| Tylosin is one of macrolide antibiotics especially for animals use. Because of its good antibacterial activities against a large number of Gram-positive bacteria, some Gram-negative bacteria and particularly against mycoplasma, tylosin has been widely used in livestock cultivation to keep out various kinds of infections and in particular, considered as the first choice for the treatment of mycoplasma disease of animals. It can be administered via i.v, i.m, oral and so forth. However, by virtue of its short elimination half-life, extensive tissue distribution, frequent drug administration and large dosage, the therapeutic effects of tylosin are not satisfactory. Therefore, there are of great concerns and significance to exploit new sustained-release and targeting formulations of tylosin for clinical applications. As a drug delivery, liposome has many excellent characteristics, e.g. sustained-release, targeting, no toxicity, no immunogenicity, biodegradability and so on. Our objective is to prepare phosphate tylosin liposome, looking forword to ameliorating the pharmacokinetics properties of drug, prolonging the duration time, reducing drug given bits and increasing bioavailability. The research provides theoretical basis for further enhancement of the preparation technolgy and clinic therapeutic efficacy.In this research, an RP-HPLC method of higher specialty for the content determination of phosphate tylosin liposome was first chosen and established. The analysis was performed on a Kromasil C18(4.6mmx 150mm,5μm) analytical column. The mobile phase was composed of a mixture of 0.05mol/LNaClO4 (pH value was adjusted to 3.0±0.1 by 1mol/L HCl) and acetonitrile (65:35, v/v) at a flow rate of 1.0mL/min. Phosphate tylosin was detected at 290nm and at a 30℃column temperature. The results indicated that the excipients and solvent in the liposome could be well separated from the drug under such a designated chromatogram condition and did not interfere with the assay. A good linear relationship was found between peak area(A) of componet A and the concentration(C) in the range of 5.0-320.0μg/mL. Linear regression equation was A=9890.364C+2431.270 (r=0.9999, n=7), the average recoveries were between 99.86% and 100.06%(n=5). RSD values of intra-day and inter-day were less than 2%(n=5). It had showed that this method had advantages of high sensitivity and good reproducibility. It could shut out the effect of excipients such as phospholipid and detect the content of drug quickly and accurately. At the same time, the refrigerated high-speed centrifugation which was used to separate free drugs from phosphate tylosin liposome for the entrapment efficiency determination was investigated. Under such a centrifugation condition as 12000r/min for 45min at 4℃C, the two parts were separated completely, the recoveries of free drug ranged from 99.40% to 101.40% with RSD of less than 2%(n=3).Moreover,the oil-water partition coefficients(lgP)of phosphate tylosin in phosphate buffered saline with different pH value were also detected preliminarily. The fact that lgP increased(-1.3-1.3)along with the hoist of pH in the range of 3-9 indicated that the state of phosphate tylosin exsisting in solution had much relativity to the pH, which giving a way for designing and optimizing the prescription and preparation process of liposome.Soybean phosphatidylcholine(SPC) and cholesterol (CH) were taken as the carrier materials in this project. The feasibilities to prepare phosphate tylosin liposome using different methods, such as thin film dispersion method, ether injection, reverse-phase evaporation method, pH gradient method and ammonium sulfate gradient method, were investigated. Entrapment efficiency was considered as the main parameter to screen the most suitable method (ammonium sulfate gradient method) which had the highest entrapment efficiency for the further studies. Single-factor test was adopted to investigate the major influencing factors on entrapment efficiency in ammonium sulfate gradient method. Meanwhile, orthogonal design duplication test was used to optimize the preparation process. As a result, the best prescription and preparation were:SPC-CH mass ratio was 4:1, drug-SPC ratio was 1:10, concentration of ammonium sulfate was 300mmol/L, incubation temperature was 50℃, incubation time was 20min, dialysis time was 6h and pH was adjusted to 7.0.Then 58.15% and 4.32% of mean entrapment efficiency and drug loading were received in the end.The quality of phosphate tylosin liposome prepared by the best prescription and preparation was overall evaluated by means of investigating such pharmaceutics properties as the shape, particle size, distribution, pH value, in vitro drug release, stability, hemolysis and so on. It had shown that the prepared liposome was milky white suspension and was spherical or ellipsoidal in shape; the mean particle size was 6.526μm and more than 90.3% of the amount were in the range of 1-12μm; the distribution was uniformity and the decentralization was fine; the character was relatively stable storaged in a cryogenic condition; there were preferable sustained-release effect in vitro drug release and no cell hemolysis. All the received results were well in corresponding with the quality requirements to liposome formulations.In order to solve the stability of liposome, phosphate tylosin liposome freeze-drying product was manufactured. In this study, freeze-drying technique was selected and freeze-drying prescription was deeply discussed. By comparison, the kind of best freeze-drying protective agent and dosage were fixed. Meanwhile, the characteristics of freeze-drying formulation were further evaluated. At last, sucrose and manito were considered as the best protective agents with the best mass ratio of sucrose-SPC-mannito as 0.5:1:2. Freeze-drying products had level, full and compact surface, no shrinkage, favorable reconstitution, little aggregation and few change in entrapment efficiency, as a result of improving the stability of liposome suspension to large extent during the storge period.Phosphate tylosin liposome was determined and compared its MIC and MBC against staphylococcus aureus(G+) and E.coli(G-)using microscale dilution method with free phosphate tylosin solution in order to study its antibacterial effect in vitro.The MIC of liposomal phosphate tylosin against the bacterias mentioned above were 1μg/mL and 4μg/mL, just as 1/4-fold and 1/2-fold as that of free drug respectively.The MBC were 4μg/mL and 8μg/mL,which were both as 1/2-fold as that of free drug. The results indicated that phosphate tylosin liposome could exhibit significant antibacterial activities in vitro compaing to the free drug. A reliable HPLC analysis method (internal standard method) was developed for the determination of drug concentration in biolgical simples. For the pharmacokinetics investigation, phosphate tylosin solution group and liposome group were set respectively and injected into rabbits via auricular vein at a single dosage of 5mg/kg.b.w., then the drug concentration in plasma at different time were detected. The results suggested that pharmacokinetics properties of phosphate tylosin solution and liposome in vivo were both fit for two compartum model with weghit coefficient as 1,C2. The pharmacokinetics equations were C(t)=67.329e-7.806t+22.649e-0.290t and C(t)=67.291e-10.082t+21.032e-0.178t, t1/2a were (0.097±0.035)h and (0.071±0.013)h, t1/2βwere (2.410±0.246)h and (3.909±0.275)h, AUC(0-48) were (93.504±9.173)(mg-h)/L and(134.048±9.034)(mg-h)/L, CLs were (0.051±0.009)L/h/kg and (0.036±0.002) L/h/kg, Vc were (0.057±0.009)L/kg and (0.057±0.007)L/kg, MRT(0-48) were (4.586±0.229)h and (11.436±0.479)h, apart. It could be clearly seen that t1/2βof the latter prolonged (P<0.05), AUC and MRT both increased remarkablely(P<0.01), Vc and CLs decreased. All indicated that phosphate tylosin made into liposome could prolong the drution time, achieving excellent sustained-release effect, thus to raise AUC and inhance efficacy, Meanwhile, for the tissue distribution study, mouse were injected via tail vein at a single dosage of 10mg/kg.b.w. and the drug concentration in tissues and plasma at different time were also required. It was found that significant changes had occurred in tissues after given phosphate tylosin liposome. Compared to solution, drug level improved in most tissues, especially in lung, and could remain for a long time, indicating that this liposomal preparation had some a lung targeting and might increase the therapeutic index of lung diseases.The whole study had showed that liposome carrying phosphate tylosin was feasible. The preparation process was simple and of good reproducibility. The resulting liposome preparations were live up to the requirements and had preferable sustained-release and lung targeting, being in favor of prolonging the acting time, improving the drug level of targeting sites, reducing the drug given times and dosage. Considered as a novel, ideal delayed-release and targeting formulation, it had better prospects. All these results were just what this project initially and finally looked forward to. |
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