【作者】 薛昊；

【导师】 杨波；

【作者基本信息】 哈尔滨商业大学 ， 药剂学， 2021， 硕士

【摘要】 疟疾是由疟原虫引发的威胁人类生命安全的重大疾病之一,其种类繁多,基因型也各不相同,极易产生耐药性,继而出现了抗药型虫株。青蒿素(Artemisinin,ART)因其独特的结构和抗疟机制成为了现今一线抗疟药物,但近些年有关青蒿素的耐药性也逐渐被报道。为明晰青蒿素体内宏观分布及代谢差异和提高药物利用度,本课题从制剂学和药动学角度出发,制备了包载青蒿素的聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米粒,并对其在青蒿素敏感/抗性伯氏疟原虫小鼠体内的药动学变化进行研究,以期为延缓及逆转青蒿素的耐药性提供新的思路和理论基础。本课题建立了青蒿素定量检测的液相色谱-串联质谱法(liquid chromatography-tandem mass spectrometry,LC-MS/MS),该法操作简便,检测迅速,灵敏度极高,专属性良好,精密度、回收率、重复性等指标均符合要求。选用PLGA作为纳米载体,以乳化溶剂挥发法成功制备了青蒿素PLGA纳米粒(PLGA Nanoparticles Loaded with Artemisinin,PLGA-ART-NPs)。以载药量和包封率为评价指标,利用单因素分析筛选出PLGA-ART-NPs制备过程中的主要影响因素为乳化剂浓度、水油两相比例、载体-药物比例。以PLGA-ART-NPs的载药量为评价指标,通过响应面法进一步优化处方,得到PLGA-ART-NPs的最优处方为:乳化剂浓度为1.7(w/v)%,水油两相比例为18:1,载体-药物比例为10:1,载药量为(4.63±0.22)%,重现性良好。使用透射电镜、马尔文粒子分析仪、LC-MS/MS等检测仪器,对PLGA-ART-NPs的粒径、Zeta电位、稳定性及体外释放特性进行评价。以最优处方制得的PLGA-ART-NPs 平均粒径为(192.2±9.13)nm,多分散指数(polydispersity index,PDI)为0.256±0.28,Zeta电位(-26.5±3.72)mV;透射电镜观察粒子圆整,形态光滑。粒径和Zeta电位的测定,提示已成功制备PLGA-ART-NPs。稳定性研究表明,PLGA-ART-NPs在人工胃液、人工肠液和含10%血浆的PBS溶液中孵育8 h时稳定性良好。在体外释放特性结果表明,青蒿素主要在胃肠道吸收,且PLGA-ART-NPs相对于原料药,表现出明显的缓释能力。本文选用对青蒿素敏感/抗性的K173基因型伯氏疟原虫,通过腹腔注射含疟原虫血液方式,建立了敏感/抗性疟原虫感染的小鼠模型。感染一段时间后,小鼠体重下降,外观变化明显,活动减少,耳朵及四肢无血色,尿黄,便稀、黏。瑞氏染色制备血涂片发现,感染后的红细胞变化明显,细胞中存在大量“圆环状”和“耳机状”的原虫滋养体(深紫色物质)。经过两次血传后,通过血涂片观察并计算小鼠的感染率,筛选出感染率为5%的小鼠作为模型给药。本课题建立了快速、灵敏、准确的小鼠血浆中青蒿素浓度的LC-MS/MS定量分析方法,分别灌胃给予青蒿素敏感/抗性疟原虫感染的小鼠以青蒿素原料药(0.5%羧甲基纤维素钠混悬)和PLGA-ART-NPs溶液,并对其药动学特征及差异进行了对比研究。本文采用液-液萃取法提取血浆中青蒿素,小鼠血浆中青蒿素在1-200 ng·mL-1浓度内线性良好,定量下限为1 ng·mL-1,方法学考察中各项指标均符合要求。以非室模型拟合分别给予原料药后敏感组和抗性组小鼠体内的药动学参数如下:峰值浓度(Cmax)分别为89.42±11.09和95.70±21.73 ng·mL-1;达峰时间(Tmax)分别为1.08±0.20和1.0 h;末端消除半衰期(t1/2)分别为3.11±1.02和3.29±2.72 h;药-时曲线下面积(AUC0-t)分别为 199.42±12.18 和 208.53±35.65 h·ng·mL-1;平均滞留时间(MRT)分别为 2.19±0.40 和 2.46±1.00 h;清除率(CL)分别为 0.20±0.01 和 0.20±0.04 L·kg·h-1;表观分布容积(Vd)分别为 0.89±0.29 和 0.92±0.68 L·kg-1。分别给予PLGA-ART-NPs后敏感组和抗性组小鼠体内的药动学参数如下:峰值浓度(Cmax)分别为 79.98±12.51 和 73.95±12.13 ng·mL-1;达峰时间(Tmax)分别为3.50±0.84 and 4.0 h;末端消除半衰期(t1/2)分别为 12.54±3.09 和 11.87±1.93 h;药-时曲线下面积(AUC0-t)分别为 580.07±106.75 和 593.56±120.09 h·ng·mL-1;平均滞留时间(MRT)分别为 8.03±0.67 和 8.04±0.72 h;清除率(CL)分别为 0.06±0.01 和 0.06±0.02 L·kg·h-1;表观分布容积(Vd)分别为 0.98±0.20 和 0.91·0.12L·kg-1。对比原料药与PLGA-ART-NPs药动学参数发现,PLGA-ART-NPs显著改善了青蒿素在模型鼠体内的药动学过程,可以起到长效抗疟作用。对比抗性/敏感组分别灌胃给予青蒿素和PLGA-ART-NPs药动学参数发现,青蒿素在抗性组和敏感组小鼠体内的吸收、分布及代谢过程无显著性差异,提示后续可从血红素相关通路阐明耐药性发生的可能机制及逆转耐药性的方法。更多还原

【Abstract】 Malaria is one of the major diseases that threaten human life safety caused by Plasmodium.Plasmodium has a wide variety of different genotypes,so it is easy to develop drug-resistant strains.Artemisinin(ART)has become the current first-line antimalarial drug due to its unique structure and antimalarial mechanism.However,resistance to artemisinin has also been gradually reported in recent years.In order to clarify the differences of macro-distribution and metabolism in the body and improve drug availability,This study from the angle of the pharmaceutics and pharmacokinetic,preparaing poly(lactic-co-glycolic acid)(PLGA)nanoparticles containing artemisinin,and their pharmacokinetic changes in mice infected with artemisinin-sensitive/resistant Plasmodium berghei were studied,expected to provide new ideas and theoretical basis for delaying and reversing artemisinin resistance.A liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the quantitative determination of artemisinin was established in this study.The method is easy to operate,quick to detect,and has highly sensitive and strong specificity,precision,recovery,repeatability meet the requirements.PLGA Nanoparticles Loaded with Artemisinin(PLGA-ART-NPs)were successfully prepared by emulsifying solvent volatilization method using PLGA as a nanocarrier.With the drug loading and encapsulation rate as evaluation indexes,the main influencing factors in the preparation process of PLGA-ART-NPs were screened by single factor analysis:the concentration of emulsifier,the ratio of water and oil two phases,and the ratio of carrier to drug.Taking the drug loading of PLGA-ART-NPs as the evaluation index,the formulation was further optimized by response surface methodology.The optimal formulation of PLGA-ARTNPs was obtained as follows:the concentration of emulsifier was 1.7(w/v)%,the ratio of water to oil was 18:1,the ratio of carrier to drug was 10:1,and the drug loading was(4.63±0.22)%,with good reproducibility.The particle size,Zeta potential,stability and in vitro release characteristics of PLGAART-NPs were evaluated by transmission electron microscopy,Malvin particle analyzer,liquid mass spectrometry and other detection instruments.The average particle size of PLGA-ARTNPs prepared according to the optimal formulation was(192.2±9.13)nm,the polydispersity index(PDI)was 0.256±0.28,and the Zeta potential was(-26.5±3.72)mV.The particle shape was round and smooth under transmission electron microscope.The determination of particle size and Zeta potential indicated that PLGA-ART-NPs had been successfully prepared.The stability study showed that PLGA-ART-NPs had good stability when incubated in artificial gastric juice,artificial intestinal juice and PBS solution containing 10%plasma for 8 h.In vitro release characteristics showed that artemisinin was mainly absorbed in the gastrointestinal tract,and PLGA-ART-NPs showed obvious sustained-release ability compared with the raw materials.In this study,the K173 genotype of Plasmodium berghei which showed sensitive/resistant to artemisinin was used to establish the mice model of sensitive/resistant Plasmodium berghei infection by intraperitoneal injection of Plasmodium berghei-containing blood.After infection for a period of time,the mice lost weight,the appearance changed significantly,the activity decreased,the ears and limbs had no color,the urine was yellow,and the stool was thin and sticky.Rick’s staining method for blood smears revealed significant changes in the infected red blood cells,with a large number of "ring-shaped" and "headset shaped" protozoa trophoblasts(dark purple substances).After twice blood transfusions,the infection rate of mice was observed and calculated by blood smear,and mice with an infection rate of 5%were selected as the model for administration.A rapid,sensitive and accurate LC-MS/MS method for quantitative analysis of artemisinin concentration in mice plasma was established in this study.Mice infected with artemisininsensitive/resistant Plasmodium berghei were traeted with oral raw materials(0.5%sodium carboxymethyl cellulose mixed)and PLGA-ART-NPs solution,respectively,and the pharmacokinetic characteristics and differences of the two groups were compared.Liquidliquid extraction method was used to extract artemisinin from plasma.The linear range of artemisinin in mice plasma was 1-200 ng·mL-1,and the lower limit of quantition was 1 ng·mL1.All the indicators of the methodological investigation met the requirements.The pharmacokinetic parameters of mice in the sensitive and resistant groups after an oral administration of raw materials were fitted by non-compartment model as follows:Cmax were 89.42±11.09 and 95.70±21.73 ng·mL-1;Tmax were 1.08±0.20 and 1.0 h;ti/2 were 3.11±1.02 and 3.29±2.72 h.AUC0-t were 199.42±12.18 and 208.53±35.65 h·ng·mL-1.MRT were 2.19±0.40 and 2.46±1.00 h;CL were 0.20±0.01 and 0.20±0.04 L·kg·h-1.Vd were 0.89±0.29 and 0.92±0.68 L·kg-1.The pharmacokinetic parameters of mice in the sensitive and resistant groups after an oral administration of PLGA-ART-NPs as follows:Cmax were 79.98±12.51 and 73.95±12.13 ng·mL-1,Tmax were 0.79±0.03 and 1.0 h;t1/2 were 12.54±3.09 and 11.87±1.93 h.AUC0-t were 580.07±106.75 and 593.56±120.09 h·ng·mL-1.MRT were 8.03±0.67 and 8.04±0.72 h;CL was 0.06±0.01 and 0.06±0.02 L·kg·h-1.Vd were 0.98±0.20 and 0.91±0.12 L·kg-1.Compared the pharmacokinetic parameters of raw materials and PLGA-ART-NPs,PLGAART-NPs significantly improved the pharmacokinetic process of artemisinin in model mice,and had a long-term antimalarial effect.Compared the pharmacokinetic parameters of raw materials and PLGA-ART-NPs in the resistant and sensitive groups,the absorption,distribution and metabolic process of artemisinin in the resistant and sensitive groups were not significantly different,suggesting that the possible mechanism of the occurrence of drug resistance and the method of reverse drug resistance can be elucidated from the heme related pathway in the future.更多还原