【作者】 张寅瑛；

【导师】 洪战英；

【作者基本信息】 第二军医大学 ， 药物分析学， 2012， 硕士

【摘要】 近年来，手性药物在临床上的应用已受到越来越广泛的关注，评价手性药物对映体间在体内的立体选择性特征，对于药物的安全、有效至关重要。手性药物对映体在药物代谢的立体选择性表现在药物的吸收、分布、代谢和排泄等各个环节。仅以消旋体的形式无法准确阐明手性药物在体内的动态变化，而应分别研究手性对映体的药动学特征、药物代谢酶影响及其代谢途径。本课题选择镇痛中药延胡索中的四氢小檗碱（tetrahydroberberine，THB）和四氢巴马汀（tetrahydropalmatine，THP）为研究对象，采用HPLC手性固定相模式建立血浆中THB对映体的定量方法，研究THB对映体在SD大鼠体内的药动学特征；利用MDCK-MDR1单层细胞模型研究THB单一对映体的跨膜转运机制；采用“cocktail”探针法评价THP对映体对大鼠肝细胞色素P450酶亚型活性的影响；利用UPLC-Q-TOF/MS和LC-MS/MS分析技术，对大鼠灌胃给药四氢巴马汀单一对映体后尿液中THP对映体代谢产物及体内代谢途径进行推测。获得此类四氢原小檗碱同类物在动物体内的手性药代动力学特征，可能产生的代谢产物以及对映体对CYP450同工酶活性的影响，具体内容包括：1.四氢小檗碱在大鼠血浆中的手性药代动力学研究建立血浆中THB对映体的HPLC手性固定相定量方法，血浆样品采用SupelcleanLC-18固相萃取柱提取，色谱柱为Chiral AD柱(250mm×4.6mm，5μm)；流动相组成为甲醇-乙醇（80:20，v/v），流速：0.4mL/min，检测波长230nm，柱温：25℃。经方法学验证，d-THB在0.010～2.5μg/mL范围内线性关系良好，l-THB在0.010～5.0μg/mL范围内线性关系良好。THB对映体的萃取回收率均大于70%，日内、日间RSD均小于15%。应用以上方法测定了SD大鼠灌胃给药50mg/kg和静注给药10mg/kg消旋体THB后的对映体血浆浓度，获得相应的药动学参数，灌胃组d-THB和l-THB的Cmax，AUC0-∞分别为1.09μg/mL，1.91μg·h/mL和2.01μg/mL，4.15μg·h/mL，均存在显著性差异（P<0.05）；静注组d-THB和l-THB的AUC0-∞分别为1.95μg·h/mL和2.79μg·h/mL，存在显著性差异（P<0.05）。结果表明，THB在大鼠血浆中药代动力学具有立体选择性特征。2. THB对映体在MDCK-MDR1细胞中的跨膜转运研究利用MDCK-MDR1单层细胞模型研究THB单一对映体的双向转运特性，采用高效液相色谱法测定THB单一对映体的含量，计算其表观渗透系数（Papp）。结果显示，l-THB从MDCK-MDR1细胞模型A面到B面的表观渗透系数Papp（A→B）为1.27×10-5nm/s，而从B面到A面的表观渗透系数Papp（B→A）为5.65×10-5nm/s，外排率为4.45；d-THB从A面到B面的表观渗透系数Papp（A→B）为1.27×10-5nm/s，而从B面到A面的表观渗透系数Papp（B→A）为4.67×10-5nm/s，外排率为3.68。由此说明，l-THB和d-THB在MDCK-MDR1单层细胞模型中的转运可能涉及到主动转运机制。3.“Cocktail”探针药物法评价THP对映体对大鼠肝细胞色素P450酶亚型活性的影响建立同时测定大鼠血浆中5种CYP450探针药物，即美托洛尔（CYP2D6）、咖啡因（CYP1A2）、氨苯砜（CYP3A4）、氯唑沙宗（CYP2E1）和甲苯磺丁脲（CYP2C9）的HPLC-MS方法，并采用“cocktail”探针药物法快速评价THP对映体对上述同工酶的影响。将SD大鼠随机分成4组，每组6只，分别给药d-THP、l-THP、dl-THP，空白对照组给予生理盐水。给药7天后静注给予5种探针药物混合溶液。采集不同时间点的血样检测各探针药物浓度，通过药动学参数来评价各组CYP450酶系的活性。结果表明，给予d-THP组的美托洛尔和咖啡因代谢减慢，半衰期t1/2延长；给予l-THP组的咖啡因代谢减慢，半衰期延长，氨苯砜和甲苯磺丁脲代谢加快，半衰期缩短；给予dl-THP组的咖啡因代谢减慢，半衰期延长，氨苯砜、氯唑沙宗和甲苯磺丁脲代谢均加快，半衰期缩短。由此推测，d-THP对CYP2D6和CYP1A2有抑制作用，l-THP对CYP1A2有抑制作用，对CYP3A4和CYP2C9有诱导作用，消旋体THP对上述同工酶的影响作用受d-型和l-型的综合影响。4.四氢巴马汀对映体在大鼠尿中代谢产物的鉴定及排泄动力学研究采用超高效液相色谱-四级杆飞行时间质谱法（UPLC-Q-TOF/MS）鉴定大鼠灌胃给药THP单一对映体后尿中的代谢产物。利用精确分子量和高碰撞能下碎片离子信息对代谢产物进行解析。结果在大鼠灌胃给药d-THP和l-THP后的尿样中，除原型药物外，分别检测到了32种和30种代谢产物，四氢巴马汀对映体在尿中代谢产物主要为一去甲基、二去甲基、羟基化、葡萄糖醛酸和硫酸化结合产物。而三去甲基化和二去甲基化羟基化产物仅在给予d-THP的尿样中检测到，提示四氢巴马汀对映体代谢可能存在立体选择性。建立了快速灵敏的LC-MS/MS定量分析方法，测定大鼠尿样中THP单一对映体浓度，并对尿样中的主要代谢产物进行半定量分析。SD大鼠灌胃给药d-THP或l-THP（60mg/kg）后，于给药后收集72h内不同时间段尿液，采用已建立的LC-MS/MS法分别测定了给药后各时间段内THP单一对映体的浓度。将原型THP对映体的尿样标准曲线方程用摩尔浓度表示，在THP尿中代谢产物定性研究的基础上，根据UPLC-Q-TOF/MS提供的各代谢产物精确分子量及各时段尿排泄体积，推算代谢产物的尿排泄量，结果表明，72h内d-THP和l-THP的累积排泄量分别为53.23±27.33μg和101.55±43.31μg，两者的比例（l/d）为2.23，累积排泄率为0.37%和0.72%；采用半定量法计算得到主要II相代谢产物，一去甲基硫酸化结合产物（M9-1和M9-2）和主要I相代谢产物，一去甲基产物（M7-2和M7-3），二去甲基产物（M3-2）的累积排泄量，I相均高于II相产物，且比较代谢产物各时间点的累积排泄量，均以给予l-THP较高，由以上结果我们推断THP对映体仅有少量经肾排泄，且尿中原型和代谢产物的排泄具有立体选择性，以l-THP占优势。更多还原

【Abstract】 In the past few years, researches on chiral drugs have drawn global attention. Theevaluation of stereoselective characteristics of enantiomers in vivo is of paramountsignificance for developing effective and safe chirality drugs. Stereoselectivity plays animportant role in pharmacokinetics involving the absorption, distribution, metabolism, andexcretion of chiral drugs. It is necessary to evaluate the pharmacokinetic behavior, effectson the main drug-metabolism enzymes and the metabolic pathway of each enantiomerrather than those of the racemate.In this paper we study on tetrahydroberberine (THB) and tetrahydropalmatine (THP),which are two biologically active components of Chinese medicinal herb named Corydalisyanhusuo W.T. Wang. An enantioselective HPLC method was developed for thequantification of tetrahydroberberine (THB) enantiomers in rat plasma and the chiralpharmacokinetics of THB was investigated in rats. Transmembrane transport mechanismof tetrahydroberberine (THB) enantiomers was studied by a MDCK-MDR1cellmonolayers. The effect of THP enantiomers on the activity of cytochrome P450isozymesin rats was evaluated using a cocktail probe drugs method. The metabolites were identifiedby UPLC and the major metabolic pathways of THP enantiomers in rat urine wereproposed after oral administration of THP enantiomers by ultra performance liquidchromatography coupled to time-of-flight mass spectrometry. These studies aimed toclarify the stereoselective characteristics of tetrahydroprotoberberines (THPBs) analoguesin pharmacokinetics and to investigate the metabolites and effects of THPBs on the activityof cytochrome P450isozymes. The detailed results were presented as follows.1. Studies on chiral pharmacokinetics of tetrahydroberberine in ratsTo develop an enantioselective HPLC method for the quantification oftetrahydroberberine (THB) enantiomers in rat plasma，Supelclean LC-18solid phaseextraction (SPE) cartridges were used to extract the enantiomers from samples and thechiral separation was carried on a Chiral AD column (250mm×4.6mm,5μm) withmethanol-ethanol (80:20, v/v) as the mobile phases at a flow rate of0.4ml/min and acolumn temperature of25℃.The detection wavelength was230nm. The linear range ford-THB was0.010～2.5μg/ml and0.010～5.0μg/ml for l-THB. The absolute recoverisfor each enantiomer were greater than70%.The intra-and inter-day variations were less than15%. The method was applied to determination the concentrations of THBenantiomers in rats after oral administration and intravenous injection at different doses ofrac-THB (50mg/kg,10mg/kg), and the pharmacokinetics parameters were then calculated,The mean Cmaxand AUC0-∞values in rats after oral administration were1.09μg/ml,1.91μg·h/ml for d-THB, and2.01μg/ml,4.15μg·h/ml for l-THB. After intravenous injectionthe mean AUC0-∞values were1.95μg·h/ml for d-THB and2.79μg·h/ml for l-THB. Therewere significant differences between the main pharmacokinetic parameters of the twoenantiomers. The results herein showed the stereoselective pharmacokinetics of THBenantiomers in rats.2. Study of transmembrane transport of tetrahydroberberine enantiomers inMDCK-MDR1cell monolayersTo study transmembrane transport mechanisms of tetrahydroberberine (THB)enantiomers by MDCK-MDR1cell monolayers. The bi-directional transport characteristicsof l-THB and d-THB were discussed. The concentration of THB enantiomers wasdetermined by HPLC and the apparent permeability coefficient (Papp) was thus calculated.The Pappof l-THB from A to B was1.27×10-5nm/s, while the Pappfrom B to A was5.65×10-5nm/s, and the efflux rate of l-THB was4.45; while the Pappof A side to B side ford-THB was1.27×10-5nm/s and the Pappof B side to A side for d-THB was4.67×10-5nm/s,the efflux rate of d-THB was3.68. The result indicated that the transmembrane transportmechanisms of l-THB and d-THB in MDCK-MDR1cell monolayer might involved activetransport.3. Evaluation of THP enantiomers on the activity of cytochrome P450isozymes inrats using a cocktail probe drugs methodA liquid chromatography-mass spectrometry method（HPLC-MS）has been developedfor simultaneous determination of five major cytochrome P450probe drugs, metoprolol(CYP2D6), caffeine(CYP1A2), dapsone(CYP3A4), chlorzoxazone(CYP2E1) andtolbutamide(CYP2C9) in rats urine. SD rats were randomly divided into groups (each n=6).Three groups of the rats was separately given d-THP, l-THP and rac-THP once daily for7days while the control group received orally saline once daily. Seven days later, a mixtureof five cocktail probe drugs including metoprolol, caffeine, dapsone, chlorzoxazone andtolbutamide were given to rats by intravenous injection. The concentrations of the five probe drugs were determined by HPLC-MS and their corresponding pharmacokineticparameters were calculated to evaluate the possible effect of THP enantiomers on CYP450isozymes activity.The metabolism of caffeine and metoprolol slowed down after receiving d-THP, withshorter t1/2. The metabolism of caffeine and dapsone slowed down after receiving l-THP,with longer t1/2,but the metabolism of tolbutamide speeded up, with shorter t1/2.Themetabolism of caffeine slowed down after receiving dl-THP, with longer t1/2,while dapsone,chlorzoxazone and tolbutamide speeded up, with shorter t1/2. There were significantlydifferent of these t1/2values from that in blank control group (P<0.05). The resultsindicated that d-THP tended to be the inhibitor of CYP2D6and CYP1A2, l-THP tended tobe the inhibitor of CYP1A2, CYP2E1and inducer of CYP2C9. The effects of dl-THP onthe activity of CYP450isozymes caused by both enantiomers.4. Identification of metabolites of THP enantiomers in rat urine and study onpharmacokinetics of THP enantiomers and their metabolitesAn ultra-performance liquid chromatography coupled to quadropole time-of-flight massspectrometry (UPLC-Q-TOF/MS) method was applied to identify the metabolites in urineafter a single oral administration of each THP enantiomers to rats. The identification wasdetermined by accurate mass measurements and fragment ions under high energy CIDmass spectra. Thus, besides the parent drug, a total of32metabolites for d-THP and30metabolites for l-THP were detected in urine after oral administration of THP enantiomersto rats. The major components in urine were demethlyzed, didesmethlyzed, hydrolyzedmetabolites, glucuronide and sulfation conjugates. Compared with l-THP, the metabolitesof tridesmethlyzed metabolites and didesmethlyzed coupled with hydrolyzed metabolitesonly were found in d-THP treated rats. The results indicated that there may existstereoselective metabolism of THP enantiomers.A sensitive and rapid LC-MS/MS method has been developed for the quantification ofTHP enantiomers in rat urine. Urine sample were collected after a single intravenous THPenantiomers at a dose of60mg/kg within72h. And the concentrations of THP enantiomersin urine at different time periods were determination by the validated LC-MS/MS method.The concentrations of the possible metabolites were calculated by the semi-quantificationwith the equations of THP enantiomers, which described as molar concentration (μmol/ml)instead of mass concentration (μg/ml). In addition, according to the accurate molecular formulae provided by UPLC-Q-TOF/MS and urine excretion volume, the amounts of themetabolites were calculated. The results showed that the cumulative excretion amounts ford-THP and l-THP within72h were53.23±27.33μg and101.55±43.31μg, respectively. Thecumulative excretion rates for d-THP and l-THP were0.37%and0.72%, respectively. Thel/d ratios of cumulative excretion were2.23. The cumulative excretion amounts of mainphase I metabolites including demethylation (M7-2and M7-3) and didesmethylation(M3-2) were more than that of phase II metabolites including demethylation and sulfation(M9-1and M9-2).Furthermore, the cumulative excretion amounts of l-THP were higherthan those of d-THP at different time periods. The results indicated that only a smallamount of THP enantiomers was excreted in urine and there existed a stereoselectiveexcretion of THP enantiomers in rat urine.更多还原