转运体介导乌苯美司发生药物相互作用和抗阿霉素多药耐药的分子药代动力学机制

Bestatin, a low molecular weight dipeptide, has been demonstrated to be a targeted drug with an antitumor activity. Our previous studies have demonstrated for the first time that bestatin was a substrate of P-gp in the rat small intestine. In addition, we have confirmed that bestatin was absorbed by the gastrointestinal oligopeptide transporter (PEPT1). Moreover, we speculate that bestatin is also a substrate of OATs in kidney. The fact indicated that the pharmacokinetics of bestatin was related to function of multi targets transporters. Thereby, we have established an LC-MS/MS method for the determination of bestatin and other drugs used in combination and also constructed the HeLa cells expressed PEPT1 stably to examine whether PEPT1 is the substrate of bestatin. The purposes of this study are (1)to investigate the drug-drug interactions (DDI)between bestatin and other coadministered drugs and clarify the molecular pharmacokinetic mechanism of DDI;(2)to clarify the mechanism for various transporters (such as P-gp, PEPT1, OAT1, OAT3 )mediated DDI for absorption and excretion of bestatin with other commonly used drugs; and (3)to identify the pharmacokinetic mechanism(s) for reversal of adriamycin resistance by bestatin, which were mediated by transporters in distribution of bestatin. We used in vivo oral administration, in situ intestinal perfusions, in vitro everted small intestinal sac preparations, in vitro kidney slices, Caco-2 cell and adriamycin-resistant leukemia cell uptake studies, qRT-PCR and Western blotting methods. This study focuses on the drug-drug interactions (DDI) and multidrug resistance which were mediated by transporters and provides important information for guiding proper clinical application for bestatin.

乌苯美司 (Bestatin)为有靶向抗癌作用的低分子二肽。前期工作首次发现其为大鼠小肠p-gp的底物，并证实其被小肠寡肽转运体PEPT1摄取，此外还为肾脏OATs的底物。这种多靶点转运体介导乌苯美司体内过程的事实驱使本研究拟建立LC-MS-MS方法同时检测乌苯美司及合用药物，构建PEPT1基因转染细胞，采用in vivo、小肠灌流、翻转小肠袢、新鲜大鼠肾切片及Caco-2细胞、阿霉素耐药白血病细胞等摄取技术和RT-PCR、Western blot等分子生物学方法，阐明多转运体靶点（p-gp、PEPT1、OAT1、OAT3等）介导乌苯美司在吸收和排泄过程与常用药物相互作用（DDI）及在分布环节转运体介导乌苯美司抗阿霉素多药耐药的分子药代动力学机制。本课题从乌苯美司DDI和抗阿霉素多药耐药二方面呼吁临床重视转运体介导的DDI和多药耐药，对指导临床安全合理用药有着重要的应用价值和临床意义。

项目的背景：乌苯美司 (Bestatin)为有靶向抗癌作用的低分子二肽。前期工作首次发现其为大鼠小肠p-gp的底物，并证实其被小肠寡肽转运体PEPT1摄取，此外还为肾脏OATs的底物。本课题的目的是阐明多转运体靶点（p-gp、PEPT1、OAT1、OAT3等）介导乌苯美司在吸收和排泄过程与常用药物相互作用（DDI）及在分布环节转运体介导乌苯美司抗阿霉素多药耐药的分子药代动力学机制。.主要研究内容、重要结果、关键数据及其科学意义：.1.建立生物样品中乌苯美司和缬沙坦、甲氨蝶呤、头孢克肟同时测定的LC-MS/MS分析方法。.2.摄取型转运体PEPT1和OATs介导的乌苯美司与缬沙坦的药物相互作用及其分子机制：用大鼠in vivo药动学实验、in vitro翻转肠摄取实验和in vitro肾切片摄取实验考察乌苯美司和缬沙坦的药动学DDI。通过hPEPT1和hOAT1/3转染细胞摄取细胞揭示转运体介导的乌苯美司与缬沙坦DDI的分子药动学机制。.3.转运体介导的抗阿霉素多药耐药的分子药代动力学机制。.通过以上研究，得出结论，缬沙坦可抑制PEPT1和 OAT1/3的转运功能，从而抑制合用的乌苯美司的肠道吸收和肾脏清除。（2）OAT1/3可介导缬沙坦的肾脏清除。（3）拟肽类药物和沙坦类药物合用时，可能诱导转运体介导的DDI。（4）乌苯美司是P-gp的底物，P-gp介导的外排转运导致乌苯美司肠道吸收减少，以及肿瘤细胞耐药性的产生。在临床使用过程中，须留意乌苯美司与其他抗肿瘤药合用时，可能发生的以P-gp为靶点的DDI。（5）当同时口服或静脉给予大鼠乌苯美司和头孢克肟时会发生DDI。（6）PEPT1和OAT1/3是乌苯美司和头孢克肟发生DDI的靶转运体，提示两药在人体发生DDI的可能。（7）Bestatin是OAT1和OAT3的底物； PAH、PCG以及JBP485通过抑制OAT1和OAT3影响bestatin的肾排泄;当bestatin和丙磺舒、PAH、PCG和JBP485同时使用时会发生药物相互作用。当MTX和bestatin联合应用时，会发生由药物转运体所介导的药物相互作用；MTX和bestatin药物发生相互作用的第一靶点位于肠道的P-gp，第二靶点位于肾脏的OAT1和OAT3。本课题的结果为临床安全合理联合用药提供了理论和实验证据。

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