A formulation of enoxaparin sodium for sheep and its preparation and use

Abstract

The application discloses a sheep enoxaparin sodium preparation and a preparation and application thereof, and the preparation is a lyophilized powder injection containing a pharmaceutical active ingredient of sheep enoxaparin sodium. The preparation method comprises the following steps: preparation of a medicinal liquid, sterilization filtration, filling, sterile freeze-drying and cover rolling. The sheep enoxaparin sodium preparation provided by the application has good stability, can be transported or stored at a high temperature, can be stored for a long time at 30 DEG C, can be stored for 6 months at 40 DEG C or below for a short time, does not need to be transported or stored at a low temperature, is particularly suitable for application in countries and regions in the international climate zone III dry-hot zone and IV humid-hot zone, and effectively improves the effectiveness, safety and accessibility of drug application. The process route of the preparation method is simple and easy to implement. The preparation can be prepared into a halal medicine and is applied in disease prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to ovine enoxaparin sodium preparation and preparation method thereof. BACKGROUND

[0002] Ovine enoxaparin sodium (OES) is a low molecular weight heparin sodium salt, which is derived from benzyl ester of ovine intestinal mucosa heparin by alkaline depolymerization, and is used as an anticoagulant and antithrombotic agent in clinical practice. Chinese patent (patent number CN 106243246 B) discloses that it meets all quality index requirements of Enoxaparin Sodium in mainstream pharmacopoeias such as the United States Pharmacopeia (USP) and the European Pharmacopoeia (EP) except for the requirement of Enoxaparin Sodium source. Ovine enoxaparin sodium has certain differences in chemical structure (disaccharide composition) and physicochemical properties from Enoxaparin Sodium from pig source as required by the above pharmacopoeias, and is an innovative drug of new source.

[0003] Ovine enoxaparin sodium has the same pharmacodynamic properties as Enoxaparin Sodium (from pig source). The source from sheep is an important feature, and it can potentially be prepared as a halal drug, while heparin products from pig source are not halal. Halal is a special name in accordance with Islamic doctrine, and Islamic doctrine has clear requirements for food and drugs. Only ruminant products such as cattle, sheep, and goats are allowed to be eaten, and non-ruminant products such as pigs and dogs are prohibited. The global Muslim population exceeded 1.6 billion in 2013, accounting for 23% of the world's 6.9 billion population in that year, and the population of Islamic countries exceeded 1.8 billion in 2021. Heparin drugs are one of the most important anticoagulant and antithrombotic agents, and are listed as life-saving drugs by the World Health Organization (WHO). However, almost all current clinical heparin drugs are from pig sources, which cannot meet the medication needs of Muslim populations and other special populations.

[0004] In April 2021, the Indonesian Food and Drug Supervisory Administration (BPOM) was the first in the world to approve the ovine enoxaparin sodium injection drug, with the English trade name "ANTITEN-A", and the holder is PT. BioFarma, one of the largest pharmaceutical production enterprises in Indonesia, which has caused a stir in the Islamic world.

[0005] Patent CN 106243246 B discloses ovine enoxaparin sodium and two types of injection solutions thereof. One is composed of ovine enoxaparin sodium and water for injection, and the other includes benzyl alcohol in addition to the above components. The ovine enoxaparin sodium preparation marketed in Indonesia is of the above Form I, which is only composed of ovine enoxaparin sodium and water for injection, and is a pre-filled injection, with a storage condition requirement of temperature not higher than 25℃. It can be seen that the storage temperature is an important factor affecting the quality of ovine enoxaparin sodium injection and the distribution of the drug, and the solution type of ovine enoxaparin sodium preparation is not resistant to high temperature.

[0006] In terms of global distribution, Muslim populations and countries are mainly distributed in Southeast Asia, South Asia, the Middle East, Central Asia and North Africa, most of which are underdeveloped regions, and are located in the Ⅲ dry tropics (annual average kinetic temperature 27.4℃, relative humidity 35%) and Ⅳ humid tropics (annual average kinetic temperature 27.4℃, relative humidity 76%) in the international climate zone, and the annual temperature is significantly higher than the current storage requirements of sheep enoxaparin sodium injection. Therefore, there is an urgent need for a stable storage form of sheep enoxaparin sodium injection that can withstand high temperatures, not only safe and effective, but also free from the harsh conditions of low-temperature transportation and storage. SUMMARY

[0007] To solve the above technical problems, the purpose of the present application is: a sheep enoxaparin sodium preparation and its preparation method, which is safe, stable and effective, greatly reduces the storage and transportation conditions, solves the problem of non-temperature storage of existing sheep enoxaparin sodium injection, facilitates the normal temperature storage, transportation and application of related drugs in Ⅲ dry tropics and Ⅳ humid tropics countries and regions, and effectively improves the effectiveness, safety and (underdeveloped areas) accessibility of the drug. The preparation method is simple and easy to operate.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is: a sheep enoxaparin sodium preparation, which is a lyophilized powder injection containing the active pharmaceutical ingredient sheep enoxaparin sodium.

[0009] In the field of biological and pharmaceutical technology, the solid preparation for injection of drugs often includes sterile lyophilized powder drug preparation, but existing enoxaparin sodium and other low molecular heparin drug preparations are almost sterile injection preparations, and the storage temperature is generally required to be not higher than 25℃ in the pharmacopoeia or the instruction manual. The sheep enoxaparin sodium drug preparation marketed in Indonesia also adopts the sterile injection preparation type, and the required storage condition is also not higher than 25℃.

[0010] The sheep enoxaparin sodium preparation proposed in the present scheme is a sterile lyophilized powder injection in solid form, which has good high temperature resistance, so that the drug can be stored and transported at high temperature for a long time, and the storage condition only needs to meet the room temperature (30℃) requirements of ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) drug stability guidelines in Ⅲ area, Ⅳa area and Ⅳb area.

[0011] The sheep enoxaparin sodium preparation proposed in the present scheme is stable in quality after 24 months of storage at 30℃ in a specific embodiment 11 but not limited to this embodiment, and is expected to be stable after a longer period of time such as 36 months and 42 months in the continuing process; in another specific embodiment 10 but not limited to this embodiment, the quality is stable after 6 months of storage at 40℃. Therefore, the sheep enoxaparin sodium preparation allows long-term storage at 30℃, and allows short-time transportation at 40℃ for up to 6 months.

[0012] Further, the foregoing enoxaparin sodium preparation, wherein the lyophilized powder injection comprises enoxaparin sodium and a pharmaceutically acceptable excipient, and the excipient comprises one or more of an excipient, an adsorbent, an isotonicity adjusting agent, and a pH adjusting agent.

[0013] Further, the foregoing enoxaparin sodium preparation, wherein the excipient is selected from one or more of mannitol, glucose, sucrose, and sorbitol; the adsorbent is activated carbon; the isotonicity adjusting agent is selected from one or both of glucose and sodium chloride; and the pH adjusting agent is selected from one or both of sodium hydroxide and hydrochloric acid.

[0014] Further, the foregoing enoxaparin sodium preparation, wherein the lyophilized powder injection is prepared by dissolving the raw material enoxaparin sodium in water or by dissolving the raw material enoxaparin sodium and an excipient in water, and then removing water by sterile freeze-drying.

[0015] Still further, the foregoing enoxaparin sodium preparation, wherein the preparation is packaged in a single vial, and the package has a specification of 2000, 4000, 6000, 8000, 10000, or 30000 units of anti-Xa factor, or 20, 40, 60, 80, 100, or 300 mg of enoxaparin sodium.

[0016] A method for preparing an enoxaparin sodium preparation, comprising the steps of:

[0017] S.1 Preparation of a liquid medicine: dissolving enoxaparin sodium with or without an excipient in water to prepare a liquid medicine having a mass concentration of 2% to 20% enoxaparin sodium;

[0018] S.2 Sterilization filtration: passing the liquid medicine through a micro-pore filter to remove microorganisms;

[0019] S.3 Filling: filling the liquid medicine from which microorganisms have been removed into a container;

[0020] S.4 Freeze-drying: filling the container with the liquid medicine with a half plug, and then sending it to a freeze-dryer for freeze-drying, and after the freeze-drying is completed, filling the container with a full plug to seal it;

[0021] S.5 Capping.

[0022] Further, the foregoing method for preparing an enoxaparin sodium preparation, wherein the freeze-drying comprises, in sequence, a pre-freezing stage in which the freeze-dryer is set to a shelf temperature of -20°C to -50°C, and after the temperature is lowered, the temperature is maintained for 2 to 6 hours, and then the vacuum is controlled to a degree of 5 to 50 Pa;

[0023] Sublimation stage: the temperature of the partition is raised to -20℃-20℃, the temperature rising time is 0.5-3 hours, the temperature is kept for 6-20 hours, and the vacuum degree is controlled to be 5-50 Pa;

[0024] Dissolution and drying stage: the temperature of the partition is raised to 20℃-50℃, the temperature rising time is 0.5-3 hours, the extreme vacuum is extracted, and the temperature is kept for more than 0.5 hours;

[0025] Pressure rising test: when the pressure rising test ΔP is less than 5 Pa / min, the drying end point is considered to be reached, the freeze drying is ended, and the vacuum is removed.

[0026] Further, the preparation method of the sheep enoxaparin sodium preparation, wherein (1) the pre-freezing stage: the temperature of the freeze dryer partition is set to -40℃, the temperature lowering time is 1 h, and the temperature is kept for 3 hours after the end, then the vacuum is extracted, and the vacuum degree is controlled to be 50 Pa.

[0027] Further, the preparation method of the sheep enoxaparin sodium preparation, wherein the sublimation stage: the temperature of the partition is raised to 0℃, the temperature rising time is 2 hours, the temperature is kept for 12 hours, and the vacuum degree is controlled to be 20±5 Pa.

[0028] Further, the preparation method of the sheep enoxaparin sodium preparation, wherein the dissolution and drying stage: the temperature of the partition is raised to 40℃, the temperature rising time is 2 hours, the temperature is kept for 2 hours, and the extreme vacuum is controlled.

[0029] Further, the preparation method of the sheep enoxaparin sodium preparation, wherein in the S.2 sterilization filtration step, the filter membrane material in the microporous filter is selected from polyether sulfone or polyvinylidene fluoride, and at least one filtration with a filter diameter less than or equal to 0.22 μm is performed.

[0030] Further, the preparation method of the sheep enoxaparin sodium preparation, wherein in the S.1 drug solution preparation step, the mass concentration of the drug solution is 8%-15%.

[0031] Further, the preparation method of the sheep enoxaparin sodium preparation, wherein in the S.3, the container for filling the drug solution is a borosilicate glass bottle; in the S.4 freeze drying, the plug in the container is a halogenated butyl rubber plug; and in the S.5 capping, the cap is an aluminum cap or an aluminum-plastic combined cap.

[0032] The sheep enoxaparin sodium preparation as a medicine for the prevention and / or treatment of diseases.

[0033] Further, the sheep enoxaparin sodium preparation, since its active pharmaceutical ingredient sheep enoxaparin sodium is derived from sheep, it can be further used as a halal medicine for the prevention and / or treatment of diseases.

[0034] The sheep enoxaparin sodium preparation provided by the application has good stability, can be transported or stored at high temperature, can be stored for a long time at 30 DEG C, and can be stored for 6 months at 40 DEG C or below, and thus does not need to be transported or stored at low temperature, and is particularly suitable for application in countries and regions in the international climate zone III dry-hot zone and IV humid-hot zone. The sheep enoxaparin sodium preparation provided by the application effectively improves the effectiveness, safety and accessibility of drug application. In addition, the preparation method of the sheep enoxaparin sodium preparation provided by the application is simple and easy to implement. DETAILED DESCRIPTION

[0035] The sheep enoxaparin sodium preparation and the preparation method thereof will be further described in detail through specific examples.

[0036] Example 1: Preparation of the sheep enoxaparin sodium preparation.

[0037] According to the analysis certificate of the sheep enoxaparin sodium raw material, the dry potency of the sheep enoxaparin sodium raw material is 104 anti-Xa factor units / mg, and the moisture is 2.5%.

[0038] 986.00 g of the sheep enoxaparin sodium raw material (total activity of 100000000 anti-Xa activity units) was accurately weighed and placed in a liquid preparation tank, and water for injection was added and pre-cooled to 25 DEG C, about 7 L, and stirred for not less than 30 minutes until completely dissolved. Water for injection was added and pre-cooled to 25 DEG C, and the volume was adjusted to 10.00 L, and stirring was continued for not less than 5 minutes. The liquid medicine was pre-filtered through a 0.45 μm PVDF filter, sterilized and filtered through a 0.22 μm PVDF filter into a temporary storage tank, and then filtered through a 0.22 μm PVDF filter. The liquid-filled Westlin bottle after loading was loaded into the box to the freeze dryer partition, and the bromobutyl rubber plug was added to the half plug; the freeze-drying was performed according to the following procedure, as shown in Table 1.

[0039] Table 1:

[0040]

[0041] After the analysis of the drying end, the results of the pressure rise test within 5 minutes were that the average ΔP was less than 1 Pa / min, the freeze-drying was stopped, the pressure was released to about 88000 Pa (-0.87 standard atmospheric pressure), the full plug was added, the box was taken out, and the aluminum plastic cover was rolled.

[0042] Result analysis:

[0043] (1) The finished product is white loose block, moisture is 0.64%, and is reconstituted with 1.0 mL of water for injection, reconstitution time is about 30 s, the clarity of the solution after reconstitution is qualified, and is colorless and clear solution (less than 0.5 turbidity standard suspension).

[0044] (2) The finished product is detected according to the United States Pharmacopoeia (USP) and the British Pharmacopoeia (BP) (porcine-derived) enoxaparin sodium injection, mainly the difference between the lyophilized powder and the injection (the sample is reconstituted with 1.0 mL of water for injection, which is equivalent to the corresponding injection), the main test items and results are shown in Table 2.

[0045] Table 2

[0046]

[0047]

[0048] Note: The acceptance criteria involved in each example are enterprise standards, which are not lower than the quality standards of enoxaparin sodium injection in the United States Pharmacopoeia (USP) and the British Pharmacopoeia (BP).

[0049] The above results show that the finished product of sheep enoxaparin sodium preparation meets the established enterprise quality standards, and is completely consistent with the (porcine-derived) enoxaparin sodium injection of USP and EP, except that the source is sheep and the corresponding is a solid dosage form.

[0050] Example 2: Formula selection investigation and preparation.

[0051] Take the same source of sheep enoxaparin sodium raw material drug in Example 1. According to Table 2, prepare different prescription liquids, and compare various pharmaceutical excipients. After bacterial filtration, each 10000 anti-Xa factor units are divided into 2 mL of borosilicate glass injection vials, each 200 bottles, 100 milliliters per bottle. The specific prescription is shown in Table 3.

[0052] Table 3: (in the table, / means not applicable or none.)

[0053]

[0054]

[0055] The freeze-drying conditions are referred to in Example 1, and the freeze-dried bottles are pressed, taken out of the box, and manually crimped. The comparison results of the different prescription liquids after freeze-drying are shown in Table 4.

[0056] Table 4

[0057]

[0058] Conclusion: The above results show that the product properties of each group after freeze-drying are similar, the moisture, reconstitution time, solution pH and clarity are similar, but the osmotic pressure ratio is better without adding isotonicity regulator and excipient. This also shows that the sheep enoxaparin sodium raw drug, as a polysaccharide material, has good freeze-dried powder, and the effect of adding excipients, isotonicity regulators, pH regulators and activated carbon and other auxiliary materials on the drug properties is limited, and even the osmotic pressure ratio of the reconstituted solution is worse than that without adding auxiliary materials.

[0059] But preferably, only water is added as a solvent during the preparation process and is removed during the freeze-drying process. The reasons are as follows.

[0060] 1. Using only water as a solvent: Sheep enoxaparin sodium is easily soluble in water and does not require the use of other solvents, which can avoid the risk of introducing other harmful or useless solvent components, and water is easily removed in the freeze-drying process.

[0061] 2. No excipient is used: Sheep enoxaparin sodium is a low molecular weight heparin sodium salt, and its solid characteristics are white or white-like amorphous powder. It is a polysaccharide material, and its freeze-dried powder or solid powder has good support. Generally, drug freeze-drying excipients use sugar materials such as glucose and mannitol, so this product does not need to add excipients, which can also reduce the risk of introducing harmful or useless components.

[0062] 3. No adsorbent is used: The sheep enoxaparin sodium raw drug and water in the drug solution formula have strict requirements for impurity limits, solution color and turbidity (particulate matter), and endotoxin limits in the corresponding pharmacopoeia quality standards. The main purpose of using activated carbon as an adsorbent is to decolorize and adsorb particulate matter to ensure the color and turbidity of the product solution. Because these items have corresponding quality control in the raw drug, activated carbon and other adsorbents are not needed, which can also reduce the risk of introducing harmful or useless components.

[0063] 4. No isotonicity regulator is used: Sheep enoxaparin sodium is a low molecular weight heparin sodium salt, and the sodium salt itself has an osmotic potential. In the corresponding commercially available sheep enoxaparin sodium injection dosage form, only sheep enoxaparin sodium and water are used, and the drug solution concentration is about 100 anti-Xa factor units per 1 mg of sheep enoxaparin sodium, which is equivalent to 100 mg. Therefore, the commercially available sheep enoxaparin sodium injection is about 10% of the drug solution concentration. In Example 2 but not limited to this example, the reconstituted drug solution of the freeze-dried powder injection is about equivalent to this 10% drug solution concentration, and the test data also shows that the osmotic pressure ratio is about 1.0. After adding isotonicity regulators such as glucose or sodium chloride, the osmotic pressure ratio exceeds 1.0, which is not isotonic, although it is still suitable for use as a drug. Therefore, not using an isotonicity regulator not only makes the drug isotonic, but also reduces the risk of introducing harmful or useless components.

[0064] 5. No pH adjuster is used: the purpose of pH adjuster is to adjust the pH of the drug solution; the prescription of commercially available Enoxaparin Sodium for Injection contains only the active pharmaceutical ingredient and water for injection, and does not contain other pH adjusters. The reason is that the active pharmaceutical ingredient Enoxaparin Sodium and water, and the water is removed in the drying process, and the pH value is strictly controlled and required in the corresponding pharmacopoeia quality standard. Since no other excipients that can easily cause changes in pH value are added, the pH of the product will not change greatly. Therefore, there is no need to use pH adjuster, and the risk of introducing harmful or useless ingredients can be reduced.

[0065] 6. In this simple scheme, water is the only excipient used, and is removed in the freeze-drying process.

[0066] In summary, the simple formula of the product is only Enoxaparin Sodium and water, and the process is filtration and aseptic freeze-drying. Water is removed in the drying process, and the active pharmaceutical ingredient Enoxaparin Sodium has strict controls on impurities, color, turbidity (particulate matter), pH and endotoxin, etc. Water can ensure the solubility, the powder properties (shape) after freeze-drying, and the color, turbidity, pH and osmolality of the solution after reconstitution, etc. Therefore, in addition to water, it is preferred not to add other solvents, excipients, adsorbents, isotonicity adjusting agents and pH adjusters. In addition, the freeze-dried powder for injection prepared by this simple formula has only Enoxaparin Sodium as a component. Before use of the drug, water is used for reconstitution, which is convenient, fast, efficient and suitable. The reconstituted drug solution is suitable for injection, and the composition is consistent with the existing commercially available Enoxaparin Sodium for Injection. Similarly, both are active pharmaceutical ingredients Enoxaparin Sodium and water for injection, and there is no other redundant component. Therefore, the drug has good compliance.

[0067] Example 3: Selection of filter membrane material.

[0068] The formula of the drug solution is: Enoxaparin Sodium 10000000 anti-Xa factor units, water for injection is added to 1000 mL, i.e. the concentration of the drug solution is 10000 anti-Xa factor units / mL.

[0069] Preparation method: at 25℃, accurately weigh the above amount of Enoxaparin Sodium active pharmaceutical ingredient into a 2L clean glass beaker, add about 750mL of pre-cooled water for injection to 25℃, mechanically stir for 25 minutes, then add about 200mL of water for injection to make up the total volume of the drug solution to 1000mL.

[0070] Filter membrane test method: the drug solution is divided into two parts of about 500mL each, and each is filtered 5 times with polyether sulfone (PES) and polyvinylidene fluoride (PVDF) filter membranes with a pore size of 0.22μm. The test results of different filter membrane materials are shown in Table 5.

[0071] Table 5 ( " / " in the table means not involved)

[0072]

[0073] Note: The acceptance criteria are the enterprise standards, which are not lower than the quality standards of enoxaparin sodium injection in the United States Pharmacopoeia (USP) and the British Pharmacopoeia (BP).

[0074] Conclusion: The experimental results show that the PES material and PVDF material filter membrane have no significant effect on the molecular weight distribution and potency of the sheep enoxaparin sodium liquid, so it can be considered that the PES material and PVDF material filter membrane are compatible with the product liquid.

[0075] Example 4: Selection of packaging materials

[0076] The formulation and preparation of the liquid are the same as in Example 3. The filtration of the liquid uses a 0.2 μm PVDF filter, and the filtrate is divided into two groups of different glass vials, each 200 vials (10,000 anti-Xa factor units per vial). The freeze-drying process is the same as in Example 1, and the hand-crimped. The product vials are placed at 40°C to investigate the stability, and the investigation time is 0 days, 10 days and 30 days. The specific packaging materials and specification information are shown in Table 6. The detection investigation results under different packaging materials at 40°C are shown in Table 7.

[0077] Table 6

[0078]

[0079] Table 7

[0080]

[0081]

[0082] Note: 1. The molecular weight tests are M 2000 , M 2000-8000 , M 8000 and MW in turn; 2. The insoluble particle tests are the number of particles ≥10 μm and the number of particles ≥25 μm in turn. The acceptance criteria are the enterprise standards, which are equivalent to the quality standards of enoxaparin sodium injection in the United States Pharmacopoeia (USP) and the British Pharmacopoeia (BP).

[0083] Conclusion: The experimental results show that different material packaging materials have no significant effect on the quality of the sheep enoxaparin sodium preparation freeze-dried powder, including color, clarity, moisture, molecular weight distribution, insoluble particles and anti-Xa factor units. In addition, compared with each other, the product quality of the injection vial made of medium borosilicate glass is better than that of the injection vial made of low borosilicate glass, and the difference between the two halogen butyl rubber stoppers is not large.

[0084] Therefore, it can be considered that borosilicate glass vials and halogenated butyl rubber stoppers have good compatibility with this product, and borosilicate glass vials are preferred.

[0085] Example 5: Investigation of the sublimation stage in the freeze-drying process.

[0086] The formulation and preparation process of the drug solution are the same as in Example 3. The drug solution was filtered using a 0.22μm PVDF filter, and the filtrate was dispensed into 2mL borosilicate glass vials (10,000 anti-Xa factor units / vial). The vials were placed in a lyophilizer with a half-stop, and after lyophilization, the pressure was released and the vials were fully stoppered, and then manually capped.

[0087] The comparison of the sublimation stage of freeze-drying processes mainly focuses on different sublimation drying temperatures, as shown in Table 8.

[0088] Table 8

[0089]

[0090]

[0091] Table 8 shows the freeze-drying results: all product bottles were in normal condition, with no spraying. The finished product was a white, loose, lumpy substance. The clarity of the solution after reconstitution was satisfactory, with a reconstitution time of approximately 30-40 seconds, and the moisture content was below 2%. This indicates that the product is suitable for sublimation freeze-drying over a wide temperature range, which indirectly demonstrates its good stability.

[0092] Example 6: Investigation of analytical drying in freeze-drying process.

[0093] The formulation and preparation of the drug solution were the same as in Example 3. The drug solution was filtered using a 0.22μm PVDF filter, and the filtrate was dispensed into 2mL borosilicate glass vials (10,000 anti-Xa factor units / vial). The vials were placed in a lyophilizer with a half-stop, and after lyophilization, the pressure was released and the vials were fully stoppered, and then manually capped.

[0094] The comparative study of the desorption process of freeze drying mainly focuses on different desorption drying temperatures, as shown in Table 9.

[0095] Table 9

[0096]

[0097] Table 9 shows the freeze-drying results: all product bottles were in normal condition, with no spraying. The finished product was a white, loose, lumpy substance. The clarity of the solution after reconstitution was satisfactory, and there was no significant difference in color at different high temperatures. The reconstitution time was approximately 30-40 seconds, and the moisture content was below 2%. This indicates that the product is suitable for desorption drying over a wide temperature range, which indirectly demonstrates its good stability.

[0098] Example 7: Effect of the concentration of the liquid prodrug before lyophilization on the quality of the finished product.

[0099] The formulation and preparation of the liquid drug were similar to those of Example 3.

[0100] Preparation method: at 25°C, three portions of ovine enoxaparin sodium were accurately weighed, each portion being 2000000 anti-Xa factor units (approximately equal to 20 g), and each portion was added to pre-cooled water for injection, stirred to dissolve, and then water for injection was added to bring the total volume of the liquid to 1000 mL, 250 mL, 200 mL, 133 mL and 100 mL, respectively, i.e. the final mass concentration was 2%, 8%, 10%, 15% and 20%, respectively. The liquid was sterilized by filtration through a 0.22 μm PVDF filter, filled into 1.0 mL to 2.0 mL boro-silicate glass vials, the vials were placed in a box, and a half plug was added; the vials were freeze-dried (pre-freezing at -40°C for 4 h; sublimation at 0°C for 14 h, vacuum control at 20 ± 5 Pa; resolution at 40°C for 4 h, extreme vacuum), after the end of the process, the pressure was released, the full plug was added, and the vials were removed from the box. The effect of the concentration of the liquid on the quality of the lyophilized finished product was investigated, and the results are shown in Table 10.

[0101] Table 10

[0102]

[0103] The results of the lyophilization in Table 10 show that when the concentration of the liquid was 2%, there was a small amount of spraying of the vials, the finished product was a relatively loose white block, and the reconstitution time, moisture and anti-Xa factor unit met the requirements; when the concentration of the liquid was 10% or more, there was no spraying of the vials; when the concentration of the liquid was 20%, the product was a relatively compact block, and the reconstitution time of the finished product was longer. In summary, the concentration of the liquid mainly affected the appearance and reconstitution time of the product, but had no significant effect on the color, clarity, moisture and potency of the product. The results show that all concentrations of the liquid can obtain a qualified lyophilized product, and a neutral and suitable concentration of 8% to 15% of the liquid is more optimal, which helps to obtain a lyophilized product with better properties.

[0104] Example 8: Investigation of different specifications of the finished product

[0105] The commercially available enoxaparin sodium injection preparations have different anti-Xa factor units of 2000 to 30000 or 20 to 300 mg specifications, usually the enoxaparin sodium injection liquid concentration is 10000 anti-Xa factor units / mL (about equivalent to 100 mg / mL), only the injection liquid volume in the pre-filled needle is different, the common specifications are: 2000 anti-Xa factor units / 0.2 mL, 4000 anti-Xa factor units / 0.4 mL, 6000 anti-Xa factor units / 0.6 mL, 8000 anti-Xa factor units / 0.8 mL and 10000 anti-Xa factor units / 1.0 mL, etc. The enoxaparin product is usually used as a disposable product in clinical, and the selection of different specifications for different body weight patients has great convenience. In addition, some enoxaparin sodium injection multi-dose forms for multiple uses, such as 30000 anti-Xa factor units / 3.0 mL, may also contain about 2% benzyl alcohol and other preservatives.

[0106] The present application investigates the sheep enoxaparin sodium preparation, different packaging specifications of a single bottle, in terms of anti-Xa factor units, 2000 anti-Xa factor units, 4000 anti-Xa factor units, 6000 anti-Xa factor units, 8000 anti-Xa factor units, 10000 anti-Xa factor units and 30000 anti-Xa factor units. The liquid composition before lyophilization of each specification is consistent, all of which are sheep enoxaparin sodium and water for injection, the concentration is 10000 anti-Xa factor units / 1.0 mL, only the filling volume is different. In addition, the 30000 anti-Xa factor units specification product uses a 5 mL borosilicate glass vial, and the rest of the specifications use a 2 mL borosilicate glass vial.

[0107] Preparation method: at 25°C, weigh 1040.0 g of sheep enoxaparin sodium raw material (analysis certificate data: LOD% is 3.8, potency 100 anti-Xa factor IU / mg), add to a 15 L liquid preparation tank; add about 8.0 L of pre-cooled to 25°C water for injection, start stirring, 150 rpm stirring for 30 minutes, take the solution sample to check that it has dissolved clear, continue to add about 1.5 L of water for injection, accurately set the liquid volume to 10.0 L, continue to stir at 150 rpm for 10 minutes; press the liquid to filter through a 0.45 μm filter and a 0.22 μm filter, collect into a temporary storage tank; adjust the filling amount and accuracy of the filling machine, control the target filling volume to be 0.2 mL; the liquid in the temporary storage tank is filtered through a 0.22 μm filter into a filling buffer tank, precision filling, to prepare a 0.2 mL (i.e. 2000 anti-Xa factor units) specification 2 mL vial liquid; the sterilized rubber stopper machine adds a half stopper, the track machine automatically sends the bottle into the freeze dryer shelf, according to the program (same as example one) freeze drying and press the full stopper out of the box, to prepare a sheep enoxaparin sodium preparation lyophilized powder (specification: 2000 anti-Xa factor units).

[0108] Other specifications are prepared, drug solution preparation according to the analysis of raw materials and LOD% data, prescription is 100000000 anti-Xa factor units and water for injection (supplemented to 10.0L), other is mainly different filling liquid volume, 4000 anti-Xa factor units, 6000 anti-Xa factor units, 8000 anti-Xa factor units and 10000 anti-Xa factor units of specification, respectively in 2mL vial 0.4mL, 0.6mL, 0.8mL and 1.0mL, while 30000 anti-Xa factor units of specification, then in 5mL vial 3.0mL.

[0109] The freeze-dried state of different specifications of products is compared, and the results are shown in Table 11.

[0110] Table 11

[0111]

[0112] The freeze-drying results of Table 11 show that the freeze-dried properties of different specifications of sheep enoxaparin sodium preparation products are consistent, and the quality is good.

[0113] The test results of different specifications of sheep enoxaparin sodium preparation samples are shown in Table 12.

[0114] Table 12

[0115]

[0116]

[0117]

[0118] Note: The acceptance criteria is the enterprise standard, which is not lower than the quality standard of enoxaparin sodium injection in the United States Pharmacopoeia (USP) and the British Pharmacopoeia (BP).

[0119] The above results show that the different specifications of finished sheep enoxaparin sodium preparation meet the established enterprise quality standards, and compared with the (porcine) enoxaparin sodium injection of USP and EP, except for the source of sheep and the corresponding solid dosage form, other quality indicators are also completely consistent.

[0120] Example 9: High temperature stress stability test.

[0121] Test sample:

[0122] Sample 1: The sheep enoxaparin sodium preparation with a specification of 6000 anti-Xa factor units prepared in Example 8.

[0123] Sample 2: self-made sheep enoxaparin sodium injection, prepared according to injection dosage form 1 and example 7 described in patent CN 106243246 B, with a prescription of sheep enoxaparin sodium and water for injection, 6000 units per package (about 60 mg / 1.0 mL).

[0124] Sample 3: self-made sheep enoxaparin sodium injection, prepared according to injection dosage form 2 described in patent CN 106243246 B, with a prescription of sheep enoxaparin sodium, water for injection and benzyl alcohol, 6000 units per package (about 60 mg / 1.0 mL).

[0125] Sample 4: commercially available (porcine source) enoxaparin sodium injection, batch number: 9S602, expiration date: 2022.06, with a prescription of (porcine source) enoxaparin sodium and water for injection, 6000 units per package (about 60 mg / 1.0 mL).

[0126] Test method: take each sample and place it in a test condition of 60±2℃, take samples at 0 days, 7 days, 15 days and 30 days respectively, and observe the changes in properties, pH, potency, molecular weight and related substances (free sulfate) and other indicators. The comparison results of high temperature stress (60℃) stability test are shown in Table 13.

[0127] Table 13

[0128]

[0129]

[0130]

[0131] Note: 1. The limit of free sulfate test item for lyophilized powder injection is ≤1.2%; 2. The limit of free sulfate test item for injection dosage form is ≤0.12% (the concentration of injection is about 10%); 3. underlined bold italicized The indicated data are out of the limit requirements.

[0132] Analysis of the above results: the sheep enoxaparin sodium preparation has good stability as a lyophilized powder. Under high temperature stress of 60℃ for 15 days, only the color is slightly darker, but still within the limit requirements. Until the 30th day, the color is close to the limit requirements, while other main quality indicators change little.

[0133] As a comparison, three groups of injection samples of the same specification, including two groups of self-made sheep enoxaparin sodium injection and commercially available (porcine source) enoxaparin sodium injection, the color is already out of the limit at the first time point (7 days) of the test, and other main quality indicators, including potency, pH, molecular weight and free sulfate, have certain changes. Some test items will exceed the limit at the 15th day or the 30th day, showing that the active ingredient of the drug has certain degradation.

[0134] The above results show that the sheep enoxaparin sodium preparation has good temperature stability, and the stability of the solid dosage form is significantly improved compared with the injection dosage form.

[0135] Example 10: ICH accelerated stability test.

[0136] Three batches of sheep enoxaparin sodium preparation pilot products were taken, which were produced according to Example 1, with a specification of 6000 anti-Xa factor units, and packaging materials of 2 mL borosilicate glass tube bottles, bromobutyl rubber stoppers and aluminum plastic combined covers. The samples were placed under the ICH accelerated conditions at a temperature of 40℃±2℃ and a relative humidity of 75%±5% for 6 months. Samples were taken at the end of the 1st month, 2nd month, 3rd month and 6th month during the test period, and were detected according to the key inspection items of stability. The accelerated stability test results are shown in Table 14.

[0137] Table 14

[0138]

[0139]

[0140] Result analysis: The color of the sheep enoxaparin sodium preparation was slightly deepened under the conditions of 40℃±2℃ and 75%±5% relative humidity for 6 months, and the color was lighter than Y5 at 6 months, which was much less than the limit requirement (Y4 / BY4, the color of BY4 is deeper), and there was no trend of deterioration in other quality indicators.

[0141] The above results show that the product as a lyophilized powder injection has good stability at high temperature (40℃), and the storage and transportation time under the condition of 40℃ can be as long as 6 months.

[0142] Example 11: ICH long-term stability test.

[0143] This test takes the long-term storage conditions of international climate zone III dry hot zone (annual average kinetic temperature of 27.4℃, relative humidity of 35%) and IV humid hot zone (annual average kinetic temperature of 27.4℃, relative humidity of 76%) as the investigation target. In this ICH long-term stability test, the temperature of 30℃±2℃ and the relative humidity of 75%±5% are selected as the storage conditions for investigation.

[0144] Test process: The same three batches of sheep enoxaparin sodium preparation in Example 10 were placed under the conditions of temperature 30℃±2℃ and relative humidity 75%±5% for 42 months. Samples were taken at the end of the 3rd month, 6th month, 9th month, 12th month, 18th month, 24th month, 36th month and 42nd month during the test period, and were detected according to the key inspection items of stability.

[0145] The current test results have been carried out for 24 months, and the long-term stability test results are shown in Table 15:

[0146] Table 15

[0147]

[0148]

[0149] Result analysis: The key quality indicators of the product enoxaparin sodium preparation have no obvious changes under the condition of 30℃±2℃ and relative humidity of 75%±5% for 24 months, especially the aforementioned solution color item which is easy to change at high temperature, and there is almost no significant deepening. It is expected that all test items should still be stable for 36 months or 42 months.

[0150] The above results show that the lyophilized powder injection of enoxaparin sodium preparation described in the present application has good stability and can be stored at 30℃ or below for a long time, which meets the requirements of international climate zone III dry and hot zone and IV humid and hot zone for drug temperature storage. The lyophilized powder injection of enoxaparin sodium preparation described in the present application can meet the storage and transportation requirements at high temperature of 40℃ for up to 6 months.

[0151] As can be seen from the above examples 1 to 11, the lyophilized powder injection of enoxaparin sodium preparation has greatly improved stability compared to the already marketed injection dosage form, and the storage and transportation time at high temperature of 40℃ can be up to 6 months, and the key indicators are not affected, and the quality is almost unchanged after long-term storage at 30℃ for 24 months. Therefore, the product can be stored at high temperature or room temperature. The lyophilized powder injection of enoxaparin sodium preparation described in the present application uses a sterile freeze-drying preparation process, which has simple and easy-to-control preparation method steps.

[0152] As a comparison with the prior art injection dosage form, the influence of high temperature (40℃) is significant, not only the color will quickly deepen beyond the standard, but also the activity (anti-Xa factor titer), molecular weight and other indicators will decrease or degrade (or trend), indicating that the active ingredient of the drug will degrade, which will seriously affect the effectiveness and safety of the drug when used. The storage condition required by the prior art enoxaparin sodium injection is "not higher than 25℃". However, for the Islamic countries and regions mainly located in international climate zone III dry and hot zone and IV humid and hot zone, this requirement has great limitations, and the room temperature condition required by these regions is usually 30℃.

[0153] Therefore, the sheep enoxaparin sodium preparation provided by the present application has good stability, can be transported or stored at high temperature, can be stored for a long time at 30 DEG C, and can be stored for 6 months at 40 DEG C or below for a short time, and does not need to be transported or stored at low temperature, and is particularly suitable for application in countries and regions in the international climate zone III dry-hot zone and IV humid-hot zone, and the sheep enoxaparin sodium preparation effectively improves the effectiveness, safety and accessibility of drug application.

[0154] Heparin drugs are one of the most important anticoagulant and antithrombotic drugs, and are listed as life-saving drugs by WHO, but current clinical almost all are pig-derived heparin drugs, which cannot meet the medication needs of Muslim population and other special population. Sheep enoxaparin sodium has the same pharmacodynamic properties as (pig-derived) enoxaparin sodium. It is an important feature that it is derived from sheep, and therefore the sheep enoxaparin sodium preparation can be prepared as a halal drug for application in disease prevention and treatment.

Claims

1. A lyophilized powder injection of sheep enoxaparin sodium, characterized in that: The active pharmaceutical ingredient in this preparation, sheep enoxaparin sodium, is derived from sheep. This lyophilized powder injection is prepared by adding water to the raw material sheep enoxaparin sodium without excipients to form a solution, which is then sterilely freeze-dried to remove water. The preparation steps include: S.1 Preparation of drug solution: Dissolve sheep enoxaparin sodium in water without excipients to prepare a drug solution with a mass concentration of 8% to 15%; S.2 Sterilization Filtration: The drug solution flows through a microporous filter to remove microorganisms; S.3 Filling: Fill the container with the filtered solution to remove microorganisms; S.4 Aseptic freeze-drying: The container filled with the drug solution is partially stoppered, then sent to a freeze dryer for freeze-drying. After freeze-drying, it is fully stoppered and sealed. The aseptic freeze-drying steps include:

1. Pre-freezing stage: Set the freeze dryer partition temperature to -20℃~-50℃, keep it warm for 2~6 hours after cooling, and then vacuum it, controlling the vacuum degree to 5~50 Pa. II. Sublimation stage: The partition is heated to -20℃ to 20℃ for 0.5 to 3 hours, and then held for 6 to 20 hours, with the vacuum level controlled at 5 to 50 Pa. III. Drying stage: The partition is heated to 20℃~50℃ for 0.5~3 hours, then vacuumed and kept warm for more than 0.5 hours; IV. Pressure Rise Test: When the pressure rise test ΔP is less than 5 Pa / min, it is considered that the drying endpoint has been reached, the freeze drying is ended, and the vacuum is removed; S.5 rolled cap.

2. The lyophilized powder injection of sheep enoxaparin sodium according to claim 1, characterized in that: Medically acceptable excipients include one or more of the following: excipients, adsorbents, isotonic adjusters, and pH adjusters.

3. The lyophilized powder injection of sheep enoxaparin sodium according to claim 1, characterized in that: The packaging specifications of the single vial of the formulation include 2000, 4000, 6000, 8000, 10000 and 30000 units of anti-Xa factor, or 20 mg, 40 mg, 60 mg, 80 mg, 100 mg and 300 mg by weight.

4. A method for preparing a lyophilized powder injection of sheep enoxaparin sodium, characterized in that: Includes the following steps: S.1 Preparation of drug solution: Dissolve sheep enoxaparin sodium in water without excipients to prepare a drug solution with a mass concentration of 8% to 15%; S.2 Sterilization Filtration: The drug solution flows through a microporous filter to remove microorganisms; S.3 Filling: Fill the container with the filtered solution to remove microorganisms; S.4 Aseptic freeze-drying: The container filled with the drug solution is partially stoppered, then sent to a freeze dryer for freeze-drying. After freeze-drying, it is fully stoppered and sealed. The aseptic freeze-drying steps include:

1. Pre-freezing stage: Set the freeze dryer partition temperature to -20℃~-50℃, keep it warm for 2~6 hours after cooling, and then vacuum it, controlling the vacuum degree to 5~50 Pa. II. Sublimation stage: The partition is heated to -20℃ to 20℃ for 0.5 to 3 hours, and then held for 6 to 20 hours, with the vacuum level controlled at 5 to 50 Pa. III. Drying stage: The partition is heated to 20℃~50℃ for 0.5~3 hours, then vacuumed and kept warm for more than 0.5 hours; IV. Pressure Rise Test: When the pressure rise test ΔP is less than 5 Pa / min, it is considered that the drying endpoint has been reached, the freeze drying is ended, and the vacuum is removed; S.5 rolled cap.

5. The method for preparing a lyophilized powder injection of sheep enoxaparin sodium according to claim 4, characterized in that: Pre-freezing stage: Set the freeze dryer partition temperature to -40℃, the cooling time to 1 hour, then keep warm for 3 hours, and finally vacuum the system, controlling the vacuum level at 50 Pa.

6. The method for preparing a lyophilized powder injection of sheep enoxaparin sodium according to claim 4, characterized in that: Sublimation stage: The partition is heated to 0°C for 2 hours and held for 12 hours, with the vacuum degree controlled at 20±5 Pa.

7. The method for preparing a lyophilized powder injection of sheep enoxaparin sodium according to claim 4, characterized in that: Analysis of the drying stage: The partition is heated for 2 hours, kept at that temperature for 2 hours, and the vacuum is controlled.

8. The method for preparing a lyophilized powder injection of sheep enoxaparin sodium according to claim 4, characterized in that: In the S.2 sterilization filtration step, the filter membrane material in the microporous filter is selected from polyethersulfone or polyvinylidene fluoride, and at least one filtration with a filtration diameter of less than or equal to 0.22 μm is performed.

9. The method for preparing a lyophilized powder injection of sheep enoxaparin sodium according to claim 4, characterized in that: In S.3, the container used for filling the liquid medicine is a borosilicate glass bottle; in S.4, the stopper in the container for aseptic freeze drying is a halogenated butyl rubber stopper; in S.5, the cap is an aluminum cap or an aluminum-plastic composite cap.

10. The use of the lyophilized sheep enoxaparin sodium powder for injection as described in any one of claims 1 to 3 in the preparation of anticoagulant and antithrombotic drugs.

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