【作者】 段明郁；

【导师】 钟大放；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2006， 博士

【摘要】 昂丹司琼结构中含有一手性碳原子，临床现以消旋体给药；昂丹司琼与5-羟色胺有相似的母核，是强效、高选择性的5-羟色胺-3受体（5-HT₃）拮抗剂，有强镇吐作用。化疗药物和放射治疗可造成小肠释放5-羟色胺（5-HT），经由5-HT₃受体激活迷走神经的传入支，触发呕吐反射。昂丹司琼能阻断这一反射的触发，临床主要用于治疗因放疗及化疗诱发的恶心、呕吐。前期对昂丹司琼的代谢和动力学的研究表明，昂丹司琼在肝脏的细胞色素P450酶催化下广泛代谢，代谢途径包括苯环羟基化、N-去甲基化、羟基化后的代谢产物与葡萄糖醛酸或硫酸结合。进一步深入研究昂丹司琼的代谢、立体选择性药物动力学及其产生的机理，对指导昂丹司琼单一光学活性药物的研究开发具有重要意义。一、目的分离并鉴定昂丹司琼的代谢产物，系统研究昂丹司琼在哺乳动物体内的代谢；建立测定生物样品中昂丹司琼及其代谢产物含量和手性固定相拆分昂丹司琼对映体的方法，研究昂丹司琼的立体选择性药物动力学，并阐明其产生的机理；研究左昂丹司琼临床前药物动力学，为手性药物的研究开发提供依据。二、方法分离纯化昂丹司琼的卷枝毛霉AS 3.3421转化液和大鼠多剂量灌胃给予昂丹司琼后累积的尿样、粪样和胆汁中的昂丹司琼代谢产物，并通过核磁共振光谱和电喷雾离子化质谱确定其结构。采用液相色谱-多级质谱联用（LC／MSⁿ）的分析方法，结合分离得到昂丹司琼代谢产物的色谱行为和多级质谱特征，研究了昂丹司琼在微生物模型中和哺乳动物体内的代谢。建立测定生物样品中昂丹司琼的LC／MS／MS法，研究了大鼠单剂量静脉给予昂丹司琼单一对映体后的药物动力学，和左昂丹司琼的临床前药物动力学，并结合手性固定相拆分昂丹司琼对映体的方法，研究了昂丹司琼在大鼠体内构型的稳定性和立体选择性药物动力学。建立测定生物样品中昂丹司琼代谢产物的LC／MS／MS法，研究了昂丹司琼的立体选择性药物动力学机理。三、结果共分离纯化并鉴定了11种昂丹司琼的代谢产物，分别为8种Ⅰ相代谢产物、3种Ⅱ相代谢产物（2种葡萄糖醛酸结合物、1种硫酸结合物），为昂丹司琼代谢和立体选择性药物动力学研究提供对照品。采用LC／MSⁿ法对昂丹司琼及其代谢产物进行多级质谱分析，总结其质谱特点为：Ⅱ相代谢产物（葡萄糖醛酸结合物、硫酸结合物）在进行二级全扫描分析时，首先丢失结合基团而得相应的母核部分；母核部分进行三级全扫描分析时所得的碎片与相对应的羟基化代谢物的二级全扫描碎片相同。采用LC／MSⁿ分析方法研究了昂丹司琼在微生物模型中和在哺乳动物（大鼠、和人）体内的代谢情况，共发现26种代谢产物。综合分析每种代谢产物的准分子离子、多级碎片离子和色谱保留时间，并与昂丹司琼及制备的11种代谢产物对照品进行比较，鉴定代谢产物的化学结构分别为昂丹司琼原形（M0）的1-羟基化代谢产物（M1、M2），1-羟基化后与葡萄糖醛酸结合代谢产物（M20）；2-羟基化代谢产物（M13）；5-羟基化代谢产物（M12），5-羟基化后与葡萄糖醛酸结合代谢产物（M19）；6-羟基化代谢产物（M6），6-羟基化后与葡萄糖醛酸结合代谢产物（M18）、与硫酸结合代谢产物（M24）；7-羟基化代谢产物（M5），7-羟基化后与葡萄糖醛酸结合代谢产物（M10）、与硫酸结合代谢产物（M23）；8-羟基化代谢产物（M7），8-羟基化后与葡萄糖醛酸结合代谢产物（M17）、与硫酸结合代谢产物（M11）；N-去甲基代谢产物（M8），同时发生1-羟基化和N-去甲基代谢产物（M15、M16）：同时发生6-羟基化和N-去甲基代谢产物（M14），而后与葡萄糖醛酸结合代谢产物（M22）；同时发生7-羟基化和N-去甲基代谢产物（M3），而后与葡萄糖醛酸结合代谢产物（M9）、与硫酸结合代谢产物（M25）；同时发生8-羟基化和N-去甲基代谢产物（M4），而后与葡萄糖醛酸结合代谢产物（M21）、与硫酸结合代谢产物（M26）。观察到昂丹司琼有10种代谢途径，其包括：1-羟基化、2-羟基化、5-羟基化、6-羟基化、7-羟基化、8-羟基化、N-去甲基、羟基化同时N-去甲基、羟基化后与葡萄糖醛酸结合和硫酸结合。其中昂丹司琼的1-位的羟基化为首次发现的代谢途径，并且证明此代谢途径是由CYP1A2催化的。大鼠静注昂丹司琼单一对映体后在体内不发生构型转化。昂丹司琼对映体在大鼠体内的药物动力学具有立体选择性，左旋昂丹司琼代谢速度比右旋昂丹司琼代谢慢；左昂丹司琼的大鼠血浆蛋白结合率比右昂丹司琼略低：大鼠肝微粒体、人肝微粒体和重组酶孵化实验证实昂丹司琼对映体经酶催化为N-去甲基昂丹司琼、7-羟基昂丹司琼和8-羟基昂丹司琼的代谢途径存在立体选择性。大鼠静注左昂丹司琼后，3个剂量组AUC_0-∞与给药剂量线性相关；大鼠静注左昂丹司琼后，在各组织中广泛分布，肺组织中含量最高：左昂丹司琼在大鼠体内消除较快，3.0 h时各组织中浓度不足0.5 h时的20％，表明它不易在各组织中蓄积；左昂丹司琼在脑组织中含量较低，表明它不易透过血脑屏障。四、讨论昂丹司琼在微生物模型中和哺乳动物体内代谢的差异一方面表现在Ⅰ相代谢产物的数量上，微生物模型中Ⅰ相代谢产物种类少而且比较专一（M1、M2、M7和M8），而在哺乳动物体内，代谢产物种类多而广泛，多达13种；另一方面在微生物模型中未检测到Ⅱ相代谢产物，而在哺乳动物体内，有13种Ⅱ相代谢产物，分别为8种葡萄糖醛酸结合物和5种硫酸结合物。昂丹司琼在大鼠体内立体选择性药物动力学主要产生于立体选择性的代谢，酶促动力学差异是昂丹司琼对映体立体选择性物动力学的主要原因。昂丹司琼对映体在人肝微粒体中的动力学也存在差异，推断昂丹司琼对映体在人体内的动力学也存在立体选择性。更多还原

【Abstract】 Ondansetron, 1, 2, 3, 9-tetrahydro-9-methyl-3-[（2-methy-1H-imidazol-1-yl）methyl] -4H-carbazol-4-one, is a 5-hydroxytryptamine type 3 （5-HT₃） receptor antagonist used in the treatment of chemotherapy- and radiotherapy-induced nausea and emesios. In previous studies on the metabolism and pharmacokinetics of ondansetron, it is extensively metabolized in the liver through the cytochrome P450 system. Ondansetron undergoes metabolic pathways including hydroxylation of benzene ring followed by glucuronide or sulphate conjugation and N-demethylation.1. ObjectiveThe thesis aimed to isolate and identify metabolites of ondansetron, to investigate ondansetron metabolism in microbial model and in mammals, to study the stereoselective pharmacokinetics of ondansetron, and to gain further understanding stereoselective pharmacokinetics mechanism of ondansetron.2. MethodMetabolites of ondansetron were isolated from the biotransformed culture sample of ondansetron by Mucor circinelloides AS 3.3421 and urine, feces and bile sample of rat after multi-dose oral administration of ondansetron and identified by NMR and ESI-MS. Metabolites of ondansetron in the microbial model and in mammals were investigated by LC/MSⁿ method. The stereoselective pharmacokinetics of ondansetron was studied by assays for determination of ondansetron, its metabolites by LC/MS/MS, the stereoselective pharmacokinetics mechanism was studied by ondansetron enantiomers chiral separated and determination of ondansetron metabolites by LC/MS/MS.3. ResultsA total of 11 metabolites of ondansetron were isolated from the biotransformed culture sample of ondansetron by Mucor circinelloides AS 3.3421 and urine, feces and bile sample of rat. They were 8 phase I metabolites, 3 phase II （2 glucuronides, 1 sulphate conjugations） metabolites, which could be used as reference substances in the studies on metabolism and stereoselective pharmacokinetics of ondansetron. Using multi-stage ion trap mass spectrometric analysis, the characteristic fragment ions of ondansetron and its metabolites were obtained.Metabolites of ondansetron in the microbial model of and in mammals （rat and human） were investigated. A total of 26 metabolites were found and identified by comparisons of their chromatographic behaviors and multi-stage mass spectra to those of ondansetron and 11 isolated standards. These metabolites included 1-hydroxy-ondansetron （M1, M2）, conjugated metabolite of M1 with glucuronic acid （M20）; 2-hydroxy-ondansetron （M13）; 5-hydroxy-ondansetron （M12）, conjugated metabolite of M12 with glucuronic acid （M19）; 6-hydroxy-ondansetron （M6）, conjugated metabolites of M6 with glucuronic acid （M18） and with sulphuric acid （M24）; 7-hydroxy-ondansetron （M5）, conjugated metabolites of M5 with glueuronic acid （M10） and with sulphurie acid （M23）; 8-hydroxy-ondansetron （M7）, conjugated metabolites of M7 with glucuronic acid （M17） and with sulphuric acid （M11）; N-demethyl-ondansetron （MS）; M1 and M2 N-demethylation （M15, M16）; M6 N-demethylation （M14）, conjugated metabolite of M14 with glucuronic acid （M22）; M5 N-demethylation （M3）, conjugated metabolites of M3 with glucuronie acid （M9） and with sulphuric acid （M25）; M7 N-demethylation （M4）, conjugated metabolites of M4 with glucuronic acid （M21） and with sulphuric acid （M26）. The metabolic pathways of ondansetron in the microbial model and in mammals were oxidation of N-demethylation, 1-hydroxylation, 5-hydroxylation, 6-hydroxylation, 7-hydroxylation, 8-hydroxylation, combination of N-demethylation and hydroxylation, hydroxylation followed by glucuronide or sulpate conjugation. One new metabolic pathway （hydroxylation on 1-position） was found in this study, and the new pathway was mediated by CYP1A2.The stereoselective pharmacokinetics showed that R-ondansetron cleared slower than S-ondansetron in rat. The stereoselective pharmacokinetics of ondansetron enantiomer was caused by stereoselecive metabolism of cytochrome P450 enzymes: CYP1A2, 2C9, 2C19, 2D6 and 3A4.4. DiscussionMore metabolites of ondansetron in mammals were detected than microbial model, and no metabolites of phaseⅡwas detected in microbial model. There should be stereoselective pharmacokinetics of ondansetron enantiomers in human, because the stereoselecive metabolism was found in human liver microsomes.更多还原