【作者】 郑健；

【导师】 王洋； 周凡凡；

【作者基本信息】 东北林业大学 ， 细胞生物学， 2013， 博士

【摘要】 喜树碱(CPT)是20世纪60年代从珙桐科植物喜树中提取的天然生物碱,由于具有显著的抗癌活性,得到了广泛的重视。甲氧基喜树碱(MCPT)和羟基喜树碱(HCPT)均是喜树碱的天然衍生物,并具有较强的抗肿瘤活性。HCPT是我国临床应用的抗肿瘤药物,在药效学以及药剂学方面均有广泛的研究。关于MCPT的研究并不多见,这可能是由于MCPT较强的毒性所致。在我们的研究中发现,HCPT是MCPT的-种主要代谢产物,这激发了我们进一步研究MCPT的兴趣。在本文中我们研究了MCPT的细胞毒活性及其与细胞转运载体之间的相互作用,并进一步对MCPT及其代谢产物HCPT在大鼠血浆的药代动力学、组织分布特征以及排泄规律进行了研究。为了初步确定MCPT的抗肿瘤活性,以CPT和HCPT为对照,研究了MCPT对人卵巢癌细胞2774、结肠癌细胞系HT29.前列腺癌细胞系DU145、肺癌细胞系NCI-H520以及人胰腺癌细胞系MPC2的体外细胞毒活性。结果表明,MCPT在所测试的所有细胞系中的细胞毒活性均高于HCPT;除HT29外,MCPT在其他细胞系中均表现出比CPT更强的细胞毒活性。考察了CPT、MCPT和HCPT与多种转运蛋白的相互作用,发现这三种物质均能够有效抑制有机阴离子转运蛋白3(OAT3)的转运功能。这说明,这三种物质有可能是OAT3的底物。另外,由于OAT3主要分布于肾脏中,在肾脏毒素的排除中起到重要作用,这有可能是喜树碱类容易产生肾脏毒性的一个原因。利用HPLC-PDA和LC-MS/MS的方法确证了HCPT为MCPT的代谢产物,并建立了一种灵敏的、可靠的同时检测大鼠血浆中MCPT及其代谢产物HCPT的高效液相色谱-荧光检测的方法。考察了大鼠尾静脉注射5mg/kg MCPT后,MCPT及其主要代谢产物HCPT在血浆中药代动力学。尾静脉注射MCPT后,MCPT立刻达到最高浓度,随后浓度很快降低；而HCPT在5-10min后达到最高浓度,且明显高于MCPT,HCPT的药时曲线下面积约为MCPT的1.5倍,说明MCPT主要通过去甲基化作用代谢为HCPT。考察了大鼠尾静脉注射5mg/kg MCPT后,MCPT及其主要代谢产物HCPT在大鼠各组织中的分布情况。结果表明,肝脏、脾脏和肾脏中MCPT的含量较高,而在脑中的含量最少；而对于HCPT,在肝脏和肾脏中的含量最高,其他组织中的含量略少。比较分析了大鼠尾静脉注射MCPT后,MCPT及其代谢产物HCPT在胆汁、尿液和粪便中的排泄特征。结果表明,MCPT主要以胆汁的形式排出体外,而HCPT则主要以粪便的形式排除体外。本文对MCPT及其代谢产物HCPT在大鼠体内的药代动力学进行了研究,明确了MCPT和HCPT在大鼠体内的吸收、分布、代谢和排泄的规律,为MCPT的临床应用及合理开发提供了一定的理论依据。更多还原

【Abstract】 Camptothecin (CPT) is a naturally occurring alkaloid first isolated from the native Chinese tree Camptotheca acuminate Dence.(Nyssaceae) in1960s. CPT has been paid considerable attention because of its significant anti-tumor activity. Both10-methoxycamptothecin and10-hydroxycamptothecin are natural bioactive derivatives of camptothecin and have been confirmed to possess high anti-cancer properties. The reduced toxicity of HCPT campared to CPT has brought the growing interesting of in vivo and clinical studies on the pharmaceutics and pharmacology of HCPT. In contrast, few studies were carried out on MCPT due to its higher toxicity. Later, HCPT was found to be one of the major metabolites of MCPT metabolites in rat pasma after intravenous administration of MCPT, which brings new interest to the potential use of MCPT in the development of novel anticancer drugs. In the present study, the antitumor activity of MCPT against five different cancer cells as well as the interaction of MCPT, HCPT and CPT with solute carrier transporters was tested here. Further the pharmacokinetic studies, the tissue distribution studies as well as the excretion studies of MCPT and its metabolite HCPT were demonstrated.To elucidate the potential activity of MCPT, we tested its antitumor activity against five cancer cell lines, including2774(human ovarian cancer cell line), HT29(human colon cancer cell line), DU145(human prostate carcinoma cell line), NCI-H520(human lung cancer cell line) and MPC2(mice pancreatic cancer cell line), compared with CPT and HCPT. The results indicated that MCPT has higher anticancer activities against all the tested cancer cell lines comparted with HCPT, and showed higher anticancer acticities aganist all the tested cancer cell lines except nearly the same cytotoxicity against HT29cell line.In order to elucidate the contribution of solute carrier (SLC) transporters to the efficacy and toxicity of CPT, MCPT and HCPT, this study investigated the interactions of these compounds with thirteen essential SLC transporters, and our data revealed that they all significantly inhibited the function of OAT3, which implied that these three CPTs might be the potential substrates of OATs. This might be a possible reason of the kidney toxicity of CPTs because OAT3is expressed at the basolateral membranes of the renal proximal tubule where it facilated the first step in the clearance of uremic toxins.HCPT was identified as the major metabolite of MCPT in rat plasma through HPLC/photodiode array detection (PDA) and LC-MS/MS analysis. A sensitive and reliable RP-HPLC method with fluorescence detection was developed and validated for the simultaneous analysis of MCPT and HCPT in rat plasma. This method was also applied to the pharmacokinetic study of MCPT and its metabolite HCPT in rat plasma after intravenous administration.In this study, the mean plasma concentration-time curves of MCPT and its metabolite HCPT were determined for six rats each receiving a single5mg/kg i.v. dose of MCPT via the tail vein. After i.v. administration, the level of MCPT reached the maximum concentration, and declined immediately. HCPT concentration reached the maximum within the first5-10min post-administration of the dose, and after that HCPT level declined promptly.. The ratio of AUC0-∞of HCPT to that of MCPT was about150%, which demonstrated that the metabolism to the demethylated HCPT was the dominant metabolic reaction for MCPT in rats.The tissue distribution of MCPT and its metabolite HCPT were determined for rats after each receiving a single5mg/kg i.v. dose of MCPT via the tail vein. These results indicated that the content of MCPT was higher in liver, spleen and kidney, and was lowest in brain, while the content of HCPT was higher in liver and kidney than in other tissues.At last, the accumulation of MCPT and and its metabolite HCPT in excretion pathway was investigated after receiving a single5mg/kg i.v. dose of MCPT via the tail vein. The results indicated that MCPT was excreted in bile mostly, while HCPT was mainly excreted in feces.In summary, a pharmacokinetic study of MCPT and and its metabolite HCPT in rats were investigated, and the absorption, distribution, metabolism, excretion of MCPT was clarified, in order to provide a theories basis for the clinical medication, reasonable development and application of MCPT.更多还原