Azacitidine compound for injection and preparation method thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and discloses an azacitidine compound for injection and a preparation method thereof, and the freeze-dried preparation comprises azacitidine, povidone and a freeze-dried proppant. The povidone is added to form the compound, so that the solubility of azacitidine in water is remarkably improved, the dissolution rate of azacitidine is increased, degradation of azacitidine in water is slowed down, and the stability and safety of azacitidine are improved. The azacitidine compound for injection is a clear and transparent solution after being redissolved, and is small in injection administration volume and small in resistance, so that 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 pharmaceutical preparations, and in particular relates to an azacitidine complex 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 drug's lyophilized preparation, 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 in the clinical treatment of acute non-lymphocytic leukemia of myelodysplastic syndrome (MDS), and can also be used to treat breast cancer, melanoma, and colon cancer.

[0004] The structural formula of azacitidine is as follows:

[0005]

[0006] Azacitidine raw materials are poorly soluble drugs with low solubility in water (about 13.8 mg / ml), and azacitidine has poor stability and is easily hydrolyzed. This drug property results in a long dissolution time of the raw materials during the batching process, resulting in a large amount of hydrolysis impurities, and the product quality is difficult to control, which has a potential impact 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 water for injection, and medical staff use their hands to rub the syringe vigorously to make azacitidine a uniform suspension for use; intravenous injection is to add 10 ml of water for injection and then dissolve it in sodium chloride injection or glucose injection for intravenous drip. When used as a suspension, there are strict requirements for the particle size, sedimentation velocity, and sedimentation volume ratio of the azacitidine suspension, which is inconvenient for clinical use.

[0007] 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. At this preparation temperature, azacitidine hydrolyzes and produces impurities at a fast rate, which is not conducive to the chemical stability of azacitidine. The industry usually uses low temperature means to effectively slow down the growth of hydrolyzed impurities, but the drug solubility is low at low temperatures and cannot reach the 10 mg / ml or 16.67 mg / ml mentioned in the patent.

[0008] 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.

[0009] 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 compliance in clinical use.

[0010] 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.

[0011] 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 with 25-500ml of 0.9% sodium chloride injection when used. 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

[0012] In order to solve the problems in the prior art that azacitidine for injection has poor stability, is easily hydrolyzed when dissolved at room temperature, is prone to precipitation and aggregation, is prone to cause needle blockage, and has unsatisfactory compliance in clinical use, the present invention aims to provide an azacitidine complex for injection that has good stability, is in a solution state after reconstitution, and is not prone to precipitation and aggregation, and a preparation method thereof. The solubility is significantly improved by forming a complex with azacitidine and povidone. The preparation method of the azacitidine complex provided in the present invention has fast dissolution speed, short preparation time, short freeze-drying cycle, simple process, and is suitable for large-scale production.

[0013] The present invention provides an azacitidine complex for injection, comprising azacitidine, povidone and a freeze-dried support agent.

[0014] Preferably, the mass ratio of azacitidine, povidone and freeze-dried proppant is 1:(4-15):1.

[0015] Preferably, the povidone is povidone K12 or povidone K17.

[0016] Preferably, the freeze-dried support agent is selected from one or more of mannitol, sucrose, glucose, lactose and trehalose in any combination.

[0017] In another aspect, the present invention provides a method for preparing an azacitidine complex solution and a lyophilized preparation thereof, comprising the following steps:

[0018] (1) Weigh the prescribed amount of povidone and stir to dissolve it in a mixed solvent of tert-butyl alcohol / water for injection;

[0019] (2) slowly adding the prescribed amount of azacitidine raw material to the solution obtained in step (1) and stirring to dissolve;

[0020] (3) adding a prescribed amount of freeze-dried proppant to the solution obtained in step (2), stirring to dissolve, and using the solvent in step (1) to adjust the volume or weight to obtain an azacitidine complex solution, and adjusting the pH to 5.5 to 7.0;

[0021] Wherein, azacitidine: volume of the complex solution after constant volume (g / mL) = 1: (50-200);

[0022] (4) The solution obtained in step (3) is sterilized and filtered through a 0.22 μm filter, and the filtrate is dispensed into vials, which are semi-stoppered; freeze-dried, stoppered, and capped.

[0023] In the preparation process of the present invention, the preparation temperature of the drug solution is controlled to be 2-8°C; preparation at a low temperature can slow down the hydrolysis of azacitidine, and at the same time, azacitidine forms a complex with povidone, thereby improving the stability of azacitidine.

[0024] Furthermore, in step (1), the volume proportion of tert-butanol in the tert-butanol / water for injection mixed solvent is 0-70%, that is, water for injection can also be used alone as the solvent.

[0025] The azacitidine raw material used in step (2) is micronized to a particle size of D 90 Not more than 50μm.

[0026] In addition, the present invention provides a freeze-drying process for a freeze-dried preparation of an azacitidine complex, the steps of which are as follows:

[0027] a. Pre-freezing: first cool the sample solution to be freeze-dried to -40~-45℃ and maintain for 1.5~3.5h; then heat it to -16℃ at 0.5℃ / min and maintain for 1h; then cool it to -40~-45℃ and maintain for 2h;

[0028] b. Sublimation drying: evacuate the freeze dryer and maintain a vacuum of 10-20 Pa, raise the temperature of the sample to -20--13°C at 0.5°C / min, and maintain for 10-20 hours;

[0029] c. Analytical drying: Evacuate the freeze dryer and maintain a vacuum of 1-15 Pa, heat the sample to 20-30 °C at a rate of 0.5 °C / min, and continue drying for 4-8 hours.

[0030] 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.

[0031] The present invention further performs infrared, DSC and XRD tests on the azacitidine complex, azacitidine and the physical mixture of azacitidine and povidone. By comparing the FTIR spectra, it is found that the peak positions of the azacitidine complex and azacitidine are basically consistent, but at 1035cm -1 The relative peak intensity of the peak (CO) at 1000 nm to other peaks is significantly lower than that of azacitidine and its physical mixture. By comparing the DSC spectra, it is found that the melting point of the azacitidine complex is significantly different from that of azacitidine, indicating that an azacitidine complex is formed. By comparing the XRD spectra, it is found that the azacitidine complex is an amorphous substance, while azacitidine is a crystalline form, indicating that an azacitidine complex is formed.

[0032] The azacitidine complex and preparation method thereof provided by the present invention have the following beneficial effects:

[0033] 1. The present invention adds azacitidine to a povidone solution to form a complex, thereby increasing the solubility of azacitidine, significantly reducing the freeze-drying volume, effectively shortening the freeze-drying cycle, and reducing production costs.

[0034] 2. The present invention adds azacitidine to a polyvidone solution to form a complex, which accelerates the dissolution rate of azacitidine, reduces the degradation of azacitidine, effectively inhibits the ring-opening hydrolysis of azacitidine, has good chemical stability, can remain stable for a long time during storage, and improves product stability.

[0035] 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

[0036] Figure 1 The morphological appearance photos of the prepared freeze-dried samples, of which the left one is Example 2 and the right one is Example 5;

[0037] Figure 2 The morphology of the samples after reconstitution is shown in Figure 2, with Example 2 on the left and Example 5 on the right.

[0038] Figure 3 is azacitidine, a physical mixture of azacitidine and povidone, and an infrared spectrum of the azacitidine complex prepared in Example 2;

[0039] Figure 4 is a differential scanning calorimetry (DSC) diagram of azacitidine in Example 2;

[0040] Figure 5 is a differential scanning calorimetry (DSC) diagram of a physical mixture of azacitidine and povidone;

[0041] Figure 6 is a differential scanning calorimetry (DSC) graph of the azacitidine complex prepared in Example 2;

[0042] Figure 7 is the X-ray powder diffraction (XRD) pattern of azacitidine in Example 2;

[0043] Figure 8 is the X-ray powder diffraction (XRD) pattern of the physical mixture of azacitidine and povidone in Example 2;

[0044] Fig. 9 This is the X-ray powder diffraction (XRD) pattern of the azacitidine complex prepared in Example 2. DETAILED DESCRIPTION

[0045] 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.

[0046] Example 1 Preparation of Azacitidine Complex for Injection

[0047] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0048]

[0049] The preparation process is as follows:

[0050] Dissolve povidone K12 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried support agent mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.37.

[0051] The above solution was sterilized and filtered through a 0.22 μm filter. After filtration, it was divided into medium borosilicate glass bottles at 20 ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying.

[0052] 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 15Pa, heat the sample to -18°C at 0.5°C / min and maintain for 20 hours; c. Desorption drying: evacuate the freeze dryer and maintain 5Pa, 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.

[0053] Example 2 Preparation of Azacitidine Complex for Injection

[0054] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0055]

[0056]

[0057] The preparation process is as follows:

[0058] Dissolve povidone K12 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried support agent mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.37.

[0059] The above solution was sterilized and filtered through a 0.22 μm filter. After filtration, it was divided into medium borosilicate glass bottles at 7.5 ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying.

[0060] 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 1.5 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 20Pa, 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.

[0061] The lyophilized azacitidine complex is a loose mass. Figure 1 Left, after reconstitution, it is a clear and transparent solution. Figure 2 Left: It can be seen that the azacitidine complex prepared in the present application does not undergo precipitation and aggregation after dissolution, and is not prone to blocking the needle when used.

[0062] The azacitidine bulk drug, the physical mixture of azacitidine and povidone, and the azacitidine complex were subjected to DSC and XRD detection, wherein the mixing ratio of the physical mixture of azacitidine and povidone was consistent with the prescription amount of the complex in this embodiment, that is, azacitidine:povidone=1:6.

[0063] DSC test results ( Figures 4 to 6 ) shows that the phase transition temperature of the azacitidine complex obtained by the present invention is significantly different from that of the physical mixture of azacitidine and povidone, and azacitidine. XRD test results ( Figures 7 to 9 ) shows that the characteristic diffraction peak of the azacitidine complex obtained in the present invention disappears, and its structure changes from a crystalline form to an amorphous form.

[0064] Figure 3 The FTIR spectra of azacitidine, a physical mixture of azacitidine and povidone, and the azacitidine complex obtained in this example are shown. By comparing the FTIR spectra, it is found that the peak positions of the azacitidine complex and azacitidine are basically consistent, except for the peak at 1035 cm -1 The relative peak intensity of the peak (CO) at α-1 and α-2 to other peaks is significantly reduced compared with azacitidine and its physical mixture, forming an azacitidine complex.

[0065] Example 3 Preparation of Azacitidine Complex for Injection

[0066] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0067]

[0068] The preparation process is as follows:

[0069] Dissolve povidone K12 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried proppant mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.36. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap to obtain. The freeze-drying procedure is the same as in Example 2.

[0070] Example 4 Preparation of Azacitidine Complex for Injection

[0071] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0072]

[0073] The preparation process is as follows:

[0074] Dissolve povidone K12 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried proppant mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.36. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap to obtain. The freeze-drying procedure is the same as in Example 2.

[0075] Example 5 Preparation of Azacitidine Complex for Injection

[0076] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0077]

[0078] The preparation process is as follows:

[0079] Dissolve povidone K17 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried proppant mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.35. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7.5ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap to obtain. The freeze-drying procedure is the same as in Example 2.

[0080] The lyophilized azacitidine complex is a loose mass. Figure 1 Right, after reconstitution, it is a clear and transparent solution. Figure 2Right, it can be seen that the azacitidine complex prepared in the present application does not undergo precipitation and aggregation after dissolution, and is not prone to blocking the needle when used.

[0081] Example 6 Preparation of Azacitidine Complex for Injection

[0082] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0083]

[0084] The preparation process is as follows:

[0085] Dissolve povidone K17 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried proppant mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.37. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap to obtain. The freeze-drying procedure is the same as in Example 2.

[0086] Example 7 Preparation of Azacitidine Complex for Injection

[0087] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0088]

[0089] The preparation process is as follows:

[0090] Dissolve povidone K17 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried proppant mannitol to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.36. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap to obtain. The freeze-drying procedure is the same as in Example 2.

[0091] Example 8 Preparation of Azacitidine Complex for Injection

[0092] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0093]

[0094] The preparation process is as follows:

[0095] Dissolve povidone K12 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried support agent glucose to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.37. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. Fill with nitrogen, stopper, unbox, and roll the cap to obtain. The freeze-drying procedure is the same as in Example 2.

[0096] Example 9 Preparation of Azacitidine Complex for Injection

[0097] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0098]

[0099] The preparation process is as follows:

[0100] Dissolve povidone K17 in 80% of the prescribed amount of 2-8°C water for injection, slowly add azacitidine API to the above solution, stir to dissolve, add freeze-dried support agent sucrose to dissolve, add 2-8°C water for injection to make up the volume, and use 0.1M sodium hydroxide or hydrochloric acid solution to adjust the pH to 6.35. The above solution is sterilized and filtered through a 0.22μm filter, and after filtration, it is divided into medium borosilicate glass bottles at 7ml / 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 2.

[0101] Example 10 Preparation of Azacitidine Complex for Injection

[0102] The composition of the lyophilized formulation of azacitidine complex is as follows:

[0103]

[0104]

[0105] The preparation process is as follows:

[0106] First, 50 ml of tert-butyl alcohol and 50 ml of water were mixed, and povidone K12 was dissolved in a mixed solvent of 2-8°C tert-butyl alcohol and water at 80% of the prescribed amount. Azacitidine API was slowly added to the above solution, stirred to dissolve, and freeze-dried proppant mannitol was added to dissolve, and then a mixed solvent of 2-8°C tert-butyl alcohol and water was added to make up the volume, and 0.1M sodium hydroxide or hydrochloric acid solution was used to adjust the pH to 6.35. The above solution was sterilized and filtered through a 0.22 μm filter, and after filtering, it was divided into medium borosilicate glass bottles at 10 ml / bottle, half-stoppered, and placed in a freeze dryer for freeze drying. 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 1.5 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 20Pa, heat the sample to -13°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 30°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.

[0107] Comparative Example 1 Preparation of Azacitidine Lyophilized Preparation

[0108] Referring to patent CN109646410A, the composition of the lyophilized preparation of azacitidine is as follows:

[0109]

[0110] The preparation process is as follows:

[0111] 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, isopropyl alcohol 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 an intermediate drug solution, and the intermediate drug solution is kept warm at 0-4°C; filtered with a 0.22 μm microporous filter membrane, divided into 24 ml / bottle, freeze-dried, plugged, rolled, and packaged to obtain a lyophilized preparation of azacitidine. The freeze-drying process is as follows: pre-freezing: -45°C for 1.5 hours, -10°C for 1 hour, -45°C for 2 hours; primary drying: -10°C for 20 hours; secondary drying: 40°C for 10 hours.

[0112] Comparative Example 2 Preparation of Azacitidine Lyophilized Preparation

[0113] Referring to patent CN104706650A, the composition of the lyophilized preparation of azacitidine is as follows:

[0114]

[0115] The preparation process is as follows:

[0116] 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 the water for injection, add water to 1000 ml, stir the liquid evenly and filter it with a 0.22 μm microporous membrane, and divide the filtered solution into glass bottles, 20 ml / bottle, half-stopper, and freeze-dry in a freeze dryer. Fill with nitrogen, stopper, unbox, and roll the cap to obtain the product. The freeze-drying procedure is the same as in Example 1.

[0117] Comparative Example 3 Preparation of Azacitidine Lyophilized Preparation

[0118] Referring to patent CN105769775A, the composition of the lyophilized preparation of azacitidine is as follows:

[0119] Azacitidine 1g

[0120] Mannitol 1g

[0121] Water for injection up to 230ml

[0122] The preparation process is as follows:

[0123] Take 80% water for injection, control the temperature at 2-6℃, add mannitol, stir until completely dissolved, add azacitidine, stir until completely dissolved, add water for injection to 230ml, filter and dispense, 23ml / bottle. Freeze-dry according to the following freeze-drying procedure: a. Pre-freeze: put the sample in after the plate temperature drops to -35±5℃, 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, 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, take out of the box.

[0124] Comparative Example 4 Preparation of Azacitidine Lyophilized Preparation

[0125] Referring to patent CN114224852A, the composition of the lyophilized preparation of azacitidine is as follows:

[0126] Azacitidine 5g

[0127] Mannitol 5g

[0128] Water for injection up to 300ml

[0129] The preparation process is as follows:

[0130] 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.

[0131] Experimental Example 1 Content and Related Substance Determination Method

[0132] Content determination

[0133] The sample content was tested by high performance liquid chromatography (HPLC) (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512):

[0134] Take 6.8g of potassium dihydrogen phosphate and 5g of sodium bisulfite as solvent, dissolve them in water and dilute to 1000ml, adjust the pH value to 3.0 with phosphoric acid, mix well, and you have it.

[0135] 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.

[0136] 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.

[0137] Related substances

[0138] The relevant substances of the samples were detected by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512):

[0139] Solvent 0.02 mol / L potassium dihydrogen phosphate solution (pH 6.5)-acetonitrile (60:40)

[0140] 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.

[0141] 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.

[0142] 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 the table below; the flow rate was 1.0 ml per minute; the detection wavelength was 214 nm; the column temperature was 25°C, and the automatic sampler temperature was 5°C.

[0143]

[0144] 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.

[0145] Limit: The total amount of impurities shall not exceed 2.0% of the labeled amount of azacitidine.

[0146] Experimental Example 2: Stability of sample reconstitution after freeze-drying

[0147] 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.

[0148] Table 1 Results of sample re-dissolution tests for Examples 1 to 10 and Comparative Examples 1 to 4

[0149]

[0150]

[0151] It can be seen that the azacitidine complex provided by the present invention forms a clear and transparent solution after re-dissolution ( Figure 2 ), which overcomes the problem of needle blockage caused by aggregation and sedimentation in the prior art, and has a short re-dissolution time and high efficiency, thereby improving the compliance of clinical use.

[0152] Table 2 Storage stability of samples of Examples 1 to 10 and Comparative Examples 1 to 4 after reconstitution (2 to 8°C)

[0153]

[0154]

[0155] Table 3 Storage stability of samples of Examples 1 to 10 and Comparative Examples 1 to 4 after reconstitution (25°C)

[0156]

[0157]

[0158] It can be seen that the azacitidine complex provided by the present invention has significantly improved chemical stability and solubility after being dissolved at room temperature, and no aggregation and sedimentation occurs. After being dissolved at room temperature and placed for a long time, the content of the active pharmaceutical ingredient can still be maintained at a stable high level.

[0159] Experimental Example 3: Long-term sample retention experiment

[0160] 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 5.

[0161] Table 4 Long-term sample test results of Examples 1 to 10

[0162]

[0163]

[0164] Experimental Example 4 Accelerated stability test

[0165] 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 (preserved at 25±2°C / 60±5%RH). The results are shown in Table 6.

[0166] Table 5 Accelerated stability test results of Examples 1 to 10

[0167]

[0168]

[0169] It can be seen from Experimental Examples 3 and 4 that the azacitidine complex lyophilized powder for injection provided by the present invention has good stability, low impurity level, good chemical stability, fast reconstitution and a clear and transparent solution after reconstitution, which is convenient for industrial production and clinical use.

[0170] The above results indicate that the present invention successfully obtains azacitidine-polyvidone complex, and 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 being dissolved at room temperature without hydrolysis. When used, it is re-dissolved into a clear and transparent solution without aggregation and precipitation, and is not prone to needle blockage, thereby improving the compliance of clinical use.

[0171] 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 complex for injection, characterized in that: The invention comprises azacitidine, povidone and a freeze-dried proppant, and the mass ratio of the azacitidine, povidone and the freeze-dried proppant is 1:(4-15):

1.

2. The azacitidine complex for injection according to claim 1, characterized in that: The povidone used is povidone K12 or povidone K17.

3. The azacitidine complex for injection according to claim 1, characterized in that: The freeze-dried support agent is selected from one or any combination of mannitol, sucrose, glucose, lactose and trehalose.

4. The method for preparing the azacitidine complex for injection according to claim 1, characterized in that: The following steps are included: (1) Weigh the prescribed amount of povidone and stir to dissolve it in a mixed solvent of tert-butyl alcohol / water for injection; (2) slowly adding the prescribed amount of azacitidine raw material to the solution obtained in step (1) and stirring to dissolve; (3) adding a prescribed amount of freeze-dried proppant to the solution obtained in step (2), stirring to dissolve, and using the solvent in step (1) to adjust the volume or weight to obtain an azacitidine complex solution, and adjusting the pH to 5.5-7.0; wherein the volume of the azacitidine complex solution after adjustment = 1: (50-200) g / mL; (4) The solution obtained in step (3) 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.

5. The method for preparing the azacitidine complex for injection according to claim 4, characterized in that: The temperature of the drug solution was controlled at 2-8°C during the entire preparation process.

6. The method for preparing the azacitidine complex for injection according to claim 4, characterized in that: In step (1), the volume proportion of tert-butanol in the tert-butanol / water for injection mixed solvent is 0 to 70%.

7. The method for preparing the azacitidine complex for injection according to claim 4, characterized in that: The azacitidine raw material used in step (2) is micronized to a particle size of D 90 Not more than 50μm.

8. The method for preparing the azacitidine complex for injection according to claim 4, characterized in that: The freeze-drying process of step (5) comprises the following steps: a. Pre-freezing: first cool the sample solution to be freeze-dried to -40~-45℃ and maintain for 1.5~3.5h; 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 10-20 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, heat 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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