Preparation method of sodium citicoline injection

Abstract

The application provides a preparation method of a sodium citicoline injection, and belongs to the technical field of preparations. The preparation method is as follows: dissolving sodium citicoline pure product in water, filtering, sequentially filtering through a redundant sterilization filter and a main sterilization filter, filling and sealing, placing the obtained liquid medicine after filling and sealing in a sterilization device preheated to 100 DEG C, sterilizing at 121 DEG C for 10 min, reducing the temperature to below 40 DEG C, and then detecting leakage, so that the sodium citicoline injection is obtained. The impurity E in the prepared sodium citicoline injection is successfully controlled below 0.5%, and the drug safety of the sodium citicoline injection is improved.

Description

Technical Field

[0001] This invention relates to a method for preparing a sodium citicoline preparation, and more particularly to a method for preparing a sodium citicoline injection. Background Technology

[0002] The main component of cytidine diphosphate choline sodium injection is cytidine diphosphate choline sodium, an important intermediate in phospholipid synthesis and metabolism. It is a nucleoside derivative and a drug that improves cerebral blood circulation and promotes nootropy. It is primarily used to treat acute traumatic brain injury and post-brain surgery-induced altered consciousness. During the preparation of cytidine diphosphate choline sodium injection, research has revealed that impurity E is generated in relatively high concentrations, and currently, there is no effective method for its removal.

[0003] In addition, the active pharmaceutical ingredient (pure cytidine diphosphate choline sodium) used to prepare cytidine diphosphate choline sodium injection is mainly obtained by three methods: 1. Chemical synthesis method, using CMP (5′-cytidine monophosphate) and phosphate choline as reactants, and p-toluenesulfonyl chloride as a condensing agent, to prepare the solution under the action of N-dimethylformamide. After preparation, it needs to be treated with 717 (Cl... - ) type anion exchange column and 711(Cl - 1. **Separation and preparation of sodium citrate choline using a concentrated column:** A drawback is the difficulty in separating sodium citrate choline from the condensing agent, resulting in insufficient product purity and unsuitability for pharmaceutical use. 2. **Enzymatic synthesis:** Sodium citrate choline is synthesized from cell extract using cytidine triphosphate (CTP) and phosphocholine. However, the synthesis requires CTP as a substrate, making the entire process costly and unsuitable for industrial production. 3. **Yeast fermentation:** This is currently the most widely used synthesis method. It utilizes CMP (5′-cytidine monophosphate) and phosphocholine as reactants to prepare sodium citrate choline through yeast fermentation. However, this method still suffers from problems in the later separation and purification process, including lengthy elution times, large eluent volume, low eluent concentration, large amounts of activated carbon used for adsorption, and relatively high column costs during elution. Summary of the Invention

[0004] To address the above problems, this invention provides a method for preparing cytidine diphosphate choline sodium injection.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a sodium citicoline injection, the method comprising the following steps:

[0007] 1) Dissolve pure sodium citicoline in water, filter, and obtain the intermediate product;

[0008] 2) The intermediate product is filtered sequentially through a redundant sterilization filter and a main sterilization filter to obtain the drug solution;

[0009] 3) The liquid medicine is filled and sealed to obtain the filled liquid medicine;

[0010] 4) Place the filled drug solution in a sterilization device preheated to 100°C, heat it to 121°C for 10 minutes, and after sterilization, cool it down to below 40°C. Then, after leak detection, the cytidine diphosphate choline sodium injection solution is obtained.

[0011] Furthermore, in step 4), after being placed in a sterilization device preheated to 100°C, the temperature is increased to 121°C in 10 minutes;

[0012] After sterilization, cool down to below 40°C within 8 minutes.

[0013] Furthermore, in step 2), the redundant sterilization filter has a filtration pressure of 0.1–0.3 MPa and a filtration velocity of 2–10 kg / min.

[0014] The main sterilization filter has a filtration pressure of 0.01–0.2 MPa and a filtration velocity of 2–10 kg / min.

[0015] Furthermore, the purification method used for the pure sodium citicoline is as follows: the crude sodium citicoline is dissolved in water, the pH value is adjusted, the color is decolorized, then a mixed solution of ethanol and acetone is added, the temperature is lowered to induce crystallization, the mixture is filtered, and the precipitate is recrystallized from ethanol to obtain the pure sodium citicoline.

[0016] Furthermore, in the purification method, the weight-to-volume ratio of crude sodium citicoline to a mixed solution of water, ethanol, and acetone is 1 kg: 4-6 L: 3-4 L.

[0017] Furthermore, in the purification method, the mixed solution of ethanol and acetone is prepared by mixing ethanol and acetone in a volume ratio of 1:5 to 6.

[0018] Furthermore, in the purification method, the crystallization temperature is 10–15°C and the time is 3–4 h.

[0019] Furthermore, in the purification method, adjusting the pH value involves adjusting the pH value to 7.5–8.0.

[0020] Furthermore, in the purification method, when recrystallizing ethanol, the amount of ethanol used is 2 to 3 times the weight of the precipitate.

[0021] Furthermore, in the purification method, the crude sodium citicoline is prepared by adding yeast, glucose, potassium hydroxide, phosphocholine and 5′-cytidine to water and reacting at room temperature (i.e., fermentation). After the reaction is complete, the resulting reaction solution is heated to inactivate the protein, filtered, the pH is adjusted to alkaline to precipitate alkaline proteins, filtered again, the pH of the filtrate is adjusted to acidic to precipitate acidic proteins, filtered again, and the filtrate is concentrated to dryness to obtain the product.

[0022] The beneficial effects of the preparation method of the cytidine diphosphate choline sodium injection of the present invention are as follows:

[0023] This invention successfully controlled the impurity E in the prepared cytidine diphosphate choline sodium injection to below 0.5% by changing the filtration and sterilization process parameters, thereby improving the medication safety of cytidine diphosphate choline sodium injection.

[0024] This invention produces a system containing sodium cytidine diphosphate choline by fermenting yeast, glucose, potassium hydroxide, phosphocholine, and 5′-cytidine. The crude sodium cytidine diphosphate choline is obtained by directly concentrating the filtrate after inactivation, basic protein, and acidic protein treatment. Using a specific ratio of water-ethanol-acetone solvent at a specific pH and temperature, sodium cytidine diphosphate choline is successfully separated from other substances in the system. In particular, sodium cytidine diphosphate choline and 5′-cytidine, which have similar physicochemical properties, are successfully separated, simplifying the post-processing.

[0025] The purification process of sodium citicoline of the present invention is simple to operate, requiring no activated carbon adsorption or elution, and no column chromatography, which effectively reduces the amount of solvent used and lowers production costs; moreover, the entire process effectively simplifies the processing steps, shortens the processing time, and saves time costs.

[0026] The purification process of this invention is simple to operate, environmentally friendly, and suitable for industrialized production. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] Example 1: A method for preparing a sodium citicoline injection.

[0029] This embodiment describes a method for preparing cytidine diphosphate choline sodium injection, specifically including the following steps:

[0030] I. Preparation of Crude Sodium Citicoline

[0031] 30 kg of yeast, 10 kg of glucose, 3 kg of potassium hydroxide, 5 kg of phosphocholine and 2 kg of 5′-cytidine monophosphate were added to 100 L of water and stirred. The mixture was stirred at room temperature for 8 h to allow it to react completely. After the reaction was complete, the resulting reaction solution was heated to 60 °C to inactivate the precipitate and then filtered. The filtrate was adjusted to pH 8.0 with 20 wt% sodium hydroxide to precipitate alkaline proteins. The filtrate was then filtered and the pH was adjusted to 2.0 with 20 wt% hydrochloric acid to precipitate acidic proteins. The filtrate was then concentrated to dryness to obtain 20.1 kg of crude phosphocholine sodium, in which the content of phosphocholine sodium was 12.28%.

[0032] II. Purification of sodium citicoline

[0033] 1 kg of crude citicoline sodium was added to 4–6 L of water (5 L in this example), stirred to dissolve, and the pH was adjusted to 8.0 with a small amount of 20 wt% sodium hydroxide. Activated carbon was added for decolorization at room temperature for 30–40 min. 3–4 L of a mixture of ethanol and acetone with a volume ratio of 1:5–6 was slowly added dropwise (3.5 L of a mixture of ethanol and acetone with a volume ratio of 1:5.5 was added in this example). The mixture was cooled to 10–15 °C and stirred to crystallize for 3–4 h (10 °C and 3.5 h in this example). The mixture was filtered, dried, and 0.1122 kg of precipitate was obtained, labeled M1, with a recovery rate of 11.22%. The actual recovery rate of precipitate M1 (based on citicoline sodium) was 90.22%, and precipitate M1 contained 98.73% citicoline sodium and 0.11% 5′-cytidine.

[0034] The precipitate M1 was recrystallized with 2 to 3 times its weight of ethanol (in this example, the amount of ethanol used was 2.5 times the weight of the precipitate) to obtain pure sodium citicoline with a purity of 99.92%.

[0035] The actual recovery rate (based on sodium citicoline, %) = weight of precipitate × content of sodium citicoline in precipitate / (amount of crude sodium citicoline × content of sodium citicoline in crude sodium citicoline) × 100%.

[0036] The contents of cytidine diphosphate choline sodium and 5′-cytidine acid were determined according to the method for determining the content of cytidine diphosphate choline sodium in Part II of the 2020 edition of the Chinese Pharmacopoeia.

[0037] III. Preparation of Sodium Citicoline Injection

[0038] 1) Add 10L of water for injection to the mixing tank. Set the stirring frequency of the mixing tank to 8Hz, turn on the stirrer, and lower the water temperature to 20-25℃. Discharge approximately 30% of the water for injection from the mixing tank into a stainless steel container for later use (20-40% can also be discharged for later use; this is not to limit the amount of water for injection discharged, but only to meet the requirement). Add 1.25kg of pure sodium citicoline to the mixing tank and stir until dissolved. Then, use the remaining water for injection in the stainless steel container to add up to the total volume of the prepared solution. Control the water temperature at 20-25℃ (i.e., the dissolution temperature is controlled at 20-25℃, in this case...). The dissolution temperature in this example is 25°C. Stirring continues for at least 20 minutes (20 minutes in this example). The pH value of the resulting liquid is controlled at 6.3-7.5 (7.0 in this example). The sanitary pump is turned on to pre-filter the liquid through a 0.22μm filter into a buffer tank to obtain an intermediate product. The intermediate product is a colorless and clear liquid with a pH value of 6.3-7.5 (7.0 in this example), a sodium citrate choline content of 0.125 g / mL, and a bacterial endotoxin content of 172 EU / mL.

[0039] 2) Take the intermediate product and filter it through a 0.22μm redundant sterilization filter at a filtration pressure of 0.1-0.3MPa and a filtration rate of 2-10kg / min into the filling chamber (in this embodiment, the filtration pressure of the redundant sterilization filter is 0.2MPa and the filtration rate is 5kg / min). Control the microbial load filtered by the redundant sterilization filter to be ≤1 microorganism / mL. Then filter it through a 0.22μm main sterilization filter at a filtration pressure of 0.01-0.2MPa and a filtration rate of 2-10kg / min into the storage tank (in this embodiment, the filtration pressure of the main sterilization filter is 0.1MPa and the filtration rate is 5kg / min) to obtain the drug solution.

[0040] The pressure difference between the two ends of the sterilization filter is ≤0.2MPa, and in this embodiment, the pressure difference between the two ends of the sterilization filter is 0.1MPa;

[0041] The pressure difference across the sterilization filter equals the filtration pressure of the redundant sterilization filter minus the filtration pressure of the main sterilization filter.

[0042] 3) The liquid medicine is filled and sealed to obtain the filled liquid medicine.

[0043] 4) Before the filled medicine enters the sterilizer, close the front door of the sterilizer. The sterilizer will automatically fill with water and heat up to 100℃ for preheating. When both T1 and T2 temperatures reach 100℃, manually depressurize until the sterilizer pressure is zero. Place the filled medicine into the sterilizer, and the program will enter the sterilization step. The steam valve will automatically open and close to control the temperature. When the temperatures of "TH, TL, T1, and T2" all reach 121℃, the sterilization program will start timing after 10 minutes. After 12 minutes, the program will automatically enter the drainage and cooling step. The drain valve will open to bring the water level to the lower level. The inlet valve will open, and the inlet pump and circulation pump will start to automatically inject cooling water below 10℃ to bring the water level in the sterilizer to the upper level. The water temperature will drop to below 40℃ within 8 minutes, and the program will automatically enter the leak detection step.

[0044] Negative pressure leak detection: The program automatically starts the vacuum pump to evacuate the inside of the sterilization cabinet. When the internal pressure of the sterilization cabinet (sterilization temperature 121℃) is less than -80kPa, the vacuum pump is turned off and maintained for 5 minutes. Then, 0.02% amaranth red water is automatically added to bring the water level in the sterilization cabinet to the upper level for negative pressure leak detection for 5 minutes. After the negative pressure leak detection is completed, it automatically enters the positive pressure leak detection.

[0045] Positive pressure leak test: Automatic compressed air inlet valve, when (sterilization temperature 121℃) the pressure is greater than 80kPa, maintain for 25 minutes to perform positive pressure leak test, open the color water drain valve, circulation pump, recover the red amaranth water into the color water tank, and end the positive pressure leak test.

[0046] After positive pressure leak detection is completed, the solution undergoes visual inspection, labeling, and packaging to obtain cytidine diphosphate choline sodium injection, labeled as N1. The impurity E content in cytidine diphosphate choline sodium injection N1 is ≤0.5% (in this embodiment, the impurity E content in cytidine diphosphate choline sodium injection N1 is 0.3%).

[0047] The method for determining the content of impurity E is as follows:

[0048] The content of impurity E was determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0512).

[0049] Test solution: Accurately measure an appropriate amount of cytidine diphosphate choline sodium injection and dilute it quantitatively with water to prepare a solution containing approximately 2.5 mg of cytidine diphosphate choline per 1 mL.

[0050] Reference solution: Accurately weigh appropriate amounts of sodium citicoline and impurity A reference standard, dissolve them in water and dilute quantitatively to prepare a mixed solution containing approximately 12.5 μg of citicoline and 5 μg of impurity A per 1 mL.

[0051] Sensitivity solution: Accurately measure 2 mL of the reference solution, place it in a 50 mL volumetric flask, dilute with water to the mark, and shake well.

[0052] System suitability solution: Accurately weigh appropriate amounts of sodium citicoline and impurity A reference standard, dissolve and dilute with water to prepare a mixed solution containing approximately 0.125 mg of citicoline and 0.05 mg of impurity A per 1 mL.

[0053] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Ultimate XB-C). 18 The chromatographic column was 4.6 mm × 250 mm and 5 μm or equivalent in performance. The mobile phase was phosphate buffer [equal volumes of 0.1 mol / L potassium dihydrogen phosphate solution and tetrabutylammonium solution (0.01 mol / L tetrabutylammonium hydroxide solution was adjusted to pH 4.5 with phosphoric acid)]-methanol (95:5). The detection wavelength was 276 nm. The injection volume of the system suitability solution was 20 μL, and the injection volume of other solutions was 10 μL.

[0054] System suitability requirements: In the system suitability solution chromatogram, the resolution between the cytidine diphosphate choline peak and the impurity A peak should meet the requirements.

[0055] In a sensitivity solution chromatogram, the signal-to-noise ratio of the peak height of the principal component should be greater than 10.

[0056] Assay: Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms up to 6 times the retention time of the main peak.

[0057] Limits: If impurity peaks are present in the chromatogram of the test solution, impurity A, calculated using the external standard method based on the peak area of ​​impurity A in the chromatogram of the reference solution, shall not exceed 0.3%; impurity E (relative retention time approximately 1.74), calculated using the external standard method based on the peak area of ​​cytidine diphosphate choline in the chromatogram of the reference solution, multiplied by a correction factor of 1.7, shall not exceed 0.85%; other impurities, calculated using the external standard method based on the peak area of ​​cytidine diphosphate choline in the chromatogram of the reference solution, shall not exceed 0.2%; except for impurity A, the total amount of impurities shall not exceed 1.05%, and peaks smaller than the area of ​​the main peak in the sensitivity solution shall be ignored.

[0058] Impurity A is 5'-cytidine acid, and impurity E is 5'-uridine diphosphate choline.

[0059] Preparation methods of sodium cytidine diphosphate choline injection in Examples 2-5

[0060] Examples 2-5 are methods for preparing cytidine diphosphate choline sodium injection. Their steps are basically the same as in Example 1, differing only in the process parameters, as detailed in Table 1.

[0061] Table 1. Summary of process parameters in Examples 2-5

[0062]

[0063]

[0064] The process steps and parameters for other parts of Examples 2 to 5 are the same as those for Example 1, and will not be repeated here.

[0065] Experiment Example 1: Comparative Experiment

[0066] Comparative Examples 1-7 are comparative experiments on the purification process of the pure cytidine diphosphate choline sodium in Example 1. All examples used the crude cytidine diphosphate choline sodium prepared in Example 1 as raw material, with the only difference being:

[0067] In Comparative Example 1, the volume ratio of the ethanol and acetone mixture was 1:10, with other parameters and amounts remaining unchanged. The resulting precipitate, labeled DM1, had an actual recovery rate (based on cytidine diphosphate choline sodium) of 90.57%, of which precipitate DM1 contained 94.31% cytidine diphosphate choline sodium and 1.23% 5′-cytidine nucleotide.

[0068] In Comparative Example 2, the volume ratio of the ethanol and acetone mixture was 1:1, and other parameters and amounts remained unchanged. The resulting precipitate was labeled DM2, and the actual recovery rate (based on sodium citicoline) was 85.76%. The precipitate DM2 contained 95.38% sodium citicoline and 1.37% 5′-cytidine.

[0069] In Comparative Example 3, the volume ratio of the ethanol and acetone mixture was 1:1, with other parameters and amounts remaining unchanged. The resulting precipitate, labeled DM3, had an actual recovery rate (based on sodium citicoline) of 85.76%, of which precipitate DM3 contained 95.38% sodium citicoline and 1.37% 5′-cytidine.

[0070] In Comparative Example 4, a mixture of 1 L of ethanol and acetone at a volume ratio of 1:5.5 was used, with other parameters and amounts remaining unchanged. The resulting precipitate was labeled DM4, and the actual recovery rate (based on sodium citicoline) was 52.73%. The precipitate DM4 contained 93.07% sodium citicoline and 1.69% 5′-cytidine.

[0071] In Comparative Example 5, an 8L mixture of ethanol and acetone at a volume ratio of 1:5.5 was used, with other parameters and amounts remaining unchanged. The resulting precipitate was labeled DM5, and the actual recovery rate (based on cytidine diphosphate choline sodium) was 94.72%. The precipitate DM5 contained 89.31% cytidine diphosphate choline sodium and 2.31% 5′-cytidine nucleotide.

[0072] In Comparative Example 6, the pH value was adjusted to 10.0 using a small amount of 20wt% sodium hydroxide, while other parameters and amounts remained unchanged. The resulting precipitate was labeled DM6, and the actual recovery rate (based on sodium citicoline) was 89.07%. The precipitate DM6 contained 97.29% sodium citicoline and 0.37% 5′-cytidine.

[0073] In Comparative Example 7, the crystallization temperature was 5℃, and other parameters and dosages remained unchanged. The resulting precipitate, labeled DM7, had an actual recovery rate (based on sodium citicoline) of 91.45%. The precipitate DM7 contained 97.07% sodium citicoline and 0.42% 5′-cytidine.

[0074] Experiment Example 2: Comparative Experiment

[0075] Comparative Examples 8 and 9 are comparative experiments on the preparation process of the cytidine diphosphate choline sodium injection in Example 1. All examples used the pure cytidine diphosphate choline sodium prepared in Example 1 as the raw material, with the only difference being:

[0076] In step 4) of Comparative Example 8, the temperature was raised to 121°C for 15 minutes, while other process parameters remained unchanged. The resulting sodium citicoline injection was labeled DN1, in which the impurity E content of sodium citicoline injection DN1 was 1.2%.

[0077] In step 4) of Comparative Example 9, the temperature was lowered to below 40°C for 30 minutes, while other process parameters remained unchanged. The resulting sodium citicoline injection was labeled DN2, in which the impurity E content of sodium citicoline injection DN2 was 1.7%.

[0078] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a sodium choline phosphate injection solution, characterized by, The preparation method comprises the following steps: 1) taking the pure cytosine sodium to dissolve in water, and filtering to obtain an intermediate product; The purification method of the pure cytosine sodium comprises the following steps: taking the crude cytosine sodium to dissolve in water, adjusting the pH value, decolorizing, then adding a mixed solution of ethanol and acetone, and crystallizing by lowering the temperature, filtering, and recrystallizing the obtained precipitate with ethanol to obtain the pure cytosine sodium; the weight-volume ratio of the crude cytosine sodium, water, and the mixed solution of ethanol and acetone is 1 kg: 4-6 L: 3-4 L; the mixed solution of ethanol and acetone is obtained by mixing ethanol and acetone at a volume ratio of 1:5-6; the crystallization temperature is 10-15℃, and the crystallization time is 3-4 h; 2) taking the intermediate product to filter through a redundant sterilization filter and a main sterilization filter in sequence to obtain a liquid medicine; 3) the liquid medicine is sealed by filling to obtain a filled liquid medicine; 4) taking the filled liquid medicine to a sterilization device preheated to 100℃, raising the temperature to 121℃ in 10 min, sterilizing at 121℃ for 10 min, reducing the temperature to below 40℃ within 8 min after the sterilization is completed, and then detecting the leakage to obtain the cytosine sodium injection.

2. The preparation method of the cytosine sodium injection according to claim 1, characterized in that, in step 2), the filtration pressure of the redundant sterilization filter is 0.1-0.3 MPa, and the filtration speed is 2-10 kg / min; the filtration pressure of the main sterilization filter is 0.01-0.2 MPa, and the filtration speed is 2-10 kg / min. In the purification method, the pH value is adjusted to 7.5-8.

0.

3. The method for preparing sodium citicoline injection according to claim 1, characterized in that, In the purification method, the amount of ethanol used in the recrystallization is 2-3 times the weight of the precipitate.

4. The method for preparing cytidine diphosphate choline sodium injection according to claim 1, characterized in that, In the purification method, the crude cytosine sodium is prepared by adding yeast, glucose, potassium hydroxide, choline phosphate, and 5'-cytidylic acid into water, reacting at room temperature, inactivating the obtained reaction solution by raising the temperature, filtering, adjusting the pH value to alkaline to precipitate alkaline proteins, filtering, adjusting the pH value of the filtrate to acid to precipitate acid proteins, filtering, and concentrating the filtrate to dryness.

5. The method for preparing sodium citicoline injection according to claim 1, characterized in that, ​

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