两种药物的手性分离和立体选择性药物动力学研究

【作者】 鹿秀梅；

【导师】 李发美；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2005， 硕士

【摘要】 手性化合物的对映体分离是手性新药研制工作的基础，寻找高效、灵敏、快速、简便的手性分离方法是药物分析领域的重要课题。本文分别采用HPCE法和HPLC法对西孟坦和马布特罗对映体进行了分离，并对马布特罗对映体在大鼠体内的药物动力学行为进行了初步研究。1．西孟坦对映体的毛细管电泳分离方法研究以β—环糊精（β-CD）为手性选择剂，采用毛细管区带电泳对西孟坦进行了手性分离研究。考察了背景电解质中硼砂缓冲液的pH及浓度、β-CD浓度、有机改性剂甲醇含量和分离电压对手性分离的影响。建立了毛细管电泳分离西孟坦对映体的方法。最佳分离条件为：20mmol／L硼砂缓冲液（pH11.0，含12mmol／Lβ-CD）-甲醇（50∶50，v／v），分离电压20kV，在此条件下西孟坦对映体可达基线分离。2．毛细管电泳法测定马布特罗离解常数利用毛细管电泳中不同pH条件下有效淌度的倒数（1／μ_e）与缓冲溶液中氢离子浓度的倒数（1／[H⁺]）之间存在着线性关系的原理，测定了马布特罗的pK_a值。以1％丙酮为中性标记物，离子强度为0.09的不同pH的缓冲液为运行缓冲液，检测波长为254nm，分离电压为10kV。结果马布特罗的pK_a为10.14，与另一种方法—等摩尔浓度分光光度法的测得值相比较，结果相近。3．毛细管电泳法同时分离分析马布特罗及其结构类似物的对映体以羟丙基-β-环糊精（HP-β-CD）为手性选择剂，应用毛细管电泳法同时对马布特罗、克仑特罗、SPFF、班布特罗等四种β₂受体激动剂类手性药物进行了对映体分离。考察了HP-β-CD浓度和背景电解质pH对分离效果的影响。最佳分离条件为100mmol／L柠檬酸—10mmol／L磷酸氢二钠—120mmol／L HP-β-CD，pH2.5，分离电压20kV，检测波长210nm，柱温为室温。在此条件下，四种药物对映体均得到良好的分离结果。对方法学进行了考察，并测定了其中两种药物的对映体纯度。4．马布特罗对映体体内分析方法的建立及其在大鼠体内的立体选择性药物动力学研究将非手性、手性色谱系统相结合，建立了大鼠血浆中马布特罗对映体的测定方法。首先用非手性色谱法将药物与血浆内源性杂质分离，然后收集检测器后药物色谱峰流分，应用大环抗生素固定相柱（Chirobiotic V），以甲醇—冰醋酸—三乙胺（100∶0.01∶0.01，v／v／v）为流动相，将两个对映体完全分离。根据血浆中马布特罗的浓度和l／d-对映体浓度比值，计算得到l-和d-马布特罗的血浆药物浓度。采用本法研究了6只大鼠单剂量灌胃给予马布特罗消旋体后，两对映体的药物动力学。结果表明，马布特罗两对映体有相似的t_max（5.33±2.1h，l-马布特罗；5.67±1.51h，d-马布特罗）和C_max（266.8±37.9ng／ml，l-马布特罗；277.9±44.6ng／ml，d-马布特罗），但配对t检验表明，两对映体的AUC_0-∞、t_1／2（k）、CL／F和MRT存在显著性差异，提示l-马布特罗比d-马布特罗作用更有效、更持久。推测两对映体的代谢存在立体选择性。更多还原

【Abstract】 Enantioseparation of chiral compound is the foundation of new chiral drug’s research and development. Searching for high efficient, sensitive, rapid and simple chiral separation method in laboratory is important in pharmaceutical analysis field. Chiral separation of simendan and mabuterol was investigated in the present work by using high performance capillary electrophoresis （HPCE） and high performance liquid chromatography （HPLC）. The pharmacokinetics of mabuterol enantiomers in rats were also studied by using the developed HPLC method.1. Chiral separation of simendan enantiomers by capillary zone electrophoresisThe enantiomers of simendan were separated by capillary zone electrophoresis （CZE） usingβ-cyclodextrin （β-CD） as the chiral selector. The influences of the concentration and pH of borate buffer solution,β-CD concentration, methanol content and separation voltage were investigated. A baseline separation of simendan enantiomers was achieved with background electrolyte of 20mmol/L borate buffer （pH 11.0） containing 12mmol/Lβ-CD-methanol （50:50, v/v） at a voltage of 20 kV.2. Determination of the dissociation constant （pK_a） value of mabuterol by capillary electrophoresisKnowledge of dissociation constant is important for prediction and understanding of the migration behavior of analytes in capillary electrophoresis. A CZE method was used to determine the pK_a value of mabuterol based on the linear relation between the reciprocal ofeffective mobility of the solute （1/μ_e） and the reciprocal of [H⁺] of the buffer solution. Acetoneof 1% was chosen as the neutral marker. The running buffer was sodium phosphate with different pH with same ion strength of 0.09. The UV detector was set at 254nm; electrophoresis was performed at a voltage of 10 kV. The dissociation constant was determined to be 10.14, which was similar with that of comparative method （Iso-molarity UV spctrophotometry）. Based on this result, chiral separation of mabuterol was investigated by HPCE.3. The simultaneous enantiomeric separation of mabuterol and its analogue by HPCE The capillary electrophoretic separation of four adrenergicβ₂-agonist （mabuterol,clenbuterol, SPFF, bambuterol） enantiomers were studied with Hydroxypropyl-β-cyclodextrin （HP-β-CD） as chiral selector. The effects of buffer pH and concentration of HP-β-CD on the electrophoretic resolution were investigated. Separation conditions were optimized to be: 100mmol/L citric acid-10mmol/L Na₂HPO₄-120mmol/L HP-β-CD buffer solution （pH 2.5）, applied voltage of 20kV, detection at 210nm. Four adrenergicβ₂-agonist enantiomers were satisfactorily separated simultaneously under this condition. The methodology was investigated and this method was used in the determination of enantiomeric purity of SPFF and mabuterol.4. Analysis of enantiomers in vivo and stereoselective pharmacokinetics of mabuterol in ratsAn achiral-chiral sequential method to determine the enantiomers of mabuterol in rat plasma was developed. Mabuteol was separated with endogenesis substances in plasma earlier by achiral chromatography system. Then the fraction of mabuterol from the achiral system was collected. The two enantiomers of mabuterol were separated by the chiral chromatography system, which was composed of Chirobiotic V column and mobile phase of MeOH-HAc-TEA （100/0.01/0.01, v/v/v）. Based on the concentration of mabuterol and the l/d ratios in the same sample, the plasma levels for l-and d-mabuterol were calculated. The method described above was used for the stereoselective pharmacokinetics study of mabuterol in six Wistar rats after intra gastric administraiton of 10mg/kg mabuterol racemate. The results showed that both enantiomers were absorbed slowly with a similar t_max of 5.33±2.1h and 5.67±1.51, and C_max, of 266.8±37.9ng/ml and 277.9±44.6ng/ml for l-mabuteol and d-mabuteol, respectively （p＞0.05）. Significant differences of AUC_0-∞、t_1／2（k）、CL/F and MRT were found between two enantiomers of mabuterol by paired Student’s t-test, which indicated that l-mabuterol was more effective and persisting longer than its antipode, showed the metabolic stereoselectivity of mabuterol. The developed method should be fundamental contributions for the preclinical study of single mabuterol enantiomer.更多还原