Tilmicosin premix and preparation method thereof

Abstract

The invention belongs to the technical field of veterinary preparations, and particularly relates to a tilmicosin premix and a preparation method thereof. The tilmicosin premix is prepared from the following raw materials: tilmicosin, a polylactic acid-glycolic acid copolymer, lecithin, modified hydroxypropyl methyl cellulose, grafted polyvinylpyrrolidone, polyethylene glycol and an antioxidant, a solid dispersion is formed through lecithin and grafted polyvinylpyrrolidone, the solubility and stability of tilmicosin are enhanced, polylactic acid-glycolic acid copolymer nano-microspheres are prepared through a double-emulsion solvent evaporation method, slow release of drugs is achieved, dodecyl modified hydroxypropyl methyl cellulose and polyethylene glycol are combined, and the sustained release effect is achieved. The solubility and the slow release characteristic are further enhanced. The activity of the granular premix is effectively preserved, and the granular premix is convenient to use and transport.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drug preparations, and particularly relates to a tilmicosin premix and a preparation method thereof. Background Art

[0002] Tilmicosin is a macrolide antibiotic widely used in livestock such as poultry and pigs, mainly for the prevention and treatment of respiratory and other infectious diseases caused by mycoplasma, Gram-negative and Gram-positive bacteria. Tilmicosin inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit of bacteria, thereby interfering with its growth and reproduction, achieving an antibacterial effect. The drug has a strong inhibitory effect on mycoplasma and common respiratory pathogens, especially Gram-negative bacteria and some Gram-positive bacteria. In clinical applications, Tilmicosin is mainly used for the treatment of respiratory diseases in livestock such as poultry, pigs, and cattle, and can effectively prevent and treat mycoplasma pneumonia, respiratory tract infections, sepsis and other diseases caused by mycoplasma, Pasteurella, etc. At present, the traditional administration form of Tilmicosin is injection or oral solution. However, these preparations have certain limitations in practical applications, such as inconvenient administration and difficult dosage control. In contrast, premixes, as a new form of drug administration, can be mixed with animal feed or drinking water, making it easier to administer drugs over a long period of time and preventive treatment. They can also evenly disperse the drug to ensure that the animal obtains a stable drug concentration. Tilmicosin premixes can not only reduce adverse drug reactions, but also accurately control the amount and time of drug administration to optimize the therapeutic effect.

[0003] At present, the preparation methods of tilmicosin premixes mainly include mechanical mixing, solution method and blending method. These methods prepare powdered premixes by mixing tilmicosin with carrier materials (such as corn flour, wheat bran, phytate, etc.). However, in the preparation process, the uniform dispersibility, solubility and stability of the drug are key issues. If tilmicosin is unevenly dispersed in the premix or chemically reacts with the carrier, it will directly affect the bioavailability and therapeutic effect of the drug. Therefore, based on the above problems, it is extremely necessary to develop a tilmicosin premix with high utilization rate and high stability. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide a tilmicosin premix and a preparation method thereof.

[0005] The technical effect described in the present invention is achieved through the following technical scheme: a tilmicosin premix, which comprises the following raw materials: tilmicosin, polylactic acid-glycolic acid copolymer, lecithin, modified hydroxypropyl methylcellulose, grafted polyvinyl pyrrolidone, polyethylene glycol and antioxidant.

[0006] Preferably, the antioxidant is any one of vitamin E, butylated hydroxyanisole, ascorbic acid, citric acid and tea polyphenols.

[0007] Preferably, the specific preparation steps of the modified hydroxypropyl methylcellulose are as follows: A1: Add hydroxypropyl methylcellulose to anhydrous ethanol, heat to 60-70°C, stir and dissolve evenly to obtain a hydroxypropyl methylcellulose solution; slowly add 0.5M NaOH solution to the hydroxypropyl methylcellulose solution, stir and mix evenly to obtain a mixed solution; A2: dodecyl chloride is slowly added to the mixed solution prepared in step A1, heated in a water bath to 60-80°C, the pH is controlled to 9-10, stirred for reaction for 4-6 hours, and then the pH is neutralized to neutral with 0.1M HCl solution, and evaporated under reduced pressure at 50-60°C and 50-100 mbar for 2-4 hours, and the evaporated product is added to a 50wt% ethanol solution, stirred and allowed to stand for 2 hours, filtered, washed with cold water at 4°C, and vacuum dried at 50°C to constant weight to obtain modified hydroxypropyl methylcellulose; Preferably, in step A1, the ratio of the amount of hydroxypropyl methylcellulose, anhydrous ethanol and NaOH solution is 2g:40-50mL:0.5-1mL; Preferably, in step A2, the mass ratio of the dodecyl chloride to the hydroxypropyl methylcellulose is 2g:1-1.4g.

[0008] Preferably, the specific preparation steps of the grafted polyvinyl pyrrolidone are as follows: B1: dissolving vinyl pyrrolidone in 75wt% ethanol solution, stirring and dissolving uniformly to obtain vinyl pyrrolidone solution; adding acrylic acid and ammonium persulfate to the vinyl pyrrolidone solution, heating the temperature to 50-70°C, stirring and reacting for 3-5h, adding an equal volume of 0.1M ammonia water to terminate the reaction, washing with deionized water, and vacuum drying at 50°C to constant weight to obtain grafted polyvinyl pyrrolidone; Preferably, in step B1, the ratio of the amount of vinyl pyrrolidone to the ethanol solution is 1 g:20 mL; the volume ratio of the vinyl pyrrolidone, acrylic acid and ammonium persulfate is 1:0.3-0.5:0.01-0.02.

[0009] Preferably, another aspect of the present invention is to provide a method for preparing a tilmicosin premix, wherein the specific preparation steps are as follows: S1: adding tilmicosin to a 75wt% ethanol solution, stirring and dissolving uniformly to obtain a tilmicosin solution; adding lecithin to the 75wt% ethanol solution, heating to 50°C in a water bath, stirring and dissolving uniformly to obtain a lecithin solution; adding grafted polyvinyl pyrrolidone to the 75wt% ethanol solution, stirring and dissolving uniformly to obtain a grafted polyvinyl pyrrolidone solution; S2: uniformly mixing the tilmicosin solution, lecithin solution and grafted polyvinyl pyrrolidone solution prepared in step S1, stirring until a uniform transparent solution is formed to obtain a mixed solution; evaporating the mixed solution under reduced pressure at 40-50° C. and 100-150 mbar for 3-5 hours, vacuum drying at 40° C. for 12 hours, and screening through a 100-mesh sieve to obtain a solid dispersion; S3: adding polylactic acid-glycolic acid copolymer to dichloromethane, stirring and dissolving uniformly to obtain a polylactic acid-glycolic acid copolymer solution; adding the solid dispersion prepared in step S2 to the polylactic acid-glycolic acid copolymer solution, stirring and mixing uniformly to obtain an oil phase; adding 10% by volume of deionized water containing 1% Tween-80 to the oil phase, stirring at a speed of 10000 rpm for 5 minutes to obtain a primary emulsion; S4: slowly pouring the primary emulsion prepared in step S3 into a 1 wt % polyvinyl alcohol aqueous solution, stirring at 10000 rpm for 10 min, stirring at room temperature for 2-4 h, then centrifuging at 8000 rpm for 10-15 min, repeatedly washing with deionized water for 3 times, and freeze-drying at -40°C for 24 h to obtain nanospheres; S5: adding modified hydroxypropyl methylcellulose to a 75wt% ethanol solution, stirring and dissolving uniformly to obtain a modified hydroxypropyl methylcellulose solution; adding polyethylene glycol to the modified hydroxypropyl methylcellulose solution, stirring and mixing uniformly, then adding the nano-microspheres prepared in step S4, stirring at 200-500rpm for 30-60min, and then spray drying, with an air inlet temperature of 80°C, an air outlet temperature of 50°C, and a pressure of 0.2MPa, to obtain dry particles; S6: mixing and stirring the dry particles prepared in step S5 and the antioxidant at a speed of 300 to 500 rpm for 30 to 60 minutes to obtain a tilmicosin premix; Preferably, in step S1, the ratio of the amount of tilmicosin to the ethanol solution is 0.5-1 g:100 mL; the ratio of the amount of lecithin to the ethanol solution is 10 mg:1-2 mL; the ratio of the amount of grafted polyvinyl pyrrolidone to the ethanol solution is 1 g:20-30 mL; Preferably, in step S2, the mass ratio of tilmicosin, lecithin and grafted polyvinyl pyrrolidone is 1:0.3-0.4:0.15-0.25; Preferably, in step S3, the ratio of the amount of the polylactic acid-glycolic acid copolymer to dichloromethane is 2-5 g:100 mL; the mass ratio of the solid dispersion to the polylactic acid-glycolic acid copolymer is 1:1.2-1.6; Preferably, in step S4, the volume of the polyvinyl alcohol solution is 1.5 to 2 times the volume of the primary emulsion; Preferably, in step S5, the polyethylene glycol is PEG-4000; the ratio of the amount of the modified hydroxypropyl methylcellulose to the ethanol solution is 0.5-1 g:100 mL; the mass ratio of the polyethylene glycol, the modified hydroxypropyl methylcellulose and the nanospheres is 0.1-0.2:0.2:1; Preferably, in step S6, the amount of the antioxidant added is 0.1-0.3% of the mass of the dry particles.

[0010] The beneficial effects of the present invention are as follows: The present invention firstly adopts lecithin and polyvinyl pyrrolidone (PVP) grafted with hydrophilic side chains to form a solid dispersion, and performs solubilization and stabilization treatment on tilmicosin. Grafted polyvinyl pyrrolidone significantly improves its water solubility and interaction ability with water molecules by introducing hydrophilic side chains, and enhances the solubilization effect on tilmicosin, which effectively prevents the drug from crystallizing and precipitating in water, maintains its amorphous state, and improves solubility and bioavailability. As an amphiphilic molecule, lecithin has a bilayer structure that can wrap tilmicosin to form liposomes, protect the drug from erosion by external environments such as gastric acid, the hydrophobic tail of lecithin interacts with the hydrophobic group of tilmicosin, and the hydrophilic head combines with the hydrophilic side chain of the grafted polyvinyl pyrrolidone. This synergistic effect enables tilmicosin to be more stably encapsulated and solubilized in the solid dispersion. The hydrophilic side chain of the grafted polyvinyl pyrrolidone not only promotes the dispersion of the lecithin-encapsulated tilmicosin in the aqueous phase, but also prevents the aggregation and precipitation of the drug, further improving the solubility and absorbability. The present invention adopts a double emulsion solvent volatilization method to prepare polylactic acid-glycolic acid copolymer (PLGA) nano-microspheres, and uniformly encapsulates the above-mentioned lecithin-grafted polyvinyl pyrrolidone solid dispersion therein, thereby ensuring the sustained release effect of the drug. Polylactic acid-glycolic acid copolymer has good biocompatibility and controllable degradation characteristics, and can gradually degrade and release drugs in the body to prolong its action time. The embedding of polylactic acid-glycolic acid copolymer microspheres not only protects tilmicosin from degradation by enzymes and acidic environments in the body, but also reduces the peak fluctuation of blood drug concentration by controlling the release rate, thereby reducing the risk of toxic and side effects.

[0011] In addition, the present invention combines dodecyl-modified hydroxypropyl methylcellulose (HPMC) with polyethylene glycol, and further enhances the solubility and sustained-release characteristics of the drug by spray drying. After dodecyl modification, hydroxypropyl methylcellulose introduces a hydrophobic alkyl chain, and forms a hydrophilic-hydrophobic balanced structure with the original hydrophilic group, so that it can form a stable polymer network with polyethylene glycol. The hydrophilic part of polyethylene glycol enhances the water solubility of the system, while the hydrophobic part of hydroxypropyl methylcellulose interacts with the hydrophobic region of the polylactic acid-glycolic acid copolymer microspheres to delay the release of the drug. Effectively synergistically improve solubility and bioavailability, and also effectively regulate the release rate, so that the drug can maintain an effective concentration for a longer time in vivo.

[0012] In summary, the present invention realizes a biphasic release mechanism by combining grafted polyvinyl pyrrolidone with polylactic acid-glycolic acid copolymer microspheres: in the early stage, the high hydrophilicity of grafted polyvinyl pyrrolidone enables the drug to dissolve and release quickly to reach an effective therapeutic concentration; in the later stage, the sustained release effect of polylactic acid-glycolic acid copolymer continuously releases the drug and prolongs the drug effect time. This biphasic release mode makes full use of the characteristics of the two materials, greatly reduces the frequency of administration, and ensures the rapid onset and lasting efficacy of the drug. In addition, the present invention effectively preserves the activity of the raw materials by preparing the tilmicosin premix into granules, and is easy to use and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 It is a graph showing the results of accelerated stability tests of the Tilmicosin premixes prepared in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3; Figure 2 It is a pharmacokinetic test result diagram of the Tilmicosin premix prepared in Example 2 of the present invention and Comparative Examples 1 to 3. DETAILED DESCRIPTION

[0015] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are purchased through conventional commercial channels.

[0016] Example 1: A tilmicosin premix, comprising the following raw materials: tilmicosin, polylactic acid-glycolic acid copolymer, lecithin, modified hydroxypropyl methylcellulose, grafted polyvinyl pyrrolidone, polyethylene glycol and an antioxidant.

[0017] The specific preparation steps of modified hydroxypropyl methylcellulose are as follows: A1: Add 10 g of hydroxypropyl methylcellulose to 200 mL of anhydrous ethanol, heat to 60°C, and stir to dissolve evenly to obtain a hydroxypropyl methylcellulose solution; slowly add 5 mL of 0.5 M NaOH solution to 200 mL of the hydroxypropyl methylcellulose solution, stir to mix evenly, and obtain a mixed solution; A2: 20 g of dodecyl chloride was slowly added to the mixed solution prepared in step A1, the temperature was heated to 60°C in a water bath, the pH was controlled to 9, and the reaction was stirred for 6 hours. Then, the pH was neutralized to neutral with 0.1 M HCl solution, and the mixture was evaporated under reduced pressure at 50°C and 100 mbar for 4 hours. The evaporated product was added to 400 mL of 50 wt% ethanol solution, stirred and allowed to stand for 2 hours, filtered, washed with cold water at 4°C, and vacuum dried at 50°C to constant weight to obtain modified hydroxypropyl methylcellulose; The specific preparation steps of grafted polyvinyl pyrrolidone are as follows: B1: Dissolve 10g of vinyl pyrrolidone in 200mL of 75wt% ethanol solution, stir and dissolve evenly to obtain a vinyl pyrrolidone solution; add 3g of acrylic acid and 0.1g of ammonium persulfate to 200mL of vinyl pyrrolidone solution, heat to 50°C, stir and react for 5h, then add 200mL of 0.1M ammonia water to terminate the reaction, wash with deionized water, and vacuum dry at 50°C to constant weight to obtain grafted polyvinyl pyrrolidone; The specific preparation steps of Tilmicosin premix are as follows: S1: Add 10 g of tilmicosin to 1000 mL of 75 wt% ethanol solution, stir and dissolve evenly to obtain a tilmicosin solution; add 3 g of lecithin to 300 mL of 75 wt% ethanol solution, heat to 50° C. in a water bath, stir and dissolve evenly to obtain a lecithin solution; add 1.5 g of grafted polyvinyl pyrrolidone to 30 mL of 75 wt% ethanol solution, stir and dissolve evenly to obtain a grafted polyvinyl pyrrolidone solution; S2: 1000 mL of the tilmicosin solution, 300 mL of the lecithin solution and 30 mL of the grafted polyvinyl pyrrolidone solution prepared in step S1 are mixed uniformly, and stirred until a uniform transparent solution is formed to obtain a mixed solution; the mixed solution is evaporated under reduced pressure at 40° C. and 150 mbar for 5 h, and then vacuum dried at 40° C. for 12 h, and sieved through a 100-mesh sieve to obtain a solid dispersion; S3: adding 20 g of polylactic acid-co-glycolic acid to 400 mL of dichloromethane, stirring and dissolving uniformly to obtain a polylactic acid-co-glycolic acid solution; adding 10 g of the solid dispersion prepared in step S2 to 240 mL of the polylactic acid-co-glycolic acid solution, stirring and mixing uniformly to obtain an oil phase; adding 24 mL of deionized water containing 1% Tween-80 to the oil phase, stirring at 10,000 rpm for 5 min to obtain a primary emulsion; S4: slowly pouring 240 mL of the primary emulsion prepared in step S3 into 360 mL of 1 wt% polyvinyl alcohol aqueous solution, stirring at 10000 rpm for 10 min, stirring at room temperature for 2 h, and then centrifuging at 8000 rpm for 10 min, repeatedly washing with deionized water for 3 times, and freeze-drying at -40°C for 24 h to obtain nanospheres; S5: adding 5 g of modified hydroxypropyl methylcellulose to 1000 mL of 75 wt% ethanol solution, stirring and dissolving uniformly to obtain a modified hydroxypropyl methylcellulose solution; adding 1 g of polyethylene glycol to 400 mL of the modified hydroxypropyl methylcellulose solution, stirring and mixing uniformly, then adding 10 g of the nano-microspheres prepared in step S4, stirring at 200 rpm for 60 min, and then spray drying, with an air inlet temperature of 80° C., an air outlet temperature of 50° C., and a pressure of 0.2 MPa, to obtain dry particles; S6: 10 g of the dry particles prepared in step S5 and 0.01 g of vitamin E were mixed and stirred at 300 rpm for 60 min to obtain a tilmicosin premix.

[0018] Example 2: A tilmicosin premix, comprising the following raw materials: tilmicosin, polylactic acid-glycolic acid copolymer, lecithin, modified hydroxypropyl methylcellulose, grafted polyvinyl pyrrolidone, polyethylene glycol and an antioxidant.

[0019] The specific preparation steps of modified hydroxypropyl methylcellulose are as follows: A1: Add 12 g of hydroxypropyl methylcellulose to 250 mL of anhydrous ethanol, heat to 65°C, and stir to dissolve evenly to obtain a hydroxypropyl methylcellulose solution; slowly add 8 mL of 0.5 M NaOH solution to 250 mL of the hydroxypropyl methylcellulose solution, stir to mix evenly, and obtain a mixed solution; A2: 20 g of dodecyl chloride was slowly added to the mixed solution prepared in step A1, the temperature was heated to 70°C in a water bath, the pH was controlled to 9.5, and the reaction was stirred for 5 h. Then, the pH was neutralized to neutral with 0.1 M HCl solution, and the mixture was evaporated under reduced pressure at 55°C and 80 mbar for 3 h. The evaporated product was added to 500 mL of 50 wt% ethanol solution, stirred and allowed to stand for 2 h, filtered, washed with cold water at 4°C, and vacuum dried at 50°C to constant weight to obtain modified hydroxypropyl methylcellulose; The specific preparation steps of grafted polyvinyl pyrrolidone are as follows: B1: Dissolve 10g of vinyl pyrrolidone in 200mL of 75wt% ethanol solution, stir and dissolve evenly to obtain a vinyl pyrrolidone solution; add 4g of acrylic acid and 0.15g of ammonium persulfate to 200mL of vinyl pyrrolidone solution, heat to 60°C, stir and react for 4h, then add 200mL of 0.1M ammonia water to terminate the reaction, wash with deionized water, and vacuum dry at 50°C to constant weight to obtain grafted polyvinyl pyrrolidone; The specific preparation steps of Tilmicosin premix are as follows: S1: Add 10 g of tilmicosin to 1500 mL of 75 wt% ethanol solution, stir and dissolve evenly to obtain a tilmicosin solution; add 4 g of lecithin to 800 mL of 75 wt% ethanol solution, heat to 50° C. in a water bath, stir and dissolve evenly to obtain a lecithin solution; add 2.5 g of grafted polyvinyl pyrrolidone to 75 mL of 75 wt% ethanol solution, stir and dissolve evenly to obtain a grafted polyvinyl pyrrolidone solution; S2: 1500 mL of the tilmicosin solution, 800 mL of the lecithin solution and 75 mL of the grafted polyvinyl pyrrolidone solution prepared in step S1 are mixed uniformly, and stirred until a uniform transparent solution is formed to obtain a mixed solution; the mixed solution is evaporated under reduced pressure at 45° C. and 120 mbar for 4 h, and then vacuum dried at 40° C. for 12 h, and sieved through a 100-mesh sieve to obtain a solid dispersion; S3: adding 20 g of polylactic acid-co-glycolic acid to 1000 mL of dichloromethane, stirring and dissolving uniformly to obtain a polylactic acid-co-glycolic acid solution; adding 10 g of the solid dispersion prepared in step S2 to 800 mL of the polylactic acid-co-glycolic acid solution, stirring and mixing uniformly to obtain an oil phase; adding 80 mL of deionized water containing 1% Tween-80 to the oil phase, stirring at 10000 rpm for 5 min to obtain a primary emulsion; S4: 800 mL of the primary emulsion prepared in step S3 was slowly poured into 1600 mL of a 1 wt% polyvinyl alcohol aqueous solution, stirred at 10000 rpm for 10 min, stirred at room temperature for 3 h, then centrifuged at 8000 rpm for 12 min, washed with deionized water for 3 times, and freeze-dried at -40°C for 24 h to obtain nanospheres; S5: adding 10 g of modified hydroxypropyl methylcellulose to 1000 mL of 75 wt% ethanol solution, stirring and dissolving uniformly to obtain a modified hydroxypropyl methylcellulose solution; adding 2 g of polyethylene glycol to 200 mL of the modified hydroxypropyl methylcellulose solution, stirring and mixing uniformly, then adding 10 g of the nano-microspheres prepared in step S4, stirring at 300 rpm for 50 min, and then spray drying, with an air inlet temperature of 80° C., an air outlet temperature of 50° C., and a pressure of 0.2 MPa, to obtain dry particles; S6: 10 g of the dry particles prepared in step S5 and 0.02 g of tea polyphenols were mixed and stirred at 400 rpm for 40 min to obtain a tilmicosin premix.

[0020] Example 3: A tilmicosin premix, comprising the following raw materials: tilmicosin, polylactic acid-glycolic acid copolymer, lecithin, modified hydroxypropyl methylcellulose, grafted polyvinyl pyrrolidone, polyethylene glycol and an antioxidant.

[0021] The specific preparation steps of modified hydroxypropyl methylcellulose are as follows: A1: Add 14 g of hydroxypropyl methylcellulose to 350 mL of anhydrous ethanol, heat to 70°C, and stir to dissolve evenly to obtain a hydroxypropyl methylcellulose solution; slowly add 10 mL of 0.5 M NaOH solution to the hydroxypropyl methylcellulose solution, stir to mix evenly, and obtain a mixed solution; A2: 20 g of dodecyl chloride was slowly added to the mixed solution prepared in step A1, heated to 80°C in a water bath, the pH was controlled to 10, stirred and reacted for 4 hours, and then the pH was neutralized to neutral with 0.1 M HCl solution, and evaporated under reduced pressure at 60°C and 50 mbar for 2 hours. The evaporated product was added to 700 mL of 50 wt% ethanol solution, stirred and allowed to stand for 2 hours, filtered, washed with cold water at 4°C, and vacuum dried at 50°C to constant weight to obtain modified hydroxypropyl methylcellulose; The specific preparation steps of grafted polyvinyl pyrrolidone are as follows: B1: Dissolve 10g of vinyl pyrrolidone in 100mL of 75wt% ethanol solution, stir and dissolve evenly to obtain a vinyl pyrrolidone solution; add 5g of acrylic acid and 0.2g of ammonium persulfate to the vinyl pyrrolidone solution, heat to 70°C, stir and react for 3h, then add 100mL of 0.1M ammonia water to terminate the reaction, wash with deionized water, and vacuum dry at 50°C to constant weight to obtain grafted polyvinyl pyrrolidone; The specific preparation steps of Tilmicosin premix are as follows: S1: Add 10 g of tilmicosin to 2000 mL of 75 wt% ethanol solution, stir and dissolve evenly to obtain a tilmicosin solution; add 3.5 g of lecithin to 600 mL of 75 wt% ethanol solution, heat to 50° C. in a water bath, stir and dissolve evenly to obtain a lecithin solution; add 2 g of grafted polyvinyl pyrrolidone to 50 mL of 75 wt% ethanol solution, stir and dissolve evenly to obtain a grafted polyvinyl pyrrolidone solution; S2: 2000 mL of the tilmicosin solution, 600 mL of the lecithin solution and 50 mL of the grafted polyvinyl pyrrolidone solution prepared in step S1 are mixed uniformly, and stirred until a uniform transparent solution is formed to obtain a mixed solution; the mixed solution is evaporated under reduced pressure at 50° C. and 100 mbar for 3 h, and then vacuum dried at 40° C. for 12 h, and sieved through a 100-mesh sieve to obtain a solid dispersion; S3: adding 20 g of polylactic acid-co-glycolic acid to 800 mL of dichloromethane, stirring and dissolving uniformly to obtain a polylactic acid-co-glycolic acid solution; adding 10 g of the solid dispersion prepared in step S2 to 560 mL of the polylactic acid-co-glycolic acid solution, stirring and mixing uniformly to obtain an oil phase; adding 56 mL of deionized water containing 1% Tween-80 to the oil phase, stirring at 10,000 rpm for 5 min to obtain a primary emulsion; S4: 550 mL of the primary emulsion prepared in step S3 was slowly poured into 1000 mL of 1 wt% polyvinyl alcohol solution, stirred at 10000 rpm for 10 min, stirred at room temperature for 4 h, then centrifuged at 8000 rpm for 15 min, washed with deionized water for 3 times, and freeze-dried at -40°C for 24 h to obtain nanospheres; S5: adding 8 g of modified hydroxypropyl methylcellulose to 1000 mL of 75 wt% ethanol solution, stirring and dissolving uniformly to obtain a modified hydroxypropyl methylcellulose solution; adding 1.5 g of polyethylene glycol to 250 mL of the modified hydroxypropyl methylcellulose solution, stirring and mixing uniformly, then adding 10 g of the nanospheres prepared in step S4, stirring at 500 rpm for 30 min, and then spray drying, with an air inlet temperature of 80° C., an air outlet temperature of 50° C., and a pressure of 0.2 MPa, to obtain dry particles; S6: 10 g of the dry particles prepared in step S5 and 0.03 g of ascorbic acid were mixed and stirred at 500 rpm for 30 min to obtain a tilmicosin premix.

[0022] Comparative Example 1: The operation of Comparative Example 1 is basically the same as that of Example 2, except that in Comparative Example 1, grafted polyvinyl pyrrolidone is not used, and only the solid dispersion formed by lecithin is used.

[0023] Comparative Example 2: The operation of Comparative Example 2 is basically the same as that of Example 2, except that in Comparative Example 2, polylactic acid-glycolic acid copolymer is not used to construct nanospheres, and the solid dispersion and modified hydroxypropyl methylcellulose solution are directly mixed and spray-dried.

[0024] Comparative Example 3: The operation of Comparative Example 3 is substantially the same as that of Example 2, except that hydroxypropyl methylcellulose is used in Comparative Example 3 instead of modified hydroxypropyl methylcellulose.

[0025] Performance Test: Toxicological test: Take mice weighing about 20g, half of which are male and half are female, and divide them into 6 groups with 10 mice in each group. Then, the tilmicosin premix prepared in Example 2 of the present invention, the tilmicosin premix prepared in Comparative Examples 1 to 3, and the positive control group (tilmicosin) are dissolved in normal saline to prepare concentrations of 10 mg / mL, 50 mg / mL, and 100 mg / mL tilmicosin concentrations. 10 mL / kg is administered orally by gavage once a day for 14 days. The negative control group is administered with the same volume of normal saline. The weight changes and deaths are recorded. No deaths are recorded. The number of deaths recorded is the cumulative total number of deaths. The results are shown in Tables 1, 2, and 3 below.

[0026] Table 1.10mg / mL mouse toxicity test results

[0027] Table 2. 50mg / mL mouse toxicity test results

[0028] Table 3. 200 mg / mL mouse toxicity test results

[0029] It can be seen from the results of Table 1, Table 2 and Table 3 that the tilmicosin premix prepared by the present invention has excellent biosafety and has lower toxic side effects than conventional tilmicosin. Even at high concentrations, the weight of mice remains basically stable and the mortality rate is extremely low. It can be seen from the results of Comparative Example 1 and Example 2 that the lack of the solubilizing effect of grafted polyvinyl pyrrolidone leads to poor dispersibility of tilmicosin in water, increased crystallization tendency, and reduced bioavailability. The lack of the synergistic effect of grafted polyvinyl pyrrolidone and lecithin makes it impossible to more stably encapsulate tilmicosin in the solid dispersion. Lecithin cannot provide such stability alone, and the drug is more easily destroyed in the gastrointestinal environment, which may lead to increased toxicity. ; From the results of Comparative Example 2 and Example 2, it can be seen that due to the lack of the sustained-release effect of the polylactic acid-glycolic acid copolymer, tilmicosin is released at a faster rate, causing the blood drug concentration to reach a peak value quickly, resulting in a large fluctuation in the peak toxicity of the drug, which may cause higher toxic side effects, leading to a significant decrease in weight at a higher concentration and a higher mortality rate; From the results of Comparative Example 3 and Example 2, it can be seen that unmodified hydroxypropyl methylcellulose cannot be effectively combined with polyethylene glycol and polylactic acid-glycolic acid copolymer, resulting in an unstable drug release rate, too fast release of tilmicosin, and large fluctuations in blood drug concentration, resulting in a significant increase in the toxicity risk at high concentrations, resulting in a significant decrease in weight and a higher mortality rate.

[0030] Accelerated stability test: The Tilmicosin premix prepared in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 and the control group (Tilmicosin) were stored at 40 ± 2°C and a relative humidity of 75 ± 5% for 6 months. Samples were taken at 1, 2, 3 and 6 months, respectively, and the color and state changes of the samples were observed and recorded. The color and state changes are shown in Table 4 below, and the percentage change of the drug content is recorded as shown in Table 4 below. Figure 1 shown.

[0031] Table 4. Accelerated stability test results

[0032] From Table 4 and Figure 1As can be seen from the results, the tilmicosin premix prepared by the present invention has excellent stability under high temperature and high humidity, can maintain good physical and chemical stability, has extremely small color amplitude change and has excellent anti-degradation property; from the results of Comparative Example 1 and Example 2, it can be seen that the lack of grafted polyvinyl pyrrolidone affects the stability and solubilization effect, and the drug is easily affected by wet and hot conditions and crystallizes, resulting in darkening of color, decreased dispersibility and further agglomeration; from the results of Comparative Example 2 and Example 2, it can be seen that the lack of sustained release and protective effects of polylactic acid-glycolic acid copolymer makes the drug more easily exposed to a wet and hot environment, which may lead to its degradation and obvious agglomeration; from the results of Comparative Example 3 and Example 2, it can be seen that since modified hydroxypropyl methylcellulose is not used, the hygroscopicity of hydroxypropyl methylcellulose with strong hydrophilicity increases under high humidity conditions, which may lead to poor particle stability, darkening of color and decreased stability. At the same time, due to the lack of protection of the network system constructed with polyethylene glycol, the drug activity is significantly degraded.

[0033] Pharmacokinetic test: 20 adult male Wistar rats weighing 200 to 250 grams were selected and divided into 4 groups, 5 rats in each group, and the rats were orally administered with the tilmicosin premix prepared in Example 2 and Comparative Examples 1 to 3, with an oral dose of 200 mg / kg of tilmicosin premix. After drug administration, blood samples were collected at different times, and plasma was separated by centrifugation. The concentration of tilmicosin in plasma was determined by high performance liquid chromatography (HPLC), and the maximum blood concentration of tilmicosin (μg / mL) was measured. The collection time points were 0h, 0.25h, 0.5h, 1h, 1.5h, 2h, 3h, 4h, 6h, 8h, 12h, 24h, 36h and 48h. The results are as follows: Figure 2 shown.

[0034] Depend on Figure 2As can be seen from the results of the comparative example 1 and the example 2, the tilmicosin premix prepared by the present invention can be quickly and effectively released in the early stage, and can achieve long-term slow release in the later stage, and has good bioavailability; as can be seen from the results of the comparative example 1 and the example 2, since only lecithin is used to form the solid dispersion, the lack of grafted polyvinyl pyrrolidone cannot achieve a biphasic mechanism of rapid release and sustained release, the drug release is slow, the initial blood drug concentration is low, and there is a lack of sustained release support later, and the overall drug effect duration is short; as can be seen from the results of the comparative example 2 and the example 2, due to the lack of the slow release of the polylactic acid-co-glycolic acid copolymer microspheres, Due to the sustained-release protection, the dissolution and absorption rate of tilmicosin is accelerated, resulting in a rapid increase in the initial blood drug concentration, but the lack of subsequent sustained-release support causes the drug concentration to drop rapidly, which may lead to its inability to maintain a stable effective concentration in the body for a long time; from the results of Comparative Example 3 and Example 2, it can be seen that the hydroxypropyl methylcellulose has not been modified, the hydrophilic-hydrophobic balance is poor, and the stable network structure formed by polyethylene glycol and modified hydroxypropyl methylcellulose is lacking, resulting in poor synergy with the polylactic acid-glycolic acid copolymer microspheres, which may lead to its poor sustained-release effect and the release rate is not stable enough.

[0035] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A tilmicosin premix, characterized in that: The composition comprises the following raw materials: tilmicosin, polylactic acid-glycolic acid copolymer, lecithin, modified hydroxypropyl methylcellulose, grafted polyvinyl pyrrolidone, polyethylene glycol and antioxidant.

2. A tilmicosin premix according to claim 1, characterized in that: The antioxidant is any one of vitamin E, butylated hydroxyanisole, ascorbic acid, citric acid and tea polyphenols.

3. A tilmicosin premix according to claim 2, characterized in that: The specific preparation steps of the modified hydroxypropyl methylcellulose are as follows: A1: Add hydroxypropyl methylcellulose to anhydrous ethanol, heat to raise the temperature, stir and dissolve evenly to obtain a hydroxypropyl methylcellulose solution; slowly add the NaOH solution to the hydroxypropyl methylcellulose solution, stir and mix evenly to obtain a mixed solution; A2: dodecyl chloride is slowly added to the mixed solution prepared in step A1, the temperature is heated in a water bath, the pH is controlled, after stirring the reaction, the pH is neutralized to neutral with HCl solution, evaporated under reduced pressure, the evaporated product is added to an ethanol solution, stirred and allowed to stand, filtered, washed with cold water, and vacuum dried to constant weight to obtain modified hydroxypropyl methylcellulose.

4. A tilmicosin premix according to claim 3, characterized in that: In step A1, the ratio of the amount of hydroxypropyl methylcellulose, anhydrous ethanol and NaOH solution is 2g:40-50mL:0.5-1mL; in step A2, the mass ratio of dodecyl chloride and hydroxypropyl methylcellulose is 2g:1-1.4g.

5. A tilmicosin premix according to claim 4, characterized in that: The specific preparation steps of the grafted polyvinyl pyrrolidone are as follows: B1: dissolving vinyl pyrrolidone in an ethanol solution, stirring and dissolving uniformly to obtain a vinyl pyrrolidone solution; adding acrylic acid and ammonium persulfate to the vinyl pyrrolidone solution, heating to a high temperature, stirring and reacting, adding ammonia water to terminate the reaction, washing with deionized water, and vacuum drying to constant weight to obtain grafted polyvinyl pyrrolidone; In step B1, the volume ratio of the polyvinyl pyrrolidone, acrylic acid and ammonium persulfate is 1:0.3-0.5:0.01-0.

02.

6. A method for preparing the tilmicosin premix according to any one of claims 1 to 5, characterized in that: The specific preparation steps are as follows: S1: adding tilmicosin to the ethanol solution, stirring and dissolving uniformly to obtain a tilmicosin solution; adding lecithin to the ethanol solution, stirring and dissolving uniformly to obtain a lecithin solution; Adding grafted polyvinyl pyrrolidone into the ethanol solution, stirring and dissolving the mixture evenly, to obtain a grafted polyvinyl pyrrolidone solution; S2: uniformly mixing the tilmicosin solution, lecithin solution and grafted polyvinyl pyrrolidone solution prepared in step S1, and stirring until a uniform transparent solution is formed to obtain a mixed solution; evaporating the mixed solution under reduced pressure, vacuum drying, and screening through a 100-mesh sieve to obtain a solid dispersion; S3: adding polylactic acid-glycolic acid copolymer to dichloromethane, stirring and dissolving uniformly to obtain a polylactic acid-glycolic acid copolymer solution; adding the solid dispersion prepared in step S2 to the polylactic acid-glycolic acid copolymer solution, stirring and mixing uniformly to obtain an oil phase; adding deionized water containing 1% Tween-80 to the oil phase, stirring at a speed of 10000 rpm for 5 minutes to obtain a primary emulsion; S4: slowly pouring the primary emulsion prepared in step S3 into the polyvinyl alcohol aqueous solution, stirring at 10000 rpm for 10 min, stirring at room temperature, and then centrifuging, repeatedly washing with deionized water, and freeze-drying to obtain nanoparticles; S5: adding modified hydroxypropyl methylcellulose to the ethanol solution, stirring and dissolving uniformly to obtain a modified hydroxypropyl methylcellulose solution; adding polyethylene glycol to the modified hydroxypropyl methylcellulose solution, stirring and mixing uniformly, and then adding the nano-microspheres prepared in step S4, stirring, and then spray drying, with an air inlet temperature of 80° C., an air outlet temperature of 50° C., and a pressure of 0.2 MPa to obtain dry particles; S6: Mix and stir the dry particles prepared in step S5 and the antioxidant to obtain a tilmicosin premix.

7. A method for preparing the tilmicosin premix according to claim 6, characterized in that: In step S1, the ratio of the amount of tilmicosin to the ethanol solution is 0.5-1 g:100 mL; the ratio of the amount of lecithin to the ethanol solution is 10 mg:1-2 mL; and the ratio of the amount of grafted polyvinyl pyrrolidone to the ethanol solution is 1 g:20-30 mL.

8. A method for preparing the tilmicosin premix according to claim 7, characterized in that: In step S2, the mass ratio of the tilmicosin, lecithin and grafted polyvinyl pyrrolidone is 1:0.3-0.4:0.15-0.25; the mass ratio of the polylactic acid-glycolic acid copolymer and dichloromethane is 2-5 g:100 mL; the mass ratio of the solid dispersion and the polylactic acid-glycolic acid copolymer is 1:1.2-1.

6.

9. A method for preparing the tilmicosin premix according to claim 8, characterized in that: In step S4, the volume of the polyvinyl alcohol solution is 1.5 to 2 times the volume of the primary emulsion; in step S5, the polyethylene glycol is PEG-4000; the ratio of the amount of the modified hydroxypropyl methylcellulose to the ethanol solution is 0.5 to 1 g:100 mL; the mass ratio of the polyethylene glycol, modified hydroxypropyl methylcellulose and nano-microspheres is 0.1 to 0.2:0.2:1; in step S6, the amount of the antioxidant added is 0.1 to 0.3% of the mass of the dry particles.

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