Disclosure of Invention
In order to improve the oil solubility of gentamicin sulfate and simultaneously realize the combination of gentamicin sulfate and Marek's vaccine, the application provides gentamicin sulfate soluble powder and a preparation method and application thereof.
In a first aspect, the application provides gentamicin sulfate soluble powder, which adopts the following technical scheme:
the gentamicin sulfate soluble powder comprises the following components in parts by weight: 30-50 parts of gentamicin sulfate, 10-30 parts of hydroxypropyl beta cyclodextrin and 15-35 parts of sucrose monohydrate.
According to the application, the hydroxypropyl beta cyclodextrin and the sucrose monohydrate are added into the gentamicin sulfate, so that the prepared gentamicin sulfate soluble powder can be dissolved in water and oil, and the gentamicin sulfate soluble powder can be used in combination with an oily vaccine; in addition, when the gentamicin sulfate soluble powder is used in combination with a Marek vaccine, the potency of the Marek vaccine is not affected.
In the application, the hydroxypropyl beta cyclodextrin has good solubility in water, small viscosity and good fluidity, and can be mixed with gentamycin sulfate to improve the water solubility of the gentamycin sulfate on one hand and to improve the stability and bioavailability of the gentamycin sulfate on the other hand. The sucrose monohydrate ester contains a crystal water and contains ester groups on a molecular chain, so that the sucrose monohydrate ester has both hydrophilicity and lipophilicity, and the sucrose monohydrate ester is added into gentamicin sulfate, and the gentamicin sulfate soluble powder obtained through emulsification has excellent oil solubility and water solubility.
In some embodiments, the hydroxypropyl beta cyclodextrin may be 10-20 parts, 10-25 parts, 20-30 parts, or 25-30 parts by weight.
In a specific embodiment, the hydroxypropyl beta cyclodextrin may also be 10 parts, 20 parts, 25 parts, or 30 parts by weight.
In some embodiments, the sucrose monohydrate ester may be 15-20 parts, 15-25 parts, 15-30 parts, 20-25 parts, 20-30 parts, 20-35 parts, 25-30 parts, 25-35 parts, or 30-35 parts by weight.
In a specific embodiment, the sucrose monohydrate ester may also be 15 parts, 20 parts, 25 parts, 30 parts, or 35 parts by weight.
Optionally, the gentamicin sulfate soluble powder comprises the following components in parts by weight: 30-50 parts of gentamicin sulfate, 20-30 parts of hydroxypropyl beta cyclodextrin and 20-30 parts of sucrose monohydrate ester.
Optionally, the gentamicin sulfate soluble powder further comprises 10-20 parts of cosolvent and 10-20 parts of solubilizer.
Optionally, the cosolvent is selected from one or more of sucrose, sodium benzoate, and sodium salicylate; the solubilizer is one or more selected from Tween 20, tween 40, polyoxyethylene castor oil, poloxamer and sorbitan fatty acid ester.
In a specific embodiment, the solubilizing agent may be tween 20, polyoxyethylated castor oil or poloxamer, and may also be a mixture of tween 20 and polyoxyethylated castor oil, a mixture of tween 20 and poloxamer, or a mixture of tween 20 and sorbitan fatty acid ester.
Optionally, the solubilizing agent is a mixture of tween 20 and polyoxyethylated castor oil.
In some embodiments, the weight ratio of tween 20 and polyoxyethylated castor oil may be 2: (0.5-0.8), 2: (0.5-1.2), 2: (0.5-1.7), 2: (0.8-1.2), 2: (0.8-1.7), 2: (0.8-2.0), 2: (1.2-1.7), 2: (1.2-2.0) or 2: (1.7-2.0).
In a specific embodiment, the weight ratio of tween 20 and polyoxyethylated castor oil may also be 2:0.5, 2:0.8, 2:1.2, 2:1.7 or 2:2.0.
Optionally, the weight ratio of tween 20 to polyoxyethylated castor oil is 2: (0.8-1.7).
In a second aspect, the application provides a method for preparing gentamicin sulfate soluble powder, comprising the following steps: dissolving gentamicin sulfate in water, adding other components, stirring uniformly, adding a pH regulator after complete emulsification, regulating the pH value to 6.9-7.0, and carrying out vacuum drying and crushing to obtain gentamicin sulfate soluble powder with the particle size of 400-500 meshes.
Optionally, the vacuum drying process parameters are as follows: the temperature is 50-70deg.C, and the time is 3-5h.
In a third aspect, the application provides a pharmaceutical composition comprising the gentamicin sulfate soluble powder.
Optionally, the pharmaceutical composition further comprises an antibacterial drug or vaccine; the vaccine is Marek's vaccine or oily vaccine.
In the application, the gentamicin sulfate soluble powder has good oil solubility on one hand, can be mutually dissolved with oily vaccines (ND and H9) and does not have layering; on the other hand, when the gentamicin sulfate soluble powder is used in combination with the Marek vaccine, the potency of the Marek vaccine is not affected.
In summary, the application has the following beneficial effects:
1. The gentamicin sulfate soluble powder which can be dissolved in water and oil is prepared by using the gentamicin sulfate, the hydroxypropyl beta cyclodextrin and the sucrose monohydrate, so that the gentamicin sulfate soluble powder can be used in combination with a water-based vaccine or an oil-based vaccine; in addition, when the gentamicin hydrochloride soluble powder is used in combination with a Marek vaccine, the pH of the Marek vaccine is basically unchanged, and the potency of the Marek vaccine is basically unaffected.
2. The application further selects the following components in weight ratio: and (0.8-1.7) Tween 20 and polyoxyethylene castor oil are used as solubilizers, and when the obtained gentamicin sulfate soluble powder is used in combination with a Marek vaccine, the osmotic pressure change of the Marek vaccine is smaller, and the influence of the vaccine titer is smaller.
Detailed Description
The application provides gentamicin sulfate soluble powder which comprises the following components in parts by weight: 30-50 parts of gentamycin sulfate, 10-30 parts of hydroxypropyl beta cyclodextrin, 15-35 parts of sucrose monohydrate, 10-20 parts of cosolvent and 10-20 parts of solubilizer. Further, the gentamicin sulfate soluble powder comprises the following components in parts by weight: 30-50 parts of gentamycin sulfate, 20-30 parts of hydroxypropyl beta cyclodextrin, 20-30 parts of sucrose monohydrate ester, 10-20 parts of cosolvent and 10-20 parts of solubilizer.
Wherein the cosolvent is selected from one or more of sucrose, sodium benzoate and sodium salicylate; the solubilizer is one or more selected from Tween 20, tween 40, polyoxyethylene castor oil, poloxamer and sorbitan fatty acid ester. Further, the solubilizer is a mixture of tween 20 and polyoxyethylated castor oil. Still further, the weight ratio of tween 20 to polyoxyethylated castor oil is 2: (0.8-1.7).
The preparation method of the gentamicin sulfate soluble powder provided by the application comprises the following steps: dissolving gentamicin sulfate in water, adding other components, stirring uniformly, adding a pH regulator after complete emulsification, regulating the pH value to 6.9-7.0, vacuum drying at 50-70 ℃ for 3-5h, and crushing to obtain gentamicin sulfate soluble powder with the particle size of 400-500 meshes.
In the application, gentamicin sulfate is purchased from the Biotechnology Co., ltd. Of Heilongjiang Games Lin Hesai, and the CAS number of the hydroxypropyl beta cyclodextrin is 12846-35-5; sucrose monohydrate ester available from Jiangsu Jiujia biotechnology Co., ltd and having a CAS number of 37318-31-3; the CAS number of the sodium benzoate is 532-32-1; tween 20 was purchased from Alatin Biochemical technologies Co., ltd; polyoxyethylated castor oil EL35, available from biotechnology limited of shanxi Emperor Yao; the poloxamer is poloxamer 407, and is purchased from the company of the pharmaceutical excipients of the Siemens; sorbitan fatty acid esters are available from Wang Biotech Inc. of river Nankang. The remaining materials, reagents, solvents, etc. of the application are also commercially available.
The present application will be described in further detail with reference to examples and performance test.
Examples 1 to 8
Examples 1-8 provide a gentamicin sulfate soluble powder, respectively.
The above-described embodiments differ in that: the addition amounts of a part of the components in the gentamicin sulfate soluble powder are shown in the following table 1.
The preparation method of the gentamicin sulfate soluble powder provided in examples 1-8 comprises the following steps: dissolving 40g of gentamicin sulfate in 20g of water, adding 15g of hydroxypropyl beta cyclodextrin, sucrose monohydrate, cosolvent (sodium benzoate) and 15g of solubilizer (Tween 20), fully and uniformly stirring, completely emulsifying, adding a pH value regulator (phosphoric acid buffer solution), regulating the pH value to 7.0, vacuum drying at 60 ℃ for 4h, and carrying out superfine grinding by a jet mill to obtain gentamicin sulfate soluble powder with the particle size of 400-500 meshes.
TABLE 1 addition amount of partial components in gentamicin sulfate soluble powder provided in examples 1 to 8
Examples 9 to 17
Examples 9-17 provide a gentamicin sulfate soluble powder, respectively.
The above embodiment differs from embodiment 3 in that: the components and proportions of the solubilizer are shown in Table 2 below.
Table 2 examples 9-17 provide solubilizing agents in gentamicin sulfate soluble powders
Comparative example 1
Comparative example 1 provides a gentamicin sulfate soluble powder.
The above comparative example is different from example 3 in that: the adding amount of sucrose monohydrate ester in the gentamycin sulfate soluble powder is 0.
Comparative example 2
Comparative example 2 provides a gentamicin sulfate soluble powder.
The above comparative example is different from example 3 in that: the sucrose monohydrate ester in the gentamicin sulfate soluble powder was replaced with xylitol anhydride monostearate (available from Chongqing day run biologicals Co.).
Comparative example 3
Comparative example 3 provides a gentamicin sulfate soluble powder.
The above comparative example is different from example 3 in that: the sucrose monohydrate ester in the gentamicin sulfate soluble powder was replaced with 15-hydroxystearic acid polyethylene glycol ester (HS 15, available from Seaman pharmaceutical excipients Co., ltd.).
The performance detection test (I) combines gentamicin sulfate soluble powder with Marek vaccine, and explores the influence of the gentamicin sulfate soluble powder on the use performance of Marek vaccine.
1. Effect of gentamicin sulfate soluble powder on Marek vaccine pH
25ML of Marek's vaccine diluent (special diluent for chicken Marek's disease frozen vaccine, provided by Beijing feather company, package: 400 mL/bottle) is used for dissolving 5g of gentamicin sulfate soluble powder, shaking for 2-3min for complete dissolution, obtaining a sample to be tested, detecting the pH value of the sample to be tested, carrying out parallel test for 5 times, and taking an average value; the results of the test using chicken Marek's disease frozen vaccine as a blank are shown in Table 3.
Note that: when the pH value of the sample to be detected is between 6.9 and 7.1, the specification is met, and the titer of the Marek vaccine is not affected.
2. Influence of gentamicin sulfate soluble powder on osmotic pressure of Marek vaccine
Dissolving 5g gentamicin sulfate soluble powder by 25mL of Marek vaccine diluent, and shaking for 2-3min to completely dissolve to obtain a sample to be tested; detecting osmotic pressure of a sample to be detected by adopting a CM320 freezing point osmotic pressure tester, carrying out parallel test for 5 times, and taking an average value; the results of the frozen vaccine against chicken Marek's disease as a blank are shown in Table 3.
Note that: when the osmotic pressure of the sample to be tested is between 250 and 450mOsm/L, the sample meets the rule, and the titer of the Marek vaccine is not affected.
3. Vaccine cell viability assay
(1) Preparing a solution:
Preparation of test solution: measuring 50mL of Marek's special diluent in a 250mL triangular flask, adding 5g of accurately weighed test drug (gentamicin sulfate soluble powder provided by the application), adding a proper amount of Marek's frozen live vaccine (chicken Marek's disease frozen vaccine, CVI 988Rupens strain, package: 2000 parts per bottle), finally enabling the cell concentration to be 105-106/mL, shaking uniformly, and performing the measurement for 0h and 1 h.
Preparation of a control solution: and (3) measuring 50mL of Marek's special diluent in a 250mL triangular flask, adding a proper amount of Marek's frozen live vaccine, and finally enabling the cell concentration to be 105-106/mL, shaking uniformly, and performing measurement by acting for 0h and 1 h.
(2) And (3) measuring: taking 6mL of a sample solution to be tested, putting the sample solution into a centrifuge tube, centrifuging for 20min at 2000r/min, discarding supernatant, adding 3mL of Marek' S special diluent, shaking uniformly, taking 10 mu L of the solution, adding 10 mu L of 0.4% trypan blue staining solution, mixing uniformly, sucking 10 mu L of the mixed solution, adding the mixed solution into a disposable counting plate, standing horizontally for 30S, inserting the mixed solution into a cell counter for focusing, performing cell counting and activity calculation, recording the reading and storing. The control solution was assayed by the same method.
(3) The results were calculated as follows, and the test solution was measured 6 times to calculate the results as an average value. The degradation results are shown in table 3 below.
TABLE 3 Performance test results in examples 1-17 and comparative examples 1-3
According to the detection results of Table 3, after the gentamicin sulfate soluble powder obtained in examples 1-17 is mixed with Marek's vaccine, the pH value is between 6.9 and 7.1, the osmotic pressure range is between about 284 and 367mOsm/L, and the cell activity ratio is more than or equal to 0.95; the osmotic pressure of the gentamicin sulfate soluble powder prepared in comparative example 1 without adding sucrose monohydrate is up to 437mOsm/L, and the cell viability ratio of 1h is only 0.89 after the gentamicin sulfate soluble powder is mixed with Marek's vaccine; comparative example 2 gentamicin sulphate soluble powder prepared by xylitol anhydride monostearate is mixed with Marek's vaccine, the osmotic pressure is up to 415mOsm/L1h, and the cell activity ratio is 0.92; comparative example 3 gentamicin sulphate soluble powder prepared by xylitol anhydride monostearate is mixed with Marek's vaccine, the osmotic pressure is up to 430mOsm/L, and the 1h cell viability ratio is 0.94; the pH of the blank was 7.0 and the osmotic pressure range was approximately 268mOsm/L. Therefore, the gentamicin sulfate soluble powder provided by the application has little influence on the pH and osmotic pressure of the Marek vaccine, the pH value and the osmotic pressure are in a reasonable range and are closer to a blank group, and the gentamicin sulfate soluble powder provided by the application has no influence on the potency of the Marek vaccine.
The test results of examples 1-7 show that, as the addition amount of hydroxypropyl beta cyclodextrin and sucrose monohydrate ester increases, the pH value of the prepared gentamicin sulfate soluble powder is basically kept unchanged after being mixed with Marek vaccine, and the osmotic pressure is in a trend of decreasing first and then increasing second; in particular, the gentamicin sulfate soluble powder obtained in examples 2 to 4 and examples 6 to 7 was mixed with Marek's vaccine, and the osmotic pressure was less than 350mOsm/L. Therefore, the application shows that the application can obtain gentamicin sulfate soluble powder with smaller influence on the osmotic pressure of the Marek vaccine by controlling the adding amount of hydroxypropyl beta cyclodextrin between 20 and 30 parts and the adding amount of sucrose monohydrate ester between 20 and 30 parts, and the use of the gentamicin sulfate soluble powder and the Marek vaccine can not influence the potency of the gentamicin sulfate soluble powder.
As can be seen from the detection results of examples 3 and examples 9 to 17, the gentamicin sulfate soluble powder obtained by using only one solvent in examples 3 and examples 9 to 10 was mixed with Marek's vaccine, the pH value was 7.05, and the osmotic pressure was 321 to 356mOsm/L; example 16 uses a weight ratio of 2:1.2, wherein the mixture of Tween 20 and poloxamer is used as a solubilizer, and the obtained gentamicin sulfate soluble powder is mixed with Marek vaccine, and has a pH value of 7.04 and an osmotic pressure of 320mOsm/L; example 17 uses a weight ratio of 2:1.2, wherein the mixture of Tween 20 and sorbitan fatty acid ester is used as a solubilizer, and the obtained gentamicin sulfate soluble powder is mixed with a Marek vaccine, and has a pH value of 7.05 and an osmotic pressure of 316mOsm/L; whereas examples 11-15 used a weight ratio of 2: (0.5-2.0) tween 20 and polyoxyethylated castor oil as solubilizer, and the obtained gentamicin sulfate soluble powder is mixed with Marek vaccine, and the osmotic pressure is 284-314mOsm/L. The application is proved to adopt the mixture of Tween 20 and polyoxyethylene castor oil as the solubilizer, the obtained gentamicin sulfate soluble powder has smaller influence on the osmotic pressure of the Marek vaccine, and the combined use of the gentamicin sulfate soluble powder and the Marek vaccine does not influence the potency of the vaccine.
Further comparison shows that examples 12-14 control the weight ratio of tween 20 and polyoxyethylated castor oil to 2: (0.8-1.7), the obtained gentamicin sulfate soluble powder is mixed with Marek vaccine, and the osmotic pressure is less than 300mOsm/L and is more similar to that of a blank group. Therefore, the application is further illustrated to employ a weight ratio of 2: tween 20 and polyoxyethylene castor oil (0.8-1.7) are used as solubilizers, so that gentamycin sulfate soluble powder with smaller influence on the vaccine titer of the Marek vaccine can be obtained.
In summary, the application controls the weight parts of each component in the gentamicin sulfate soluble powder within the following range: 30-50 parts of gentamycin sulfate, 10-30 parts of hydroxypropyl beta cyclodextrin, 15-35 parts of sucrose monohydrate ester, 10-20 parts of cosolvent and 10-20 parts of solubilizer; the gentamicin sulfate soluble powder which is used together with the Marek vaccine and does not influence the potency of the Marek vaccine can be obtained; further, the weight ratio of the application is 2: the mixture of Tween 20 and polyoxyethylene castor oil (0.8-1.7) is used as a solubilizer, and the obtained gentamicin sulfate soluble powder has smaller osmotic pressure influence on the Marek vaccine.
(II) solubility test
(1) Firstly, 20g of gentamicin sulfate soluble powder is placed in 25mL of nano dispersion liquid provided by Rui first agriculture and sea company, and is vibrated for 2-3min to be completely dissolved, so as to obtain diluent; standing the diluted solution for 4 hours without layering and precipitation;
(2) And (2) adding 22mL of the diluent obtained in the step (1) into 500mL of ND Newcastle disease inactivated vaccine, repeatedly reversing to fully dissolve the diluent, standing for 192h, and observing whether layering and precipitation occur. The results are shown in Table 4 below.
TABLE 4 solubility test results
According to the test results of the solubility test in Table 4, the gentamicin sulfate soluble powder provided by the application can be dissolved in an oily vaccine (ND Newcastle disease inactivated vaccine), and does not have layering or precipitation after standing for 192 hours.
And thirdly, combining the gentamicin sulfate soluble powder with an oily vaccine, and exploring the influence of the gentamicin sulfate soluble powder on the potency of the oily vaccine.
1. Preparing injection:
(1) First, 20g of the gentamicin sulfate soluble powder of example 13 was dissolved in 1 bottle (25 mL) of a nano-dispersion (supplied by Rui first agriculture, shake and sea Co., ltd.) and shaken well for 3 minutes, and after complete dissolution, a diluted drug solution was obtained.
(2) And (3) extracting 11mL of the diluted liquid medicine by using a syringe, and injecting the diluted liquid medicine into 250mL of ND Newcastle disease inactivated vaccine to obtain gentamicin sulfate-ND combined injection.
11ML of the diluted liquid medicine is extracted by a syringe and injected into 250mL of H9 inactivated vaccine, and gentamicin sulfate-H9 combined injection is obtained.
2. Broiler test
(1) 400 Feather chickens are selected in a standardized white feather chicken cage farm and are divided into 4 groups of 100 chickens. Wherein, 1 group of feather chicken necks are subcutaneously injected with gentamicin sulfate-ND combined injection; the 1 group of feather chicken necks are subcutaneously injected with gentamicin sulfate-H9 combined injection; subcutaneously injecting an ND Newcastle disease inactivated vaccine into the chicken neck of the 1 group; subcutaneously injecting an H9 inactivated vaccine into the chicken neck of the 1 group; then, the breeding under the same conditions was performed under the same environment.
(2) Blood was collected at 1d, 7d, 14d, 21d, 28d, 35d, 42d days after injection, respectively, and standard antigens were assayed for ND antibody, H9 antibody, and HI antibody for H9,
3. The detection results are shown in Table 5 below.
TABLE 5 results of detection of oily vaccine titers
As can be seen from the detection results in Table 5, the potency of the ND Newcastle disease inactivated vaccine and the H9 inactivated vaccine is basically unaffected after the gentamycin sulfate soluble powder provided by the application is added into the ND Newcastle disease inactivated vaccine and the H9 inactivated vaccine. Therefore, the gentamicin sulfate soluble powder provided by the application can be used for combined injection with various oily vaccines, and the potency of the oily vaccines cannot be influenced. Furthermore, the combination reduces the number of injections to the birds, thereby reducing stress.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.