【作者】 王冠；

【导师】 王唯红；

【作者基本信息】 山东大学 ， 药物分析学， 2013， 硕士

【摘要】 肿瘤学研究表明,细胞周期失控是癌变的重要原因,在肿瘤细胞分化增殖信号通路中的许多关键酶现已成为药物筛选的重要靶点,其中,细胞周期蛋白依赖性激酶(CDKs)抑制剂可有效阻滞肿瘤细胞周期,抑制肿瘤细胞增殖。目前,处于新药开发后期和进入临床研究的CDKs抑制剂已有近20种,具有广阔的发展前景。抗肿瘤候选化合物L41是由山东大学药学院药物化学研究所徐文方教授课题组首次合成的查尔酮类新型CDKs抑制剂,前期药效学实验证实,L41对人结肠癌、肺癌、乳腺癌等细胞株的体外抗肿瘤活性以及抗荷肝癌H22小鼠血道转移、裸鼠异植HCT116肿瘤生长抑制的体内实验中,均表现出高效低毒的抗肿瘤作用。L41作为新型抗肿瘤候选化合物在国家科技重大专项“十二五”计划项目的支持下已进入新药研发早期的临床前药代动力学研究阶段。L41早期的药代动力学研究是其系统成药性研究的重要组成部分,本文旨在研究其临床前前药代动力学性质及代谢情况,以揭示L41在动物体内的动态变化规律,为L41的进一步开发和其他查尔酮类CDKs抑制剂的药代动力学研究提供参考依据。1.本文建立了用于测定大鼠血浆中L41浓度的高效液相色谱分析方法；血浆样品以液液萃取法提取L41与内标苯磺酸氨氯地平。色谱分析采用Agela Venusil MP-ODS色谱柱(250mm×4.6mm I.D.5μm),流动相为甲醇-50mM乙酸铵(77：23,V／V),流速为1mL/min,柱温30℃,检测波长343nm,进样量20μL。方法学验证结果显示标准曲线在15～1200ng/mL范围内线性关系良好,相关系数r为0.9990,定量下限为15ng/mL,该方法的批内、批间精密度(RSD)均小于6%,准确度的相对误差(RE)在±9%以内,可满足体内药物分析的要求。2.该分析方法用于大鼠血浆中L41浓度的测定及药代动力学研究。大鼠禁食12h后,分别单次灌胃(15,30和60mg/kg)和单次静注(6mg/kg)给予L41,测定给药后不同时间点血浆中L41的浓度,绘制血药浓度-时间曲线,采用DAS2.0软件计算主要药代动力学参数。结果表明,大鼠灌胃给药后三个剂量组的Cmax,AUC0-∞与给药剂量呈线性相关(r＞0.99,p＜0.05),其它药代动力学参数(Tmax、t1/2、CL/F和V／F)均无显著性差异(p＞0.05)。因此,在15～60mg/kg剂量范围内,L41在大鼠体内呈现一级动力学过程。尾静脉注给药后,计算出L41的平均绝对生物利用度为32.8%,250g大鼠的CL为7.8mL/min,与大鼠的肝血浆流量(13.8mL/min)接近,并远小于大鼠的心输出量(74mL/min),说明肝首过效应是L41口服绝对生物利用度的减少的主要原因。3.本文综合利用液相色谱与离子阱、三重四级杆质谱联用技术研究了L41在大鼠体内的代谢情况,在血浆与尿液中发现7个代谢产物。单次灌胃给予L41后,收集大鼠血.浆与尿液样本,用沉淀蛋白-冷冻分离-固相萃取法处理样品,经色谱分离后对代谢产物进行定性分析。所发现的7个代谢物情况如下：M5,M6为L41去甲基化代谢产物,M7为C-4’,6’双去甲基化后C-2’羟基甲基化代谢物,M1-M3为M5-M7葡萄糖醛酸化后Ⅱ相代谢物,M4为L41葡萄糖醛酸化Ⅱ相代谢物。更多还原

【Abstract】 Oncology research shows, the nature of malignant tumor is cell cycle out of control. Nowadays, gene products that regulate the cell cycle differentiation and proliferation signaling pathways have been investigated as anticancer drug targets. Cyclin-dependent kinases (CDKs) inhibitors could limit the progression of a tumor cell cycle and inhibit tumor cell proliferation effectively. At present, about20kinds of CDKs inhibitors are undergoing clinical studies and exploitation of CDKs inhibitors will make it a new highlight in anticancer field.L41as an antitumor candidate compound was first synthesized by Prof. Wenfang research group, Shandong University. The pharmacodynamic study of L41has shown anticancer activities on many kinds of cancer cells in vitro, such as human colon cancer cell, human lung cancer cell, human breast cancer cell, human cervical cancer cell, and human ovarian cancer cell. In addition, further research in vivo has demonstrated that L41shows potent tumor-inhibiting effects in BALB/cASlac-nu mice and H22transplanted rats. This study was supported by grants from the12th Five Years National Science and Technology Major Project of China. In order to early look insight into its metabolic and pharmacokinetic properties of this novel compound, and to lay foundations for further investigation, the present dissertation studied the metabolism and pharmacokinetics of L41by using several advanced methods for the fist time. 1. A high-performance liquid chromatography (HPLC) has been developed and validated for the determination of L41in rat plasma. The analyte and amlodipine besylate (IS) were extracted from plasma using liquid-liquid extraction. Chromatographic analysis performed by an Agela Venusil MP-ODS column (5μm,250mm×4.6mm I.D.) using methanol-50mM ammonium acetate (77:23, v/v) at a flow rate of1mL/min. Injection volume is20μL, column temperature maintained at30℃, UV absorbance was recorded at343nm. The assay was linear over the concentration range of15-1200ng/mL, correlation coefficient r was0.9990, the lower limit of quantification was15ng/mL in rat plasma with intra-and inter-day precision (RSD) less than6%and accurate as relative error (RE) within±9%.2. The method was successfully applied to determine L41in rat plasma, and the pharmacokinetics of L41was studied in rats after oral and intravenous administration. After fasted for12h, rats were given orally (15,30and60mg/kg) and a single intravenous (6mg/kg) of L41. Concentration of L41was measured at different time point. After that, concentration-time curves were drawn and DAS2.0software was used to calculate the main pharmacokinetic parameters. The results showed that the Cmax, AUC0-∞were proportional to the doses (r>0.99,p<0.05), other pharmacokinetic parameters (Tmax, t1/2, CL/F and V/F) showed no significant difference (p>0.05). After intravenous administration, the average absolute bioavailability of L41in rats was32.8%; the clearance value determined in the intravenous study was about7.8mL/min in a250g rat, this is very close to hepatic plasma flow value13.8mL/min and was considerably lower than the cardiac output value74mL/min in rats. This result indicated that if metabolism is hepatic, the reduced bioavailability may be due to a significant hepatic first-pass effect and the lung and/or heart first-pass effect of L41might be negligible, if any.3. In this study, we proposed a strategy to systematically characterize associated phase Ⅰ and phase Ⅱ metabolites of L41in rat plasma and urine. Biological samples were treated with an optimized sample preparation approach involving protein precipitation, liquid-liquid extraction and solid-phase extraction prior to LC-MS/MS analysis. Both an Agilent LC/MSD Trap VL and an AB SCIEX API5000triple quadrupole mass spectrometer were used for LC-MS/MS analysis. Based on proposed strategy,3phase I and4phase Ⅱ metabolites were detected by a highly sensitive MRM method. The major metabolites of L41to be demethylation (M5, M6), C-4’,6’di-demethylation followed by C-2’(OH) methylation (M7) and their glucuronide-conjugated species. These results provide evidence for in vivo L41metabolism, and they may be applied to further analysis of L41and its metabolites in biological samples.更多还原