung function than the original drug and the positive control drug dexamethasone (see [link]). Figure 12 ).

[0201] Compared to the Con group, the lungs of the Mod group were inflated, congested, swollen, and less elastic. All treatment groups showed improvement in lung tissue damage. Compared to the Con group, the lung index of the Mod group was significantly increased. Both the 200 mg / kg drug solid dispersion and the original drug significantly reduced the lung index (see...). Figure 13 a, b).

[0202] HE staining results showed that, compared with the Con group, the lung tissue of the Mod group mice exhibited bronchial wall thickening and damage, alveolar enlargement, alveolar wall destruction, and obvious inflammatory cell infiltration in the alveolar septa. All drug-treated groups alleviated these pathological manifestations. Compared with the Mod group, all drug-treated groups significantly reduced inflammation scores and mLI. The drug solid dispersion had a better effect on mLI than the original drug and dexamethasone. The drug solid dispersion group increased MAN (see...). Figure 13 c, d, e, g, h).

[0203] Masson staining results showed that, compared with the Con group, the Mod group exhibited significant peri-airway collagen deposition and inflammatory cell infiltration, with a significantly increased collagen deposition area. All drug administration groups effectively alleviated the aforementioned pathological features. The high-dose group of the drug solid dispersion significantly reduced the collagen deposition area, superior to the original drug and dexamethasone (see...). Figure 13 f, i).

[0204] The results of inflammatory factor assays showed that, compared with the Con group, the Mod group showed significant increases in TNF-α, IL-6, and IL-1β; compared with the Mod group, both the drug solid dispersion and the original drug group significantly inhibited the increase of TNF-α and IL-1β; the high-dose drug solid dispersion group significantly inhibited the increase of IL-6, while DEX only significantly inhibited the increase of IL-1β. The anti-inflammatory effect of the high-dose drug solid dispersion was superior to that of DEX (see...). Figure 14 ).

[0205] Example 7: Pharmacodynamic evaluation of a rat model of osteoarthritis constructed by intra-articular injection of drug microspheres for anterior cruciate ligament transection combined with medial meniscectomy.

[0206] Animals: 36 healthy male SPF-grade SD rats, 8 weeks old, weighing approximately 200-220 g.

[0207] Drug: The drug microspheres obtained in Example 12 were dispersed in physiological saline and administered via intra-articular injection.

[0208] Grouping: Animals were randomly divided into 6 groups according to body weight: sham operation group (Sham), model group (Mod), triamcinolone group (TA, 0.060 mg / 100 μL), low, medium and high dose microsphere groups (LMPs 0.055 mg / 100 μL, MMPs 0.11 mg / 100 μL, HMPs 0.22 mg / 100 μL).

[0209] Protocol: Except for the Sham group, all rats in other groups underwent anterior cruciate ligament transection combined with medial meniscectomy to establish a rat osteoarthritis model. Week 0 was defined as the completion of the surgery. During week 1, all treatment groups received intra-articular injections of 100 μL per rat, while the Mod group received the same volume of saline intra-articularly. The experiment ended at week 8. After euthanasia by cervical dislocation, the right hind limb of each rat was dissected, and the skin and muscles around the joint were removed and placed in a neutral fixative. Three rats were randomly selected, and their knee joints were scanned using a Quantum GX2 Micro-CT scanner. The scanner parameters were: voltage 50 kV, current 100 μA, and resolution 36 μm / pixel. After scanning, three-dimensional reconstruction and bone parameter analysis of the tibial plateau were performed. Specific parameters measured included bone volume (BV), bone volume fraction (BV / TV), trabecular bone thickness (Tb.Th), trabecular bone pattern factor (Tb.Pf), subchondral bone plate thickness (SBP.Th), and osteophyte volume (OV).

[0210] Knee joints obtained from dissection were decalcified and embedded in paraffin for sectioning. Hematoxylin and eosin (H&E), safranin O and fast green (S&F) staining, and immunohistochemical staining were performed, including COL2, MMP-13, IL-6 and TNF-α. The sections were observed and photographed under a microscope, and the images were quantitatively analyzed by ImageJ.

[0211] A double-blind method was used to score rat knee joint tissue sections to assess the severity of cartilage damage. The scoring criteria followed the 2010 OARSI histopathology recommendations. The cartilage damage score was based on the area of ​​loss of cartilage matrix or chondrocytes, ranging from 0 to 5, with higher scores reflecting more severe cartilage damage.

[0212] Results: Compared with the Sham group, the Mod group showed increased joint surface roughness and a large number of osteophytes. Coronal views showed erosion and thinning of the subchondral bone plate structure, disordered trabecular bone network and increased porosity, indicating that the osteoarthritis model was successfully constructed. The joint roughness of each drug-treated group was significantly improved and the loss of subchondral bone plate was reduced. Quantitative analysis showed that compared with the Sham group, the Mod group had significantly reduced bone volume and volume fraction (P < 0.05), indicating that this stage was in the early stage of osteoarthritis, with increased bone resorption and severe bone loss. Simultaneously, decreased trabecular thickness, increased pattern factor, and reduced subchondral bone plate thickness indicated damage to the subchondral bone microstructure, disruption of the structural network, and decreased bone strength and stability. In the TA group and the low-dose drug microsphere group, joint surface roughness was significantly reduced, osteophyte formation was decreased, and the trend of subchondral bone plate thinning and loss could be reversed. However, bone loss and trabecular pathological changes were not significantly improved. The medium- and high-dose drug microsphere groups, in addition to reducing osteophyte formation and subchondral bone damage, also improved bone loss and trabecular pathological changes, and enhanced bone strength and stability (P < 0.01) (see...). Figure 15 ).

[0213] Compared to the Sham group, the Mod group showed severe damage to cartilage and subchondral bone, significant loss of articular cartilage layer, disordered structure, abnormal chondrocyte morphology, reduced number, and synovial invasion. The TA group and the low-dose drug microsphere group could inhibit synovial invasion and improve cartilage loss and subchondral bone damage to some extent; the medium- and high-dose drug microsphere groups significantly promoted chondrocyte proliferation, more orderly arrangement, and a more intact cartilage layer. Statistical analysis of the scores showed that the medium- and high-dose drug microsphere groups significantly reduced cartilage damage scores, superior to the TA group (see [link to TA]). Figure 16 ).

[0214] Compared with the Sham group, the Mod group showed a significant decrease in COL2 expression and a significant upregulation of MMP-13, IL-6, and TNF-α expression. The TA group and the low- and medium-dose drug microsphere groups significantly inhibited the expression of MMP-13, IL-6, and TNF-α. The high-dose drug microsphere group further significantly increased COL2 expression, showing superior performance compared to the TA group (see [link to TA group]). Figure 17 ).

[0215] The above description is only a preferred embodiment of the present invention and is 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 pharmaceutical composition, characterized in that: It contains one or more of the following crystal forms: 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone; The crystalline forms of the 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone include crystalline form I, crystalline form II, crystalline form III, crystalline form IV, and amorphous form.

2. The pharmaceutical composition according to claim 1, characterized in that: The crystal form I, determined by X-ray powder diffraction, has characteristic peaks with a relative peak height ≥ 5%. The specific positions, diffraction angles (2θ), and relative peak heights of these characteristic diffraction peaks are shown below: ； The crystal form II, as determined by X-ray powder diffraction, has characteristic peaks with a relative peak height ≥ 5%. The specific positions, diffraction angles (2θ), and relative peak heights of these characteristic diffraction peaks are shown below: ； The crystal form III, as determined by X-ray powder diffraction, has characteristic peaks with a relative peak height ≥5%. The specific positions, diffraction angles (2θ), and relative peak heights of these characteristic diffraction peaks are shown below: ； The crystal form IV, determined by X-ray powder diffraction, has characteristic peaks with a relative peak height ≥ 5%. The specific positions, diffraction angles (°), and relative peak heights of these characteristic diffraction peaks are shown below: ； The amorphous material was measured using X-ray powder diffraction, and the diffraction pattern showed diffuse, irregular ridges without sharp diffraction peaks.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that: Crystal form I was prepared by rapid evaporation via atomization. 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone was dissolved in ethanol under stirring, followed by rapid evaporation via atomization to obtain a solid powder. The operating parameters for rapid evaporation were: atomization pressure of 0.15 MPa, solution flow rate of 5 mL / min, nitrogen gas as the evaporation medium, and a flow rate of 0.4 m³ / min. 3 / min, inlet temperature 100℃.

4. The pharmaceutical composition according to claim 1 or 2, characterized in that: The crystal form II is prepared by cooling crystallization or dissolution crystallization. The cooling crystallization method involves dissolving 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone in heated tetrahydrofuran, then cooling to 4°C to precipitate crystals, filtering and collecting, and drying at 40°C to obtain the final product. The solution-precipitation crystallization method involves dissolving 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone in a main solvent, then adding it to a stirred precipitation solvent. The precipitated solid is collected by filtration and dried at 40°C to obtain the final product. The main solvent is tetrahydrofuran, and the precipitation solvent is n-hexane or purified water. The volume ratio of the main solvent to the precipitation solvent is 1:1 to 1:

10.

5. The pharmaceutical composition according to claim 1 or 2, characterized in that: The crystal form III is obtained by any one of the following methods: cooling crystallization, dissolution crystallization, and slow solvent evaporation crystallization. The steps of the cooling crystallization method are as follows: 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone is dissolved in a hot solvent, and then the resulting solution is cooled to 4°C to precipitate crystals. The crystals are collected by filtration and dried at 40°C to obtain the final product. The solvent is selected from any one of n-hexane, diethyl ether, dichloromethane, n-butanol, ethanol, ethyl acetate, isopropanol, dioxane, acetonitrile, methanol, and dimethyl sulfoxide. The steps of the solution-precipitation crystallization method are as follows: 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone is dissolved in a main solvent, and then the resulting solution is added to a stirred precipitation solvent, a solid is precipitated, filtered and collected, and dried at 40°C to obtain the product; the main solvent is selected from one of p-xylene, dichloromethane, ethanol, ethyl acetate, dioxane, acetonitrile, methanol and dimethyl sulfoxide; the precipitation solvent is selected from n-hexane or purified water; The steps of the solvent slow evaporation crystallization method are as follows: 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone is dissolved in a solvent, placed at room temperature, and the solvent is slowly evaporated to precipitate a solid. The solid is collected and dried at 40°C to obtain the product. The solvent is selected from any one of dichloromethane, ethanol, and acetonitrile.

6. The pharmaceutical composition according to claim 1 or 2, characterized in that: The crystal form IV was prepared by a cooling crystallization method; The steps of the cooling crystallization method are as follows: 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone is stirred and dissolved in hot acetone, then cooled to 4°C to precipitate crystals, which are then collected by filtration and dried at 40°C to obtain the final product.

7. The pharmaceutical composition according to claim 1 or 2, characterized in that: The amorphous material was prepared by melt-quench crystallization method; The steps of the melt-cold crystallization method are as follows: 4-(acetoxy)-3-[2-[(1R,4aS,5R,6R,8aS)-6-(acetoxy)-5-[(acetoxy)methyl]-5,8a-dimethyl-2-methylene-1-decahydronaphthalene]ethylene]dihydro-2(3H)-furanone is melted at 140°C into a molten liquid, and then placed at -20°C for rapid cooling and solidification to obtain the final product.

8. A pharmaceutical preparation, characterized in that: The pharmaceutical composition comprising the claims 1 or 2.

9. The pharmaceutical preparation according to claim 8, characterized in that: The dosage form of the pharmaceutical preparation is solid, liquid, or semi-solid.

10. The use of the pharmaceutical composition of claim 1 or 2, or the pharmaceutical preparation of claim 8 or 9, in the preparation of anti-inflammatory and antitumor drugs.

Images