【作者】 郑维思；

【导师】 陈一岳；

【作者基本信息】 广东药学院 ， 药剂学， 2007， 硕士

【摘要】 研究背景手性（Chirality）是一种化学结构特征,它引起分子的不对称性。近年来,药物手性的临床意义已引起了人们的注意。人体的手性环境和特异的对映体相互作用,导致手性药物对映体药代动力学和药效学的立体选择性（stereo-selectivity）差异。在药理手性差异中研究较多的是药代动力学的手性特征。它是基于药物吸收、分布、代谢和排泄过程的对映体选择性。其中代谢性对映体选择性发生率最高（约占40%）,且临床意义更大。以往由于没有认识到手性药物各对映体的药动学行为,特别是代谢的手性差异,所以在药物相互作用研究中常常将消旋体药物当作单一化合物来处理。由此得出的结论常与临床疗效或不良反应的发生存在着不一致的现象,甚至会错误地指导临床用药。药物代谢主要在肝脏,依赖于细胞色素P450 （ cytochrome P450, CYP 450） ,它是药物进入体内进行生物转化的重要代谢酶,参与各种内源性和外源性化合物在体内的代谢过程。该酶系具有专一性不强的特点,即同一种酶可以代谢多种药物,而同一药物又可被多个肝药酶所代谢。由于在肝细胞内蕴含有大量不同种类的肝药酶,每一种药物进入体内后,都可能由多种酶参与其代谢。因此宏观的血药浓度只能检测出总体结果,不能考察每种酶对药物的代谢情况。在联合用药过程中,特别是其中某一药物使参与催化代谢的酶活性显著增强或减弱时,就会引起其他合用药物的毒副作用出现或未达疗效的情况。对于对映体来说,酶还表现出立体选择性的差异,从而可能使其中占主药效的一个对映体的代谢速度改变甚至逆转。因此,了解各种酶参与对映体的代谢的过程,可为临床用药提供参考依据。本实验研究的手性药物是普萘洛尔（Propranolol, PPL）对映体。普萘洛尔为非选择性β-肾上腺素受体阻滞剂,口服后药物达峰时间为1～3小时,t1/2为2～5小时。主要在肝脏代谢,首过效应60%～70%,生物利用度仅为30%。临床上使用的普萘洛尔是左旋异构体S（-）- PPL和右旋异构体R（+）- PPL等量混合的消旋品。目前已证实S（- ）型对映体的β受体阻断作用要比R （+）型约强100倍。普萘洛尔在CYP450酶系中代谢,各亚族均有不同程度的催化作用,文献显示以CYP 2D6和CYP 1A2作用较强。以往的研究主要是集中在两个酶对普萘洛尔的代谢,而对其它酶参与其代谢的研究则甚少涉及。本课题选择CYP 1A1和CYP 3A4进行考察。其主要的原因是CYP 1A1虽在肝脏中的含量很低,但此酶极容易被诱导、活化,使肝脏含量迅速增加。许多外源性化合物经CYP 1A1代谢后可产生有毒的代谢产物,能诱发肿瘤的产生与发展;CYP 3A4是CYP 450酶系中最重要的亚型,约占成人肝微粒体CYP450总量的30 %～40 %。因此,有必要对这两种具代表性的肝药酶在普萘洛尔对映体的代谢过程中的参与情况进行研究和探讨。肝细胞是在人类中检测CYP 450酶活性诱导或抑制最合适的实验研究平台,是最接近临床的研究系统。普萘洛尔甚少体外药动学资料,特别是其对映体的药代动力学参数资料。因此,本实验设计以体外培养人肝细胞作为代谢反应系统。对普萘洛尔对映体在经诱导的肝细胞中代谢过程进行了研究,其目的是明确肝药酶CYP 1A1和CYP 3A4是否参与普萘洛尔对映体的代谢,并探究其酶动力学和药代动力学的特征,同时为分子生物学基础研究和临床合理用药提供参考和实验依据。本实验包括四方面的内容。一、肝细胞培养以及肝药酶CYP 1A1、CYP 3A4活性的测定目的采用人肝细胞作为体外代谢系统,测定经不同浓度的诱导剂诱导后酶代谢底物的情况,以此来描述酶活性的变化,并确定诱导剂的最佳诱导浓度。方法通过肝细胞的传代培养,用MTT法测定细胞存活率;再分别以7-ER和睾酮为底物,测定CYP 1A1和CYP 3A4的活性变化,以确定BNF诱导CYP1A1和RIF诱导CYP3A4的最佳浓度。结果BNF诱导CYP 1A1的最佳药物浓度为0.8μmol·L^-1,而RIF诱导CYP 3A4的最佳药物浓度为15μmol·L^-1。二、普萘洛尔对映体的手性拆分目的采用光学纯对映体分别加入细胞中进行代谢,但对映体在代谢过程中可能发生转化,需要将经过代谢后的对映体进行拆分来验证。方法选用GITC作为柱前衍生化试剂,进行对映体的柱前衍生化,并通过反相高效液相色谱法检测。结果在本实验的衍生条件下,消旋体可被完全衍生;普萘洛尔对映体在肝细胞系统代谢过程中没有发生结构转化,可以直接向肝细胞中分别加入R（+）-PPL和S（-）-PPL进行代谢,检测液无需再进行拆分前处理。三、HPLC中检测细胞液中普萘洛尔对映体的浓度目的建立一种用于体外细胞实验检测药物浓度的高效、简便、快速、专属性强的分析方法。方法1.反相高效液相色谱-荧光检测法定量检测普萘洛尔对映体的浓度。2.通过指定标准曲线,测定萃取回收率、方法回收率和精密度,对HPLC方法学进行确证。结果1. S（-）-PPL和R（+）-PPL在测定范围内（0.5～20μmol·L-1）线性关系良好,它们的回归方程分别为Y = 0.5627X + 0.0581和Y = 0.6699X - 0.2068,相关系数（r）分别为0.9993和0.9994。最低定量浓度（LOD,S/N≥9）均为0.5μmol·L-1。2. S（-）-PPL和R（+）-PPL萃取回收率均大于80%,方法回收率均大于90%。日内差均小于8.8%,日间差均小于13.1%。3.本方法快捷、准确、专一性强,可用于细胞液样品中的药物研究。四、普萘洛尔对映体的代谢特征目的通过处理酶动力学参数,考察CYP 1A1和CYP 3A4是否参与普萘洛尔的代谢,并且探究对映体在代谢过程中的立体选择性;同时处理和归纳药代动力学参数,为临床合理用药提供参考。方法1.测定在BNF和INF的诱导下,以及不经诱导时普萘洛尔酶动力学参数。2.分别测定不同时间点时经BNF和RIF的诱导以及不经诱导空白对照的普萘洛尔剩余浓度,建立底物浓度-反应时间曲线。结果1.BNF诱导的CYP1A1明显增强了两对映体催化能力,尤其是对S（-）型对映体的选择性。CYPP 3A4在普萘洛尔的代谢中作用较大,并表现出对R（+）-普萘洛尔作用较强的立体选择性。2.R（+）-PPL比S（-）-PPL的代谢消除快。经过诱导后,两对映体的代谢均加快。结论1.本实验所选取的两个酶均参与了普萘洛尔对映体的代谢。2.肝药酶CYP 1A1对S（-）-PPL有较强的立体选择性,而肝药酶CYP 3A4则对R（+）-PPL有显著的立体选择性。3. R（+）-PPL比S（-）-PPL的代谢消除快。诱导CYP 3A4活性的药物能显著增加R（+）-PPL的代谢,而诱导CYP 1A1活性的药物能显著增加S（-）-PPL的代谢。更多还原

【Abstract】 Chirality is a structural characteristics of chemical substance. Almost 75% drugs used in clinical practice exist molecular asymmetry, so called as chiral drugs. Recently, the clinical significance of drug chirality has been closely remarked. The interaction of demic chiral environment and specific enantiomers has resulted in pharmacokinetic or/and pharmacodynamic stereoselectivity differences between various enantiomers. Chiral characteristics of pharmacokinetics are studied much more than of pharmacodynamics. The former is based on the stereoselective processes of drug absorption, distribution, metabolism and excretion. Among them, the incidence rate of metabolic stereoselectivity of drugs is the highest (ca. 40%). However, the researches on drug-interaction were usually dealt with racemics as single compounds owing to ignoring the differences in metabolic stereoselectivity between enantiomers. As a result, the obtained conclusions were probably discordancy in curative effect and adverse reaction, and can not correctly guide rational use of clinical medication.Drugs are mainly metabolized in liver, depending on cytochrome P450 (CYP 450). It is essential enzyme system of drug biotransformation and participates metabolic processes of endogenous and exogenous compounds in vivo. There are generous liver drug enzymes in organelle of liver cells. Thus every drug will be metabolized by various kinds of enzymes. Blood drug level could just figure out total results without detecting metabolic participation of enzyme. There would be side effects or inadequacy of therapeutic effects during drug combination, especially when the drugs could strikingly strengthen or reduce enzyme activity. And enzymes would also show up stereoselectivity to entiomers, altering or even deteriorating the accretion rate of one of them.The chiral drug of this assay is propranolol(PPL). Propranolol is nonselective blocking agent ofβ-adrenoceptor. Cmax after oral administration is 1-3 hours, and t1/2 is 2-5 hours. It is mainly metabolized in liver, with 60%-70% first pass effect and 30% bioavailability. Propranolol is used clinically as racemics with partes aequales of S(-)-PPL and R(+)-PPL, but theβ-adrenoceptor blocking effects of S(-)-PPL is 100 times more than R(+)-PPL. There are various kinds of enzyme participating metabolism of propranolol in CYP 450, especially CYP 2D6 and CYP 1A2. Nevertheless, the previous researches just concentrate on portrait study of them. This essay selected CYP 1A1 and CYP 3A4 to investigate. The contents of CYP 1A1 are very low in liver. But it is very easily induced to create tumors. CYP 3A4 is the major components of CYP 450, occupying 30%-40%. Therefore, it is necessary to study these two enzymes participation in metabolism of propranolol enantiomers.Hepatocytes are suitable for studying in CYP 450 enzyme activity induction and inhibiton. This essay was used human hepatocyte as reaction system, and did the research in metabolic characteristics of the enantiomers of propranolol in the human hepatocytes treated with different inducers, including enzymatics and pharmacokinetics, in order to identify whether the enzymes participate the metabolism of enantiomers.I. The culture of human hepatocytes and the activity detection of CYP 1A1, CYP 3A4Object To observe metabolism with enzyme in hepatocytes. To describe enzyme activity through detection of substrates metabolism by enzyme treated with inducers and define the best induced concentration.Method 1. The culture of human hepatocytes.2. Detection of cells survival rate with MTT.3. Using 7-ER and Rifampicine as specific substrates to determine the activities of CYP1A1 and CYP3A4 and to define the best induced concentration.Result 1. The best induced concentration of BNF is 0.8μmol·L-1. 2. The best induced concentration of RIF is 15μmol·L-1.II. Chiral separation of propranolol enantiomersObject To verificate whether the enantiomers would convert structure after metabolism.Method The enantiomers were reacted with a pre-column chiral derivatization reagent 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate(GITC) and separated on HPLC.Result The enantiomers could be separated completely with this method.And they don’t convert after metabolism. They could be detected without separation.III. HPLC detection of propranolol enantiomers in cell culturesObject To establish a high performance, convenient, fast method for drug detection in medium in vitro.Method 1. Preparation of standard solution and sample pretreatment.2. HPLC detection of propranolol enantiomers and methodology corroboration.Result 1. The regression equation of S(-)-PPL and R(+)-PPL are Y = 0.5627X + 0.0581 and Y = 0.6699X - 0.2068. correlation coefficient are 0.9993 and 0.9994. The minimum quantitated concentration(LOD,S/N≥9)is 0.5μmol·L-12. The extraction are larger than 80% and method recovery are 90%. The intra-day and inter-day precision.3. This method is sensitive, accurate, and could be used as study of medium sample.IV. Metabolic characteristics of the enantiomers of propranololin the human hepatocytesObject To inspect whether the enzyme participate propranolol metabolism.Method R(+) , S(-) propranolol are metabolized by BNF and RIF induced cells. Then the substrate concentration-time curves and enzyme parameters (Km , V max) of carvedilol enantiomers were provided.Result Enantiomers were metabolized faster after induction. Enzyme catalytic abilities had a stereoselectivity to R(-)-PPL in control group and RIF induced group, while there is stereoselectivity to S(-)-PPL in BNF induced group.Conclusion1. Both these two enzymes are partipated in metabolism of propranolol enantiomers.2. their catalytic abilities had a stereoselectivity to R(-)-PPL in control group and RIF induced group, while there is stereoselectivity to S(-)-PPL in BNF induced group.3. Metabolism of R(-)-PPL will be increased when used the drugs which can strikingly strengthen the activity of CYP 3A4, while metabolism of S(+)-PPL will be increased when used the drugs which can sharply increase the activity of CYP 1A1.更多还原