Long-acting microcrystalline pharmaceutical composition of relugolix and preparation method therefor

Abstract

Provided are a long-acting microcrystalline pharmaceutical composition of relugolix and a preparation method therefor. A relugolix raw material is subjected to a specific recrystallization process, and the obtained crystal has a uniform relative particle size and a good crystal habit morphology. The long-acting microcrystalline pharmaceutical composition of relugolix is administered by means of intramuscular injection, and can provide sustained release in vivo for 1 month or more, 2 months or more, 3 months or more, or even 4 months or more, which can effectively reduce dosing frequency, maintain a stable plasma concentration, improve clinical efficacy, and better meet clinical needs.

Description

A long-acting microcrystalline pharmaceutical composition of retinoic acid and its preparation method Technical Field

[0001] This invention relates to the field of pharmaceutical formulations, specifically to a long-acting microcrystalline pharmaceutical composition of retinoblastine and its preparation method. Background Technology

[0002] Relugolix, a thienopyrimidine compound, chemically named N-(4-(1-(2,6-difluorobenzyl)-5-((dimethylamino)methyl)-3-(6-methoxy-3-pyridazinyl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl)phenyl)-N'-methoxyurea, is a small molecule gonadotropin-releasing hormone (GnRH) receptor antagonist co-developed by Myovant and Takeda for the treatment and symptom relief of uterine fibroids and for the treatment of advanced prostate cancer in adult patients.

[0003] Currently, various crystalline compounds of regrugoli or its salts have been prepared. For example, WO2021069711A1 discloses a series of regrugoli crystalline compounds, including anhydrous, hemihydrate, and amorphous forms I and II, as well as solvates of tetrahydrofuran, toluene, anisole, isopropanol, dioxane, trifluorotoluene, trifluoroethanol, DMF, and acetone. Among them, the anhydrous form of regrugoli I (hereinafter referred to as crystalline form I) has strong stability. The specification also discloses a method for preparing crystalline form I: Form I of compound 1 can be prepared by dissolving form V of compound 1 in DMSO at a temperature of about 35℃±5℃. Then, ethanol is added to the mixture. Then, while maintaining the temperature of the mixture at about 35℃±5℃, the mixture is filtered and the solid is washed with ethanol. The mixture is stirred at about 35℃±5℃ for about one hour, cooled to about 25℃±5℃ and stirred for about 12 hours or more. The resulting precipitate provides form I of compound 1. Meanwhile, Example 8 of CN104703992B also discloses a method for preparing regrugoli crystal form I: Crystals of dimethyl sulfoxide (DMSO, 20 mL) and regrugoli (8.93 g) in a tetrahydrofuran solvate are added to a reactor and heated to an internal temperature of 35 ± 5 °C to dissolve the mixture. After confirming the mixture is dissolved, ethanol (20 mL) is added to it at an internal temperature of 35 ± 5 °C. The mixture is filtered through a dust filter and washed with ethanol (8 mL). Ethanol (112 mL) is added to the filtrate at an internal temperature of 35 ± 5 °C, and the mixture is stirred at an internal temperature of 35 ± 5 °C for 1 hour or longer, cooled to an internal temperature of 25 ± 5 °C, and stirred again at the same temperature for 12 hours or longer. The crystals are collected by filtration, washed with ethanol (16 mL), and dried under reduced pressure at an external temperature of 50 ± 10 °C to obtain regrugoli crystal form I (7.33 g, yield 91.6%) white crystals.

[0004] Although both methods can produce regoragline crystal form I, the inventors found, upon particle size analysis and electron microscopy, that the regoragline crystal form I products prepared according to the instructions had extremely uneven particle size distribution, numerous small particles, and irregular crystal shapes. This resulted in the following problems in formulation preparation: the formulation had a wide particle size distribution and contained many small particles, leading to a significant burst release phenomenon in animals. Initially, excessively high blood drug concentrations could cause adverse reactions, while later, low blood drug concentrations resulted in a short overall duration of drug concentration, failing to meet the goal of stable and slow release in vivo. Furthermore, the purchased regoragline crystal form I raw materials also exhibited the same problems of excessive small particles and irregular crystal shapes upon particle size analysis and electron microscopy, further affecting the pharmacokinetics of the formulation and the duration of effective blood drug concentration.

[0005] The currently marketed retinoglitazone is an oral formulation (trade name: Orgovyx), which requires daily administration. For patients requiring long-term medication, there may be missed doses, and the blood drug concentration fluctuates significantly after each dose, affecting drug efficacy and patient compliance. Therefore, there is still a need to develop a long-acting retinoglitazone formulation that reduces the frequency of administration and maintains a stable release of blood drug concentration. Summary of the Invention

[0006] This invention provides a long-acting microcrystalline drug composition for retinoglitazone, wherein the retinoglitazone raw material is subjected to a specific recrystallization process, resulting in crystals with relatively uniform particle size and good crystal morphology, which can achieve stable release of blood drug concentration, reduce the frequency of administration, improve efficacy and patient compliance, and better meet clinical needs.

[0007] This invention provides a long-acting microcrystalline pharmaceutical composition of regrugoline, comprising, by weight percentage: 40%-90% regrugoline, 1%-10% sodium carboxymethyl cellulose, 5%-30% mannitol, 0.1%-10% sodium dihydrogen phosphate, and 0.1%-10% disodium hydrogen phosphate. The regrugoline used is regrugoline crystal form I, with a particle size not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization is performed using the following method: [The text abruptly ends here, so the translation stops.] Methyl sulfoxide (DMSO) and rellugoline were added to a reaction flask and heated until the reaction solution became clear. The mixture was then filtered. The filtrate was heated to 50-55°C, and preheated ethanol was added. The mixture was stirred until a solid precipitated, and then an equal volume of preheated ethanol was added. The mass-volume ratio of rellugoline: DMSO: preheated ethanol was 1:1.5:18. The mixture was kept at this temperature and stirred for 120 min. Then, the temperature was lowered to 20-30°C and stirred for another 1 h. The mixture was then filtered, and the filter cake was washed with ethanol and dried under vacuum at 50°C to obtain the final product.

[0008] The relugoline long-acting microcrystalline drug composition provided by this invention uses relugoline crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization is performed using the following method: Dimethyl sulfoxide (DMSO) and relugoline are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55°C, and preheated ethanol is added and stirred until homogeneous. Twice the amount of preheated ethanol is added again, and the mixture is kept warm and stirred until solid precipitates. The same volume of preheated ethanol as the first addition is then added, wherein the mass-volume ratio of relugoline:DMSO:preheated ethanol is 1:1.5:18. The mixture is kept warm and stirred for 120 min, cooled to 20-30°C, and kept warm and stirred for 1 h. After filtration, the filter cake is washed with ethanol and then vacuum dried at 50°C to obtain the final product.

[0009] The relugoline long-acting microcrystalline drug composition provided by this invention uses relugoline crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization is performed using the following method: Dimethyl sulfoxide (DMSO) and relugoline are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55°C, preheated ethanol is added and stirred until homogeneous, then an equal volume of preheated ethanol and seed crystals are added and stirred until solid precipitates. The mass-volume ratio of relugoline: DMSO: preheated ethanol: seed crystals is 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature is lowered to 20-30°C and stirred again for 1 h. After filtration, the filter cake is washed with ethanol and then vacuum dried at 50°C to obtain the final product.

[0010] The relugoline long-acting microcrystalline drug composition provided by this invention uses relugoline crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization is performed using the following method: Dimethyl sulfoxide (DMSO) and relugoline are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55°C, and preheated ethanol is added and stirred until homogeneous under the heat-preserving condition. Preheated ethanol and seed crystals are added again and stirred until solid precipitates. Preheated ethanol is then added again, with the volume ratio of the three added preheated ethanols being 1:2:1, and the mass-volume ratio of relugoline: DMSO: preheated ethanol: seed crystals being 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature is lowered to 20-30°C and stirred again for 1 h. After filtration, the filter cake is washed with ethanol and then vacuum dried at 50°C to obtain the final product.

[0011] The relugoline long-acting microcrystalline pharmaceutical composition provided by the present invention, wherein the relugoline crystal form I has characteristic peaks at diffraction angles of approximately 7.6°, 9.1°, 10.1°, 12.2°, 15.0°, 16.7°, 17.4°, 18.8°, 19.5°, 20.1°, 22.2°, 22.8°, 26.7°, and 27.5° as indicated by X-ray powder diffraction.

[0012] The present invention provides a long-acting microcrystalline pharmaceutical composition of retinoglitazone, wherein the average particle size of retinoglitazone is 2-25 μm, preferably 3-15 μm, and more preferably 4-10 μm.

[0013] This invention provides a method for preparing a long-acting microcrystalline pharmaceutical composition of regrugolide, comprising the following steps: sequentially weighing purified water, sodium carboxymethyl cellulose, mannitol, sodium dihydrogen phosphate, and disodium hydrogen phosphate, and stirring to dissolve them to obtain a dispersion solvent; adding the weighed regrugolide raw material to the dispersion solvent for initial suspension to obtain an initial suspension; adding zirconium oxide grinding beads to the grinding mill cavity, setting an appropriate grinding mill speed and feed rate, and circulating and grinding the initial suspension until the average particle size of regrugolide is 2-25 μm; then filling the mixture into vials at an appropriate volume for freeze-drying; characterized in that: the regrugolide... Relugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape, was prepared by recrystallization using the following method: Dimethyl sulfoxide (DMSO) and relugoli were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55℃, and preheated ethanol was added. The mixture was stirred until solid precipitated, and then an equal volume of preheated ethanol was added. The mass-volume ratio of relugoli:DMSO:preheated ethanol was 1:1.5:18. The mixture was kept at this temperature and stirred for 120 min, then cooled to 20-30℃ and stirred again for 1 h. After filtration, the filter cake was washed with ethanol and dried under vacuum at 50℃ to obtain the final product.

[0014] The preferred method for preparing the long-acting microcrystalline pharmaceutical composition of regrugoli provided by the present invention is as follows: the regrugoli used is regrugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. The following preparation method is used for recrystallization: dimethyl sulfoxide (DMSO) and regrugoli are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55°C, preheated ethanol is added and stirred evenly, and twice the amount of preheated ethanol is added again and stirred while maintaining the temperature until solid precipitates. The same volume of preheated ethanol as the first addition is added, wherein the mass-volume ratio of regrugoli: DMSO: preheated ethanol is 1:1.5:18, and the mixture is stirred while maintaining the temperature for 120 min. The temperature is then lowered to 20-30°C and stirred while maintaining the temperature for 1 h. The mixture is filtered, the filter cake is washed with ethanol, and then dried under vacuum at 50°C to obtain the final product.

[0015] The preferred method for preparing the long-acting microcrystalline pharmaceutical composition of regrugoli provided by the present invention is as follows: the regrugoli is regrugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular block shape. Recrystallization is performed using the following method: dimethyl sulfoxide (DMSO) and regrugoli are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55°C, preheated ethanol is added and stirred evenly, and then an equal volume of preheated ethanol and seed crystals are added and stirred until solid precipitates. The mass-volume ratio of regrugoli: DMSO: preheated ethanol: seed crystals is 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature is lowered to 20-30°C and stirred again for 1 h. After filtration, the filter cake is washed with ethanol and then vacuum dried at 50°C to obtain the final product.

[0016] The preferred method for preparing the long-acting microcrystalline pharmaceutical composition of regrugoli provided by the present invention is as follows: the regrugoli used is regrugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization is performed using the following method: dimethyl sulfoxide (DMSO) and regrugoli are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55°C, and preheated ethanol is added and stirred evenly under the heat-preserving condition. Preheated ethanol and seed crystals are added again and stirred until solid precipitates. Preheated ethanol is added again, wherein the volume ratio of the three added preheated ethanol is 1:2:1, and the mass-volume ratio of regrugoli: DMSO: preheated ethanol: seed crystals is 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature is lowered to 20-30°C and stirred again for 1 h. After filtration, the filter cake is washed with ethanol and then vacuum dried at 50°C to obtain the final product.

[0017] The present invention provides a method for preparing a long-acting microcrystalline pharmaceutical composition of retinoblastine, wherein: the particle size of the grinding beads is 0.3-3 mm, the grinding mill speed is 900-3000 rpm, and the feeding speed is 1000-3000 ml / min.

[0018] This invention provides the use of a long-acting microcrystalline pharmaceutical composition of retinoblastine in the preparation of a medicament for the treatment of uterine fibroids and advanced prostate cancer in adult patients.

[0019] The long-acting microcrystalline pharmaceutical composition of retinoblastine provided by the present invention is administered by intramuscular injection at a dose of 200-1200 mg, preferably 300-1000 mg, and more preferably 400-800 mg.

[0020] The long-acting microcrystalline drug composition of retinoblastine provided by the present invention can be continuously released in vivo for at least 1 month, or more than 2 months, more than 3 months, or even more than 4 months, which can effectively reduce the frequency of administration, maintain stable blood drug concentration, improve clinical efficacy, and better meet clinical needs.

[0021] In the process of developing a long-acting microcrystalline drug composition of regoragrib, the inventors discovered that the regoragrib active pharmaceutical ingredient (API) has a significant impact on the preparation of the formulation. This impact extends beyond the API's crystal form itself; it also includes specific crystal morphology and particle size distribution. Through extensive experimental research, the inventors ultimately determined that the specific recrystallization process provided in this application yields regoragrib API of crystal form I with relatively regular blocky crystals. The resulting regoragrib particles are all at least 2 μm in size and exhibit relatively uniform particle size. Long-acting microcrystalline formulations of regoragrib prepared using the regoragrib API provided by this invention exhibit stable drug release and a long duration of effective blood drug concentration. Attached Figure Description

[0022] Figure 1. X-ray powder diffraction pattern of Regulus V crystal form obtained in Experiment 1.

[0023] Figure 2. X-ray powder diffraction pattern of Regulugoli crystal form I obtained from Experiment Example 1.

[0024] Figure 3. Electron micrograph of Regulus I obtained from Experiment 1.

[0025] Figure 4. X-ray powder diffraction pattern of Regulugoli crystal form I obtained from Experiment Example 2.

[0026] Figure 5. Electron micrograph of Regulus I obtained from Experiment 2.

[0027] Figure 6. Axis powder diffraction pattern of the product obtained in Experiment Example 3.

[0028] Figure 7. Electron micrograph of product A obtained from Experiment Example 3.

[0029] Figure 8. BX-ray powder diffraction pattern of the product obtained in Experiment Example 3.

[0030] Figure 9. Electron micrograph of product B obtained from Experiment Example 3.

[0031] Figure 10. CX-ray powder diffraction pattern of the product obtained in Experiment Example 3

[0032] Figure 11 Electron micrograph of product C obtained in Experiment Example 3

[0033] Figure 12. DX-ray powder diffraction pattern of the product obtained in Experiment Example 3

[0034] Figure 13 Electron micrograph of product D obtained in Experiment Example 3

[0035] Figure 14. EX-ray powder diffraction pattern of the product obtained in Experiment Example 3

[0036] Figure 15 Electron micrograph of product E obtained from Experiment Example 3

[0037] Figure 16. FX-ray powder diffraction pattern of the product obtained in Experiment Example 4

[0038] Figure 17 Electron micrograph of product F obtained in Experiment Example 4

[0039] Figure 18. GX-ray powder diffraction pattern of the product obtained in Experiment Example 4

[0040] Figure 19 Electron micrograph of product G obtained from Experiment Example 4

[0041] Figure 20. HX-ray powder diffraction pattern of the product obtained in Experiment Example 4

[0042] Figure 21 Electron micrograph of product H obtained in Experiment Example 4

[0043] Figure 22. IX-ray powder diffraction pattern of the product obtained in Experiment Example 4

[0044] Figure 23 Electron micrograph of product I obtained from Experiment Example 4

[0045] Figure 24. X-ray powder diffraction pattern of the product obtained in Experiment Example 4.

[0046] Figure 25 Electron micrograph of product J obtained in Experiment Example 4

[0047] Figure 26. KX-ray powder diffraction pattern of the product obtained in Experiment 5.

[0048] Figure 27 LX-ray powder diffraction pattern of the product obtained in Experiment Example 5

[0049] Figure 28 Electron micrograph of product L obtained in Experiment Example 5

[0050] Figure 29. MX-ray powder diffraction pattern of the product obtained in Experiment 5.

[0051] Figure 30 Electron micrograph of product M obtained from Experiment Example 5

[0052] Figure 31 N-X powder diffraction pattern of the product obtained in Experiment Example 6

[0053] Figure 32 Electron micrograph of product N obtained from Experiment Example 6

[0054] Figure 33. Oxy-ray powder diffraction pattern of the product obtained in Experiment Example 6

[0055] Figure 34 Electron micrograph of product O obtained from Experiment Example 6

[0056] Figure 35. PX-ray powder diffraction pattern of the product obtained in Experiment Example 6

[0057] Figure 36 Electron micrograph of product P obtained from Experiment 6.

[0058] Figure 37 shows the QX-ray powder diffraction pattern of the product obtained in Experiment 6.

[0059] Figure 38 Electron micrograph of product Q obtained in Experiment Example 6

[0060] Figure 39 Release curves of long-acting retinoic acid microcrystalline formulations with different particle sizes in rats in Experiment 1.

[0061] Figure 40 Release curves of long-acting retinoic acid microcrystalline formulations with different particle sizes in dogs in Experiment 2.

[0062] Figure 41 Release curves of long-acting retinoic acid microcrystalline formulations with different particle sizes in monkeys in Experiment 3.

[0063] Figure 42. Electron micrograph of purchased regulus crystal type I in Experiment Example 4.

[0064] Figure 43 shows the X-ray powder diffraction pattern of the purchased rilugor crystal form in Experiment Example 4.

[0065] Figure 44. Experimental Example 4: Long-acting microcrystalline formulations of reglugoli prepared from different sources of reglugoli crystal form I raw materials in rats.

[0066] Release curve in the body Detailed Implementation

[0067] To better understand the present invention, the present invention will be further illustrated below with reference to specific embodiments, but the content of the present invention is not limited thereto.

[0068] Example 1: Preparation of Relugoli Crystal Form I

[0069] Toluene (200 ml) and a tetrahydrofuran solvate of 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothiopheno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (4.0 g, 1 eq) were added to a reaction flask. The mixture was heated to 55±5 °C. The reaction solution became turbid. The mixture was stirred at this temperature for 5 h, then cooled to 20±5 °C and stirred at this temperature for 2 h or longer. The mixture was filtered, and the filter cake was dried under vacuum at 50 °C to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo- A white solid, 3.90 g, 97.5% yield, of toluene solvate of 1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea, was detected by XRD as crystal form V. Characteristic diffraction peaks were observed at 2θ angles: 7.412°, 7.740°, 9.273°, 10.753°, 11.340°, 15.493°, 16.332°, 16.848°, 16.973°, 18.398°, 18.892°, 20.057°, 21.371°, 21.655°, 23.907°, 24.152°, and 27.085°. The X-ray powder diffraction pattern is shown in Figure 1.

[0070] Dimethyl sulfoxide (DMSO, 4.5 ml) and the toluene solvate of 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (2.0 g, 1 eq) were added to a reaction flask and heated to 50 °C. The reaction solution became clear, and ethanol (4 ml) was added. 5 ml), stir well, filter, wash with ethanol (6 ml), add ethanol (21.0 ml) to the filtrate at 50±5℃, keep warm and stir for 1 h, a white solid precipitates in the reaction solution, cool to 20±5℃, keep warm and stir for 12 h, filter, wash the filter cake with ethanol (8 ml), dry the filter cake under vacuum at 50℃ to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazine-3- A white solid, 1.55 g (77.5% yield), of [2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea, was obtained. XRD analysis showed it to be crystal form I, with characteristic diffraction peaks at 2θ angles: 7.697°, 9.245°, 10.243°, 11.668°, 12.400°, 15.174°, 16.905°, and 17.6°. The X-ray powder diffraction patterns are shown in Figure 2, and the electron microscope images are shown in Figure 3. The obtained Relugoli crystal form I particle sizes are: D10 = 8.2 μm, D50 = 98.7 μm, and D90 = 275 μm.

[0071] As can be seen from the electron micrograph, the size distribution of Relugoli I crystals obtained by this recrystallization process is extremely uneven, with a large number of fine fragments and irregular crystal shapes.

[0072] Example 2: Preparation of Relugoli Crystal Form I

[0073] Acetonitrile (70 ml, 3.5 ml / g), water (180 ml, 9 ml / g), and 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (20 g, 1 eq) were added to a reaction flask and heated to 50±5℃. Concentrated hydrochloric acid (5 ml) was added until the reaction solution became clear and the pH was 4. Triethylamine (10 ml) was added to adjust the pH to 8-9. A large amount of white solid precipitated in the reaction solution. Water (200 ml, 10 ml / g) was added dropwise. After the addition was complete, the temperature was lowered to 20±5℃ and kept warm. After crystallization for 1 hour, the mixture was filtered, and the filter cake was washed with water (32 ml) and acetonitrile (8 ml). The wet product was transferred to a reaction flask, and tetrahydrofuran (100 ml) and water (0.6 ml) were added. The mixture was heated to 50 ± 5 °C and stirred for 1 hour. The mixture was then cooled to 5 ± 5 °C and stirred for 2 hours. After filtration, the filter cake was washed with tetrahydrofuran (20 ml) and dried under vacuum at 50 °C to obtain a tetrahydrofuran solvate of 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (19.0 g, yield 95.0%).

[0074] Dimethyl sulfoxide (DMSO, 4.5 ml) and the tetrahydrofuran solvate of 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (2.0 g, 1 eq) were added to a reaction flask and heated to 40 °C. The reaction solution became clear, and ethanol was added. (4.5 ml), stir well, filter, wash with ethanol (6 ml), add ethanol (21.0 ml) to the filtrate at 40±5℃, keep warm and stir for 1 h, a white solid precipitates in the reaction solution, cool to 20±5℃, keep warm and stir for 12 h, filter, wash the filter cake with ethanol (8 ml), dry the filter cake under vacuum at 50℃ to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridyl)] A white solid, 1.34 g (67.0% yield), of azinon-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea, was determined by XRD to be crystal form I. Characteristic diffraction peaks were observed at 2θ angles: 7.580°, 9.112°, 10.109°, 11.538°, 12.259°, 15.036°, and 16.768°. The X-ray powder diffraction patterns are shown in Figure 4, and the electron microscope images are shown in Figure 5. The obtained Relugoli crystal form I particle sizes are: D10 = 3.70 μm, D50 = 81.1 μm, and D90 = 198 μm.

[0075] As can be seen from the electron micrograph, the size distribution of Relugoli I crystals obtained by this recrystallization process is extremely uneven, with a large number of fine fragments and irregular crystal shapes.

[0076] Experimental Example 3: Preparation of Rellugoli Crystal Form I at Different Crystallization Temperatures

[0077] Dimethyl sulfoxide (DMSO, 25 ml), anhydrous ethanol (EtOH, 25 ml), and 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (10.0 g, 1 eq) were added to a reaction flask. The mixture was heated until the reaction solution became clear. After cooling to the appropriate temperature, 175 ml of preheated ethanol and 20 mg of crystal form I were added. The mixture was stirred until homogeneous and kept at a constant temperature until solid precipitates. The cooling temperature and stirring time are shown in Table 1. The mixture was then cooled to 20-30℃ and stirred for 1 hour. After filtration, the filter cake was washed with ethanol (20 ml) and dried under vacuum at 50℃ to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea.

[0078] Table 1. Recrystallized products at different cooling temperatures and stirring times.

[0079] Product A, as determined by XRD analysis, is a mixed crystal of crystal form I and crystal form II. Characteristic diffraction peaks are observed at the 2θ angle: 7.2°, 13.1°, and 15.7° are the main characteristic peaks of crystal form II, while 7.4°, 8.9°, 12.1°, 16.6°, and 17.3° are characteristic peaks of crystal form I. The X-ray powder diffraction pattern is shown in Figure 6, and the electron micrograph is shown in Figure 7. The product exhibits flat, elongated crystals with particle sizes of D10 (6.32 μm), D50 (66.0 μm), and D90 (438 μm).

[0080] XRD analysis of Product B showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.523°, 9.063°, 10.045°, 12.202°, ​​14.980°, 16.681°, 17.418°, 18.811°, 19.474°, 20.122°, 22.062°, 22.265°, 22.861°, 23.082°, 26.678°, 27.482°, 29.042°, 29.735°. The X-ray powder diffraction pattern is shown in Figure 8, and the electron micrograph is shown in Figure 9. It is a massive crystal with particle sizes of D10 of 45.2 μm, D50 of 96.0 μm, and D90 of 166 μm.

[0081] XRD analysis of product C showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.496°, 9.017°, 10.006°, 12.174°, 14.955°, 16.686°, 17.393°, 18.747°, 19.466°, 20.066°, 21.909°, 22.252°, 22.817°, 23.031°, 26.632°, 27.434°, 28.984°, 29.710°. The X-ray powder diffraction pattern is shown in Figure 10, and the electron micrograph is shown in Figure 11. It is a massive crystal with particle sizes of D10 of 61.4 μm, D50 of 121 μm, and D90 of 242 μm.

[0082] XRD analysis of product D showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.559°, 9.077°, 10.091°, 12.240°, 15.008°, 16.687°, 17.456°, 18.825°, 19.523°, 20.160°, 22.094°, 22.317°, 22.902°, 26.726°, 27.509°, 29.057°, 29.773°. The X-ray powder diffraction pattern is shown in Figure 12, and the electron micrograph is shown in Figure 13. It is a massive crystal with particle sizes of D10 (77.7 μm), D50 (129 μm), and D90 (210 μm).

[0083] XRD analysis of product E showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.492°, 9.041°, 10.009°, 12.164°, 14.949°, 16.661°, 17.384°, 18.750°, 19.448°, 20.085°, 21.957°, 22.248°, 22.812°, 22.895°, 26.634°, 27.462°, 29.007°, 29.705°. The X-ray powder diffraction pattern is shown in Figure 14, and the electron micrograph is shown in Figure 15. It is a massive crystal with particle sizes of D10 of 70.5 μm, D50 of 128 μm, and D90 of 225 μm.

[0084] Results: The above experimental results show that the crystallization temperature affects the crystal form of the obtained product: a crystallization temperature of 30℃ produces a mixed crystal product; crystallization temperatures of 40℃, 50℃, and 60℃ all yield crystal form I products, and the particle size of the product increases with increasing temperature, with product yields around 85%. To reduce the risk of mixed crystal formation after process scale-up, a crystallization temperature of 40–60℃ is selected, preferably 50–60℃.

[0085] Experimental Example 4: Relugoli Crystal Form I Obtained with Different Solvent Ratios

[0086] The experimental procedure is the same as in Experiment Example 3. The different solvent ratios, whether seed crystals were added, and the experimental results are shown in Table 2.

[0087] Table 2. Relugoli crystal form I obtained with different solvent ratios.

[0088] XRD analysis of product F showed it to be crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.563°, 9.122°, 10.077°, 12.170°, 14.923°, 16.581°, 17.399°, 18.778°, 19.351°, 20.124°, 22.063°, 22.296°, 22.822°, 26.636°, 27.403°, 28.997°, 29.661°. The X-ray powder diffraction pattern is shown in Figure 16, and the electron micrograph is shown in Figure 17. It is a massive crystal with particle sizes of D10 (103 μm), D50 (182 μm), and D90 (300 μm).

[0089] XRD analysis of product G showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.544°, 9.096°, 10.054°, 12.229°, 15.013°, 16.620°, 17.438°, 18.819°, 19.503°, 20.150°, 22.092°, 22.294°, 22.827°, 26.741°, 27.485°, 29.047°, 29.730°. The X-ray powder diffraction pattern is shown in Figure 18, and the electron micrograph is shown in Figure 19. It is a massive crystal with particle sizes of D10 (98.5 μm), D50 (169 μm), and D90 (294 μm).

[0090] XRD analysis of product H showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.870°, 9.345°, 10.282°, 12.391°, 15.139°, 16.860°, 17.581°, 18.893°, 19.618°, 20.276°, 21.489°, 22.186°, 22.978°, 26.753°, 27.611°, 29.138°, 29.829°. The X-ray powder diffraction pattern is shown in Figure 20, and the electron micrograph is shown in Figure 21. It is a massive crystal with particle sizes of D10 (85.7 μm), D50 (129 μm), and D90 (190 μm).

[0091] XRD analysis of Product I showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.634°, 9.130°, 10.102°, 12.247°, 15.032°, 16.717°, 17.456°, 18.806°, 19.533°, 20.136°, 21.347°, 22.093°, 22.891°, 26.736°, 27.447°, 29.014°, 29.786°. The X-ray powder diffraction pattern is shown in Figure 22, and the electron micrograph is shown in Figure 23. It is a massive crystal with particle sizes of D10 (76.7 μm), D50 (184 μm), and D90 (397 μm).

[0092] XRD analysis of product J showed it to be crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.507°, 9.028°, 10.012°, 12.168°, 14.945°, 16.620°, 17.392°, 18.764°, 19.434°, 20.087°, 21.344°, 22.262°, 22.818°, 26.665°, 27.478°, 29.007°, 29.714°. The X-ray powder diffraction pattern is shown in Figure 24, and the electron micrograph is shown in Figure 25. It is a massive crystal with particle sizes of D10 (84 μm), D50 (129 μm), and D90 (195 μm).

[0093] result:

[0094] The above experimental results show that when the DMSO:EtOH solvent ratio increases from 1:4 to 1:12, the obtained regrugoli (sterile) is all of crystal form I, with a slight decrease in particle size; when the solvent ratio is between 1:5 and 1:12, the yield of the obtained regrugoli (sterile) is above 80%. Therefore, the optimal DMSO:EtOH solvent ratio is determined to be 1:5 to 1:12.

[0095] Experiments with and without seed crystals yielded products of crystal form I, with a slightly lower yield without seed crystals. Without seed crystals, the crystallization process is less controllable, potentially leading to uneven particle size in the crystallized product, thus affecting the subsequent production of regrangoli microcrystalline formulations. With seed crystals, the crystallization process is induced, resulting in better particle size uniformity, which is more beneficial for subsequent microcrystalline formulation production and product quality control. Therefore, considering recrystallization yield and product quality control, the preferred method for regrangoli crystallization is induced crystallization with seed crystals.

[0096] Experimental Example 5: Relugoli crystal form I obtained from different ratios of relugoli and DMSO

[0097] The experimental procedure is the same as in Experiment Example 3. The experimental results obtained by using different proportions of relugoline and DMSO solvent are shown in Table 3.

[0098] Table 3. Relugoli obtained from different proportions of relugoli and DMSO

[0099] XRD analysis of product K showed that it was a mixed crystal of crystal form I and crystal form II, with characteristic diffraction peaks at the 2θ angle: 7.451°, 13.324°, and 16.051° were the main characteristic peaks of crystal form II, while 7.812°, 9.292°, 12.463°, 16.970°, and 17.706° were characteristic peaks of crystal form I. The X-ray powder diffraction pattern is shown in Figure 26.

[0100] XRD analysis of product L showed it to be crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.855°, 9.412°, 10.402°, 12.564°, 15.314°, 16.984°, 17.802°, 19.171°, 19.848°, 20.471°, 21.855°, 22.367°, 23.176°, 26.719°, 27.840°, 29.454°, 30.111°. The X-ray powder diffraction pattern is shown in Figure 27, and the electron micrograph is shown in Figure 28. It is a massive crystal with particle sizes of D10 (156 μm), D50 (237 μm), and D90 (349 μm).

[0101] XRD analysis of product M showed that it is crystal form I, with characteristic diffraction peaks at the 2θ angle: 7.728°, 9.245°, 10.234°, 12.398°, 15.123°, 16.867°, 17.584°, 18.981°, 19.608°, 20.257°, ​​21.584°, 22.125°, 23.044°, 26.513°, 27.677°, 29.223°, 29.917°. The X-ray powder diffraction pattern is shown in Figure 29, and the electron micrograph is shown in Figure 30. It is a massive crystal with particle sizes of D10 (99.1 μm), D50 (163 μm), and D90 (268 μm).

[0102] Results: The above experimental results show that when the ratio of rectulgoline to DMSO is 1:1.2, the product obtained is a mixed crystal of crystal form I and crystal form II. When the ratio of rectulgoline to DMSO is 1:1.5 to 1:3, the rectulgoline (sterile) obtained is all crystal form I. As the amount of DMSO increases, the product yield decreases. The optimal ratio of rectulgoline to DMSO is determined to be 1:1.5 to 1:3, with a preferred ratio of 1:1.5 to 1:2.5.

[0103] Experimental Example 6: Relugoli Crystal Form I Obtained by Different Methods of Ethanol Addition

[0104] (1) Relugoli crystal form I (product N): Relugoli:DMSO:ethanol = 1:1.5:18, added all at once.

[0105] Dimethyl sulfoxide (DMSO, 15 ml) and 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothiopheno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (10 g, 1 eq) were added to a reaction flask and heated until the reaction solution was clear. The mixture was filtered, and the mother liquor was heated to 50-55°C. Preheated ethanol (180 ml) was added and stirred until homogeneous. Solid precipitated. The mixture was kept at this temperature and stirred for 120 min, and a large amount of solid precipitated. The temperature was lowered to 20-30°C and stirred for 1 h. The mixture was filtered, and the filter cake was washed with ethanol (10 ml). The filter cake was dried under vacuum at 50°C to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxy] A white solid, 9.1 g (91% yield), consisting of pyridazine-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea, was determined by XRD to be a mixed crystal of crystal form I and II. Characteristic diffraction peaks at 2θ angles of 7.273°, 13.286°, and 15.861° were observed, indicating that crystal form II was the primary characteristic crystal. The peaks 7.654°, 9.117°, 12.305°, 15.077°, 16.739°, and 17.519° are characteristic peaks of crystal form I. The X-ray powder diffraction pattern is shown in Figure 31, and the electron micrograph is shown in Figure 32. Product N includes two crystal forms: massive crystals and slender needle-like crystals. The particle sizes are: D10 is 6.19 μm, D50 is 51.8 μm, and D90 is 213 μm.

[0106] (2) Relugoli crystal form I (product O): Relugoli:DMSO:ethanol = 1:1.5:18, added in portions.

[0107] Add dimethyl sulfoxide (DMSO, 15 ml) and 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (10 g, 1 eq) to a reaction flask, heat until the reaction solution is clear, filter, and heat the mother liquor to 50-55°C. At ℃, add preheated ethanol (90 ml), stir until homogeneous, and solid precipitates. Add more preheated ethanol (90 ml), and a large amount of solid precipitates. Keep warm and stir for 120 min, then cool to 20-30℃ and keep warm and stir for 1 h. Filter, wash the filter cake with ethanol (10 ml), and dry the filter cake under vacuum at 50℃ to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazine-3-yl)] A white solid (8.3 g, yield 83%) of [-2,4-dioxo-1,2,3,4-tetrahydrothiopheno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea, as determined by XRD, is crystal form I. Characteristic diffraction peaks are observed at 2θ angles: 7.573°, 9.063°, 10.065°, 12.195°, 14.945°, 16.627°, 17.393°, 1... The X-ray powder diffraction patterns are shown in Figure 33, and the electron micrographs are shown in Figure 34. Product O is a blocky crystal with particle sizes of D10 of 75.9 μm, D50 of 126 μm, and D90 of 193 μm. The X-ray powder diffraction patterns are 8.779°, 19.446°, 20.144°, 21.375°, 22.009°, 22.806°, 26.648°, 27.489°, 29.024°, and 39.678°. Product O is a blocky crystal with particle sizes of D10 of 75.9 μm, D50 of 126 μm, and D90 of 193 μm.

[0108] (3) Relugoli crystal form I (product P): Relugoli:DMSO:ethanol = 1:1.5:18, added in portions.

[0109] Add dimethyl sulfoxide (DMSO, 15 ml) and 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (10 g, 1 eq) to a reaction flask, heat until the reaction solution is clear, filter, and heat the mother liquor to 50-55℃, then pour it into a pre-mixed container. Add 45 ml of hot ethanol and stir well. Add 90 ml of preheated ethanol and stir while maintaining the temperature until a large amount of solid precipitates. Add another 45 ml of preheated ethanol and stir while maintaining the temperature at 50-55°C for 120 min. Cool down to 20-30°C and stir while maintaining the temperature for 1 h. Filter and wash the filter cake with 10 ml of ethanol. Dry the filter cake under vacuum at 50°C to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-dimethylaminomethyl ...-3-dimethylaminomethyl-3-(6-dimethylaminomethyl)-5-dimethylaminomethyl-3-(6-dimethylaminomethyl)-5-dimethylaminomethyl-3-(6-dimethylaminomethyl-3-di [-Methoxypyridazine-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (8.37 g, yield 83.7%) is a white solid. XRD analysis showed it to be crystal form I, with characteristic diffraction peaks at 2θ angles: 7.592°, 9.099°, 10.073°, 12.193°, 14.901°, 16.662°, 17°. The X-ray powder diffraction patterns are shown in Figure 35, and the electron micrographs are shown in Figure 36. Product P is a blocky crystal with particle sizes of D10 of 115 μm, D50 of 171 μm, and D90 of 248 μm.

[0110] (4) Relugoli crystal form I (product Q): Relugoli:DMSO:ethanol:seed crystals = 1:1.5:18:0.025, added in portions.

[0111] Add dimethyl sulfoxide (DMSO, 15 ml) and 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (10 g, 1 eq) to a reaction flask, heat until the reaction solution is clear, filter, and heat the mother liquor to 50-55°C, then pour it into preheated ethanol. In 45 ml of alcohol, stir well, then add 90 ml of preheated ethanol and 25 mg of seed crystals. Stir and maintain the temperature until a large amount of solid precipitates. Add another 45 ml of preheated ethanol, and stir and maintain the temperature at 50-55°C for 120 min. Cool to 20-30°C and stir and maintain the temperature for 1 h. Filter, wash the filter cake with 10 ml of ethanol, and dry the filter cake under vacuum at 50°C to obtain 1-[4-[1-(2,6-difluorobenzyl)-5-dimethylaminomethyl-3- -(6-methoxypyridazine-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl]-3-methoxyurea (8.34 g, yield 83.4%) is a white solid. XRD analysis showed it to be crystal form I, with characteristic diffraction peaks at 2θ angles: 7.762°, 9.317°, 10.268°, 12.464°, 15.184°, 16.864°, 1 The X-ray powder diffraction pattern is shown in Figure 37, and the electron micrograph is shown in Figure 38. Product Q is a blocky crystal with particle sizes of D10 of 98.3 μm, D50 of 162 μm, and D90 of 258 μm. The X-ray powder diffraction pattern is 7.668°, 19.097°, 19.749°, 20.393°, 21.615°, 22.266°, 23.080°, 26.648°, 27.693°, 29.257°, ​​and 29.959°. Product Q is a blocky crystal with particle sizes of D10 of 98.3 μm, D50 of 162 μm, and D90 of 258 μm.

[0112] result:

[0113] The above experimental results show that when ethanol is added all at once during the recrystallization process, the resulting relugoli product N is a mixed crystal product. When ethanol is added in stages, the resulting products O, P, and Q are all crystal form I products. With the increase in the number of ethanol additions, the particle size of the relugoli (sterile) tends to increase. Product Q, obtained by adding seed crystals to induce crystallization, has fewer crystal agglomerates, and the crystal shape of the resulting product is more controllable. Therefore, the optimal method for adding ethanol is in stages, preferably in three stages, and more preferably by adding 0.25% seed crystals to induce crystallization.

[0114] Example 1: Preparation of a long-acting microcrystalline formulation of retinoic acid

[0115] Accurately weigh 90.0 g of purified water, 0.5 g of sodium carboxymethyl cellulose, 1.8 g of mannitol, 0.12 g of sodium dihydrogen phosphate, and 0.10 g of disodium hydrogen phosphate. Add each excipient to the purified water in sequence and stir to dissolve to obtain a dispersion solvent. Accurately weigh 10.0 g of product Q prepared in Example 6 and add it to the dispersion solvent to obtain a preliminary suspension. Load 0.8 mm grinding beads into the grinding mill chamber, set the grinding mill speed to 1500 rpm and the feed rate to 1200 ml / min, and circulate the preliminary suspension through the grinding mill for grinding. Stop grinding when the target particle size is 8.0 μm. Fill the grinding solution into vials and freeze-dry to obtain the long-acting microcrystalline formulation of retinoic acid.

[0116] Example 2: Preparation of a long-acting microcrystalline formulation of retinoic acid

[0117] Accurately weigh 80.0 g of purified water, 0.8 g of sodium carboxymethyl cellulose, 2.6 g of mannitol, 0.12 g of sodium dihydrogen phosphate, and 0.15 g of disodium hydrogen phosphate. Add each excipient to the purified water in sequence and stir to dissolve to obtain a dispersion solvent. Accurately weigh 20.0 g of product O prepared in Example 6 and add it to the dispersion solvent to obtain a preliminary suspension. Load 0.4 mm grinding beads into the grinding mill chamber, set the grinding mill speed to 1200 rpm and the feed rate to 1500 ml / min, and circulate and grind the preliminary suspension through the grinding mill. Stop grinding when the target particle size is 5.1 μm. Fill the grinding solution into vials and freeze-dry to obtain the long-acting microcrystalline formulation of retinoic acid.

[0118] Example 3: Preparation of a long-acting microcrystalline formulation of retinoic acid

[0119] Accurately weigh 85.0 g of purified water, 1.1 g of sodium carboxymethyl cellulose, 2.5 g of mannitol, 0.30 g of sodium dihydrogen phosphate, and 0.25 g of disodium hydrogen phosphate. Add each excipient to the purified water and stir to dissolve, obtaining a dispersion solvent. Accurately weigh 15.0 g of product P prepared in Example 6 and add it to the dispersion solvent to obtain a preliminary suspension. Load 2.0 mm grinding beads into the grinding mill chamber, set the grinding mill speed to 2500 rpm and the feed rate to 2000 ml / min, and circulate the preliminary suspension through the grinding mill for grinding. Stop grinding when the target particle size is 10.3 μm. Fill the grinding solution into vials and freeze-dry to obtain the long-acting microcrystalline formulation of retinoic acid.

[0120] Example 4: Preparation of a long-acting microcrystalline formulation of retinoic acid

[0121] Accurately weigh 40.0 g of purified water, 1.7 g of sodium carboxymethyl cellulose, 5.1 g of mannitol, 0.18 g of sodium dihydrogen phosphate, and 0.15 g of disodium hydrogen phosphate. Slowly add each excipient to the purified water and stir to dissolve, obtaining a dispersion solvent. Accurately weigh 10.0 g of product Q prepared in Example 6 and add it to the dispersion solvent to obtain a preliminary suspension. Load 1.5 mm grinding beads into the grinding mill chamber, set the grinding mill speed to 1500 rpm and the feed rate to 2500 ml / min, and circulate the preliminary suspension through the grinding mill for grinding. Stop grinding when the target particle size is 15.2 μm. Fill the grinding solution into vials and freeze-dry to obtain the long-acting microcrystalline formulation of retinoic acid.

[0122] Example 1: Comparison of release of long-acting retinoic acid microcrystalline formulations with different particle sizes in rats.

[0123] 1. Information on laboratory animals and samples

[0124] SD rats: Jinan Pengyue Experimental Animal Co., Ltd.

[0125] Experimental samples: Relugoline long-acting microcrystalline formulations with target particle sizes of 2.0 μm, 3.8 μm, 5.1 μm, 8.0 μm, 10.3 μm, 16.8 μm and 24.5 μm were prepared according to Example 1.

[0126] 2. Experimental Procedure

[0127] The experimental rats were weighed, and the actual dosage was calculated based on a dose of 40 mg / kg. The long-acting microcrystalline formulations of retinoic acid with different particle sizes were suspended in water for injection and injected into the rats via intramuscular injection. Plasma samples were collected before administration and at 0.25 h, 1 h, 6 h, 1 d, 3 d, 5 d, 7 d, 9 d, 11 d, 14 d, 17 d, 21 d, 24 d, 28 d, 35 d, and 42 d after administration (the collection time could be reduced or extended depending on the drug concentration in the body). Approximately 0.5 mL of blood was collected from the orbital venous plexus of the rats and placed in a heparinized EP tube. The tubes were centrifuged at 13500 rpm for 10 minutes, and the supernatant was transferred to another EP tube and stored at -40°C for analysis. The collected plasma samples were pretreated and analyzed by LC-MS / MS to determine the concentration.

[0128] 3. Experimental Results

[0129] Relugoline long-acting microcrystalline formulations with different particle sizes can be released sustainably for varying durations in rats (release curves shown in Figure 39), and can be used to prepare long-acting formulations with different sustained-release periods according to development goals. The time to peak concentration for microcrystalline formulations with different particle sizes ranges from 5 to 11 days. As the particle size increases, the peak drug concentration gradually decreases, while the time to peak concentration gradually increases, resulting in a gradually prolonged overall release time in rats. When the particle size is 24.5 μm, the drug can be released sustainably in rats for more than 42 days.

[0130] Example 2: Comparison of release of long-acting retinoic acid microcrystalline formulations with different particle sizes in dogs.

[0131] 1. Information on laboratory animals and samples

[0132] Beagle Dog: Qingdao Bolong Co., Ltd.

[0133] Experimental samples: Relugoline long-acting microcrystalline formulations with target particle sizes of 4.2 μm, 8.0 μm, and 10.8 μm were prepared according to Example 1.

[0134] 2. Experimental Procedure

[0135] The experimental dogs were weighed, and the actual dosage was calculated based on a dose of 20 mg / kg. The long-acting microcrystalline formulations of retinoic acid with different particle sizes were suspended in water for injection and injected into the dogs via intramuscular injection. Plasma samples were collected before administration and at 0.25 h, 1 h, 6 h, 1 d, 3 d, 5 d, 7 d, 9 d, 13 d, 17 d, 21 d, 24 d, 28 d, 35 d, and 42 d after administration (the collection time could be reduced or extended depending on the drug concentration in the body). Approximately 0.5 mL of blood was collected from the venous plexus of the dog's forelimb and placed in a heparinized EP tube. The tube was centrifuged at 13500 rpm for 10 minutes, and the supernatant was transferred to another EP tube and stored at -40°C for analysis. The collected plasma samples were pretreated and analyzed by LC-MS / MS to determine the concentration.

[0136] 3. Experimental Results

[0137] Relugoline long-acting microcrystalline formulations with different particle sizes can be released sustainably in dogs for varying durations (release curves shown in Figure 40), allowing for the development of long-acting formulations with different sustained-release periods, depending on the development goals. The time to peak concentration for microcrystalline formulations of different particle sizes ranges from 7 to 13 days. With increasing particle size, the peak drug concentration decreases slightly, while the time to peak concentration gradually lengthens, and the overall release time in dogs also gradually increases. When the particle size is 10.8 μm, the drug can be released sustainably in dogs for more than 42 days.

[0138] Example 3: Comparison of release of long-acting retinoic acid microcrystalline formulations with different particle sizes in monkeys.

[0139] 1. Information on laboratory animals and samples

[0140] Crab-eating macaques: Guangxi Guidong Primate Development and Experiment Co., Ltd.

[0141] Experimental samples: Relugoline long-acting microcrystalline formulations with target particle sizes of 6.2 μm and 9.3 μm were prepared according to Example 1.

[0142] 2. Experimental Procedure

[0143] Weigh the cynomolgus monkeys and calculate the actual dosage based on a dose of 50 mg / kg. Suspend the above-mentioned long-acting microcrystalline formulations of retinoic acid with different particle sizes in water for injection and inject them into the experimental monkeys via intramuscular injection. Collect plasma samples before administration and at 0.25 h, 1 h, 6 h, 1 d, 3 d, 5 d, 7 d, 9 d, 11 d, 14 d, 17 d, 21 d, 24 d, 28 d, 35 d, 42 d, 49 d, 56 d, 70 d, 84 d, 98 d, and 112 d after administration (the collection time can be reduced or extended depending on the drug concentration in the body). Approximately 0.5 mL of blood was collected from the venous plexus of the monkey's upper limb and placed in an EDTA-K2 anticoagulant tube. The tube was placed on wet ice and centrifuged at 3000g for 10 minutes at 4°C within 30 minutes after blood collection. The plasma was then transferred to a clean EP tube and stored at -70°C for later analysis. The collected plasma samples were pretreated and analyzed by LC-MS / MS to determine the concentration.

[0144] 3. Experimental Results

[0145] Two groups of long-acting microcrystalline formulations of regorafenib with different particle sizes were able to continuously release the drug in monkeys for more than three months, even up to four months, with peak plasma concentrations reaching approximately 14-17 days (see Figure 41 for specific release curves). As the particle size increased, the peak drug concentration decreased slightly, but the overall release time in monkeys significantly prolonged. With a particle size of 9.3 μm, the drug could be continuously released in monkeys for more than 112 days.

[0146] Example 4: Comparison of particle size distribution, electron micrographs, and release behavior in rats of long-acting microcrystalline formulations of relugoli prepared from different sources and relugoli crystal form I.

[0147] 1. Information on laboratory animals and samples

[0148] SD rats: Jinan Pengyue Experimental Animal Co., Ltd.

[0149] Experimental Samples: Referring to Example 1, a long-acting microcrystalline formulation of retinoic acid with a target particle size of approximately 8 μm was prepared using purchased crystal form I raw material (Sichuan Ren'an Pharmaceutical Co., Ltd., batch number: 338-2209001, its electron micrograph is shown in Figure 42, and its X-ray powder diffraction pattern is shown in Figure 43), raw material prepared in Example 1, raw material prepared in Example 2, and product Q raw material prepared in Example 6.

[0150] 2. Experimental Procedure

[0151] (1) Particle size detection and electron microscopy observation of microcrystalline formulations

[0152] Long-acting microcrystalline formulations of retinoglitazone prepared from retinoglitazone crystal form I raw materials from different sources were subjected to particle size distribution determination and electron microscopy observation.

[0153] (2) Pharmacokinetic experiments of microcrystalline formulations in rats

[0154] The experimental rats were weighed, and the actual dosage was calculated according to a dose of 40 mg / kg. The long-acting microcrystalline formulations of regoragline prepared from different sources of regoragline crystal form I were suspended in water for injection and injected into the rats via intramuscular injection. Plasma samples were collected before administration and at 0.25 h, 1 h, 6 h, 1 d, 3 d, 5 d, 7 d, 9 d, 11 d, 14 d, 17 d, 21 d, 24 d, and 28 d after administration (the collection time could be reduced or extended depending on the drug concentration in the body). Approximately 0.5 mL of blood was collected from the orbital venous plexus of the rats and placed in a heparinized EP tube. The tubes were centrifuged at 13500 rpm for 10 minutes, and the supernatant was transferred to another EP tube and stored at -40℃ for analysis. The collected plasma samples were pretreated and analyzed by LC-MS / MS to determine the concentration.

[0155] 3. Experimental Results

[0156] Table 4 shows the particle size distribution and electron micrographs of long-acting microcrystalline formulations of relugoli prepared using relugoli crystal form I raw materials from different sources. The results indicate that the particle morphology of the long-acting microcrystalline formulations of relugoli prepared using purchased raw materials, and those obtained from Experimental Example 1 and Experimental Example 2, is relatively irregular, with a wide particle size distribution and a large Span value, indicating uneven particle size distribution. When the particle size D50 is close, the smaller D10 indicates a higher number of small particles in the formulation; the larger D90 indicates the presence of some larger particles. When using product Q raw material prepared in Experimental Example 6 to prepare the long-acting microcrystalline formulation of relugoli, the particle morphology and particle size distribution were significantly improved, with a significantly narrower particle size distribution and a smaller Span value, indicating a more uniform particle size distribution. This is therefore more beneficial for quality control of the microcrystalline formulation and improvement of its release behavior in animals.

[0157] Table 4. Particle size distribution and electron micrographs of microcrystalline formulations prepared from regoraciform I raw materials from different sources.

[0158] Figure 44 compares the release behavior of relugoline long-acting microcrystalline formulations prepared from relugoline crystal form I raw materials from different sources in rats. The relugoline long-acting microcrystalline formulations prepared using purchased raw materials, as well as those from Experimental Example 1 and Experimental Example 2, exhibited a significant burst release behavior in rats due to the high number of small particles in the formulation. The initially high blood drug concentration may lead to adverse reactions, while the later blood drug concentration tends to be low, resulting in a shorter overall duration of release and failing to meet the goal of stable and slow release in vivo. In contrast, the relugoline microcrystals prepared using product Q raw material from Experimental Example 6 exhibited a more uniform particle size distribution, resulting in better release behavior in animals, more stable blood drug concentrations, and a longer duration of release, thus better meeting the goal of long-acting sustained release.

Claims

1. A long-acting microcrystalline pharmaceutical composition of regrugoli, comprising, by weight percentage: 40%-90% regrugoli, 1%-10% sodium carboxymethyl cellulose, 5%-30% mannitol, 0.1%-10% sodium dihydrogen phosphate, and 0.1%-10% disodium hydrogen phosphate, wherein the regrugoli used is regrugoli crystal form I, with a particle size not less than 2 μm and a relatively regular blocky crystal shape, and is recrystallized using the following preparation method: dimethyl sulfoxide... DMSO and rilugoline were added to a reaction flask and heated until the reaction solution became clear. The mixture was then filtered. The filtrate was heated to 50-55°C, and preheated ethanol was added. The mixture was stirred until solid precipitated, and then an equal volume of preheated ethanol was added. The mass-volume ratio of rilugoline:dimethyl sulfoxide:preheated ethanol was 1:1.5:

18. The mixture was kept warm and stirred for 120 min, then cooled to 20-30°C and kept warm and stirred for 1 h. The mixture was then filtered, and the filter cake was washed with ethanol and dried under vacuum at 50°C to obtain the final product.

2. The pharmaceutical composition according to claim 1, characterized in that: wherein... The rellugoline used was rellugoline crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization was performed using the following method: Dimethyl sulfoxide (DMSO) and rellugoline were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55°C, and preheated ethanol was added and stirred until homogeneous. Twice the amount of preheated ethanol was added again, and the mixture was kept warm and stirred until solid precipitated. The same volume of preheated ethanol as the first addition was then added, with a mass-to-volume ratio of rellugoline:DMSO:preheated ethanol of 1:1.5:

18. The mixture was kept warm and stirred for 120 min, then cooled to 20-30°C and kept warm and stirred for 1 h. After filtration, the filter cake was washed with ethanol and dried under vacuum at 50°C to obtain the final product.

3. The pharmaceutical composition according to claim 1, characterized in that: wherein... The rellugoli used was rellugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization was performed using the following method: Dimethyl sulfoxide (DMSO) and rellugoli were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55℃, and preheated ethanol was added and stirred until homogeneous. Then, an equal volume of preheated ethanol and seed crystals were added and stirred until solid precipitated. The mass-volume ratio of rellugoli: DMSO: preheated ethanol: seed crystals was 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature was lowered to 20-30℃ and stirred again for 1 h. After filtration, the filter cake was washed with ethanol and dried under vacuum at 50℃ to obtain the final product.

4. The pharmaceutical composition according to claim 1, characterized in that: wherein... The relugoli used was relugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization was performed using the following method: Dimethyl sulfoxide (DMSO) and relugoli were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55℃, and preheated ethanol was added and stirred until homogeneous under the same temperature. Preheated ethanol and seed crystals were added again and stirred until solid precipitated. More preheated ethanol was added, with the volume ratio of the three additions being 1:2:1, and the mass-volume ratio of relugoli: DMSO: preheated ethanol: seed crystals being 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature was lowered to 20-30℃ and stirred again for 1 h. The mixture was then filtered, the filter cake was washed with ethanol, and dried under vacuum at 50℃ to obtain the final product.

5. A method for preparing a long-acting microcrystalline pharmaceutical composition of retinoic acid, comprising the following steps: Purified water, sodium carboxymethyl cellulose, mannitol, sodium dihydrogen phosphate, and disodium hydrogen phosphate were weighed sequentially and dissolved by stirring to obtain a dispersion solvent. The weighed regrugolide active pharmaceutical ingredient was slowly added to the dispersion solvent for initial suspension to obtain a preliminary suspension. Zirconia grinding beads were added to the grinding mill chamber, and a suitable grinding mill speed and feed rate were set. The preliminary suspension was circulated and ground until the average particle size of regrugolide was 2-25 μm. Then, a suitable filling volume was set and the mixture was filled into vials for lyophilization. The characteristic feature is that the regrugolide active pharmaceutical ingredient is regrugolide crystal form I, with particle size... The crystals, with a diameter of not less than 2 μm and a relatively regular blocky shape, are prepared and recrystallized using the following method: Dimethyl sulfoxide and rellugoline are added to a reaction flask, heated until the reaction solution is clear, and then filtered. The filtrate is heated to 50-55℃, and preheated ethanol is added. The mixture is stirred until solid precipitates, and then an equal volume of preheated ethanol is added. The mass-volume ratio of rellugoline:dimethyl sulfoxide:preheated ethanol is 1:1.5:

18. The mixture is kept at this temperature and stirred for 120 min, then cooled to 20-30℃ and stirred again for 1 h. After filtration, the filter cake is washed with ethanol and then dried under vacuum at 50℃ to obtain the final product.

6. The preparation method according to claim 5, characterized in that: wherein... The rellugoline used was rellugoline crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization was performed using the following method: Dimethyl sulfoxide (DMSO) and rellugoline were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55°C, and preheated ethanol was added and stirred until homogeneous. Twice the amount of preheated ethanol was added again, and the mixture was kept warm and stirred until solid precipitated. The same volume of preheated ethanol as the first addition was then added, with a mass-to-volume ratio of rellugoline:DMSO:preheated ethanol of 1:1.5:

18. The mixture was kept warm and stirred for 120 min, then cooled to 20-30°C and kept warm and stirred for 1 h. After filtration, the filter cake was washed with ethanol and dried under vacuum at 50°C to obtain the final product.

7. The preparation method according to claim 5, characterized in that: wherein... The rellugoli used was rellugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization was performed using the following method: Dimethyl sulfoxide (DMSO) and rellugoli were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55℃, and preheated ethanol was added and stirred until homogeneous. Then, an equal volume of preheated ethanol and seed crystals were added and stirred until solid precipitated. The mass-volume ratio of rellugoli: DMSO: preheated ethanol: seed crystals was 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature was lowered to 20-30℃ and stirred again for 1 h. After filtration, the filter cake was washed with ethanol and dried under vacuum at 50℃ to obtain the final product.

8. The preparation method according to claim 5, characterized in that: wherein... The relugoli used was relugoli crystal form I, with a particle size of not less than 2 μm and a relatively regular blocky crystal shape. Recrystallization was performed using the following method: Dimethyl sulfoxide (DMSO) and relugoli were added to a reaction flask, heated until the reaction solution became clear, and then filtered. The filtrate was heated to 50-55℃, and preheated ethanol was added and stirred until homogeneous under the same temperature. Preheated ethanol and seed crystals were added again and stirred until solid precipitated. More preheated ethanol was added, with the volume ratio of the three additions being 1:2:1, and the mass-volume ratio of relugoli: DMSO: preheated ethanol: seed crystals being 1:1.5:18:0.0025. After stirring at this temperature for 120 min, the temperature was lowered to 20-30℃ and stirred again for 1 h. The mixture was then filtered, the filter cake was washed with ethanol, and dried under vacuum at 50℃ to obtain the final product.

9. The preparation method according to any one of claims 5 to 8, characterized in that: The grinding beads have a particle size of 0.3-3mm, the grinding mill speed is 900-3000rpm, and the feeding speed is 1000-3000ml / min.

10. The use of the retinoic acid long-acting microcrystalline pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for the treatment of prostate cancer or endometriosis.

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