【作者】 孙伟；

【导师】 祖元刚； 赵修华；

【作者基本信息】 东北林业大学 ， 生药学， 2014， 硕士

【摘要】 两性霉素B(Amphotericin B,AmB)是一种多烯类抗生素,在1995年从链霉菌的培养液中分离而得来的。AmB是可运用于全身性真菌感染的广谱抗真菌药,它一直是治疗真菌感染的黄金标准。它的高效抗真菌机制是因为与真菌细胞膜内的甾醇结合,破坏细胞膜的完整性。细胞膜通透的破坏使真菌细胞内重要物质外漏,导致真菌细胞死亡。可是,由于AmB的低水溶性限制了其应用。因此,关键问题是解决AmB的应用,提高它的水溶性、口服吸收和体内生物利用度。纳米粒载药系统是近些年兴起的一种新型制剂技术,是21世纪战略技术的制高点。它不仅能够增加难溶性药物的溶解度,提高药物在体内的生物利用度,而且药物制成纳米粒后,还具有靶向、缓释、增强疗效以及减少毒副作用等特点。本课题主要是应用纳米技术提高AmB的水溶性、生物利用度以及口服吸收,使AmB成为更加有效的抗真菌口服药物。本课题主要是解决口服AmB的水溶性问题,采用液体反溶剂重结晶技术和冷冻干燥法制备AmB纳米制剂,从而提高AmB的水溶性。通过单因素试验对液体反溶剂法进行优化,确定了表面活性剂及浓度、搅拌时间、沉积温度、搅拌强度、药物浓度和反溶剂-溶剂的体积比对AmB纳米粒平均粒径的影响程度。然后,对最优条件下获得的AmB纳米粒进行理化性质检测和生物功能检测,主要包括扫描电子显微镜检测(SEM),质谱检测(MS),傅里叶红外光谱检测(FTIR),X-射线衍射检测(XRD),差示扫描量热检测(DSC),热重分析检测(TG),溶剂残留检测,纯度检测,溶出度实验和生物利用度实验。1.通过单因素试验对液体反溶剂法进行优化,AmB纳米粒最优制备条件如下:0.15%吐温-80,搅拌时间为30 min,沉积温度为12 ℃C,搅拌强度为2000 r/min,药物浓度为20mg/mL,反溶剂-溶剂的体积比为13/1。实验结果表明:随着搅拌强度和沉积时间的增加,AmB纳米粒的平均粒径逐渐减小,但是当沉积时间超过30 min后,粒径不再减小,基本保持不变;随之吐温-80浓度和药物浓度的增加,AmB纳米粒的粒径逐渐增大;随着沉积温度和反溶剂-溶剂的体积比的增加,AmB纳米粒的粒径呈现先减小后增加的趋势。最优条件下制备得到的AmB纳米粒的平均粒径为135.1 nm。2.理化性质检测:扫描电子显微镜检测结果说明制备后AmB纳米粒呈圆球状。AmB纳米粒与原药相比粒径明显减小,形状也更加规则,SEM结果暗示着AmB纳米粒会有更高的溶解度。FTIR和MS的检测结果表明:通过液体反溶剂制备后得到AmB纳米粒化学结构没有发生改变。XRD和DSC的检测结果表明:通过液体反溶剂制备的AmB纳米粒结构从晶体转变成为非晶体。溶剂残留结构表明:AmB纳米粒中DMSO的残留量为0.24%,低于人用药物注册技术要求国际协调会对三类溶剂的要求。溶解度和溶出度结果表明:重结晶后的AmB纳米粒的溶出速率是AmB原粉的2.1倍,平衡溶解度是AmB原粉的13倍。如上的结果都证明了,AmB纳米粒有提高其口服生物利用度的潜力。3.对大鼠进行生物利用度研究,采用高效液相色谱法检测大鼠血浆中AmB的含量。结果表明,AmB原料药口服在动物体内几乎不吸收,经过纳米化后的AmB粉体在体内的吸收明显增加,相对生物利用度是原料药的4.95倍。通过以上实验结果表明,本实验所制备的AmB纳米粒粒径分布均匀,形状规则呈球形,并且其理化性质稳定。AmB的水溶性得到了很大改善,同时生物利用度也得到了很大的提高。更多还原

【Abstract】 Amphotericin B(AmB)is a polyene macrolide antibiotic,which was isolated from Streptomyces nodosus in 1955.AmB is the classic broad-spectrum agent for systemic fungal infections and it remains the gold standard therapy for fungal infections.The mechanism of action of this highly effective antifungal is binding to the ergosterol in the cell membrane of fungi and some protozoan parasites,which disrupts cell membrane integrity.Membrane pore formation damages membrane permeability,which causes the leakage of important materials,leading to fungal cell death.However,the poor water solubility of AmB limits its application.Therefore,the key issues need to be resolved in AmB applications are to improve its water solubility,increase its oral absorption,and enhance its vivo bioavailability.In recent years,nanotechnology is a new formulation technology and the commanding point in the 21st century.It is not only to increase the solubility of poorly soluble drugs,to improve the bioavailability of drugs in the body,but also has targeted,sustained-release characteristic to enhance the drug efficacy and reduce side effects,etc.This project tried to improve the water solubility of AmB using nanotechnology,improve drug bioavailability and enhance the oral absorption,making AmB a more effective antifungal oral drug.This project focused on the solubility of AmB for oral administration and prepared AmB nanoparticles through liquid antisolvent precipitation and by freeze-drying.The liquid antisolvent precipitation was optimized trough a single-factor experiment.We determined the effects of surfactants and their concentration,the stirring time,the precipitation temperature,the stirring intensity,the drug concentration and the volume ratio of antisolvent to solvent on the mean particle size(MPS)of the AmB nanoparticles.For the obtained optimal AmB nanoparticles through single-factor experiment conduct physicochemical property testing and other test,including scanning electron microscopy(SEM),mass spectrometry(MS),Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),differential scanning calorimetry(DSC),thermal gravimetric analysis(TG),solvent residue,drug purity test,dissolution test and bioavailability.1.The liquid antisolvent precipitation was optimized through a single-factor experiment.the optimal conditions for AmB nanoparticles preparation are as follows:0.15%Tween-80,stirring time of 30 min,precipitation temperature of 12℃,stirring intensity of 2000 r/min,drug concentration of 20mg/mL,and antisolvent to solvent volume ratio of 13/1.The experimental results show that increased stirring intensity and precipitation time favored AmB nanoparticles with smaller MPS,but precipitation times exceeding 30 min did not further reduce the MPS and almost remain unchanged.Increased Tween-80 concentration and the drug concentration increased the MPS of the AmB nanoparticles.Increased precipitation temperature and antisolvent to solvent volume ratio initially decreased the MPS of the AmB nanoparticles,which increased thereafter.Optimum conditions produced AmB nanoparticles with an MPS of 135.1 nm.2.Physicochemical property testing and other test:The SEM demonstrated that AmB nanoparticles produced through were almost spherical.It were significantly smaller and more uniform than the raw AmB,which indicates better solubility.The FTIR and MS analyses demonstrated that the molecular structure of the AmB nanoparticles was unchanged after liquid antisolvent precipitation.The XRD and DSC analyses indicated that the prepared AmB nanoparticles were changed from crystalline to amorphous.The solvent residue result demonstrated that the residual DMSO in the AmB nanoparticles was 0.24%lower than the the International Conference on Harmonization(ICH)limit for class Ⅲ solvents.The AmB nanoparticles exhibited 2.1 times faster dissolution rate and 13 times equilibrium solubility compared with the raw drug.These detection results indicate that the AmB nanoparticles potentially improved the oral absorption of AmB.3.In the bioavailability studies of rats,the AmB content in rat plasma was detected by high performance liquid chromatography(HPLC).The results show that the raw AmB almost without being absorbed in animals.However,AmB nanoparticles have an abvious increase.The relative bioavailability was 4.95 times more than the raw AmB.Through the above experimental results,the prepared AmB nanoparticles had uniform particle size distribution,shapeed rules spherical.And the physical and chemical properties of AmB nanoparticles were stability.The water-soluble of AmB get abvously improved,and the bioavailability of AmB has an abvious increase.更多还原