Azacitidine clathrate compound for injection and preparation method of azacitidine clathrate compound

Abstract

The invention belongs to the field of novel pharmaceutical preparations, discloses an azacitidine clathrate compound for injection and a preparation method thereof, and particularly relates to an azacitidine clathrate compound freeze-dried preparation with stable properties, the freeze-dried preparation is composed of azacitidine and cyclodextrin, the clathrate compound is formed by adding cyclodextrin, and the freeze-dried preparation is prepared from the azacitidine and the cyclodextrin. According to the azacitidine preparation, the solubility of azacitidine in water is remarkably improved, and the dissolution rate of azacitidine is increased, so that the degradation of azacitidine in water is slowed down, and the stability and safety of azacitidine are improved. The azacitidine clathrate compound for injection is a clear and transparent solution after being redissolved, the subcutaneous injection administration volume is small, the resistance is small, and the clinical compliance is remarkably improved. The product prepared by the technical scheme is stable in quality and good in resolubility, and visible foreign matters and related substances are qualified.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel pharmaceutical preparations, and in particular relates to an azacitidine inclusion compound for injection and a preparation method thereof. Background Art

[0002] Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal diseases originating from hematopoietic stem cells, characterized by abnormal development of myeloid cells, manifested as ineffective hematopoiesis, refractory cytopenia, and a high risk of transformation to acute myeloid leukemia (AML). According to the International Prognostic Scoring System, the median survival of MDS patients classified as intermediate-2 and high-risk (collectively referred to as high-risk) is only 1.2 and 0.4 years, respectively, and the risk of progression to AML is high. Previously, there was no other treatment method other than allogeneic hematopoietic stem cell transplantation (HSCT) that could effectively intervene in the natural course of MDS.

[0003] Azacitidine, chemically known as 1-(β-D-ribofuranosyl)-4-amino-1,3,5-triazine-2(1H)-one, is a DNA methyltransferase inhibitor that can cause DNA hypomethylation and has direct cytotoxic effects. It was developed by Pharmion Pharmaceuticals in the United States. In May 2004, the U.S. FDA approved the marketing of the lyophilized preparation of the drug, Vidaza. Its mechanism of action is that azacitidine binds to DNA molecules after phosphorylation, and DNA methyltransferase reacts with azacitidine to form a covalently bound product. The activity of DNA methyltransferase is inhibited and degraded, causing a decrease in the level of DNA methylation in tumor tissues, demethylation of hypermethylated tumor suppressor genes, and restoration of gene expression, thereby inhibiting tumor cells. It is mainly used clinically to treat myelodysplastic syndrome (MDS) acute non-lymphocytic leukemia, and can also be used to treat breast cancer, melanoma, and colon cancer. The structural formula of azacitidine is as follows: .

[0004] The solubility of azacitidine raw materials is extremely low, about 13.8 mg / ml, and it is a poorly soluble drug. This drug characteristic results in a long dissolution time of the raw materials during the batching process, resulting in a large amount of hydrolysis impurities, making it difficult to control the product quality, and thus causing potential impacts on the safety and effectiveness of clinical medication. According to the information in the original research instructions, azacitidine for injection has two uses: subcutaneous injection and intravenous injection. Subcutaneous injection requires the addition of 4 ml of injection water to make azacitidine a uniform suspension. Intravenous injection is to add 10 ml of injection water to dissolve it and then add it to sodium chloride injection or glucose injection for intravenous drip. When used as a suspension, there are strict requirements on the particle size, sedimentation velocity, and sedimentation volume ratio of the azacitidine suspension, which is inconvenient for clinical use.

[0005] Patents CN104739778A, CN108721222A, and CN114224852A reduce the production of impurities and improve product stability by reducing the freeze-drying volume and shortening the freeze-drying cycle, but ignore the significant effect of temperature on the generation of hydrolyzed impurities during the preparation process. Low temperature is usually used in the industry to effectively slow down the growth of hydrolyzed impurities, but the solubility of the drug is low at low temperatures and cannot reach the 10 mg / ml or 16.67 mg / ml mentioned in the patents.

[0006] Patent CN101632643A discloses an azacitidine for injection and a preparation method thereof, and uses vitamin C to increase the stability of azacitidine in aqueous solution. However, the main related substances of azacitidine for injection come from hydrolysis products, and vitamin C, as a reducing protective agent, cannot inhibit the decomposition of this product in water; at the same time, vitamin C is a strong acidic substance, and azacitidine is easily hydrolyzed in an acidic environment, so vitamin C cannot increase the stability of azacitidine in water.

[0007] Patents CN111297809A, CN103251564A, CN104706650A, CN105769775A, CN109646410A, WO2013012135A1, WO2019231225A1, CN108295034A and CN109820827A disclose azacitidine powder for injection, all of which have improvements in the preparation process: by controlling the preparation temperature and pH of the drug solution, adding organic solvents for dispersion, shortening the dissolution time and other process measures to reduce the generation of hydrolysis impurities. The above patents still have some unresolved technical problems: (1) Process problems: the filling volume is 20-25 ml, the freeze-drying cycle is long, freeze-drying is difficult, and the production cost is high, which indirectly reduces the stability of the product; (2) The powder injection becomes a suspension after reconstitution, which is prone to sedimentation and aggregation, easy to block the needle, inaccurate dosage, and poor clinical compliance.

[0008] Patents CN107961216A and CN108451901A disclose a preparation of azacitidine and a preparation method thereof, wherein the solubility of the product is improved by adding a flow aid, poloxamer 188, and an antioxidant, sodium pyrosulfite, and the product is filtered and sterilized into a borosilicate glass ampoule. Poloxamer 188 is a nonionic surfactant with limited solubilization ability; sodium pyrosulfite is an antioxidant and has no inhibitory effect on the hydrolysis of azacitidine. The patent is for an azacitidine injection, and water for injection is used as a solvent. Azacitidine is easily hydrolyzed, so the injection has poor storage stability.

[0009] Patent US2022096507A1 discloses dissolving azacitidine in a non-aqueous solvent DMSO to reduce the generation of azacitidine hydrolysis impurities. The azacitidine dosage form designed in this patent is a concentrated solution for injection, which needs to be administered intravenously in combination with 25-500ml 0.9% sodium chloride injection. At present, the main route of administration of azacitidine powder injection is subcutaneous injection. This patent is administered by intravenous drip, and the drip solution contains DMSO, which poses a safety hazard and causes allergic reactions in some patients. Therefore, compared with the original powder injection, it has no obvious clinical advantages and does not improve the technical defects of the marketed products. Summary of the invention

[0010] Based on the drawbacks of the above prior art such as unsatisfactory compliance for clinical use, poor chemical stability, easy hydrolysis, poor solubility and resolubility, and the difficulties in the research and development of this product, the present invention aims to provide a more stable azacitidine inclusion compound for injection in a solution state after reconstitution and a preparation method thereof, wherein azacitidine is combined with cyclodextrin to form an inclusion compound with high solubility, and the method has fast dissolution speed, short preparation time, short freeze-drying cycle, simple process, and is suitable for large-scale production.

[0011] The present invention provides an azacitidine inclusion compound for injection, comprising azacitidine and cyclodextrin.

[0012] In the azacitidine inclusion compound lyophilized preparation of the present invention, the cyclodextrin is hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin.

[0013] In the azacitidine inclusion compound lyophilized preparation of the present invention, the mass ratio of azacitidine to cyclodextrin is 1:10-30.

[0014] In another aspect, the present invention provides a method for preparing an azacitidine inclusion complex solution and a lyophilized preparation thereof, comprising the following steps: (1) Weigh the prescribed amount of cyclodextrin and dissolve it in water for injection; (2) Slowly add the prescribed amount of azacitidine raw material to the solution obtained in step (1), stir to dissolve, make up to volume or weight with water for injection, and adjust the pH to 5.5-7.0; Wherein, azacitidine: volume after constant volume (g / mL) = 1: (40~150); (3) The solution obtained in step (2) is sterilized and filtered through a 0.22 μm filter, and the filtrate is dispensed into vials, which are half-stoppered; freeze-dried, stoppered, and capped.

[0015] During the whole preparation process, the preparation temperature of the drug solution is controlled at 2-8°C. Preparation at low temperature can slow down the hydrolysis of azacitidine. At the same time, azacitidine forms an inclusion complex with cyclodextrin, thereby improving the stability of azacitidine.

[0016] Furthermore, the azacitidine API used in step (2) is micronized to a particle size D 90 Not more than 50μm.

[0017] In addition, the inventors also provided a freeze-drying process for a freeze-dried preparation of azacitidine inclusion compound, the steps of which are as follows: a. Pre-freezing: first cool the sample solution to be freeze-dried to -40~-45℃ and maintain for 1.5h~3.5h; then heat it to -16℃ at 0.5℃ / min and maintain for 1h; then cool it to -40~-45℃ and maintain for 2h; b. Sublimation drying: evacuate the freeze dryer and maintain a vacuum of 5-20 Pa, raise the temperature of the sample to -20--13°C at 0.5°C / min, and maintain for 10-20 hours; c. Drying: Evacuate the freeze dryer and maintain a vacuum of 1-15 Pa. Raise the temperature of the sample to 20-30 °C at a rate of 0.5 °C / min and continue drying for 4-8 hours.

[0018] The solution is freeze-dried through the above steps, and the freeze-dried sample is made easier to sublimate and dry through annealing, and more loose, thereby shortening the freeze-drying cycle. The gaps in the freeze-dried product are larger, which is more conducive to its re-dissolution.

[0019] The present invention further performs infrared, DSC, and XRD detection on azacitidine inclusion complex, azacitidine, and a physical mixture of azacitidine and cyclodextrin. By comparing the FTIR spectra, it is found that the FTIR spectrum of the azacitidine inclusion complex has new peaks relative to azacitidine and the physical mixture of azacitidine cyclodextrin. By comparing the DSC spectra, it is found that the melting point of the azacitidine inclusion complex is significantly different from the melting point of azacitidine, indicating that an azacitidine inclusion complex has been formed. By comparing the XRD spectra, it is found that the azacitidine inclusion complex is an amorphous substance, while azacitidine is a crystalline form, indicating that an azacitidine inclusion complex has been formed.

[0020] The azacitidine inclusion compound for injection and the preparation method thereof provided by the present invention have the beneficial effects of: 1. The present invention adds azacitidine to a cyclodextrin solution to form an inclusion compound, thereby increasing the solubility of azacitidine, significantly reducing the lyophilization volume, effectively shortening the lyophilization cycle, and reducing production costs; 2. After azacitidine is dissolved at room temperature, the triazole ring in its chemical structure is easily hydrolyzed and ring-opened. In the present invention, azacitidine is added to a cyclodextrin solution to form an inclusion complex, which accelerates the dissolution rate of azacitidine, improves the chemical stability of azacitidine, reduces the degradation of azacitidine, and improves the product stability; 3. The solubility of the currently marketed azacitidine powder injection for injection is about 13.8 mg / ml at room temperature. After being reconstituted with 4 ml of water for injection, it is a suspension, which is prone to sedimentation and aggregation, easy to block the needle, and inaccurate dosage. The azacitidine powder injection provided by the present invention has a short reconstitution time and is a clear and transparent solution after reconstitution, which meets the clinical use requirement of 25 mg / ml and improves the compliance of the preparation for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphological appearance photos of the prepared freeze-dried samples, of which the left one is Example 4 and the right one is Example 8; Figure 2 The morphology of the samples after the freeze-dried samples were reconstituted is shown in Figure 4 on the left and in Figure 8 on the right; Figure 3 is azacitidine, a mixture of azacitidine and cyclodextrin, and the infrared spectrum of the azacitidine inclusion complex prepared in Example 4; Figure 4 is the differential scanning calorimetry graph of azacitidine; Figure 5 is a differential scanning calorimetry graph of a physical mixture of azacitidine and cyclodextrin; Figure 6 is a differential scanning calorimetry diagram of the azacitidine inclusion complex prepared in Example 4; Figure 7 is the X-ray powder diffraction pattern of azacitidine; Figure 8 is the X-ray powder diffraction pattern of a physical mixture of azacitidine and cyclodextrin; Fig. 9 This is the X-ray powder diffraction pattern of the azacitidine inclusion complex prepared in Example 4. DETAILED DESCRIPTION

[0022] The beneficial effects of the present invention are further described by the following examples. The examples are only for illustrative purposes and do not limit the scope of the present invention. At the same time, obvious changes and modifications made by ordinary technicians in the field according to the present invention are also included in the scope of the present invention.

[0023] Example 1 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Sulfonbutyl-β-cyclodextrin 10g Water for injection to 60ml.

[0024] The preparation process is as follows: Dissolve sulfobutyl-β-cyclodextrin in 80% of the prescription amount of 2-8℃ water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8℃ water for injection to make up to volume, and adjust the pH to 6.47 with 0.1M sodium hydroxide or hydrochloric acid solution. Sterilize and filter the above solution through a 0.22μm filter, and after filtration, dispense into medium borosilicate glass bottles at 6ml / bottle, half-stopper, and freeze-dry in a freeze dryer.

[0025] Freeze drying is performed according to the following freeze drying procedures: a. Pre-freezing: first cool the sample solution to -40°C and maintain for 2 hours; then heat it to -16°C at 0.5°C / min and maintain for 1 hour; then cool it to -40°C and maintain for 2 hours; b. Sublimation drying: evacuate the freeze dryer and maintain 10Pa, heat the sample to -13°C at 0.5°C / min and maintain for 10 hours; c. Desorption drying: evacuate the freeze dryer and maintain 1Pa, heat the sample to 25°C at 0.5°C / min and continue drying for 4 hours. Fill with nitrogen, press the stopper, take it out of the box, and roll the cap to obtain it.

[0026] Example 2 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Sulfonbutyl-β-cyclodextrin 15g Water for injection up to 50 ml.

[0027] The preparation process is as follows: Dissolve sulfobutyl-β-cyclodextrin in 80% of the prescription amount of 2-8℃ water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8℃ water for injection to make up to volume, and adjust the pH to 6.47 with 0.1M sodium hydroxide or hydrochloric acid solution. Sterilize and filter the above solution through a 0.22μm filter, and after filtration, divide it into medium borosilicate glass bottles at 5ml / bottle, half-stopper it, and put it into a freeze dryer for freeze drying.

[0028] Freeze drying is performed according to the following freeze drying procedures: a. Pre-freezing: first cool the sample solution to -45°C and maintain for 2 hours; then heat it to -16°C at 0.5°C / min and maintain for 1 hour; then cool it to -45°C and maintain for 2 hours; b. Sublimation drying: evacuate the freeze dryer and maintain 5Pa, heat the sample to -18°C at 0.5°C / min and maintain for 15 hours; c. Desorption drying: evacuate the freeze dryer and maintain 1Pa, heat the sample to 25°C at 0.5°C / min and continue drying for 4 hours. Fill with nitrogen, press the stopper, take it out of the box, and roll the cap to obtain it.

[0029] Example 3 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Sulfonbutyl-β-cyclodextrin 20g Water for injection up to 50 ml.

[0030] The preparation process is as follows: Dissolve sulfobutyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.46. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 5ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0031] Example 4 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Sulfonbutyl-β-cyclodextrin 20g Water for injection to 40ml.

[0032] The preparation process is as follows: Dissolve sulfobutyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.46. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 4ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0033] The lyophilized azacitidine inclusion complex is a loose mass. Figure 1 Left, after reconstitution, it is a clear and transparent solution. Figure 2 Left: Azacitidine inclusion complex does not precipitate or aggregate after dissolution, and is not likely to clog the needle during use.

[0034] The azacitidine bulk drug, the physical mixture of azacitidine and sulfobutyl-β-cyclodextrin, and the azacitidine inclusion complex were subjected to DSC and XRD detection. The mixing ratio of the physical mixture of azacitidine and sulfobutyl-β-cyclodextrin was consistent with the prescription amount of the inclusion complex in this embodiment, that is, azacitidine:sulfobutyl-β-cyclodextrin=1:20.

[0035] DSC test results ( Figures 4 to 6 ) shows that the phase transition temperature of the azacitidine inclusion complex obtained by the present invention is significantly different from that of the physical mixture of azacitidine and sulfobutyl-β-cyclodextrin and azacitidine. XRD test results ( Figures 7 to 9) shows that the characteristic diffraction peak of the azacitidine inclusion complex obtained in the present invention disappears, and its structure changes from a crystalline form to an amorphous form.

[0036] Figure 3 The azacitidine bulk drug, a physical mixture of azacitidine and sulfobutyl-β-cyclodextrin, and the FTIR spectrum of the azacitidine inclusion complex in this embodiment. It can be clearly seen from the figure that the inclusion complex has a peak at 1159.1 cm -1 and 1206.9cm -1 A peak (CO) that did not appear in the raw material drug and the physical mixture appeared, indicating that the inclusion complex of azacitidine and sulfobutyl-β-cyclodextrin was successfully obtained by the method.

[0037] Example 5 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Sulfonbutyl-β-cyclodextrin 25g Water for injection to 40ml.

[0038] The preparation process is as follows: Dissolve sulfobutyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.45. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 4ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0039] Example 6 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Sulfonbutyl-β-cyclodextrin 30g Water for injection to 40ml.

[0040] The preparation process is as follows: Dissolve sulfobutyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.47. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 4ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0041] Example 7 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Hydroxypropyl-β-cyclodextrin 15g Water for injection up to 50 ml.

[0042] The preparation process is as follows: Dissolve hydroxypropyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.46. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 5ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0043] Example 8 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Hydroxypropyl-β-cyclodextrin 20g Water for injection to 40ml.

[0044] The preparation process is as follows: Dissolve hydroxypropyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.47. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 4ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0045] The lyophilized azacitidine inclusion complex is a loose mass. Figure 1 Right, after reconstitution, it is a clear and transparent solution. Figure 2 Right: Azacitidine inclusion complex does not precipitate or aggregate after dissolution and is not likely to clog the needle during use.

[0046] Example 9 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Hydroxypropyl-β-cyclodextrin 25g Water for injection to 40ml.

[0047] The preparation process is as follows: Dissolve hydroxypropyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.45. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 4ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0048] Example 10 Preparation of Azacitidine Inclusion Complex for Injection: The composition of the lyophilized formulation of azacitidine inclusion complex is as follows: Azacitidine 1g Hydroxypropyl-β-cyclodextrin 30g Water for injection to 40ml.

[0049] The preparation process is as follows: Dissolve hydroxypropyl-β-cyclodextrin in 80% of the prescription amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add 2-8°C water for injection to make up to volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.45. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 4ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cover to obtain. The freeze-drying procedure is the same as in Example 1.

[0050] Comparative Example 1 Preparation of Azacitidine Lyophilized Preparation: Referring to patent CN109646410A, the composition of the lyophilized preparation of azacitidine is as follows: Azacitidine 1g Mannitol 3g Tert-butyl alcohol: isopropyl alcohol: propylene glycol (volume ratio) = 1:2:1 48ml Water for injection up to 240 ml.

[0051] The preparation process is as follows: 80% of the water for injection is measured, and the prescribed amount of mannitol is added to the water for injection, stirred and dissolved, and cooled to 0-4°C, and the prescribed amount of azacitidine is added to the mixed solution of tert-butyl alcohol, isopropanol and propylene glycol, stirred and dispersed; the mannitol aqueous solution is mixed with the azacitidine tert-butyl alcohol dispersion, and the remaining amount of water for injection is added to obtain the intermediate drug solution, and the intermediate drug solution is kept warm at 0-4°C; filtered with a 0.22μm microporous filter membrane, divided into 24ml / bottle, freeze-dried, plugged, rolled, and packaged to obtain azacitidine lyophilized preparation. The freeze-drying process is as follows: pre-freezing: -45°C for 1.5h, -10°C for 1h, -45°C for 2h; primary drying: -10°C for 20h; secondary drying: 40°C for 10h.

[0052] Comparative Example 2 Preparation of Azacitidine Lyophilized Preparation: Referring to patent CN104706650A, the composition of the lyophilized preparation of azacitidine is as follows: Azacitidine 5g Mannitol 5g Ethanol 20ml Add water for injection to 1000ml.

[0053] The preparation process is as follows: Measure 950 ml of water for injection, cool to -3 to -1 ° C, weigh 5 g of mannitol and dissolve it in the water for injection, weigh 5 g of azacitidine, stir and disperse azacitidine in 20 ml of ethanol, mix and dissolve the azacitidine ethanol dispersion with water for injection, add water to 1000 ml, stir the liquid evenly and filter it with a 0.22 μm microporous filter membrane, and divide the filtered solution into glass bottles, 20 ml / bottle. Half-stopper, put into a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap. The freeze-drying procedure is the same as in Example 1.

[0054] Comparative Example 3 Preparation of Azacitidine Lyophilized Preparation: Referring to patent CN105769775A, the composition of the lyophilized preparation of azacitidine is as follows: Azacitidine 1g Mannitol 1g Water for injection to 230ml.

[0055] The preparation process is as follows: Measure 80% water for injection, control the temperature at 2-6°C, add mannitol, stir until completely dissolved, add azacitidine, stir until completely dissolved, then add water for injection to make up to 230 ml, filter and package, 23 ml / bottle.

[0056] Freeze-drying was carried out according to the following freeze-drying procedure: a. Pre-freezing: put the sample in after the plate temperature drops to -35±5℃ and keep warm for 2 hours; b. Primary sublimation drying: reduce the vacuum degree to 50-150μbar, increase the shelf temperature to -10℃ at 15℃ / h, and keep warm for 15 hours; c. Secondary sublimation drying: maintain the vacuum degree to 50-150μbar, increase the shelf temperature to 30℃ at 20℃ / h, and keep warm for 18 hours. After nitrogen filling, press the stopper and take out of the box.

[0057] Comparative Example 4 Preparation of Azacitidine Lyophilized Preparation: Referring to patent CN114224852A, the composition of the lyophilized preparation of azacitidine is as follows: Azacitidine 5g Mannitol 5g Add water for injection to 300ml.

[0058] The preparation process is as follows: Accurately weigh 90% of the prescribed amount of water for injection; weigh the prescribed amount of azacitidine (particle size of 100μm) and mannitol, mix in a sterile bag for 10 minutes, slowly add water for injection to 300ml, and stir to dissolve. After filtering, divide into 6ml / bottle. Freeze-dry according to the following freeze-drying procedures: a. Pre-freeze: put the sample in after the plate temperature drops to -35±5℃, and keep warm for 2 hours; b. Primary sublimation drying: reduce the vacuum degree to 50-150μbar, increase the shelf temperature to -10℃ at 15℃ / h, and keep warm for 15 hours; c. Secondary sublimation drying: maintain the vacuum degree to 50-150μbar, increase the shelf temperature to 30℃ at 20℃ / h, keep warm for 18 hours, fill with nitrogen and then press the plug, and take out of the box.

[0059] Experimental Example 1 Content and related substances determination method.

[0060] Content determination: The sample content was tested by high performance liquid chromatography. The analytical method used is given below. Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512): Solvent: Take 6.8g of potassium dihydrogen phosphate and 5g of sodium bisulfite, dissolve in water and dilute to 1000ml, adjust the pH value to 3.0 with phosphoric acid, mix well, and you have it.

[0061] Chromatographic conditions: octylsilane bonded silica gel was used as the filler (YMC Triart C8, 4.6 mm × 250 mm); 0.02 mol / L potassium dihydrogen phosphate solution (pH 6.5)-methanol (90:10) was used as the mobile phase for isocratic elution, with a flow rate of 1.0 ml per minute; the detection wavelength was 243 nm; the column temperature was 15 °C, the automatic sampler temperature was 5 °C, and the injection volume was 20 μl.

[0062] Determination method: Take 5 vials of this product, dissolve the contents with solvent and transfer completely to a 250ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure an appropriate amount, dilute with solvent to make a solution containing approximately 40μg of azacitidine per 1ml, as the test solution, accurately measure 20μl, inject into liquid chromatograph, and calculate by peak area using the external standard method.

[0063] Related substances: The sample-related substances were detected by high performance liquid chromatography. The analytical method used is given below. Determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512): Solvent: 0.02 mol / L potassium dihydrogen phosphate solution (pH 6.5)-acetonitrile (60:40) Sample solution: Take a bottle of sample, add 4 ml of water to dissolve, add refrigerated solvent to dissolve and dilute to make a solution containing about 0.035 mg per 1 ml.

[0064] Reference solution: Accurately weigh an appropriate amount of azacitidine reference substance, dissolve it in refrigerated solvent and dilute it to make a solution containing approximately 0.035 mg per 1 ml.

[0065] Chromatographic conditions: octylsilane bonded silica gel was used as filler (YMC Triart C8, 4.6 mm × 250 mm); 0.02 mol / L potassium dihydrogen phosphate solution (pH 6.5) was used as mobile phase A, and mobile phase A-acetonitrile (60:40) was used as mobile phase B; gradient elution was performed according to Table 0 below; the flow rate was 1.0 ml per minute; the detection wavelength was 214 nm; the column temperature was 25 °C, and the autosampler temperature was 5 °C.

[0066] Table 0 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 10 100 0 12 0 100 20 0 100 20.1 100 0 45 100 0 .

[0067] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the peak area according to the external standard method.

[0068] Limit: The total amount of impurities shall not exceed 2.0% of the labeled amount of azacitidine.

[0069] Experimental Example 2 Stability of samples after reconstitution after freeze-drying.

[0070] 4 ml of water for injection was added to the freeze-dried preparation samples prepared in Examples 1 to 10 and Comparative Examples 1 to 4 for reconstitution. The reconstitution results are shown in Table 1 below.

[0071] Table 1 Results of re-dissolution test of samples in Examples 1 to 10 and Comparative Examples 1 to 4

[0072] It can be seen from the experimental results in Table 1 that the azacitidine inclusion complex provided by the present invention has good resolubility, short resolubility time and fast resolubility rate, and is a clear and transparent solution after resolubility, and is not prone to problems such as needle blockage due to aggregation and precipitation, thereby improving the compliance of the preparation for clinical use.

[0073] Table 2 Storage stability of samples of Examples 1 to 10 and Comparative Examples 1 to 4 after reconstitution (2 to 8°C)

[0074] Table 3 Storage stability of samples of Examples 1 to 10 and Comparative Examples 1 to 4 after reconstitution (25°C)

[0075] According to the experimental results in Table 2 and Table 3, it can be seen that the azacitidine inclusion complex obtained by the present invention effectively improves the stability of azacitidine. Compared with Comparative Examples 1-4, the stability of azacitidine after reconstitution is significantly improved.

[0076] Experimental Example 3: Long-term sample retention experiment.

[0077] The freeze-dried preparations prepared in Examples 1 to 10 were subjected to long-term sample retention experiments, and the inspection indicators included: properties, pH value, detection of related substances and content (stored at 2 to 8° C.). The results are shown in Table 4.

[0078] Table 4 Long-term sample test results of Examples 1 to 10

[0079] Experimental Example 4 Accelerated stability test The freeze-dried preparations prepared in Examples 1 to 10 were subjected to long-term sample retention experiments, and the inspection indicators included: properties, pH value, detection of related substances and content (stored at 25±2°C / 60±5%RH). The results are shown in Table 5.

[0080] Table 5 Accelerated stability test results of Examples 1 to 10

[0081] Experimental Example 5: Experiment on influencing factors The freeze-dried preparations prepared in Examples 1 to 10 were experimentally investigated for influencing factors, and the investigation indicators included: properties, pH value, detection of related substances and content (stored at 40±2°C / 75±5%RH). The results are shown in Table 6.

[0082] Table 6 Test results of influencing factors of Examples 1 to 10

[0083] Based on the above experimental examples, it can be seen that the azacitidine inclusion compound lyophilized powder for injection provided by the present invention has good stability, can still maintain a high level of active drug content after long-term storage, successfully improves the chemical stability and solubility of azacitidine, accelerates the dissolution rate of azacitidine, and enables it to remain stable for a long time after dissolution at room temperature without hydrolysis, and has a low impurity level, fast resolubility and a clear and transparent solution after resolubility, without aggregation and precipitation, and is not prone to needle blockage, thereby improving the compliance of clinical use and facilitating industrial production and clinical use.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent substitutions, modifications, etc. made by technicians in this field without any creative work within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An azacitidine inclusion compound for injection, characterized in that: The invention comprises azacitidine and cyclodextrin, wherein the mass ratio of azacitidine to cyclodextrin is 1:10-30.

2. The azacitidine inclusion compound for injection according to claim 1, characterized in that: The cyclodextrin used is hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin.

3. The method for preparing the azacitidine inclusion compound for injection according to claim 1, characterized in that: The following steps are included: (1) Weigh the prescribed amount of cyclodextrin and dissolve it in water for injection; (2) Slowly add the prescribed amount of azacitidine raw material to the solution obtained in step (1), stir to dissolve, and make up to volume or weight with water for injection, and adjust the pH to 5.5-7.0; wherein azacitidine: volume after making up to volume = 1: (40-150) g / mL; (3) The solution obtained in step (2) is sterilized and filtered through a 0.22 μm filter, and the filtrate is dispensed into vials, which are half-stoppered; freeze-dried, stoppered, and capped.

4. The method for preparing the azacitidine inclusion compound for injection according to claim 3, characterized in that: During the entire preparation process, the temperature of the drug solution was controlled at 2~8°C.

5. The method for preparing the azacitidine inclusion compound for injection according to claim 3, characterized in that: Step (2) Azacitidine is micronized to a particle size of D 90 Not more than 50μm.

6. The method for preparing the azacitidine inclusion compound for injection according to claim 3, characterized in that: The freeze drying in step (3) comprises the following steps: a. Pre-freezing: first cool the sample solution to be freeze-dried to -40~-45℃ and maintain for 1.5h~3.5h; then heat it to -16℃ at 0.5℃ / min and maintain for 1h; then cool it to -40~-45℃ and maintain for 2h; b. Sublimation drying: evacuate the freeze dryer and maintain a vacuum of 5-20 Pa, raise the temperature of the sample to -20--13°C at 0.5°C / min, and maintain for 10-20 hours; c. Analytical drying: Evacuate the freeze dryer and maintain a vacuum of 1-15 Pa. Raise the temperature of the sample to 20-30 °C at a rate of 0.5 °C / min and continue drying for 4-8 hours.

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