CN114259556A - Human interferon alpha 2b spray and preparation method thereof - Google Patents
Human interferon alpha 2b spray and preparation method thereof Download PDFInfo
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- CN114259556A CN114259556A CN202111600304.8A CN202111600304A CN114259556A CN 114259556 A CN114259556 A CN 114259556A CN 202111600304 A CN202111600304 A CN 202111600304A CN 114259556 A CN114259556 A CN 114259556A
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Abstract
The invention relates to a human interferon alpha 2b spray and a preparation method thereof. The human interferon alpha 2b spray comprises human interferon alpha 2b, a protective agent, a bacteriostatic agent, a thickening agent and a buffering agent, wherein the protective agent is selected from at least one of trehalose, sucrose, human serum albumin and edetate disodium, and the bacteriostatic agent is selected from at least one of m-cresol, phenol and benzyl alcohol. The human interferon alpha 2b spray has the advantages of weak irritation, no bad smell, quick bacteriostatic action without cosolvent and long bioactivity maintaining time.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a human interferon alpha 2b spray and a preparation method thereof.
Background
The human interferon alpha 2b spray is a local external sterile liquid preparation. The human interferon alpha 2b spray is administrated through skin and mucous membrane, is mainly used for treating the virus infection of mouth, nose, cavity respiratory tract and skin surface, and can ensure that the systemic side effect of the interferon is reduced or not caused while the local effective virus infection resistance can be realized by using a smaller dose. However, human interferon α 2b is an active protein drug, has an unstable structure, is easily inactivated by temperature, storage time and other factors during production and storage, and does not achieve the expected therapeutic effect. Meanwhile, the human interferon alpha 2b spray is used as a sterile liquid preparation packaged in multiple doses, so that the medicament is easy to deteriorate and the biological activity is reduced due to the pollution and the reproduction of microorganisms in the process of multiple use, and the use of a patient is not facilitated.
Therefore, in the development of a spray containing human interferon α 2b, the most important issue is how to maintain interferon activity and prevent microbial contamination during multiple use. A part of the traditional human interferon alpha 2b spray adopts human serum albumin as a protective agent and chlorobutanol as a bacteriostatic agent, the biological activity is basically kept consistent when the spray is stored for 12 months at the temperature of 2-8 ℃, but whether the biological activity of the spray maintains higher level for a longer time (for example, 12-24 months) is still to be researched. A part of the traditional human interferon alpha 2b spray adopts gelatin and hydroxybenzene methyl cellulose as protective agents, sodium benzoate and benzalkonium bromide as bacteriostatic agents, and the bacteriostatic agents are respectively stored for 24 months at 6 +/-2 ℃, and the biological activities of the bacteriostatic agents are 80% and 70% of the biological activities of 0 day. In contrast, sodium benzoate as a bacteriostatic agent has advantages over benzalkonium bromide in maintaining interferon biological activity. However, sodium benzoate is mostly used in common oral solutions, and sodium benzoate used in sterile preparations has slow bacteriostatic effect and is difficult to meet the requirement of bacteriostatic effect. Benzalkonium bromide has larger irritation and peculiar smell, and the compliance of patients is poorer when the medicine is applied to oral mucosa and other parts. In addition, gelatin and the hydroxybenzene methyl cellulose which are protective agents are difficult to dissolve in water, and the difficulty in the preparation process is increased. The other part of the traditional human interferon alpha 2b spray adopts sucrose, edetate disodium and histidine as protective agents, methylparaben and propylparaben as bacteriostatic agents, and the biological activity can be basically kept consistent after being stored for 24 months at the temperature of 2-8 ℃. However, the hydroxypropyl bacteriostat has a scorched smell, which is not beneficial for the use of the patients with oral and nasal mucosa, and the methylparaben has low solubility, and ethanol or glycerol is usually added as a cosolvent, and if the sterile preparation does not contain ethanol, glycerol and the like, the quick bacteriostat efficacy required by the sterile preparation can not be achieved due to low solubility.
Disclosure of Invention
Based on the above, it is necessary to provide a human interferon alpha 2b spray which has weak irritation, no bad smell, quick bacteriostatic action without using a cosolvent, and long retention time of biological activity, aiming at the problems of strong irritation, slow bacteriostatic action and peculiar smell of the traditional human interferon alpha 2b spray.
In addition, a preparation method of the human interferon alpha 2b spray is also provided.
A human interferon alpha 2b spray comprises human interferon alpha 2b, a protective agent, a bacteriostatic agent, a thickening agent and a buffering agent, wherein the protective agent is at least two of trehalose, sucrose, human serum albumin and edetate disodium, and the bacteriostatic agent is selected from at least one of m-cresol, phenol and benzyl alcohol.
The human interferon alpha 2b spray adopts at least one of trehalose, sucrose, human serum albumin and edetate disodium as a protective agent, and at least one of m-cresol, phenol and benzyl alcohol as a bacteriostatic agent, so that the human interferon alpha 2b spray has the advantages of weak irritation, no bad smell (such as burnt soil smell), quick bacteriostatic action without a cosolvent and longer biological activity maintaining time.
In one embodiment, the ratio of the mass of the human interferon alpha 2b to the mass of the protective agent, the mass of the bacteriostatic agent, the mass of the thickening agent and the molar weight of the buffering agent is (5 mg-1000 mg): (0.01 g-60 g): (1 g-20 g): (1 g-40 g): (5 mmol-40 mmol).
In one embodiment, in the human interferon alpha 2b spray, the concentration of the human interferon alpha 2b is 5-1000 mug/mL, the concentration of the protective agent is 0.01-60 g/L, the concentration of the bacteriostatic agent is 1-20 g/L, and the concentration of the thickening agent is 1-40 g/L.
In one embodiment, the protective agent is a mixture of trehalose, human serum albumin, and edetate disodium.
In one embodiment, in the human interferon alpha 2b spray, the concentration of the trehalose is 0.5-60 g/L, the concentration of the human serum albumin is 0.1-50 g/L, and the concentration of the edetate disodium is 0.01-10 g/L.
In one embodiment, the buffer is selected from one of phosphate buffer salt, citrate buffer salt and acetate buffer salt.
In one embodiment, the pH of the human interferon alpha 2b spray is 5.8-8.
In one embodiment, the thickening agent is selected from at least one of methylcellulose and hypromellose.
In one embodiment, the thickening agent is hypromellose E5.
The preparation method of the human interferon alpha 2b spray comprises the following steps:
mixing the thickening agent with water, and heating to prepare clear thickening liquid;
mixing the clarified thickening solution with the buffering agent, the protective agent and the bacteriostatic agent to prepare an auxiliary material; and
and mixing the auxiliary material with the human interferon alpha 2b, and sterilizing to obtain the human interferon alpha 2b spray.
In one embodiment, the thickener is hypromellose E5, and the step of preparing the clear thickener comprises: heating the mixture of the thickening agent and the water to 60-80 ℃.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. The term "and/or" includes any and all combinations of one or more of the associated listed items. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "hypromellose" as used herein refers to "hypromellose".
One embodiment of the application provides a human interferon alpha 2b spray, which comprises human interferon alpha 2b, a protective agent, a bacteriostatic agent, a thickening agent and a buffering agent, wherein the protective agent is at least two of trehalose, sucrose, human serum albumin and edetate disodium, and the bacteriostatic agent is at least one selected from m-cresol, phenol and benzyl alcohol. The human interferon alpha 2b spray is a sterile liquid spray preparation for local external use. The human interferon alpha 2b spray adopts at least two of trehalose, sucrose, human serum albumin and edetate disodium as protective agents, and at least one of m-cresol, phenol and benzyl alcohol as bacteriostatic agents, so that the human interferon alpha 2b spray has the advantages of weak irritation, no bad smell, quick bacteriostatic action without cosolvent and long biological activity maintaining time.
Specifically, human interferon alpha 2b is used as an active ingredient. In some embodiments, the human interferon α 2b is a recombinant human interferon α 2 b. Specifically, the recombinant human interferon alpha 2b is obtained by separating and purifying after being expressed by recombinant bacteria expressing human interferon alpha 2b genes.
In some embodiments, the ratio of the mass of human interferon alpha 2b to the mass of the protective agent, the mass of the bacteriostatic agent, the mass of the thickening agent and the molar amount of the buffer is (5mg to 1000 mg): (0.01 g-60 g): (1 g-20 g): (1 g-40 g): (5 mmol-40 mmol). Furthermore, the ratio of the mass of the human interferon alpha 2b to the mass of the protective agent, the mass of the bacteriostatic agent, the mass of the thickening agent and the molar weight of the buffering agent is (5 mg-100 mg): (0.05 g-20 g): (9 g-20 g): (5 g-20 g): (10 mmol-30 mmol).
In some embodiments, the concentration of human interferon alpha 2b is between 5 μ g/mL and 1000 μ g/mL (i.e., between 5 ten thousand IU/mL and 1000 ten thousand IU/mL). In an alternative specific example, the concentration of human interferon alpha 2b is 5. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL, 500. mu.g/mL, 800. mu.g/mL, or 1000. mu.g/mL. Furthermore, the concentration of the human interferon alpha 2b is 5 mu g/mL-100 mu g/mL (namely 5 ten thousand IU/mL-100 ten thousand IU/mL).
The protective agent is used for maintaining the activity of human interferon alpha 2 b. In some embodiments, the concentration of the protectant is from 0.01g/L to 60 g/L. In an alternative embodiment, the concentration of the protectant is 0.05g/L, 0.1g/L, 0.5g/L, 1g/L, 2.5g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 35g/L, 45g/L, or 55 g/L. Furthermore, the concentration of the protective agent is 0.05 g/L-20 g/L.
In some embodiments, the protectant is selected from at least two of trehalose, sucrose, human serum albumin, and edetate disodium. In other embodiments, the protectant is selected from at least three of trehalose, sucrose, human serum albumin, and edetate disodium.
In some embodiments, the protectant is a mixture of trehalose, human serum albumin, and edetate disodium. The inventor finds that the interferon alpha 2b is difficult to maintain high biological activity and store for more than 24 months by using single human serum albumin. The combination of human serum albumin, trehalose and edetate disodium is used as a protective agent, so that the interferon alpha 2b can maintain high biological activity and can be stored for more than 24 months. Wherein, human serum albumin can prevent the interferon alpha 2b from being absorbed and aggregated, trehalose can effectively protect the molecular structure of the interferon alpha 2b and prevent the interferon alpha 2b from being degraded, and edetate disodium effectively combines metal ions and can prevent the interferon alpha 2b from being degraded and aggregated. The combined use of human serum albumin, trehalose and edetate disodium has synergistic effect, and enhances the protection effect on interferon alpha 2 b. In addition, the combined use of the human serum albumin, the trehalose and the edetate disodium can also reduce the consumption of the human serum albumin, and save the economic cost.
Optionally, the trehalose concentration is 0.5g/L to 60 g/L. In an alternative specific example, the concentration of trehalose is 0.5g/L, 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 30g/L, 40g/L, 50g/L, or 60 g/L. Further, the concentration of the trehalose is 5 g/L-20 g/L. Optionally, the concentration of human serum albumin is 0.1 g/L-50 g/L. In an alternative specific example, the concentration of human serum albumin is 0.1g/L, 0.5g/L, 2g/L, 5g/L, 10g/L, 15g/L, 20g/L, 30g/L, 40g/L, or 50 g/L. Furthermore, the concentration of the human serum albumin is 0.5 g/L-20 g/L. Optionally, the concentration of edetate disodium is 0.01g/L to 10 g/L. In an alternative specific example, the concentration of edetate disodium is 0.01g/L, 0.05g/L, 0.1g/L, 0.5g/L, 1g/L, 2g/L, 5g/L, 8g/L, or 10 g/L. Furthermore, the concentration of the edetate disodium is 0.05 g/L-10 g/L.
The bacteriostatic agent is used for inhibiting the growth of microorganisms and preventing microbial contamination in the process of multiple use. In some embodiments, the concentration of bacteriostatic agent is 1g/L to 20 g/L. Furthermore, the concentration of the bacteriostatic agent is 9 g/L-20 g/L. The inventor researches and discovers that the bacteriostatic agent containing the benzene ring and the hydroxyl simultaneously has good water solubility, quick bacteriostatic action, good safety and small influence on the activity of human interferon alpha 2 b. In some embodiments, the bacteriostatic agent is benzyl alcohol, m-cresol, or phenol. It is verified that the benzyl alcohol has little influence on the activity of human interferon alpha 2b, and other bacteriostats, such as sodium benzoate, methyl hydroxybenzoate and propyl hydroxybenzoate, have different degrees of influence on the activity of human interferon alpha 2b, especially sodium benzoate. In addition, the benzyl alcohol used as the bacteriostatic agent has no peculiar smell and weak irritation, and has quick bacteriostatic action without a cosolvent. In an alternative specific example, the concentration of benzyl alcohol is 1g/L, 2.5g/L, 5g/L, 8g/L, 10g/L, 15g/L, or 20 g/L. Further, the concentration of the benzyl alcohol is 9 g/L-20 g/L.
The thickening agent is used for prolonging the detention time of the human interferon alpha 2b at the spraying position, softening the cuticle layer and promoting the absorption of the human interferon alpha 2 b. In some embodiments, the concentration of the thickener is 1g/L to 40 g/L. In an alternative specific example, the concentration of the thickener is 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 30g/L, or 40 g/L. Further, the concentration of the thickener is 5g/L to 20 g/L. Optionally, the thickening agent is selected from at least one of methylcellulose and hypromellose. In some embodiments, the thickening agent is low viscosity hypromellose. Optionally, the low viscosity hypromellose has a viscosity of 2 to 15mpa.s at 25 ℃. In one optionally specific example, the thickening agent is hypromellose E5. It is understood that in other embodiments, the thickening agent is not limited to the above, but may be other substances.
The buffer serves to maintain the pH of the system. In some embodiments, the human interferon alpha 2b spray has a pH of 5.8 to 8. Further, the pH value of the human interferon alpha 2b spray is 6.5-7.5. In some embodiments, the buffer is selected from one of phosphate buffer salts, citrate buffer salts, and acetate buffer salts. Optionally, the concentration of the buffer is 5mmol/L to 40 mmol/L. In one specific example, the buffer concentration is 5mmol/L, 10mmol/L, 20mmol/L, 30mmol/L, or 40 mmol/L. Further, the concentration of the buffer is 10mmol/L to 30 mmol/L. It is understood that in other embodiments, the buffer is not limited to the above, but may be other substances.
In some embodiments, the human interferon alpha 2b spray does not contain a cosolvent.
It will be appreciated that the human interferon alpha 2b spray described above also contains water. In some embodiments, the water is water for injection.
In addition, an embodiment of the present application further provides a preparation method of the human interferon α 2b spray, which includes step S110, step S120, and step S130.
Step S110: dissolving the thickener in water, heating, and making into clear thickening solution.
Specifically, the thickener is as described above and will not be described herein in detail. Because the thickening agent is insoluble in water at normal temperature, the thickening agent is mixed with water and then heated, so that the thickening agent is more easily dissolved in water to prepare a clear solution (thickening solution) containing the thickening agent, and the problem that the thickening agent in the human interferon alpha 2b spray is insoluble is solved skillfully. In some embodiments, the step of preparing a clarified thickener comprises: mixing the thickening agent with water, heating to 60-80 ℃, and preparing clear thickening liquid. It will be appreciated that stirring the thickener with water during heating is also included to accelerate the dissolution of the thickener. In an alternative specific example, the temperature of heating is 60 ℃, 65 ℃, 70 ℃, 75 ℃ or 80 ℃.
Step S120: mixing the clarified thickening solution with a buffering agent, a protective agent and a bacteriostatic agent to prepare an auxiliary material.
Specifically, the buffering agent, the protective agent and the bacteriostatic agent are as described above and will not be described herein.
In one embodiment, the buffer, the protective agent and the bacteriostatic agent are added into the clarified thickening solution, and stirred to dissolve the buffer, the protective agent and the bacteriostatic agent in the thickening solution to prepare the auxiliary material. It will be appreciated that when the clarified thickener is mixed with the other components, the clarified thickener has cooled sufficiently to below 40 ℃.
Step S130: mixing the auxiliary material with human interferon alpha 2b, and sterilizing to obtain the human interferon alpha 2b spray.
In some embodiments, the bacteria are removed by filtration through a filter. In an alternative embodiment, a filter membrane with a particle size of 0.22 μm is used to filter a mixture of the adjunct with human interferon α 2 b. It is understood that in other embodiments, other means of sterilization may be used. Of course, the amounts of human interferon alpha 2b, thickener, water, buffer, protectant and bacteriostatic were weighed according to the respective concentrations in the human interferon alpha 2b spray to be prepared.
In some embodiments, after the step of sterilizing, a step of dispensing is also included.
The preparation method of the human interferon alpha 2b spray comprises the steps of preparing the indissolvable thickening agent into clear thickening liquid in a heating mode, and then mixing the clear thickening liquid with other auxiliary material components and the human interferon alpha 2b, so that the problem that the thickening agent is indissolvable in water is solved, the operation is simple and convenient, and the preparation method is a stable and efficient preparation method.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.
Example 1
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
trehalose: 5g of the total weight of the mixture;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
2.14g of monobasic sodium phosphate monohydrate;
hypromellose E5: 10g of a mixture;
benzyl alcohol: 10g of a mixture;
water for injection is added to: 1000 mL;
the preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 with the formula amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: and (3) 30 min. Standing and cooling to room temperature (25 ℃, the same below), sequentially adding the disodium hydrogen phosphate dihydrate, the sodium dihydrogen phosphate monohydrate, the trehalose and the benzyl alcohol according to the prescription amount, uniformly stirring, finally adding the human interferon alpha 2b, adding the water for injection to a constant volume of 1000mL, uniformly stirring, sterilizing and filtering with a 0.22 mu m filter membrane, and aseptically subpackaging in a container.
Example 2
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
disodium edetate: 0.1 g;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hypromellose E5: 10g of a mixture;
benzyl alcohol: 10g of a mixture;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 with the formula amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: standing for 30min, cooling to room temperature, sequentially adding disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, disodium edetate and benzyl alcohol, stirring, adding human interferon alpha 2b, adding water for injection to a constant volume of 1000mL, stirring, sterilizing with 0.22 μm filter membrane, filtering, and packaging in containers.
Example 3
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
trehalose: 5g of the total weight of the mixture;
disodium edetate: 0.1 g;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hypromellose E5: 10g of a mixture;
benzyl alcohol: 10g of a mixture;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 according to the prescription amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: 30min, standing, cooling to room temperature, sequentially adding disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, trehalose, disodium edetate and benzyl alcohol in the prescribed amount, stirring uniformly, finally adding human interferon alpha 2b, adding water for injection to a constant volume of 1000mL, stirring uniformly, sterilizing and filtering with a 0.22 mu m filter membrane, and aseptically subpackaging in a container.
Example 4
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
trehalose: 5g of the total weight of the mixture;
disodium edetate: 0.1 g;
human serum albumin: 10g of a mixture;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hypromellose E5: 10g of a mixture;
benzyl alcohol: 10g of a mixture;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 according to the prescription amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: 30min, standing, cooling to room temperature, sequentially adding disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, trehalose, edetate disodium, human serum albumin and benzyl alcohol according to the prescription amount, stirring uniformly, finally adding human interferon alpha 2b, adding water for injection to a constant volume of 1000mL, stirring uniformly, sterilizing and filtering with a 0.22 mu m filter membrane, and aseptically subpackaging in a container.
Example 5
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
trehalose: 5g of the total weight of the mixture;
disodium edetate: 0.1 g;
human serum albumin: 10g of a mixture;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hypromellose E5: 10g of a mixture;
methyl hydroxybenzoate: 2.5 g;
hydroxy phenyl propyl ester: 0.2 g;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 according to the prescription amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: 30min, standing and cooling to room temperature, sequentially adding the disodium hydrogen phosphate dihydrate, the sodium dihydrogen phosphate monohydrate, the trehalose, the disodium edetate, the human serum albumin, the methyl hydroxybenzoate and the propyl hydroxybenzoate in the prescribed amount, uniformly stirring, finally adding the human interferon alpha 2b, adding the water for injection to a constant volume of 1000mL, uniformly stirring, sterilizing and filtering with a 0.22 mu m filter membrane, and aseptically subpackaging in containers.
Example 6
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
trehalose: 5g of the total weight of the mixture;
disodium edetate: 0.1 g;
human serum albumin: 10g of a mixture;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hypromellose E5: 10g of a mixture;
sodium benzoate: 10g of a mixture;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 according to the prescription amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: 30min, standing, cooling to room temperature, sequentially adding disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, trehalose, edetate disodium, human serum albumin and sodium benzoate, stirring, finally adding human interferon alpha 2b, adding water for injection to a constant volume of 1000mL, stirring, sterilizing with a 0.22 μm filter membrane, filtering, and aseptically subpackaging in containers.
Example 7
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
human serum albumin: 10g of a mixture;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hydroxypropyl methylcellulose: 10g of a mixture;
benzyl alcohol: 10g of a mixture;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 according to the prescription amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: standing for 30min, cooling to room temperature, sequentially adding disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, human serum albumin and benzyl alcohol, stirring, adding human interferon alpha 2b, adding water for injection to a constant volume of 1000mL, stirring, sterilizing with 0.22 μm filter membrane, filtering, and packaging in containers.
Example 8
The formulation of the human interferon alpha 2b spray of this example is as follows:
interferon α 2 b: 1g of a compound;
trehalose: 5g of the total weight of the mixture;
disodium edetate: 0.1 g;
human serum albumin: 10g of a mixture;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
sodium dihydrogen phosphate monohydrate: 2.14 g;
hypromellose E5: 10g of a mixture;
water for injection is added to: 1000 mL.
The preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 according to the prescription amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: 30min, standing, cooling to room temperature, sequentially adding disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, trehalose, edetate disodium and human serum albumin, stirring uniformly, finally adding human interferon alpha 2b, adding water for injection to a constant volume of 1000mL, stirring uniformly, sterilizing with a 0.22 μm filter membrane, filtering, and aseptically subpackaging in containers.
Example 9
The formulation of the human interferon alpha 2b spray of this example is as follows:
human interferon α 2 b: 1g of a compound;
disodium hydrogen phosphate dodecahydrate: 1.61 g;
2.14g of monobasic sodium phosphate monohydrate;
hypromellose E5: 10g of a mixture;
benzyl alcohol: 10g of a mixture;
water for injection is added to: 1000 mL;
the preparation method of the human interferon alpha 2b spray of the embodiment comprises the following steps:
weighing hydroxypropyl methylcellulose E5 with the formula amount, pouring into a beaker with certain injection water, heating to 75 ℃, and stirring at the frequency: 25Hz, stirring time: and (3) 30 min. Standing and cooling to room temperature, sequentially adding the disodium hydrogen phosphate dodecahydrate, the sodium dihydrogen phosphate monohydrate and the benzyl alcohol according to the prescription amount, uniformly stirring, finally adding the human interferon alpha 2b, adding the water for injection to a constant volume of 1000mL, uniformly stirring, performing aseptic subpackaging in a container by a 0.22-micrometer filter membrane for sterilization and filtration.
And (3) testing:
1. the human interferon alpha 2b sprays prepared in each example were examined for bacteriostatic efficacy, and some results are shown in table 1. In Table 1, N/A: indicating no bacteriostatic effect; NI: indicating no increase in test bacteria.
TABLE 1
According to the judgment standard of the bacteriostatic efficacy of the skin administration preparation, the three bacteria are reduced by 2lg and 3lg respectively in 2 days and 7 days to meet the requirement. As can be seen from Table 1, the human interferon alpha 2b spray of example 4 using benzyl alcohol as the bacteriostatic agent meets the bacteriostatic standard, and has no obvious odor, fast bacteriostatic action and strong acting force.
2. The interferon alpha 2b sprays prepared in each example were tested for activity at different time points for interferon alpha 2b to determine the effect of different bacteriostats on interferon alpha 2b activity, and some results are shown in table 2.
TABLE 2
As can be seen from Table 2, benzyl alcohol had almost no effect on the biological activity of interferon-. alpha.2b.
3. The human interferon alpha 2b spray prepared in each example is placed at 2-8 ℃ for a long time, the stability of the spray is examined for 24 months, and partial results are shown in table 3.
TABLE 3
As can be seen from table 3, the human interferon α 2b spray using the mixture of trehalose, human serum albumin and edetate disodium as the protective agent has better stability and better protection effect, and is more favorable for the long-term storage of interferon α 2 b.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions obtained by logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent of the invention is subject to the content of the appended claims, and the description can be used for explaining the content of the claims.
Claims (10)
1. The human interferon alpha 2b spray is characterized by comprising human interferon alpha 2b, a protective agent, a bacteriostatic agent, a thickening agent and a buffering agent, wherein the protective agent is selected from at least one of trehalose, sucrose, human serum albumin and edetate disodium, and the bacteriostatic agent is selected from at least one of m-cresol, phenol and benzyl alcohol.
2. The human interferon alpha 2b spray according to claim 1, wherein the ratio of the mass of human interferon alpha 2b to the mass of the protective agent, the mass of the bacteriostatic agent, the mass of the thickening agent and the molar amount of the buffer is (5 mg-1000 mg): (0.01 g-60 g): (1 g-20 g): (1 g-40 g): (5 mmol-40 mmol).
3. The human interferon alpha 2b spray according to claim 2, wherein in the human interferon alpha 2b spray, the concentration of the human interferon alpha 2b is 5 to 1000 μ g/mL, the concentration of the protective agent is 0.01 to 60g/L, the concentration of the bacteriostatic agent is 1 to 20g/L, and the concentration of the thickening agent is 1 to 40 g/L.
4. The human interferon alpha 2b spray according to any one of claims 1 to 3, wherein the protective agent is a mixture of trehalose, human serum albumin and edetate disodium.
5. The human interferon alpha 2b spray according to claim 4, wherein the concentration of trehalose is 0.5g/L to 60g/L, the concentration of human albumin is 0.1g/L to 50g/L, and the concentration of edetate disodium is 0.01g/L to 10 g/L.
6. The human interferon alpha 2b spray according to any one of claims 1 to 5, wherein the buffering agent is selected from one of phosphate buffer salt, citrate buffer salt and acetate buffer salt; preferably, the pH value of the human interferon alpha 2b spray is 5.8-8.
7. The human interferon alpha 2b spray according to any one of claims 1 to 3, 5 and 6, wherein the thickener is at least one selected from methylcellulose and hypromellose.
8. The human interferon alpha 2b spray according to claim 7, wherein the thickener is hypromellose E5.
9. The method for preparing human interferon alpha 2b spray according to any one of claims 1 to 8, comprising the steps of:
mixing the thickening agent with water, and heating to prepare clear thickening liquid;
mixing the clarified thickening solution with the buffering agent, the protective agent and the bacteriostatic agent to prepare an auxiliary material; and
and mixing the auxiliary material with the human interferon alpha 2b, and sterilizing to obtain the human interferon alpha 2b spray.
10. The method of claim 9, wherein the thickening agent is hypromellose E5, and the step of preparing the clear thickening solution comprises: heating the mixture of the thickening agent and the water to 60-80 ℃.
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