Stable coenzyme A freeze-dried preparation for injection
Through lyophilized protective agent and static magnetic field assisted technology, combined with phosphate buffer and multi-stage drying treatment, the problem of poor stability of Coenzyme A is solved, and a high stability and safety lyophilized preparation for injection is achieved, which simplifies the Production and reduce costs.
Patent Information
- Application Number
- CN202411312830.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2024-12-31
AI Technical Summary
Coenzyme A has poor stability and is prone to degradation under drying and refrigeration conditions. The pH value fluctuates greatly during the lyophilization process, resulting in unstable product quality and may cause adverse reactions.
Using lyophilized protective agents such as trehalose, β-glucan, etc., the static magnetic field assists in dissolving Coenzyme A, adjusts the pH value through phosphate buffer, and undergoes multi-stage drying and back-temperature treatment during the lyophilization process to form a stable Protective layer, enhances water solubility and moisture resistance.
It significantly improves the stability and safety of Coenzyme A, reduces the risk of adverse reactions, ensures the efficacy and safety of the drug, and simplifies the production process and reduces costs.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medicines, in particular to a stable coenzyme A freeze-dried preparation for injection. Background Art
[0002] Coenzyme A (CoA) is a nucleotide coenzyme, composed of pantothenic acid, adenosine, phosphoric acid and aminoethanethiol. It is a coenzyme of acetylase in organisms and one of the most important enzymes in the human body. It participates in the tricarboxylic acid cycle, plays the role of transferring acetyl groups, and plays an important role in the metabolism of sugars, proteins and lipids. Coenzyme A can promote the normal metabolism of lipids, inhibit the formation of lipid peroxides, prevent platelet aggregation, and prevent cholesterol deposition in blood vessel walls and vascular atherosclerosis. Coenzyme A deficiency in the human body can cause depression, anxiety, irritability, loss of appetite, fatigue, frequent respiratory infections, numbness, muscle weakness, cramps, paresthesia (such as tingling, tingling, burning sensation in the feet), adrenal failure and other symptoms.
[0003] Pure coenzyme A is a white or light yellow powder with a maximum purity of 95%, generally 70% to 80%. Because of the thiol group in the molecule, it has a garlic-like odor and is easily soluble in water, but insoluble in acetone, ether and ethanol. Coenzyme A has the common properties of nucleotides and thiols and is a strong acid.
[0004] According to the existing technical literature - Research on Coenzyme A Quality Standard (Lian Ying, Master's thesis of Zhengzhou University, 2009-10-15), Coenzyme A has poor stability. Under dry and refrigerated conditions, the content decreases by 1% to 2% per month. In a neutral, alkaline, or acidic aqueous solution environment, Coenzyme A is extremely unstable, and at a temperature of 120°C, the content decreases by 23% in 30 minutes. When vacuum dried, the content decreases by 14.3% after 4 hours at a temperature of 40°C, and by 15% in 24 hours. High-purity Coenzyme A has strong hygroscopicity and quickly absorbs moisture and degrades when exposed to air. In the market, it is usually produced through aseptic freeze-drying technology, and its production process is: liquid preparation, sterilization filtration, filling, freeze-drying, plugging and capping, light inspection, and packaging. The current process has the following problems: (1) Since Coenzyme A must first be prepared into an aqueous solution, it is always in a non-dry state before freeze-drying, which is easy to degrade and the product quality is unstable. (2) After coenzyme A reaches the specified pH standard during the preparation process, its pH value may fluctuate up to ±0.8 after freeze-drying and the final clinical reconstitution step. This may not only cause the pH value of the product to exceed the preset internal control standard, but also increase the degradation rate, thereby leading to an increase in the content of related impurities. (3) The coenzyme A obtained by ordinary vacuum drying is an amorphous powder with low purity (≤85%) and poor stability. The coenzyme A preparation processed in this way may have adverse reactions during clinical use.
[0005] Therefore, the market urgently needs a lyophilized preparation of coenzyme A with good stability, low content of related substances and suitable for industrial production. Summary of the invention
[0006] To solve the above problems, the present invention aims to provide a stable lyophilized preparation of coenzyme A for injection.
[0007] The invention is realized by the following technical measures, and provides a stable coenzyme A lyophilized preparation for injection, wherein the prescription raw materials include, by weight: 220,000 units of coenzyme A, 4 to 7 parts of lyophilization protective agent, 10 to 60 parts of mannitol, and 1 to 6 parts of phosphate buffer.
[0008] Furthermore, the lyoprotectant is one or more combinations of trehalose, β-glucan, gelatin, hyaluronic acid, β-cyclodextrin, γ-polyglutamic acid, polyaspartic acid, threonine, L-cysteine, sodium thiosulfate, sodium pyrosulfite, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate.
[0009] The following steps are involved: S1. Dissolve the prescribed amount of lyophilized protective agent with water for injection; dissolve the prescribed amount of coenzyme A in the lyophilized protective agent solution under a static magnetic field device, mix for 10-30 minutes, and obtain a coenzyme A mixed solution for standby use; S2. Add the mannitol solution in the formula into the water for injection and stir to dissolve for later use; S3. The coenzyme A mixed solution in step S1 is combined with the mannitol solution in step S2, and the pH value is adjusted to 5.0 to 6.0 by adding phosphate buffer; S4. Under sterile conditions, the solution in S3 is filtered three times and filled to obtain a filling solution; S5. freeze-drying the filling solution obtained in S4, comprising the following steps: (1) Prefreezing: cool to -45~-35℃ and keep warm for 1~2h; Raise the temperature to -12~-8℃ and keep warm for 2~3h; Cool down to -45~-35℃, keep warm for 1~2h; (2) Sublimation drying: heat to -2~2℃ and keep warm for 2~3h; (3) Freeze drying: Heat to above 26-36°C, keep warm for 5-6 hours, end freeze drying, and set aside; S6. The filled lyophilized powder obtained in step S5 is capped to obtain a lyophilized preparation of coenzyme A.
[0010] Furthermore, in S1, the static magnetic field device is composed of a neodymium iron boron magnet, and the average static magnetic field intensity on the surface of the N pole and the S pole is 3-10 mT.
[0011] Furthermore, the filter membranes used for filtration in S4 are all polyethersulfone filter membranes, the pore size of the filter membranes used for primary filtration is 0.3-0.45 μm, and the pore sizes of the filter membranes used for secondary filtration and tertiary filtration are uniformly 0.2-0.22 μm.
[0012] Furthermore, in S5 (1), the pre-freezing cooling or heating rate is 1-2°C / min, and the vacuum degree is 5-10Pa.
[0013] Furthermore, in S5 (2) sublimation drying: the heating rate is 0.1~1°C / min, and the vacuum degree is 30~35Pa.
[0014] Furthermore, in S5 (3) analytical drying: the heating rate is 0.1~1°C / min, and the vacuum degree is 40~45Pa.
[0015] The beneficial effects of the present invention are: (1) The coenzyme A for injection prepared using the above new formula forms a protective layer that stabilizes coenzyme A during the preparation process. Polysaccharides can form a sol in an aqueous solution, and amino acids are dispersed in the sol to form a sol-like protective layer, which further increases the stability period of coenzyme A in aqueous solvents and improves the moisture-proof performance after freeze-drying. At the same time, the addition of antioxidants reduces the related substances of oxidized coenzyme A, 3'-dephosphorylated coenzyme A and 5'-adenosine phosphate, thereby improving the safety of the drug, reducing adverse drug reactions, and ensuring a definite therapeutic effect.
[0016] (2) The present invention uses a static magnetic field to assist in the preparation of coenzyme A for injection. Under the action of the magnetic field, the lone pair of electrons of the methylene and pyranoside bonds of the amino acids / polysaccharides are acted upon by the magnetic torque in the direction of the magnetic field, resulting in orderly stretching vibrations, thereby enhancing the sol protective layer of the amino acids / polysaccharides and significantly improving the stability of coenzyme A.
[0017] (3) In the static magnetic field assisted process of the present invention, the outer protective layer of coenzyme A is strengthened, and at the same time, the static magnetic field has an inhibitory effect on the bacteria in the liquid medicine, which acts on the free radicals produced by the degradation of long-chain fatty acids during bacterial metabolism, triggering bacterial oxidative stress, thereby inhibiting bacterial growth. This technology can improve the sterilization effect of the system and ensure the safety of clinical medication.
[0018] (4) The preparation process of the coenzyme A lyophilized preparation of the present invention uses phosphate buffer as an acid-base regulator, which can maintain a relatively stable pH value of coenzyme A in the solution. At the same time, since coenzyme A contains a phosphate group in its structure, phosphate buffer is beneficial to maintaining the stability of its molecular structure and reducing the content of related substances. Phosphate buffer is added to the liquid formula of the present invention, and the pH value of the re-dissolved product after lyophilization does not exceed ±0.3, which further improves the stability of coenzyme A.
[0019] (5) The freeze-drying process of the present invention adds a temperature-returning step, and performs tempering treatment at a temperature 5 to 15°C higher than the eutectic point, which promotes the recrystallization of the freeze-dried coenzyme A, allowing small ice crystal molecules to migrate to large ice crystal molecules to form more sublimation channels, thereby improving the sublimation efficiency; finally, the water content in the freeze-dried product of coenzyme A is further reduced by sublimation drying and analytical drying at a specific temperature, and the appearance and stability of the finished product can be improved, ultimately ensuring that the prepared freeze-dried coenzyme A preparation for injection meets the requirements of relevant products. The freeze-drying process of the coenzyme A for injection provided by the present invention is simpler to operate, shortens the freeze-drying time, has a low content of related substances, and reduces production costs. DETAILED DESCRIPTION
[0020] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially. Example 1
[0021] A stable coenzyme A lyophilized preparation for injection comprises the following steps: S1. Dissolve 3 parts of trehalose, 1 part of γ-polyglutamic acid, and 0.2 parts of sodium thiosulfate in water for injection respectively, dissolve 220,000 units of coenzyme A in the trehalose aqueous solution under a static magnetic field strength of 3 mT, add γ-polyglutamic acid solution and sodium thiosulfate solution, mix for 30 minutes, and obtain a coenzyme A mixed solution for standby use; S2. Add 25 parts of mannitol into water for injection and stir to dissolve for later use; S3. The coenzyme A mixed solution in step S1 is combined with the mannitol solution in step S2, and 4 parts of phosphate buffer are added to adjust the pH value to 5.5; S4. Under a sterile condition, the solution obtained in S3 is filtered three times and then filled to obtain a filling solution, wherein the filter membranes used for filtration are all polyethersulfone filter membranes, the pore size of the filter membrane for primary filtration is 0.45 μm, the pore size of the filter membrane for secondary filtration and the pore size of the filter membrane for tertiary filtration are uniformly 0.2 μm; S5. The filling solution obtained in S4 was cooled to -45°C at a rate of 1°C / min and a vacuum degree of 5Pa, kept warm for 1h, heated to -12°C, kept warm for 2h, cooled to -45°C, kept warm for 1h; heated to -2°C at a rate of 0.1°C / min and a vacuum degree of 30Pa, kept warm for 2h; heated to 26°C at a rate of 0.1°C / min and a vacuum degree of 40Pa, kept warm for 5h, and freeze-drying was terminated; S6. The filled lyophilized powder obtained in step S5 is capped to obtain a lyophilized preparation of coenzyme A. Example 2
[0022] A stable coenzyme A lyophilized preparation for injection comprises the following steps: S1. Dissolve 4 parts of β-cyclodextrin, 2 parts of threonine, and 1 part of sodium pyrosulfite in water for injection, respectively. Dissolve 220,000 units of coenzyme A in the β-cyclodextrin aqueous solution under a static magnetic field strength of 10 mT, add threonine solution and sodium pyrosulfite solution, mix for 30 minutes, and obtain a coenzyme A mixed solution for standby use; S2. 15 parts of mannitol were added to water for injection and stirred for dissolution and set aside; S3. The coenzyme A mixed solution in step S1 is combined with the mannitol solution in step S2, and 1 portion of phosphate buffer is added to adjust the pH value to 5.5; S4. Under a sterile condition, the solution obtained in S3 is filtered three times and then filled to obtain a filling solution, wherein the filter membranes used for filtration are all polyethersulfone filter membranes, the pore size of the filter membrane for primary filtration is 0.45 μm, the pore size of the filter membrane for secondary filtration and the pore size of the filter membrane for tertiary filtration are uniformly 0.2 μm; S5. The filling solution obtained in S4 was cooled to -35°C at a rate of 2°C / min and a vacuum degree of 10Pa, kept warm for 2h, heated to -8°C, kept warm for 3h, cooled to -35°C, kept warm for 2h; heated to 2°C at a rate of 1°C / min and a vacuum degree of 35Pa, kept warm for 3h; heated to 36°C at a rate of 1°C / min and a vacuum degree of 35Pa, kept warm for 6h, and freeze-drying was terminated; S6. The filled lyophilized powder obtained in step S5 is capped to obtain a lyophilized preparation of coenzyme A. Example 3
[0023] A stable coenzyme A lyophilized preparation for injection comprises the following steps: S1. Dissolve 2.5 parts of hyaluronic acid, 2.5 parts of polyaspartic acid, and 0.5 parts of butylated hydroxytoluene in water for injection, respectively, dissolve 220,000 units of coenzyme A in the hyaluronic acid aqueous solution under a static magnetic field strength of 6 mT, add the polyaspartic acid solution and the butylated hydroxytoluene solution, mix for 30 minutes, and obtain a coenzyme A mixed solution for standby use; S2. Add 20 parts of mannitol into water for injection and stir to dissolve for later use; S3. The coenzyme A mixed solution in step S1 is combined with the mannitol solution in step S2, and 2.5 parts of phosphate buffer are added to adjust the pH value to 5.5; S4. Under a sterile condition, the solution obtained in S3 is filtered three times and then filled to obtain a filling solution, wherein the filter membranes used for filtration are all polyethersulfone filter membranes, the pore size of the filter membrane for primary filtration is 0.45 μm, the pore size of the filter membrane for secondary filtration and the pore size of the filter membrane for tertiary filtration are uniformly 0.2 μm; S5. The filling solution obtained in S4 was cooled to -40°C at a rate of 1.5°C / min and a vacuum degree of 8Pa, kept warm for 1.5h, heated to -10°C, kept warm for 2.5h, cooled to -40°C, kept warm for 1.5h; heated to 0°C at a rate of 0.5°C / min and a vacuum degree of 32Pa, kept warm for 2.5h; heated to 30°C at a rate of 0.5°C / min and a vacuum degree of 32Pa, kept warm for 5.5h, and freeze-drying was terminated; S6. The filled lyophilized powder obtained in step S5 is capped to obtain a lyophilized preparation of coenzyme A. Comparative Example 1
[0024] The production process is partially the same as that of Example 3, and the difference from Example 3 is that S1 does not add lyoprotectant and static magnetic field treatment; S3 phosphate buffer is replaced with sodium hydroxide; S5. The filling solution obtained in S4 is cooled to -45°C and kept warm for 4 hours; heated to -18°C and kept warm for 6 hours; heated to 0°C and kept warm for 4 hours; heated to 20°C and kept warm for 6 hours, and freeze-drying is terminated; and the coenzyme A lyophilized preparation is obtained by capping. Comparative Example 2
[0025] The production process is partially the same as that of Example 3, and the difference from Example 3 is that S1 does not add lyoprotectant and static magnetic field treatment; S5. The filling solution obtained in S4 is cooled to -45°C and kept warm for 4 hours; heated to -18°C and kept warm for 6 hours; heated to 0°C and kept warm for 4 hours; heated to 20°C and kept warm for 6 hours, and freeze-drying is terminated; and the coenzyme A lyophilized preparation is obtained by capping. Comparative Example 3
[0026] The production process is partially the same as that of Example 3, and the difference from Example 3 is that S1 does not add a lyophilization protective agent and static magnetic field treatment. Comparative Example 4
[0027] The production process is partially the same as that of Example 3, but different from Example 3 in that the static magnetic field treatment is removed in S1.
[0028] Test Example 1 Test on reconstitution of the lyophilized coenzyme A preparation for injection prepared by the present invention Table 1 Test results of each group of coenzyme A lyophilized preparations for injection
[0029] According to Table 1, it can be seen that the reconstitution time of the coenzyme A lyophilized preparations for injection prepared in Examples 1 to 3 is within 50s, and the dissolution rate is relatively fast; the reconstitution time of Comparative Examples 1 to 4 is within 80s, among which the reconstitution time of Comparative Examples 1-2 is relatively long, and the solution is turbid, indicating that the lyophilization protectant, static magnetic field treatment and inappropriate lyophilization procedure have an effect on the reconstitution of the lyophilized agent. The pH value of the liquid before and after reconstitution of Examples 1 to 3 and Comparative Examples 2 to 4 does not change by more than ±0.3, and the coenzyme A content of Comparative Example 2 is higher than that of Comparative Example 1, which may be due to the presence of phosphate buffer salt, which can absorb a certain amount of H+ and OH- without causing a significant change in pH through the effective balance of conjugate acid-base pairs, keeping coenzyme A in a solution with a relatively constant pH value and alleviating the degradation rate. The qualified microorganisms of Examples 1 to 3 may be due to the fact that the polysaccharides in the lyophilization protectant have a certain antibacterial effect, and the static magnetic field has an inhibitory effect on the bacteria in the liquid medicine. The superposition of the two technologies enhances the sterilization effect and ensures that the product meets the relevant regulations.
[0030] Test Example 2 Influencing factors and stability test of the lyophilized coenzyme A preparation for injection prepared by the present invention (1) High temperature test Take the preparation, remove the outer packaging, place it in a sealed clean container, place it at 40°C for 10 days, and test various indicators.
[0031] (2) High humidity test Take the preparation, remove the outer packaging, place it in a constant humidity sealed container, place it at 25°C and humidity RH of 90%±5% for 10 days, and test various indicators.
[0032] (3) Strong light exposure test Take the preparation, remove the outer packaging, place it in a light box, and place it under the condition of illumination of 4500lx±500lx for 10 days, and test various indicators. The results are as follows: Table 2 Test results of influencing factors of Comparative Examples 1-4 and Examples 1-3
[0033] (4) Accelerated test The coenzyme A lyophilized preparation for injection prepared by the present invention was placed in a simulated market packaging at a temperature of (40±2)°C and a relative humidity of (75%±5)5 for 12 months, and samples were taken and tested every month. The results are as follows: Table 3 Six-month accelerated test results of Comparative Example 1 and Examples 1-3
[0034] (5) Long-term trials The coenzyme A lyophilized preparation for injection prepared by the present invention was placed in a simulated market packaging at a temperature of (30±2)°C and a relative humidity of (65%±5)% for 24 months. Samples were taken every 3 months for the first 12 months, and samples were taken at 18 and 24 months after 12 months. The results are as follows: Table 4 24-month long-term test results of Comparative Example 1 and Examples 1-3
[0035] According to the above test results, the properties and content of the coenzyme A lyophilized preparation for injection prepared according to the present invention are in compliance with the regulations during storage, and the changes are not obvious with the increase of time, indicating that it has high stability and few related substances. The present invention uses a lyophilization protective agent and is assisted by a static magnetic field to prepare coenzyme A for injection, increases the stability of coenzyme A in water solvents, and reduces the generation of related substances; the freeze-drying process adopts a rewarming process to improve the crystallization rate, the crystal size becomes larger, the crystal form and the distribution size are more uniform, so that the gaps formed after drying are more uniform, effectively improving the drying efficiency, improving the appearance of the finished product and the stability of the finished product; during the sublimation drying process, a lower drying temperature is first used to remove most of the water, and then the temperature is increased and the vacuum degree is adjusted to maintain a steady sublimation rate, effectively reducing the internal airflow disturbance of the powder cake, ensuring that the powder cake that has been evaporated does not collapse, so that its final moisture content is about 1%, and further effectively reducing the risk of degradation of the finished product during storage.
[0036] In summary, the preparation process of the lyophilized coenzyme A preparation for injection of the present invention is simple to operate, has high stability, contains less related substances, and improves drug safety.
[0037] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.
Claims
1. A stable lyophilized preparation of coenzyme A for injection, characterized in that: The prescription raw materials include by weight: 220,000 units of coenzyme A, 4 to 7 parts of lyophilization protectant, 10 to 60 parts of mannitol, and 1 to 6 parts of phosphate buffer (disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, potassium chloride).
2. A stable lyophilized preparation of coenzyme A for injection according to claim 1, characterized in that: The freeze-drying protective agent is one or more combinations of trehalose, β-glucan, gelatin, hyaluronic acid, β-cyclodextrin, γ-polyglutamic acid, polyaspartic acid, threonine, L-cysteine, sodium thiosulfate, sodium pyrosulfite, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate.
3. A stable lyophilized preparation of coenzyme A for injection, characterized in that: The following steps are involved: S1. Dissolve the prescribed amount of lyophilized protective agent with water for injection; dissolve the prescribed amount of coenzyme A in the lyophilized protective agent solution under a static magnetic field device, mix for 10-30 minutes, and obtain a coenzyme A mixed solution for standby use; S2. Add the prescribed amount of mannitol into water for injection, stir and dissolve, and set aside; S3. The coenzyme A mixed solution in step S1 is combined with the mannitol solution in step S2, phosphate buffer is added, and the pH value is adjusted to 5.0~6.0; S4. Under sterile conditions, the solution in S3 is filtered three times and filled to obtain a filling solution; S5. freeze-drying the filling solution obtained in S4, comprising the following steps: (1) Prefreezing: cool to -45~-35℃ and keep warm for 1~2h; Raise the temperature to -12~-8℃ and keep warm for 2~3h; Cool down to -45~-35℃, keep warm for 1~2h; (2) Sublimation drying: heat to -2~2℃ and keep warm for 2~3h; (3) Freeze drying: Heat to above 26-36°C, keep warm for 5-6 hours, end freeze drying, and set aside; S6. The filled lyophilized powder obtained in step S5 is capped to obtain a lyophilized preparation of coenzyme A.
4. A stable lyophilized preparation of coenzyme A for injection according to claim 3, characterized in that: The static magnetic field device is composed of a neodymium iron boron magnet, and the average static magnetic field intensity on the N pole and S pole surface is 3-10 mT.
5. A stable lyophilized preparation of coenzyme A for injection according to claim 3, characterized in that: The filter membranes used in S4 are all polyethersulfone filter membranes. The pore size of the filter membrane used for primary filtration is 0.3~0.45μm, and the pore size of the filter membrane used for secondary filtration and the pore size of the filter membrane used for tertiary filtration are uniformly 0.2~0.22μm.
6. A stable lyophilized preparation of coenzyme A for injection according to claim 3, characterized in that: In S5 (1), the pre-freezing cooling or heating rate is 1~2℃ / min, and the vacuum degree is 5~10Pa.
7. A stable lyophilized preparation of coenzyme A for injection according to claim 3, characterized in that: (2) Sublimation drying in S5: heating rate is 0.1~1℃ / min, vacuum degree is 30~35Pa.
8. The stable lyophilized preparation of coenzyme A for injection according to claim 3, characterized in that: In S5 (3) analytical drying: the heating rate is 0.1~1℃ / min, and the vacuum degree is 40~45Pa.