【作者】 刘骅；

【导师】 金涌； 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 硕士

【摘要】 A771726是免疫调节药来氟米特（leflunomide,LEF）在机体内的活性代谢物,后者在临床上主要用于治疗类风湿性关节炎、系统性红斑狼疮、抗移植排斥反应和银屑病。A771726是原形药物,将其制成口服制剂在体内直接吸收起作用,省略了前体药物转化的环节,目前正在申报一类新药。本实验旨在观察A771726在大鼠体内的吸收、血浆蛋白结合、分布、排泄特点,并与来氟米特的相关药动学参数进行比较,进行合理评价,提供新药临床前研究资料,以支持人体药代动力学研究并为临床用药提供参考依据。1.生物样品中A771726分析方法的建立本文采用沉淀蛋白处理血浆样品和液液萃取处理生物样品,建立A771726的HPLC分析方法。色谱分离条件为:色谱柱:Kromasil ODS C₁₈（150×4.6mm）;流动相为0.01mol/L磷酸盐缓冲液（pH4.5）和甲醇（43:57）;检测波长288nm;柱温:30℃。采用本文建立的生物样品预处理方法和反相高效液相色谱法分离测定生物样品中A771726的含量,生物样品预处理方法回收率高,色谱分离选择性好。本法的准确度、精密度、灵敏度、专属性和定量线性范围均达到了体内药物分析的要求。该方法成功运用于A771726的大鼠药代动力学研究。2.A771726在大鼠体内的药代动力学研究SD大鼠单次口服给药（3、6、12mg/kg）后不同时间点取血,用上述建立的HPLC法测定血浆中A771726的浓度,根据所得的血浆药物浓度—时间曲线,计算药动学参数,T_1/2Ke（h）:16.71±2.34、14.69±1.94、16.45±2.49;AUC_（0-tn）（mg/L·h）:271.38±78.87、785.57±170.92、994.13±165.43;C_max（mg/L）:11.42±2.59、29.38±5.41、42.10±5.73;T_max（h）:4.00±0.82、4.10±0.74、4.20±1.03;MRT_（0-tn）（h）:22.07±1.80、21.91±1.60、20.71±1.88。单剂量口服A771726三个剂量组的药物吸收均成线性关系,其血药浓度—时间曲线符合一级吸收的一房室模型。3.A771726的血浆蛋白结合率研究血浆中加入A771726使最终浓度分别达到3,6,12μg/ml,每份加药血浆样品用平衡透析法在4℃条件下透析108h,透析结束后,用HPLC法分别测定血浆及缓冲液样品中的药物浓度,计算药物的血浆蛋白结合率,结果表明在上述范围内A771726与人的血浆蛋白结合率约97.7%,无浓度依赖性。4.A771726在大鼠体内的组织分布研究SD大鼠口服给药A771726（6mg/kg）后,分别于给药后2h、12h、24h股动脉放血处死,立即取出各脏器组织,滤纸吸干浮血,称重后用生理盐水制成匀浆,取匀浆液处理,用HPLC法测定各组织中药物浓度。结果显示,给药后A771726分布于主要组织,各脏器组织中的药物含量均低于血浆,肝肾组织中A771726浓度较高,大多数组织在12h达高峰,脑、骨髓、胸腺、睾丸中含量较低。5.A771726在大鼠体内的排泄研究SD大鼠口服给药A771726（6mg/kg）后收集胆汁、粪便、尿液样品,记录体积和重量,用HPLC法分析样品中A771726的浓度,将胆汁、粪便、尿液中的药物浓度乘以相应的胆汁排泄量、粪便量、尿量,并累加求和,计算药物的累积排泄量。结果显示大鼠单次灌胃给药后,粪便的排泄结果显示,0～48h排泄量占总给药量的1.61%;尿液中的排泄结果显示,0～48h排泄量占总给药量的0.87%;0～24h内从胆汁排泄的A771726量占总给药量的13.88%。更多还原

【Abstract】 A771726 is the active metabolic product of leflunomide,the later is a type of immunoregulatory agents.Leflunomide educes the pharmacological action by metabolized into A771726,and it has been extensively exerted on rheumatoid arthritis, systemic lupus erythematosus,anti-rejection organ transplantation and psoriasis. A771726 is an active compound,which is directly absorbed in vivo,abbreviates the transformation of prodrug.The works of this paper were designed to evaluate the pharmacokinetics features of A771726 in rats,compare to the parameters of leflunomide,so as to support the pharmacokinetics study in human and offer the reference for clinical dosage and dosage form changing.Part one:The establishment of analytical method of A771726 in biological matrixA reversed-phase high-performance liquid chromatographic（RP-HPLC） method has been developed for the determination of A771726 in rat plasma,bile,urine,feces and tissue homogenates.The sample pretreatment included deproteinization for plasma samples and a liquid-liquid extraction for bile,urine,feces and tissue homogenates. Separation was obtained on a Kromasil C18 column（150×4.6mm）,using an excitation wavelength of 288nm.The isocratic mobile phase consisted of methanol-0.01mol/L potassium dihydrogen phosphate buffer（pH 4.5）（57:43,v/v） was run at a flow rate of 1 ml/min for different biological matrix.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substance.The calibration curves were linear in each sample range with correlation coefficient above 0.999.The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 0.7～2.3%and 0.7～6.4%,respectively.The method has been successfully used to support the pharmacokinetics study of A771726.Part two:The pharmacokineties study of A771726 in ratsFollowing a single oral administration with different doses of A771726 to SD rats （3,6,12mg/kg）,the blood samples were collected at different time after dosing.All collected blood samples were centrifuged to obtain plasma and the concentrations of A771726 in plasma were determined by HPLC method described as above,then calculated corresponding pharmacokinetic parameters based on the time process of blood drug concentration.The blood plasma Cot curve of A771726 conformed to one compartment model of the first order absorption.The main pharmacokinetic parameters of A771726（3,6,12mg/kg） were T_1/2K2（h）:16.71±2.34,14.69±1.94,16.45±2.49, AUC_（0.tn）（mg/L·h）:271.38±78.87,785.57±170.92,994.13±165.43,C_max（mg/L）: 11.42±2.59,29.38±5.41,42.10±5.73,T_max（h）:4.00±0.82,4.10±0.74,4.20±1.03, MRT_（0-tn）（h）:22.07±1.80,21.91±1.60,20.71±1.88.The metabolism of A771726 following oral administration accords with the linear relation,and its blood plasma Cot curve consists with the first order kinetics of one compartment model.Part three:In vitro binding of A771726 to human plasma proteinsPlasma protein binding was determined by equilibrium dialysis（108h） at 4℃with final concentrations of 3,6 and 12μg/ml.After the dialysis,the concentrations of A771726 were determined by HPLC method.The binding ratio was calculated as percent of total concentration.The results indicated that the mean extent of binding to plasma proteins in human appeared to be in dependent of concentration used.The binding percentage of the drug was about 97.7%.Part four:The tissue distribution study of A771726 in ratsSD rats were killed by exsanguination at 2h,12h and 24h after oral administration （6mg/kg）.Tissues were taken out and made into homogenates preparation with normal sodium and the concentrations of A771726 in the tissues were determined by HPLC method.It was shown that A771726 was distributed to the major organism after oral administration.The A771726 concentrations were.high in liver,kidney,and low in brain, bone marrow,thymus and testis/uterus.The drug seems not accumulate in rats.Part five:Urinary,fecal and biliary excretion of A771726 after oral administrationBile,urine and feces of rats were collected after oral administration（6mg/kg）,and then the concentrations of A771726 were determined by HPLC method.Accumulative excretion amount of A771726 were 13.88%in bile,0.87%in urine and 1.61%in feces, respectively.更多还原