【作者】 佟金；

【导师】 顾景凯；

【作者基本信息】 吉林大学 ， 药物分析， 2019， 硕士

【摘要】 研究目的随着糖尿病发病率在全球范围内的快速上升,针对糖尿病并发症的治疗药物层出不穷。达瑞司他钾是我国根据传统药物研发出来的一种通过抑制葡萄糖代谢过程中的多元醇通路,缓解糖尿病并发症的醛糖还原酶抑制剂类创新型药物。为了保证达瑞司他钾的安全性与有效性,需对达瑞司他钾进行临床前药代动力学研究。研究方案本研究以SD大鼠作为达瑞司他钾临床前研究试验对象。1)为了准确测定大鼠血浆中达瑞司他钾浓度,本课题基于液质联用技术（LC-MS/MS）建立了一种快速、高效、灵敏度高的定量方法:血浆样品中的达瑞司他钾经液液萃取法被提取出来;应用梯度洗脱法对达瑞司他钾进行色谱分离;采用ESI源,负离子模式,在三重四极杆质谱API4000的MRM扫描方式下,选择m/z 390.0→m/z 346.0（达瑞司他钾）和m/z 478.2→434.2（内标）作为监测离子对实现对达瑞司他钾的定量分析,定量范围为10³000 ng/mL。为了保证方法的可靠性,本课题根据NMPA指导原则进行了方法学验证。2)为了研究达瑞司他钾在大鼠血浆中的药代动力学行为,本课题分别对大鼠进行静脉给药、空腹灌胃给药和进食后灌胃给药,并用上述定量方法测定血药浓度,分析饮食影响。3)应用飞行时间高分辨质谱TripleTOF^TM5600结合IDA技术对达瑞司他钾在大鼠尿液、粪便样品中的代谢产物进行鉴定,揭示达瑞司他钾在大鼠体内的生物转化过程。4)为了能对大鼠尿液、粪便样品中达瑞司他钾原形药和主要代谢产物做出准确测定并计算累积排泄率,我们使用QTRAP^?5500离子阱质谱,建立了一种多组分定量分析方法:对生物样品进行沉淀蛋白法预处理,达瑞司他钾原形药及主要代谢产物通过梯度洗脱方式在色谱柱上被依次分离开来。在ESI源正离子模式下,采用MRM扫描方式,对原形药及代谢产物进行准确定量,定量范围为1⁵00 ng/mL。研究结果1)方法学验证结果表明,本课题所建立的大鼠血浆中达瑞司他钾的定量方法具备灵敏度高、回收率高、专属性强、无残留、样品稀释可靠的特点,可以用于达瑞司他钾的药代动力学研究。2)经灌胃给药后,不同大鼠对于达瑞司他钾的吸收差异极大,且食物对达瑞司他钾的吸收有显著影响,进食可以加快达瑞司他钾在大鼠体内的消除。进食后灌胃给药达瑞司他钾（10 mg/kg）的绝对生物利用度为6.08%,结果偏低。3)在大鼠的尿液和粪便样品中,主要发现原形药经氧化、脱羧基、去甲基、脱羧基后去甲基的4种I相代谢产物。4)经累积排泄率计算,60.467%的达瑞司他钾从粪便中排出,7.821%的达瑞司他钾从尿液中排出且其中56.09%是以原形药的形式排出。所有排泄物样品中主要检测到的代谢产物为达瑞司他钾氧化和去甲基后的代谢产物,所占比例分别为5.14%和6.84%。更多还原

【Abstract】 PurposeAs the incidence of diabetes has risen rapidly around the world,treatments for diabetes complications have come out one after the other.Darirestat potassium is an aldose reductase inhibitor developed by Chinese experts based on traditional medicines,alleviating diabetic complications by inhibiting the polyol pathway in glucose metabolism process.In order to ensure the safety and effectiveness of darisstatal potassium,preclinical studies on darirestat potassium are required.MethodIn this study,SD rats were used as subjects for pre-clinical studies of darirestat potassium.1)In order to determine the concentration of darirestat potassium in rat plasma accurately,a rapid,efficient and sensitive quantitative method based on LC-MS/MS was established:The darirestat potassium in plasma samples was extracted by liquid-liquid extraction;it was effectively separated by gradient elution;The negative ion mode of the ESI source was used in the triple quadrupole mass spectrometry API4000,and under the MRM scanning mode,select m/z 390.0→m/z346.0（daristat potassium）and m/z 478.2→434.2（internal standard）as the monitoring ion pairs to achieve the quantitative analysis of darisita potassium,the quantitative range is 10³000 ng/mL.In order to ensure the reliability of the method,the method has been verified by the NMPA guidelines.2)In order to study the pharmacokinetic behavior of darirestat potassium in rat plasma,rats were administered intravenously,fasting intragastric administration and intragastric administration after eating,and the blood concentration was determined by the above quantitative method for analyzing dietary effects.3)Using the time-of-flight,high resolution mass spectrometry TripleTOF^TM5600 combined with IDA technology to identify the metabolites of darirestat potassium in rat urine and fecal samples for analyzing the biotransformation process of darirestat potassium in rats.4)In order to measure the darirestat potassium parent drug with major metabolites in rat urine or fecal samples accurately and calculate the cumulative excretion rate,we established a quantification method for multi-components by using QTRAP^?5500 ion trap mass spectrometry:The sample was pretreated by precipitation protein method,and the darirestat potassium parent drug and main metabolites were separated by gradient elution from column.In the positive ion mode of the ESI source,we use the MRM scanning method to determine the concentration of darirestat potassium parent drug and its main metabolites.The quantitation range is from 1 to 500 ng/mL.Results1)Methodological verification results show:The established quantification method in rat plasma for darirestat potassium has been characterized by high sensitivity,high recovery rate,strong specificity,no residue,and reliable dilution.So it can be used for pharmacokinetic study of darisitarine potassium.2)By intragastrically administered rats,we found the absorption of darirestat potassium was significantly different in different rats,and the food had a significant effect on the absorption of darirestat potassium.Eating can accelerate the elimination of darirestat potassium in rats.The absolute bioavailability of darirestat potassium by intragastric administration（10 mg/kg）after eating was 6.08%,and the result was low.3)In the urine and feces samples of rats,four kinds of darirestat potassium phase I metabolites were found,respectively by oxidation,decarboxylation,demethylation,and demethylation after decarboxylation.4)According to the cumulative excretion rate,60.467%of darirestat potassium was excreted from the feces,7.821%of darirestat potassium was excreted from the urine and 56.09%of it was excreted in the form of parent drug.The main metabolites detected in all excreta samples were the metabolites of oxidation and demethylation,with a ratio of 5.14%and 6.84%.更多还原