达托霉素和克拉霉素共载药脂质体的研究
发布时间：2019-05-24 01:48
【摘要】：耐甲氧西林金黄色葡萄球菌(MRSA)引起的感染严重影响了公众的健康,甚至是已用某些抗生素治疗过MRSA引起的感染还会导致高的死亡率,随着病原体耐药防控难度的持续增大,除了不断研发新型抗生素之外,对已有抗生素进行新制剂创制与联合给药具有同等的重要性。本研究旨在评估达托霉素和克拉霉素联用对耐甲氧西林金黄色葡萄球菌(MRSA)感染的协同作用效果。 本文采用聚乙二醇修饰的脂质体作为载体,考察并系统评价经优化制备的达托霉素、克拉霉素共载药脂质体(PL[CD])抗耐甲氧西林金葡菌(MRSA252)全身感染。通过优化制备得到的达托霉素、克拉霉素共载药脂质体(PL[CD])平均粒径(98.2±2.21)nm,多分散系数为0.251,包封率分别为(92.94±1.21,94.71±1.37)%,载药量为(8.45±0.11,0.29±0.043)%,粒径大小均一,并具有良好的稳定性。在临床剂量下,体内外评价结果显示,达托霉素和克拉霉素共载药脂质体(PL[CD])与达托霉素脂质体及克拉霉素脂质体制剂相比,对MRSA252有更持久的抑制效果,并能显著提高耐甲氧西林金葡菌全身感染模型小鼠的存活率上述研究结果表明,达托霉素和克拉霉素共载药脂质体在体外能够显著增强对抗MRSA的活性,在体内能显著减少MRSA的定植量并能增加宿主的生存率。因此,可为今后临床应用提供相关的依据。 本课题的主要研究内容如下： (1)达托霉素、克拉霉素体外协同优选方案 通过棋盘法对达托霉素、克拉霉素在体外进行联合,通过计算,联合抑菌指数(FIC)为0.5,可初步判定达托霉素与克拉霉素体外联用有协同作用。 (2)单药脂质体的制备及体外协同方案优化 克拉霉素高效液相色谱法在45℃条件下,使用C18色谱分离柱,流速为0.7mL/min,流动相是含有0.1%三氟乙酸的乙腈与3.5mM的HC1调pH至3.5±0.2的0.033nol/L KH2PO4(40/60,v/v),检测波长为205nm；达托霉素的高效液相色谱法在35℃条件下,流速为1mL/min,流动相是乙腈与0.5%的NH4H2PO4(36/64,v/v),检测波长为221nrn。 通过考察制备达托霉素长循环脂质体的几个影响因素：氢化卵磷脂(HSPC)与胆固醇的摩尔比、脂药质量比,最终得出制备达托霉素长循环脂质体的最优处方,就能得到稳定的脂质体剂型。在4℃的冰箱中保存2个月后几乎无泄漏、无沉淀现象发生。成功制备的克拉霉素、达托霉素单药脂质体联合抑菌指数主要采用棋盘法来测定,得到PL[C]与PL[D]最优化的比率为32：1,此比值用于后续制备共载药脂质体。 (3)共载药脂质体的制备与评价 通过优化制备得到的达托霉素、克拉霉素共载药脂质体平均粒径(98.2+2.21)nm,多分散系数为0.251,包封率分别为(92.94±1.21,94.71±1.37)%,载药量为(8.45±0.11,0.29±0.043)%,粒径大小均一,并具有良好的稳定性。 体外抗耐甲氧西林金葡菌实验主要考察达托霉素三个制剂：克拉霉素脂质体(PL[C])、达托霉素脂质体(PL[D])、达托霉素、克拉霉素共载药脂质体(PL[CD])对MRSA252生长的影响,结果表明,PL[CD]对MRSA252的抑制效果更持久。生物膜的定性和定量结果的分析表明,PL(CD)比PL[C], PL[D]抑制MRSA的效果更强。 通过比较PL[C]、PL[D]、PL[CD]对MRSA252致全身感染KM小鼠的治疗效果,结果显示,临床剂量下,达托霉素长循环脂质体能使感染KM小鼠的存活率在观察期(7天)提高到90%。安全性评价结果表明共载药脂质体未对KM小鼠没有明显毒性。
[Abstract]:The infection caused by methicillin-resistant Staphylococcus aureus (MRSA) seriously affects the health of the public, and even the infection that has been caused by the treatment of MRSA with certain antibiotics can also lead to high mortality, and as the prevention and control difficulty of the pathogen increases continuously, In addition to the constant development of new types of antibiotics, the new formulation of existing antibiotics has the same importance as the combination. The purpose of this study was to assess the synergistic effect of daptomycin and clarithromycin on the infection of methicillin-resistant Staphylococcus aureus (MRSA). In this paper, polyethylene glycol modified liposomes were used as carrier to study and evaluate the systemic sense of daptomycin and clarithromycin co-carrier liposome (PL[CD]) against methicillin-resistant Staphylococcus aureus (MRSA252). The average particle size (98.2-2.21) nm, the multi-dispersion coefficient of 0.251 and the encapsulation rate of the daptomycin and the clarithromycin co-carrier liposome (PL[CD]) were (92.94-1.21, 94.71-1.37)%, (8.45-0.11, 0.29-0.043)% and the particle-size-size of the liposome (PL[CD]). I. It has good stability. Sex. In the clinical dose, the in-vivo evaluation results show that the daptomycin and clarithromycin co-carrier liposomes (PL[CD]) have a more durable inhibitory effect on the MRSA252 compared to the daptomycin liposome and the clarithromycin liposome formulation. The results of the study show that the co-carrier of daptomycin and clarithromycin can significantly enhance the activity against MRSA in vitro, and can significantly reduce the colonization of MRSA in vivo and increase the survival of the host. Rate. Therefore, it can be relevant to the clinical application in the future According to the main research of the subject The volume is as follows: (1) in vitro of daptomycin and clarithromycin in a synergistic prefer scheme, daptomycin and clarithromycin are combined in vitro by a checkerboard method, 0.5. It can be used to determine the in vitro of daptomycin and clarithromycin in vitro. co-acting. (2) The preparation of a single-agent liposome The preparation and in vitro synergistic scheme optimizes the clarithromycin high performance liquid chromatography to use a C18 chromatographic separation column under the condition of 45 DEG C, the flow rate is 0.7 mL/ min, the mobile phase is acetonitrile with 0.1% trifluoroacetic acid and 3.5 mM HC1 to adjust the pH to 0.033 nol/ L KH2PO4 of 3.5 to 0.2 (40/60, v/ v) with a detection wavelength of 205 nm; a high performance liquid chromatography of daptomycin at a flow rate of 1 mL/ min at 35 C and a mobile phase of acetonitrile and 0.5% NH4H2PO4 (36/64, v/ v), The detection wavelength was 221 nrn. Several factors influencing the preparation of daptomycin long-cycle liposomes were investigated: the molar ratio of hydrogenated lecithin (HSPC) to cholesterol, the mass ratio of lipid, and finally the optimal formulation of the preparation of daptomycin long-cycle liposomes was obtained. and a stable liposome dosage form can be obtained, and after two months are stored in a refrigerator at 4 DEG C, The combined antibacterial index of clarithromycin and daptomycin as a single agent was determined by a checkerboard method, and the ratio of PL[C] to PL[D] was 32:1. The value is used for subsequent preparation of the co-carrier liposome. (3) The preparation and evaluation of the co-carrier liposomes were optimized to prepare the daptomycin, the clarithromycin co-carrier liposome average particle size (98.2 + 2.21) nm, the polydispersity index of 0.251, the encapsulation rate (92.94% 1.21, 94.71% 1.37)%, and the drug loading (8.45%). 0.11, 0.29 (0.043)% The anti-methicillin-resistant Staphylococcus aureus in vitro mainly studied the three preparations of daptomycin: clarithromycin liposome (PL[C]), daptomycin liposome (PL[D]), daptomycin, and clarithromycin co-carrier liposome (PL[CD]). ) Effect on the growth of MRSA252, the results show that PL[ CD] The inhibition of MRSA252 is more durable. The analysis of the qualitative and quantitative results of the biofilm shows that the PL (CD) ratio PL[ [C], PL[D] inhibited the efficacy of MRSA. By comparing PL[C], PL[D], and PL[CD] to the therapeutic effect of MRSA252 on the whole body infected KM mice, the results showed that the long circulating liposomes of daptomycin can make the infection K The survival of M mice increased to 90% in the observation period (7 days). The safety evaluation junction
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
本文编号：2484440
[Abstract]:The infection caused by methicillin-resistant Staphylococcus aureus (MRSA) seriously affects the health of the public, and even the infection that has been caused by the treatment of MRSA with certain antibiotics can also lead to high mortality, and as the prevention and control difficulty of the pathogen increases continuously, In addition to the constant development of new types of antibiotics, the new formulation of existing antibiotics has the same importance as the combination. The purpose of this study was to assess the synergistic effect of daptomycin and clarithromycin on the infection of methicillin-resistant Staphylococcus aureus (MRSA). In this paper, polyethylene glycol modified liposomes were used as carrier to study and evaluate the systemic sense of daptomycin and clarithromycin co-carrier liposome (PL[CD]) against methicillin-resistant Staphylococcus aureus (MRSA252). The average particle size (98.2-2.21) nm, the multi-dispersion coefficient of 0.251 and the encapsulation rate of the daptomycin and the clarithromycin co-carrier liposome (PL[CD]) were (92.94-1.21, 94.71-1.37)%, (8.45-0.11, 0.29-0.043)% and the particle-size-size of the liposome (PL[CD]). I. It has good stability. Sex. In the clinical dose, the in-vivo evaluation results show that the daptomycin and clarithromycin co-carrier liposomes (PL[CD]) have a more durable inhibitory effect on the MRSA252 compared to the daptomycin liposome and the clarithromycin liposome formulation. The results of the study show that the co-carrier of daptomycin and clarithromycin can significantly enhance the activity against MRSA in vitro, and can significantly reduce the colonization of MRSA in vivo and increase the survival of the host. Rate. Therefore, it can be relevant to the clinical application in the future According to the main research of the subject The volume is as follows: (1) in vitro of daptomycin and clarithromycin in a synergistic prefer scheme, daptomycin and clarithromycin are combined in vitro by a checkerboard method, 0.5. It can be used to determine the in vitro of daptomycin and clarithromycin in vitro. co-acting. (2) The preparation of a single-agent liposome The preparation and in vitro synergistic scheme optimizes the clarithromycin high performance liquid chromatography to use a C18 chromatographic separation column under the condition of 45 DEG C, the flow rate is 0.7 mL/ min, the mobile phase is acetonitrile with 0.1% trifluoroacetic acid and 3.5 mM HC1 to adjust the pH to 0.033 nol/ L KH2PO4 of 3.5 to 0.2 (40/60, v/ v) with a detection wavelength of 205 nm; a high performance liquid chromatography of daptomycin at a flow rate of 1 mL/ min at 35 C and a mobile phase of acetonitrile and 0.5% NH4H2PO4 (36/64, v/ v), The detection wavelength was 221 nrn. Several factors influencing the preparation of daptomycin long-cycle liposomes were investigated: the molar ratio of hydrogenated lecithin (HSPC) to cholesterol, the mass ratio of lipid, and finally the optimal formulation of the preparation of daptomycin long-cycle liposomes was obtained. and a stable liposome dosage form can be obtained, and after two months are stored in a refrigerator at 4 DEG C, The combined antibacterial index of clarithromycin and daptomycin as a single agent was determined by a checkerboard method, and the ratio of PL[C] to PL[D] was 32:1. The value is used for subsequent preparation of the co-carrier liposome. (3) The preparation and evaluation of the co-carrier liposomes were optimized to prepare the daptomycin, the clarithromycin co-carrier liposome average particle size (98.2 + 2.21) nm, the polydispersity index of 0.251, the encapsulation rate (92.94% 1.21, 94.71% 1.37)%, and the drug loading (8.45%). 0.11, 0.29 (0.043)% The anti-methicillin-resistant Staphylococcus aureus in vitro mainly studied the three preparations of daptomycin: clarithromycin liposome (PL[C]), daptomycin liposome (PL[D]), daptomycin, and clarithromycin co-carrier liposome (PL[CD]). ) Effect on the growth of MRSA252, the results show that PL[ CD] The inhibition of MRSA252 is more durable. The analysis of the qualitative and quantitative results of the biofilm shows that the PL (CD) ratio PL[ [C], PL[D] inhibited the efficacy of MRSA. By comparing PL[C], PL[D], and PL[CD] to the therapeutic effect of MRSA252 on the whole body infected KM mice, the results showed that the long circulating liposomes of daptomycin can make the infection K The survival of M mice increased to 90% in the observation period (7 days). The safety evaluation junction
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943
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