A carbetocin injection and its preparation method

Abstract

The present invention relates to a carbetocin injection and a preparation method thereof, belonging to the technical field of pharmaceutical preparations. A stabilizing adjuvant is added to the carbetocin injection. Based on 10 L of the carbetocin injection, the addition amount of the stabilizing adjuvant is 0.25 g to 1.0 g, and the stabilizing adjuvant is selected from at least one of apigenin, catechin, luteolin, troxerutin, puerarin, and baicalin. The present invention discovers that by introducing an appropriate amount of specific natural substances such as puerarin as a stabilizing adjuvant into the carbetocin injection, it can effectively slow down the increase in the impurity content during the storage of the carbetocin injection and reduce the types and dosages of excipients in the prescription. This not only improves the storage stability of the carbetocin injection but also avoids the risks of increased microbial levels and incompatibility with raw materials brought about by the introduction of excessive excipients, thereby enhancing the safety of drug use.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and particularly relates to a carbetocin injection and a preparation method thereof. Background Art

[0002] Carbetocin, chemically named deamino-2-oxo-methyltyrosine-1-κ-oxytocin, has significant long-acting characteristics. It can be administered intravenously as a single dose immediately after cesarean section under epidural or spinal anesthesia and after vaginal delivery, effectively preventing uterine hypotonia and postpartum hemorrhage, which greatly facilitates clinical use. Due to the convenient use, rapid onset, and long half-life of its injection dosage form, it has been widely used clinically.

[0003] The structural formula of carbetocin is as follows:

[0004] 。

[0005] However, as a synthetic polypeptide drug, carbetocin faces certain challenges in terms of stability. Especially when stored for a long time in an environment with high temperature, oxidation, and high microbial levels, the peptide bonds of carbetocin are prone to breakage and condensation reactions, resulting in an increase in degradation impurities and affecting its drug formation and clinical efficacy. Therefore, it is necessary to improve its stability through fine formulation design and pharmaceutical process optimization to ensure the quality of the drug during storage and use.

[0006] In addition, carbetocin cannot tolerate high temperature for a long time, so it is not suitable to be sterilized by terminal sterilization process. At the same time, no obvious antibacterial property has been found in the aqueous solution of carbetocin, which makes the risk of microbial contamination higher during the production process, thus bringing potential hidden dangers to clinical use. How to effectively control microbial contamination and ensure the sterility of the drug is a key problem to be solved in the preparation process of carbetocin injection.

[0007] On the other hand, puerarin and the like, as reducing secondary metabolites widely distributed in plants, have various biological activities, such as immunity and metabolic regulation, anti-inflammatory, antibacterial, antiviral, antioxidant, etc. Moreover, compared with synthetic drugs, they have a wider range of indications, fewer side effects, and better tolerance.

[0008] At present, injection products such as puerarin injection have been industrialized and used clinically for a long time, and their clinical safety has been effectively confirmed. However, there is no relevant report in the existing technology on adding natural substances such as puerarin as components to carbetocin injection. Summary of the Invention

[0009] To solve the above problems, the present invention provides a carbetocin injection and its preparation method. It is first discovered that applying an appropriate amount of specific natural substances such as puerarin as a stabilizing adjuvant in the preparation of carbetocin injection can effectively slow down the increase in the impurity content during the storage of carbetocin injection, so as to improve the storage stability of carbetocin injection.

[0010] In the first aspect, the present invention provides a carbetocin injection, which contains carbetocin, a stabilizing adjuvant and water for injection;

[0011] Based on 10 L of the carbetocin injection, the addition amount of the stabilizing adjuvant is 0.25 g to 1.0 g;

[0012] The stabilizing adjuvant is selected from at least one of apigenin, catechin, luteolin, troxerutin, puerarin and baicalin.

[0013] Further, based on 10 L of the carbetocin injection, the addition amount of the stabilizing adjuvant is 0.25 g to 0.5 g; the stabilizing adjuvant is puerarin.

[0014] Further, the carbetocin injection is composed of carbetocin, the stabilizing adjuvant, an osmotic pressure regulator, a pH regulator and water for injection;

[0015] Based on 10 L of the carbetocin injection, the addition amount of carbetocin is 0.9 g to 1.1 g;

[0016] Based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 88 g to 92 g;

[0017] The dosage of the pH regulator is such that the pH value of the carbetocin injection is 4.0 to 6.5;

[0018] The dissolved oxygen content of the water for injection ≤ 3.0 mg / L.

[0019] Further, based on 10 L of the carbetocin injection, the addition amount of carbetocin is 1.0 g; based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 90 g; the dosage of the pH regulator is such that the pH value of the carbetocin injection is 5.0 to 6.0; the dissolved oxygen content of the water for injection ≤ 1.0 mg / L.

[0020] Further, the osmotic pressure regulator includes sodium chloride.

[0021] Further, the pH regulator includes at least one of sodium hydroxide, triethylamine, hydrochloric acid and glacial acetic acid.

[0022] In a second aspect, based on the same inventive concept, the present invention provides a method for preparing the carbetocin injection according to any one of the first aspect, the preparation method comprising the following steps:

[0023] Introduce a protective gas into the water for injection to obtain water for injection with a dissolved oxygen content ≤ 3.0 mg / L;

[0024] Add other components except the pH regulator to the water for injection with a dissolved oxygen content ≤ 3.0 mg / L for dissolution, then make up the volume to 95 vol% of the formulation amount, adjust the pH of the system to 4.0 - 6.5 with the pH regulator and then make up the volume to full amount to obtain the carbetocin injection.

[0025] Further, in terms of volume percentage, the protective gas is a sterile inert gas with a purity > 90.0%, and the sterile inert gas includes at least one of nitrogen and helium.

[0026] Further, the preparation method further comprises:

[0027] Subject the carbetocin injection to sterile filtration and perform bottling and sealing under an inert gas atmosphere, and then perform leak detection and lamp inspection to obtain the finished product of the carbetocin injection.

[0028] Further, in terms of volume percentage, the residual oxygen content in the reagent bottle containing the carbetocin injection after bottling and sealing ≤ 5%, preferably ≤ 2%; the sterile filtration is performed using a two-stage 0.22 μm filter, and the filter material includes at least one of polyethersulfone, polyvinylidene fluoride and polytetrafluoroethylene.

[0029] The above technical solutions provided by the embodiments of the present invention have at least the following advantages compared with the prior art:

[0030] The embodiments of the present invention provide a carbetocin injection and a preparation method thereof. The present invention discovers that by introducing an appropriate amount of specific natural substances such as puerarin as a stabilizing aid into the carbetocin injection, it can effectively slow down the increase in the content of impurities such as impurity Gly 9 -OH, impurity sulfoxide I, impurity sulfoxide II, impurity D-Asn 5 and other impurities, and reduce the types and amounts of excipients in the formulation, which not only improves the storage stability of the carbetocin injection, but also avoids the risk of increased microbial level and the risk of incompatibility with the raw material brought about by excessive introduction of excipients, thereby enhancing the safety of its medication. Specific Embodiments

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0032] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0033] In a first aspect, the present invention provides a carbetocin injection, and the carbetocin injection contains carbetocin, a stabilizing adjuvant, and water for injection;

[0034] Based on 10 L of the carbetocin injection, the addition amount of the stabilizing adjuvant is 0.25 g to 1.0 g;

[0035] The stabilizing adjuvant is selected from at least one of apigenin, catechin, luteolin, troxerutin, puerarin, and baicalin.

[0036] The embodiments of the present invention provide a carbetocin injection. The present invention discovers that by introducing an appropriate amount of specific natural substances such as puerarin as a stabilizing adjuvant into the carbetocin injection, it can effectively slow down the increase in the content of impurities such as impurity Gly 9 -OH, impurity sulfoxide I, impurity sulfoxide II, impurity D-Asn 5 and other impurities, and reduce the types and amounts of excipients in the prescription. This not only improves the storage stability of the carbetocin injection, but also avoids the risks of increased microbial levels and compatibility with raw materials brought about by excessive introduction of excipients, thereby enhancing the safety of its use.

[0037] In the present invention, the CAS number of apigenin is 520-36-5, the CAS number of catechin is 7295-85-4, the CAS number of luteolin is 491-70-3, the CAS number of troxerutin is 7085-55-4, the CAS number of puerarin is 3681-99-0, and the CAS number of baicalin is 21967-41-9. Among the above natural substances, injection products such as troxerutin injection and puerarin injection are existing industrialized and long-term clinically used injection products, and their clinical safety has been effectively proven.

[0038] In some specific embodiments, based on 10 L of the carbetocin injection, the addition amount of the stabilizing adjuvant is 0.25 g to 0.5 g; the stabilizing adjuvant is puerarin.

[0039] In some specific embodiments, the carbetocin injection is composed of carbetocin, the stabilizing adjuvant, the osmotic pressure regulator, the pH regulator, and water for injection;

[0040] Based on 10 L of the carbetocin injection, the addition amount of carbetocin is 0.9 g to 1.1 g;

[0041] Based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 88 g to 92 g;

[0042] The dosage of the pH regulator is such that the pH value of the carbetocin injection is 4.0 to 6.5;

[0043] The dissolved oxygen content of the water for injection is ≤ 3.0 mg / L.

[0044] After adding carbetocin, the stabilizing adjuvant, the osmotic pressure regulator, and the pH regulator to the carbetocin injection provided by the present invention according to the above parameter requirements, the balance is water for injection.

[0045] In some specific embodiments, based on 10 L of the carbetocin injection, the addition amount of carbetocin is 1.0 g; based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 90 g; the dosage of the pH regulator is such that the pH value of the carbetocin injection is 5.0 to 6.0; the dissolved oxygen content of the water for injection is ≤ 1.0 mg / L.

[0046] In some specific embodiments, the osmotic pressure regulator includes sodium chloride.

[0047] In some specific embodiments, the pH regulator includes at least one of sodium hydroxide, triethylamine, hydrochloric acid, and glacial acetic acid.

[0048] In a second aspect, based on the same inventive concept, the present invention provides a preparation method of the carbetocin injection according to any one of the first aspect, and the preparation method includes the following steps:

[0049] Introduce a protective gas into the water for injection to obtain water for injection with a dissolved oxygen content ≤ 3.0 mg / L;

[0050] Add other components except the pH regulator to the water for injection with a dissolved oxygen content ≤ 3.0 mg / L for dissolution, and then adjust the pH of the system to 4.0 to 6.5 with the pH regulator to obtain the carbetocin injection.

[0051] In the preparation method of the carbetocin injection provided by the present invention, by strictly controlling the oxygen content in the preparation process, the chemical stability and the aseptic guarantee level of this product are further ensured. At the same time, this preparation method is implemented based on the carbetocin injection described in any item of the first aspect, so it has at least the beneficial effects described in any item of the first aspect, which will not be elaborated here.

[0052] In some specific embodiments, in terms of volume percentage, the protective gas is a sterile inert gas with a purity > 90.0%, and the sterile inert gas includes at least one of nitrogen and helium.

[0053] In some specific embodiments, the preparation method further includes:

[0054] Subject the carbetocin injection to aseptic filtration and perform bottling and capping under an inert gas atmosphere, and then perform leak detection and lamp inspection to obtain the finished product of carbetocin injection.

[0055] In some specific embodiments, in terms of volume percentage, the residual oxygen content in the reagent bottle containing the carbetocin injection after bottling and capping ≤ 5%, preferably ≤ 2%. In the present invention, the aseptically filtered liquid medicine is filled into a medium-borosilicate glass ampoule bottle; during the filling process, first use the protective gas to purge and fill the empty ampoule bottle, then fill the liquid medicine into the ampoule bottle, the filling volume is 1 - 1.6 ml, and before sealing, use the inert gas to fill the ampoule bottle containing the filled liquid medicine so that the residual oxygen content reaches the preset value; during the bottling and capping process, maintain the laminar flow of the protective gas.

[0056] In some specific embodiments, the aseptic filtration uses a two-stage 0.22 μm filter, and the filter material includes at least one of polyethersulfone, polyvinylidene fluoride, and polytetrafluoroethylene.

[0057] The two-stage 0.22 μm filter in the present invention is a precision filtration device commonly used in liquid or gas purification. It is composed of two filtration units with a pore size of 0.22 microns in series, which can provide a higher level of filtration effect, and commercially available equipment can be directly used.

[0058] It should be noted that for the component raw materials involved in the carbetocin injection and its preparation method provided by the embodiments of the present invention, if there is no special limitation or specific description, commercially available products can be directly used or self-made by using existing publicly disclosed preparation methods; at the same time, for the steps and parameters involved, if there is no special limitation or specific description, they can be carried out according to the existing processing technology of carbetocin injection or directly use existing equipment, and the present invention document will not elaborate one by one.

[0059] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions in the following embodiments, they are usually determined according to national standards. If there is no corresponding national standard, they are carried out according to general international standards, conventional conditions or the conditions recommended by the manufacturer.

[0060] The impurity information in the following examples and comparative examples is as follows:

[0061] Impurity Gly 9 -OH (or [Gly 9 -OH] carbetocin) has the following chemical structure:

[0062] .

[0063] The chemical structure of impurity sulfoxide I (or carbetocin sulfoxide I) is as follows:

[0064] .

[0065] The chemical structure of impurity sulfoxide II (or carbetocin sulfoxide II) is as follows:

[0066] .

[0067] Impurity D-Asn 5 (or D-Asn 5 carbetocin) has the following chemical structure:

[0068] .

[0069] The chemical structure of unknown impurity 1 is shown as follows:

[0070] .

[0071] Example 1

[0072] This example provides a carbetocin injection, which is composed of carbetocin, a stabilizing aid, an osmotic pressure regulator, a pH regulator and water for injection;

[0073] Based on 10 L of the carbetocin injection, the addition amount of carbetocin is 1.0 g;

[0074] Based on 10 L of the carbetocin injection, the addition amount of the stabilizing aid is 0.25 g, and the stabilizing aid is troxerutin;

[0075] Based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 90 g, and the osmotic pressure regulator is sodium chloride;

[0076] The dosage of the pH regulator is such that the pH value of the carbetocin injection is 5.3.

[0077] The preparation method of the above-mentioned carbetocin injection comprises the following steps:

[0078] Introduce a protective gas (nitrogen) into the water for injection to obtain water for injection with a dissolved oxygen content of 1.48 mg / L;

[0079] Add 0.25 g of troxerutin, 90 g of sodium chloride, and 1 g of carbetocin to the water for injection with a dissolved oxygen content of 1.48 mg / L, stir and dissolve, make up the volume to 95 vol% of the preparation amount, then adjust the pH of the system to 5.3 with glacial acetic acid at a concentration of 1 mol / L and sodium hydroxide at a concentration of 2 mol / L, and then make up the volume to the full amount to obtain 10 L of the carbetocin injection;

[0080] Filter the carbetocin injection through a secondary 0.22 μm PVDF filter element for sterilization and perform sealing under a nitrogen inert gas atmosphere. The filling volume is 1.2 ml / branch, the residual oxygen content detection is 3.22%, and then perform leak detection and lamp inspection to obtain qualified finished products of the carbetocin injection.

[0081] On the basis of Example 1, Examples 2 to 6 provide a carbetocin injection and its preparation method. The differences between Examples 2 to 6 and Example 1 are only as follows: the types of the stabilizing aids are different; among them, the stabilizing aid in Example 2 is baicalin, the stabilizing aid in Example 3 is puerarin, the stabilizing aid in Example 4 is apigenin, the stabilizing aid in Example 5 is catechin, and the stabilizing aid in Example 6 is luteolin.

[0082] On the basis of Example 1, Examples 7 to 8 provide a carbetocin injection and its preparation method. The differences between Examples 7 to 8 and Example 1 are only as follows: the dosages of the stabilizing aids are different; among them, based on 10 L of the carbetocin injection, the addition amount of the stabilizing aid in Example 7 is 0.5 g (that is, the addition amount of the stabilizing aid in the preparation process in Example 1 is increased from 0.25 g to 0.5 g), and the addition amount of the stabilizing aid in Example 8 is 1.0 g based on 10 L of the carbetocin injection (that is, the addition amount of the stabilizing aid in the preparation process in Example 1 is increased from 0.25 g to 1.0 g).

[0083] Comparative Example 1

[0084] This example provides a carbetocin injection and its preparation method. The difference from Example 1 is only that: no stabilizing adjuvant is added (that is, the addition amount of the stabilizing adjuvant in the preparation process in Example 1 is adjusted from 0.25 g to 0 g); the remaining steps and parameters are the same.

[0085] Comparative Example 2

[0086] This example provides a carbetocin injection and its preparation method. The difference from Example 1 is only that: based on 10 L of the carbetocin injection, the addition amount of the stabilizing adjuvant is 0.1 g (that is, the addition amount of the stabilizing adjuvant in the preparation process in Example 1 is adjusted from 0.25 g to 0.1 g); the remaining steps and parameters are the same.

[0087] Comparative Example 3

[0088] This example provides a carbetocin injection and its preparation method. The difference from Example 1 is only that: the stabilizing adjuvant is adjusted to bamboo leaf flavonoids (that is, troxerutin in the example is replaced by bamboo leaf flavonoids); the remaining steps and parameters are the same.

[0089] Comparative Example 4

[0090] This example provides a reported carbetocin injection, and its preparation method includes the following steps:

[0091] Add 10 g of methionine, 470 g of mannitol, 11.9 g of succinic acid, and 1 g of carbetocin to 10 L of water for injection without filling protective gas (nitrogen), stir and dissolve, and then adjust the pH of the system to 5.3 with 1 mol / L glacial acetic acid and 2 mol / L sodium hydroxide to obtain the carbetocin injection;

[0092] Seal the carbetocin injection with a secondary 0.22 μm PVDF filter element, and the filling volume is 1.2 ml / branch. After sealing, perform leak detection and lamp inspection to obtain the carbetocin injection sample.

[0093] Comparative Example 5

[0094] This example provides a carbetocin injection and its preparation method. The difference from Example 1 is only that: no protective gas is used to control the dissolved oxygen and residual oxygen; the remaining steps and parameters are the same.

[0095] Test Example

[0096] In this example, the samples of carbetocin injection obtained from the above-mentioned examples and comparative examples were subjected to storage stability investigation for 6 months under the conditions of 40°C ± 2°C and relative humidity of 75% ± 5%. At the investigation time points of 0 month (i.e., when the experiment started), 1 month, 3 months, and 6 months, the impurity content was detected by high performance liquid chromatography. The detection results are shown in Table 1, Table 2, and Table 3. Among them, Table 1 and Table 2 show the comparative results of the effects of different types of stabilizing agents on the storage stability of carbetocin injection (the stabilizing agent in Example 1 is troxerutin, the stabilizing agent in Example 2 is baicalin, the stabilizing agent in Example 3 is puerarin, the stabilizing agent in Example 4 is apigenin, the stabilizing agent in Example 5 is catechin, the stabilizing agent in Example 6 is luteolin, and the stabilizing agent in Comparative Example 3 is bamboo leaf flavonoid; Comparative Example 4 is the reported carbetocin injection), and Table 3 shows the comparative results of the effects of different addition amounts of stabilizing agents on the storage stability of carbetocin injection (the stabilizing agent in Example 7 is added at 0.5 g, the stabilizing agent in Example 8 is added at 1 g, the stabilizing agent in Comparative Example 1 is added at 0 g, and the stabilizing agent in Comparative Example 2 is added at 0.1 g); Table 4 shows the comparative results of the effects of protective gas on the storage stability of carbetocin injection (nitrogen is used as the protective gas in Example 1, Comparative Example 4 is the reported carbetocin injection, and Comparative Example 5 is without using protective gas).

[0097] Table 1 Comparative Results of the Effects of Different Types of Stabilizing Agents on the Storage Stability of Carbetocin Injection - I

[0098]

[0099] Table 2 Comparative Results of the Effects of Different Types of Stabilizing Agents on the Storage Stability of Carbetocin Injection - II

[0100]

[0101] Table 3 Comparative Results of the Effects of Different Addition Amounts of Stabilizing Agents on the Storage Stability of Carbetocin Injection

[0102]

[0103] Table 4 Comparative Results of the Effects of Protective Gas on the Storage Stability of Carbetocin Injection

[0104]

[0105] It can be seen from Table 1, Table 2, Table 3, and Table 4 that:

[0106] 1) Different types of stabilizing agents with the same addition amount have different stabilizing effects on carbetocin injection. Among them, in Example 3, when the stabilizer is puerarin, the stabilizing effect is better than that of other types of stabilizers. The total impurity growth in Examples 1 - 6 is less than that in Comparative Example 3 and Comparative Example 4. When the stabilizers are troxerutin, baicalin, puerarin, apigenin, catechin, and luteolin, they have good stabilizing effects.

[0107] 2) When the dosage of the stabilizer increases from 0.25 g / 10 L to 0.5 g / 10 L, the growth of total impurities in 6 - month stability decreases from 1.14% to 0.80%. When it continues to increase to 1.0 g / 10 L, the growth of total impurities in 6 - month stability is 0.77%, showing no obvious change compared with 0.5 g / 10 L. When the dosage of the stabilizer is reduced to 0.1 g / 10 L, the growth of total impurities in 6 - month stability is 2.55%, which is significantly increased compared with Example 1. When the dosage of the stabilizer continues to be reduced to 0 g / 10 L, the growth of total impurities in 6 - month stability is 3.11%. When the addition amount of the stabilizer is in the range of 0.25 g - 1.0 g, it has good stability.

[0108] 3) When no protective gas is added to control dissolved oxygen and residual oxygen, sulfoxide I and sulfoxide II increase significantly. In 6 - month stability, the growth of total impurities is 4.94%, which is significantly increased compared with Example 1 and Comparative Example 4. Therefore, adding a protective gas to control dissolved oxygen and residual oxygen can effectively improve the stability of this product.

[0109] In addition, in the present invention, the clinical minimum dose of troxerutin is 240 mg / time, and the maximum addition amount in this experiment is 0.1 mg, which is 1 / 2400 of the clinical minimum use dose. Its dosage is lower than 1 / 1000 of the minimum dose required to evaluate drug cross - contamination, and the dosage is safe, with no obvious medication risk in clinical use. And to further confirm the safety of carbetocin injection after adding the stabilizer, a rabbit vascular irritation test, a guinea pig systemic active anaphylaxis test, a rat passive cutaneous anaphylaxis test, and a hemolysis test were carried out using the carbetocin injection with puerarin as the stabilizer in Example 3. The test results showed no irritation, no allergy, and no hemolysis, indicating that the carbetocin injection prepared by the present invention has safety.

[0110] (1) Rabbit vascular irritation test

[0111] Seven qualified rabbits were selected for quarantine and adaptability observation. First, the two lightest rabbits (one female and one male) were eliminated. Then, five rabbits with remaining body weights of 2.2669 - 2.7233 kg for females and 2.4232 - 2.7370 kg for males were selected, including 3 females and 2 males, with no pregnancy in females. They were sorted by weight from light to heavy according to gender. The study was conducted using the self - comparison method on the left and right sides of the same body. That is, the test article or the marketed preparation at a concentration of 100 μg / ml was injected into the left ear marginal vein of the rabbit, and an equal volume of negative control (i.e., 0.9% sodium chloride injection) was injected into the right ear marginal vein. The drug was administered once a day for 7 consecutive days, and the blood vessels at the administration site were observed for any irritant reactions.

[0112] After the general condition was observed for 96 h after the last administration, three rabbits (2 females and 1 male) were euthanized by carbon dioxide. The blood vessels at the administration site and the surrounding tissues were taken for histopathological examination. The remaining two rabbits were observed for 14 days for recovery. After the observation, the administration site was observed macroscopically and histopathologically using the same method as above to understand the reversibility of the irritant reaction.

[0113] Table 5 Results of rabbit vascular irritation test

[0114]

[0115] No irritant reactions were found during the drug administration period, after the last administration, and during the recovery period in the experimental group. The results of the rabbit vascular irritation test of this product showed no vascular irritation.

[0116] (2) Guinea pig active systemic anaphylaxis test

[0117] Thirty-three SPF guinea pigs that passed quarantine and adaptability observation were selected, including 18 females and 15 males, with no pregnant females. First, the animals with the lightest (3 females and 2 males) and heaviest (1 male) weights were selected as spare animals. The remaining 27 animals, with female weights ranging from 335.4 to 376.9 g and male weights ranging from 341.6 to 378.6 g, were sorted by weight from smallest to largest by gender and divided into a negative control group, a positive control group, and a test article group, a total of 3 groups, with 9 animals in each group, 5 females and 4 males. The test article group was intraperitoneally injected with 100 μg / ml × 0.5 ml / animal of the test article for sensitization, the positive control group was sensitized with 5 mg / ml × 0.5 ml / animal of ovalbumin, and the negative control group was sensitized with 0.5 ml / animal of 0.9% sodium chloride injection. Sensitization was performed once every other day for 3 consecutive times. Three animals (2 females and 1 male) from each group were challenged by a single rapid intravenous injection with a volume twice that of the sensitization dose on the 14th day after the last sensitization injection; if the challenge result of the test article group was negative on the 14th day after the last sensitization, the remaining animals were challenged on the 21st day after the last sensitization injection; if the challenge result of the test article group was positive on the 14th day after the last sensitization, all 9 animals in each group were challenged on the 14th day after the last sensitization injection. The allergic reaction symptoms of the guinea pigs were observed, the degree of allergic reaction was judged, and the incidence of allergic reaction was calculated.

[0118] Table 6 Body weight examination results of guinea pigs in each test article group during sensitization and challenge

[0119]

[0120] Table 7 Results of systemic allergic reaction test after challenge on the 14th day after the last sensitization

[0121]

[0122] Table 8 Results of systemic allergic reaction test after challenge on the 21st day after the last sensitization

[0123]

[0124] Conclusion: The results showed that: ① During the sensitization period, no obvious abnormalities were observed in the general conditions of the guinea pigs in each group. ② Compared with the negative control group, there were no significant differences in the body weights of the guinea pigs in the test article group during the three sensitizations and at the time of challenge on the 14th and 21st days after the last sensitization (p > 0.05). ③ No allergic reaction symptoms occurred within 3 hours after challenge on the 14th and 21st days after the last sensitization in the negative control group and the test article group, and their incidences of allergic reaction were both 0%, and the allergic reactions were both judged as negative. The positive control group immediately showed allergic reaction symptoms after challenge, and its incidence of allergic reaction was 100%, and the allergic reaction was judged as extremely strong positive. The results of the guinea pig systemic active allergic test for this product showed no active allergic properties.

[0125] (3) Rat passive cutaneous anaphylaxis test

[0126] Select 14 rats that have passed quarantine and adaptation observation, with 7 males and 7 females, and the females are not pregnant. First, eliminate the lightest (1 female) and heaviest (1 male) animals as reserve animals. For the remaining 12 rats, with female body weights ranging from 181.4 to 194.3 g and male body weights ranging from 206.5 to 218.6 g, sort them by body weight from lightest to heaviest by gender, and divide them into a negative control group, a positive control group, and a test article group according to the randomized block design method, with 4 rats in each group. The rats in the test article group are sensitized by intraperitoneal injection of 100 μg / ml × 0.5 ml / rat of the test article. Each rat in the positive control group is sensitized by intraperitoneal injection of a mixture of 5 mg / ml × 0.5 ml of ovalbumin plus 0.5 ml of 4% aluminum hydroxide gel. The rats in the negative control group are sensitized by intraperitoneal injection of 0.5 ml / rat of 0.9% sodium chloride injection. Sensitize once every other day for 3 consecutive times, and prepare sensitized serum on the 14th day after the last sensitization. Another 22 SPF-grade SD rats are selected, with 11 males and 11 females, and the females are not pregnant. First, eliminate the heaviest (2 females and 2 males) animals as reserve animals. For the remaining 18 rats, with female body weights ranging from 183.5 to 207.6 g and male body weights ranging from 210.7 to 219.8 g, sort them by body weight from lightest to heaviest by gender, and divide them into a negative control group, a positive control group, and a test article group according to the randomized block design method, with 6 rats in each group, 3 males and 3 females. Each group is passively sensitized by intradermal injection of 0.1 ml of antiserum diluted 1:2, 1:4, and 1:8. 48 hours later, the antigen dose equal to the sensitizing dose for preparing the antiserum plus 0.5 ml / rat of 1% Evans blue solution is injected intravenously for challenge. 30 minutes later, sacrifice the animals in each group, cut the skin on the back, observe whether blue spots appear in the inner layer of the corresponding injection site skin, and measure the diameter of the blue reaction spots with an electronic digital caliper to observe whether the rats have passive cutaneous anaphylaxis reactions.

[0127] Table 9 Blue spot determination of carbetocin injection in rat passive cutaneous anaphylaxis test

[0128]

[0129] Conclusion: The test results show that ① after challenge, the number of rats with blue spot diameters greater than 5 mm in the inner layer of the skin of the negative control group and the test article group rats is zero, and the positive rate is 0.0%. ② In the positive control group, typical blue spots are formed. Among them, the number of rats with blue spot diameters greater than 5 mm in the inner layer of the skin of the 1:2, 1:4, and 1:8 antiserum is 4, 6, and 5 respectively, and the positive rates are 66.7%, 100.0%, and 83.3% respectively. The results of the rat passive cutaneous anaphylaxis test of this product show that this product has no passive anaphylaxis.

[0130] (4) Hemolysis test

[0131] During the test, take 7 clean test tubes and number them. Tubes 1 to 5 are the test tubes for the test article, tube 6 is the negative control tube, and tube 7 is the positive control tube. Add 2% rabbit red blood cell suspension, 0.9% sodium chloride injection or purified water, and the test article respectively according to the requirements. Gently shake each tube and incubate it in a water bath at 37°C ± 0.5°C. Observe the hemolysis or aggregation conditions 15 min to 3 h after adding 2% rabbit red blood cell suspension, and observe at 0 min and 3 h. To further observe the hemolysis situation, centrifuge the solutions in all test tubes at 1500 rpm for 15 min in a 4°C centrifuge, observe the color of the supernatant, and confirm the hemolysis situation as shown in Table 10.

[0132] Table 10 Determination results of the hemolytic test of carbetocin injection

[0133]

[0134] Note: “+” indicates hemolysis or aggregation, “-” indicates no hemolysis or aggregation

[0135] Conclusion: The test results show that ① all hemolysis occurred in the positive control tube (tube 7) within 15 min after adding the liquid, and no hemolysis or aggregation occurred in the negative control tube (tube 6) within 3 h. ② No aggregation and hemolysis occurred in the test article tubes (tubes 1 to 5) within 3 h. ③ After centrifugation and observation at 3 h, the supernatants of the test article tubes (tubes 1 to 5) and the negative control tube (tube 6) were colorless and clear. Therefore, the test article does not cause hemolytic reaction.

[0136] The various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the described range description has specifically disclosed all possible sub-ranges and single values within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, which applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0137] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A carbetocin injection, characterized in that, The carbetocin injection contains carbetocin, a stabilizing agent, and water for injection; Based on 10 L of the carbetocin injection, the addition amount of the stabilizing agent is 0.25 g to 1.0 g; The stabilizing agent is selected from at least one of apigenin, catechin, luteolin, troxerutin, puerarin, and baicalin; The preparation method of the carbetocin injection includes the following steps: Introduce a protective gas into the water for injection to obtain water for injection with a dissolved oxygen content ≤ 3.0 mg / L; Add other components except the pH regulator to the water for injection with a dissolved oxygen content ≤ 3.0 mg / L for dissolution, then make up the volume to 95 vol% of the formulated amount, adjust the pH of the system to 4.0 - 6.5 with the pH regulator, and then make up the volume to the full amount to obtain the carbetocin injection; Filter the carbetocin injection through sterilization and seal it under an inert gas atmosphere, and then conduct leak detection and lamp inspection to obtain the finished product of the carbetocin injection; By volume percentage, the residual oxygen content in the ampoule containing the carbetocin injection after sealing ≤ 5%; 2. The carbetocin injection according to claim 1, wherein Based on 10 L of the carbetocin injection, the addition amount of the stabilizing agent is 0.25 g to 0.5 g; the stabilizing agent is puerarin.

3. The carbetocin injection according to claim 1 or 2, characterized in that, The carbetocin injection is composed of carbetocin, the stabilizing agent, an osmotic pressure regulator, a pH regulator, and water for injection; Based on 10 L of the carbetocin injection, the addition amount of carbetocin is 0.9 g to 1.1 g; Based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 88 g to 92 g; The dosage of the pH regulator is such that the pH value of the carbetocin injection is 4.0 - 6.5; The dissolved oxygen content of the water for injection ≤ 3.0 mg / L.

4. The carbetocin injection according to claim 3, wherein, Based on 10 L of the carbetocin injection, the addition amount of carbetocin is 1.0 g; based on 10 L of the carbetocin injection, the addition amount of the osmotic pressure regulator is 90 g; the dosage of the pH regulator is such that the pH value of the carbetocin injection is 5.0 - 6.0; the dissolved oxygen content of the water for injection ≤ 1.0 mg / L.

5. The carbetocin injection according to claim 3, wherein, The osmotic pressure regulator includes sodium chloride.

6. The carbetocin injection according to claim 3, wherein The pH regulator includes at least one of sodium hydroxide, triethylamine, hydrochloric acid, and glacial acetic acid.

7. The carbetocin injection according to claim 1, wherein The protective gas includes at least one of carbon dioxide, nitrogen, and helium; the purity of the protective gas > 95 vol%.

8. The carbetocin injection according to claim 1, characterized in that, The sterilization filtration is carried out using a two-stage 0.22 μm filter, and the filter material includes at least one of polyethersulfone, polyvinylidene fluoride, and polytetrafluoroethylene.

Images