达托霉素长循环脂质体抗耐甲氧西林金葡菌的研究

发布时间：2018-04-30 11:57

本文选题：达托霉素 + 长循环脂质体　；参考：《西南大学》2014年硕士论文

【摘要】：随着病原体耐药防控难度的持续增大,除不断研发新型抗生素之外,对已有抗生素进行递送研究与新制剂创制具有同等重要性。达托霉素是近年来上市的-种新型脂肽类抗生素,目前仅有其普通溶液型注射剂用于临床。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长循环达托霉素脂质体(PLD)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脂质体粒径(111.5±15.4)nm、载药量较高(5.81±0.19)%,并具有良好稳定性。体内外评价结果显示,在相同剂量下PLD与达托霉素原料药及普通脂质体制剂相比,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。此外,PLD能显著提高达托霉素在肺部的分布并显示良好的安全性。上述研究结果提示,长循环脂质化达托霉素对抗MRSA严重感染(血源性肺炎等)具有良好的应用前景,可为达托霉素新制剂的研发及临床相关研究提供重要参考。本课题主要研究内容如下：(1)达托霉素长循分析方法的建立确定达托霉素的最大吸收波长为221nm,并且还确定了HPLC检测达托霉素的色谱条件：色谱柱：Inertsil ODS-SP C18柱(150mm×5mm,5μm) 流动相：乙腈-0.5%磷酸二氢铵缓冲液=40：60 流速：0.7mL/min;检测波长：221nm进样量：20μ L；柱温：30℃出峰时间：6.5±0.2min最低检测限：0.60ng还通过回收率实验、精密度实验得出此种检测方法准确且重现性好。 (2)达托霉素长循环脂质体的制备及相关性质考察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、脂药质量比以及高压均质机循环次数。最终得出制备达托霉素长循环脂质体的最优处方为,氢化卵磷脂：胆固醇：mPEG2ooo-DSPE=18：6：1(摩尔比),脂药比=15：1(质量比),水化液经高压均质机1000bars循环均质10次,就能得到稳定的达托霉素长循环脂质体。由最优制备工艺所制得的脂质体,粒径为(111.5±15.4)nm, Zeta电位为(-15.1±2.8)mV,载药量为(5.81±0.19)%,包封率为(92.56±3.15)%,并且性质稳定,在4-C的冰箱中保存2个月后几乎无泄漏、无沉淀现象发生。 (3)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：达托霉素原料药水溶液DS、普通达托霉素脂质体CLD、达托霉素长循环脂质体PLD对MRSA252生长的影响,结果表明,PLD对MRSA252的抑制效果更持久。 (4)药动学及组织分布研究结果表面达托霉素长循环脂质体延长了达托霉素在大鼠血液里的循环时间,且明显的增加了达托霉素在肝、脾、肺的分布量。 (5)体内药效学、安全性评价通过比较DS、CLD、PLD对MRSA252致全身感染KM小鼠的治疗效果比较,结果显示,尾静脉注射单次次给药达托霉素长循环脂质体就能使感染KM小鼠的存活率在观察期(7天)提高到80%。安全性评价结果表明达托霉素长循环脂质体未对KM小鼠有明显毒性。
[Abstract]:With the increasing difficulty of drug resistance and prevention and control, in addition to the continuous development of new antibiotics, the research on the delivery of the existing antibiotics is of equal importance to the creation of new preparations. DDT is a new type of lipopeptide antibiotic in recent years. At present, only the common solution injection is used in clinical. This paper uses poly (two) The alcohol modified liposome was used as a carrier to evaluate and systematically evaluate the optimized long circulation dacostin liposome (PLD) against methicillin resistant Staphylococcus aureus (MRSA252) systemic infection. Through the optimization of the size of the liposomes (111.5 + 15.4) nm, the drug loading is higher (5.81 + 0.19)%, and has good stability. The results showed that, at the same dose, PLD had a more persistent inhibitory effect on MRSA252 compared with the common liposomal drug and the common liposome agent, and could significantly increase the survival rate of the model mice with methicillin-resistant Staphylococcus aureus systemic infection. In addition, PLD could significantly improve the distribution of DTM in the lung and show good safety. The results suggest that long circulatory liposomes have good application prospects against MRSA severe infection (hematogenous pneumonia, etc.), and can provide an important reference for the research and development of the new preparation of dalamycin and clinical related research.
The main contents of this research are as follows: (1) establishment of DAC analysis method
The maximum absorption wavelength of Das was determined to be 221nm, and the chromatographic conditions for HPLC detection of Das were also determined.
Chromatographic column: Inertsil ODS-SP C18 column (150mm x 5mm, 5 m)
Mobile phase: acetonitrile -0.5% phosphoric acid two hydrogen ammonium buffer =40:60
Flow rate: 0.7mL/min; detection wavelength: 221nm sampling amount: 20 mu L; column temperature: 30 C peak time: 6.5 + 0.2min minimum detection limit: 0.60ng is also through recovery experiment, precision experiment results show that this method is accurate and reproducible.
(2) preparation and related properties of long circulating liposomes of DTaP
Several factors affecting the preparation of the long circulating liposomes of dalochomycin: the molar ratio of HSPC to cholesterol, the mass ratio of lipoid drugs and the cycles of high pressure homogenizer. The optimal formulation for the preparation of the long circulating liposome of dalotamiin was obtained, and the hydrogenated oophospholipid: cholesterol: mPEG2ooo-DSPE=18:6:1 (mole ratio) When the liposomes are compared with the =15:1 (mass ratio), the hydrated liquid is homogenized 10 times through the high pressure homogenizer 1000bars, and the stable dapycin long circulating liposome can be obtained. The liposomes obtained by the optimal preparation process are (111.5 + 15.4) nm, the Zeta potential is (-15.1 2.8) mV, the drug loading is (5.81 + 0.19)%, the encapsulation rate is (92.56 + 3.15)%, and the property is stable. It has been stored in the refrigerator of 4-C for 2 months, and there is almost no leakage.
(3) the experiment of anti methicillin resistant Staphylococcus aureus in vitro
Three preparations of dalotomiin were mainly studied: DS, CLD of dalotomiin liposome, and the effect of PLD on the growth of MRSA252. The results showed that the inhibition effect of PLD to MRSA252 was more lasting.
(4) study on pharmacokinetics and tissue distribution
Results DDT lengthened the circulation time of dalotamicin in the blood of rats, and significantly increased the distribution of dalamycin in the liver, spleen and lung.
(5) in vivo pharmacodynamics, safety evaluation
By comparing the therapeutic effects of DS, CLD, and PLD on KM mice induced by MRSA252, the results showed that the single dose of tataycin long circulating liposome injected into the tail vein could increase the survival rate of the infected KM mice in the observation period (7 days) to the 80%. security evaluation results, indicating that the long circulating liposomes of tataycin did not have obvious toxicity to KM mice. Sex.

【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】：R978.1

【参考文献】