手性黄皮酰胺生物转化的立体选择性

以相同的肝微粒体温孵体系对(+) 和(-) 黄皮酰胺(Cla)进行了温孵,以HPLC分析比较了代谢结果,发现(+)-和(-)-Cla所产生的主要代谢产物基本相同,但主要代谢产物在(+)-和(-)-Cla温孵体系中含量差别较大,左旋体代谢产生的CM1和CM5的量比右旋体大;CM2是左旋Cla的主要代谢产物之一,右旋体产生极小的量;而CM3是右旋Cla的主要代谢产物,左旋体产生的量较少. CM4, CM6是由CM3进一步代谢所产生的双羟基代谢产物,右旋体产生的量多于左旋体. 定量分析(+)-,(-)-Cla在肝微粒体中不同时间的代谢情况,发现(+)-,(-)-Cla在实验浓度下均为非线性动力学,二者代谢速率基本相同,不同时间代谢的立体选择性基本相同.     

左旋黄皮酰胺在大鼠肝微粒体中的代谢转化研究

用大鼠肝微粒体体外温孵法进行了左旋黄皮酰胺[(-)-clausenamide]代谢转化研究,优化了温孵体系,建立了反相HPLC-DAD同时分离检测左旋黄皮酰胺及其体外代谢产物的分析方法。用硅胶低压柱色谱、制备TLC及制备HPLC分离纯化了两个代谢产物并进行了光谱鉴定。结果表明,两个代谢物分别确定为6-和5-位羟基取代的黄皮酰胺。     

黄皮酰胺对映体在大鼠肝微粒体中的酶促反应动力学黄皮酰胺对映体在大鼠肝微粒体中的酶促反应动力学

目的研究黄皮酰胺（clausenamide,Clau）对映体在大鼠肝微粒体中的酶促反应动力学并比较其立体选择性差异。方法应用反相HPLC法测定Clau对映体在体外代谢系统中的产物，并用Eadie-Hofstee作图法分析数据、求算酶促反应动力学参数K_m和V_max以及肝代谢速率V_max/K_m。结果在体外代谢系统中，左旋黄皮酰胺主要生成7-羟-Clau、5-羟-Clau和4-羟-Clau，其优势代谢途径为7位羟化；7位羟化代谢的V_max/K_m值高于5位和4位。右旋黄皮酰胺的4位羟化反应K_m最小、V_max最大, 因此代谢速率最高，是左旋体4位羟化的8倍；而其7-羟-Clau和5-羟-Clau 的产生量很小。结论黄皮酰胺对映体在大鼠肝微粒体中的羟化代谢存在明显的底物立体选择性差异。     

右旋和左旋黄皮酰胺在大鼠体内代谢转化的研究

目的　研究黄皮酰胺在大鼠体内的代谢转化途径。方法　收集ip给药后大鼠的尿液、粪便及血液进行分析,寻找已知的代谢产物并通过HPLC-DAD和HPLC-MS分析寻找未知的代谢产物,确定大鼠体内的主要代谢途径。并通过比较(+),(-)-黄皮酰胺代谢的差异,初步研究其代谢转化的立体选择性。结果　HPLC分析发现,大鼠肝微粒体中所分离得到的6个主要代谢产物均在体内存在,(+),(-)-黄皮酰胺的代谢有明显的差异,根据MS碎片信息确定了一个新的代谢产物的结构,即N-去甲黄皮酰胺。结论　手性黄皮酰胺主要在肝脏中发生羟基化代谢反应,并有明显的立体选择性。     

抗焦虑新药AF-5及其代谢物在人肝微粒体体外温孵体系中代谢研究

目的研究一类抗焦虑新药AF-5及其代谢物(I,II)在人肝微粒体体外温孵体系中代谢情况.方法自制人肝微粒体,用Lowry法测定酶活性为8.79mg·mL^-1.以此配制人肝微粒体体外温孵体系,加入药物,温孵后,提取分离,GC-MS测定.结果鉴定了AF-5在人肝微粒体体外温孵体系中的两个主要代谢物,并阐明了其体外代谢途径为AF-5的4位首先氧化为羟基,然后氧化成羰基.结论AF-5在体外人肝微粒体温孵体系中,100min后完全代谢成羟基代谢物I及羰基代谢物II,以羟基代谢物为主要代谢产物.AF-5代谢物I在人肝微粒体温孵体系中,可转化为代谢物II,而代谢物II在人肝中则不再代谢.     

氯代正丁苯酞在大鼠肝微粒体中的代谢研究

用大鼠肝微粒体体外温孵方法进行了氯代正丁苯酞(CBP)代谢转化研究,优化了温孵体系的组成,建立了反相HPLC-DAD在线同时分离检测CBP及其4个体外代谢产物的分析方法,并比较了在苯巴比妥(PB)与3-甲基胆蒽(3-MC)诱导的微粒体中代谢差别。利用TLC、柱色谱与制备HPLC分离纯化了3个主要代谢产物,并进行了NMR,MS,UV等光谱鉴定,确定了其主要代谢产物为γ-羟基氯代正丁苯酞与β-羟基氯代正丁苯酞及它们的立体异构体。     

双环醇在大鼠和人肝微粒体的代谢

目的研究参与双环醇代谢的主要药物代谢酶及代谢动力学参数，分离鉴定双环醇代谢产物。方法双环醇与大鼠和人肝微粒体进行温孵，以高效液相色谱、质谱、核磁共振技术检测并分离鉴定双环醇及其代谢产物。结果双环醇在地塞米松诱导大鼠肝微粒体中的代谢速率显著高于正常大鼠肝微粒体，酮康唑可显著抑制双环醇的代谢。双环醇主要代谢产物为:4-羟基-4′-甲氧基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯和4-甲氧基-4′-羟基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯。结论双环醇在大鼠和人肝微粒体的主要代谢产物为4-羟基-4′-甲氧基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯和4-甲氧基-4′-羟基-6-羟甲基-6′-甲氧羰基-2，3，2′，3′-双亚甲二氧基联苯，细胞色素P450 3A主要参与双环醇代谢。     

左旋一叶碱的代谢转化

目的研究一叶碱［securinine,(-)SE］在大鼠体内外的代谢转化。方法采用大鼠肝微粒体体外温孵法对(-)SE的代谢转化进行了研究,优化了代谢体系,建立了反相HPLC法同时分离检测(-)SE及其体外代谢产物的分析方法。用液液萃取,制备TLC及半制备HPLC分离纯化了4个代谢产物并进行了光谱鉴定。在此基础上,建立了生物体液中(-)SE及其代谢物的反相HPLC分析方法,并用该法检测了ip给药后大鼠的胆汁、尿样及其经β-葡糖醛酸苷酶水解后的样品。结果代谢物分别鉴定为6-位羟基,6-位羰基及5-位α及β羟基取代的(-)SE,还证实了体内6-位羟基代谢物进一步形成了二相结合型产物。结论基本阐明(-)SE在大鼠体内外代谢转化的途径。     

苯环喹溴铵在大鼠肝微粒体中的代谢转化研究

目的:建立检测大鼠肝微粒体中苯环喹溴铵代谢产物的方法,验证其在大鼠体内的代谢途径。方法:采用大鼠肝微粒体体外温孵法,建立液相色谱-质谱法测定并分析肝微粒体中苯环喹溴铵及其代谢物。色谱及质谱条件如下:色谱柱为TSK-gelODS-80Ts,流动相为甲醇-40mmol·L-1的乙酸铵水溶液(含0.1%甲酸)梯度洗脱;正离子模式,扫描型离子检测。结果:在体外代谢系统中,根据质谱碎片信息检测出6个代谢产物,分别是苯环喹溴铵的二羟基、单羟基和氧化产物。结论:建立的液相色谱-质谱法能够准确灵敏地测定大鼠肝微粒体中苯环喹溴铵的代谢产物,验证了在大鼠体内苯环喹溴铵的代谢部位在环戊烷基上。     

左旋一叶萩碱的代谢转化

目的　研究一叶碱 [securinine ,( - )SE]在大鼠体内外的代谢转化。方法　采用大鼠肝微粒体体外温孵法对 ( - )SE的代谢转化进行了研究 ,优化了代谢体系 ,建立了反相HPLC法同时分离检测 ( - )SE及其体外代谢产物的分析方法。用液液萃取 ,制备TLC及半制备HPLC分离纯化了 4个代谢产物并进行了光谱鉴定。在此基础上 ,建立了生物体液中 ( - )SE及其代谢物的反相HPLC分析方法 ,并用该法检测了ip给药后大鼠的胆汁、尿样及其经 β 葡糖醛酸苷酶水解后的样品。结果　代谢物分别鉴定为 6 位羟基 ,6 位羰基及 5 位α及 β羟基取代的 ( - )SE ,还证实了体内 6 位羟基代谢物进一步形成了二相结合型产物。结论　基本阐明 ( - )SE在大鼠体内外代谢转化的途径     

蓝萼甲素在大鼠体内外的代谢转化

目的研究蓝萼甲素在大鼠体内外的代谢转化。方法采用大鼠肝微粒体体外温孵法,研究对蓝萼甲素的代谢转化。采用RP-HPLC法同时分离检测蓝萼甲素及其体外代谢产物。结果用液-液萃取、制备HPLC法,从大鼠胆汁中分离了一个代谢产物,经质谱分析推测结构为羟基化蓝萼甲素,并采用HPLC-MS连用,分析了肝微粒体体外温孵样品中的代谢产物,推测了蓝萼甲素的可能代谢转化途径。结论蓝萼甲素在大鼠肝微粒体和胆汁中可被代谢转化,主要代谢产物为羟基化蓝萼甲素。     

Metabolism of lovastatin by rat and human liver microsomes in vitro

The metabolism of lovastatin (Mevacor) was examined using isolated microsomes derived from the livers of normal and phenobarbital-treated rats and from human liver samples. Incubation of lovastatin with rat liver microsomes resulted in the formation of several polar metabolites of lovastatin. The metabolites were isolated by HPLC and identified by NMR and mass spectrometry. One fraction consisted of a 2:1 mixture of 6-hydroxy-lovastatin and the rearrangement product delta 4,5-3-hydroxy lovastatin. Addition of a trace of acid to this mixture resulted in the formation of a single aromatized product, the desacyl-delta 4a,6,8-dehydro analog of lovastatin. Another microsomal metabolite was determined to be the delta 4,8a,1-3-hydroxy-lovastatin derivative. The chromatographic pattern of metabolites produced from lovastatin by human liver microsomes was similar to that obtained with rat liver microsomes. Metabolism of lovastatin by rat liver microsomes was both time and concentration dependent; optimal microsomal metabolism occurred with 0.1 mM lovastatin, whereas higher lovastatin concentrations inhibited the reaction. The open acid form of lovastatin was poorly metabolized by both the rat and the human liver microsomes.     

大鼠肝微粒体温孵体系中（＋）,（—）—黄皮酰胺及其代谢产物的LC—MS分析

目的：建立黄皮酰胺及其代谢产物的ＨＰＬＣ－ＥＳＩ－ＭＳ在线检测分析方法,并对未分离得到的微量代谢产物进行分析确证,探索ＬＣ－ＭＳ在代谢转化研究中的应用。方法：利用柱后补偿技术,采用正离子检测对大鼠肝微体中（＋）,（－）－黄皮酰胺及其代谢产物进行ＨＰＬＣ－ＥＳＩ－ＭＳ分析,根据ＭＳ的碎片信息检测主要的代谢产物,特别是对未分离得到的代谢产物的结构碎片进行分析,确定其结构。结果：除检测出主要已知代谢产物     

Hydroxylation of 4,4'-methylenebis(2-chloroaniline) by canine, guinea pig, and rat liver microsomes

An in vitro microsomal mixed function oxidase enzyme system was used to study the phase I metabolism of 4,4'-methylenebis(2-chloroaniline) (MBOCA) by dog, guinea pig, and rat liver. TLC with color development and autoradiography, and HPLC with detection by UV absorbance and radioactivity flow monitoring were utilized to isolate metabolites. Reference standards of the N-oxidized metabolites were prepared by oxidation of MBOCA with 3-chloroperoxybenzoic acid and structures confirmed by mass spectrometry and proton NMR. These were utilized to identify the N-hydroxy and nitroso metabolites of MBOCA isolated from the microsomal incubations by comparison of their HPLC retention times and mass spectra. The structure of the o-hydroxy metabolite (ring, ortho to the amine) isolated from the microsomal incubations was elucidated by mass spectrometry and proton NMR. N- and o-hydroxylations of MBOCA were shown to increase with incubation time, microsomal protein, substrate, and NADPH concentration, and were inhibited by 2,3-dichloro-6-phenylphenoxyethylamine, an inhibitor of the microsomal mixed function oxidase enzyme system. Guinea pig liver microsomes oxidized MBOCA to the N-hydroxy metabolite predominantly, whereas the dog liver formed predominantly the o-hydroxylated metabolite, with significant amounts of the hydroxylamine as well. The rat liver formed lesser amounts of the N- and o-hydroxylated metabolites, but larger numbers of other polar compounds.     

In vitro metabolism of the antianxiety drug buspirone as a predictor of its metabolism in vivo

1. Metabolism of the antianxiety drug buspirone was studied by in vitro incubations with rat liver microsomes and hepatocytes. Metabolites were isolated and purified by h.p.l.c. The purified metabolites were identified by co-elution on h.p.l.c. with authentic standards and by g.l.c.-electron impact mass spectrometry of their trimethylsilyl (TMS) derivatives. 2. Five metabolites of buspirone were identified in the microsomal incubates and seven in the hepatocyte incubates. The major metabolites arose from aromatic hydroxylation at C-5, N-dealkylation of the butyl chain, and hydroxylation at C-6' and C-3' on the azaspirodecanedione moiety. 3. Metabolism of buspirone by rat liver microsomes was NADPH-dependent and was completely inhibited by cytochrome P-450 inhibitors SKF-525A and metyrapone. 4. Metabolites of buspirone formed in vitro were good predictors of the primary metabolites formed in vivo. 5. Hepatocytes and phenobarbital-induced rat liver microsomes were better predictors of in vivo metabolism of buspirone than non-induced rat liver microsomes. These in vitro systems should provide excellent models for studying the metabolism of other azaspirodecanedione-containing drugs.     

In vitro biotransformation of xanthohumol, a flavonoid from hops (Humulus lupulus), by rat liver microsomes.

Xanthohumol (XN) is the major prenylated flavonoid of the female inflorescences (cones) of the hop plant (Humulus lupulus). It is also a constituent of beer, the major dietary source of prenylated flavonoids. Recent studies have suggested that XN may have potential cancer-chemopreventive activity, but little is known about its metabolism. We investigated the biotransformation of XN by rat liver microsomes. Three major polar metabolites were produced by liver microsomes from either untreated rats or phenobarbital-pretreated rats as detected by reverse-phase high-performance liquid chromatography analysis. Liver microsomes from isosafrole- and beta-naphthoflavone-pretreated rats formed another major nonpolar metabolite in addition to the three polar metabolites. As determined by liquid chromatography/mass spectrometry and (1)H NMR analyses, the three major polar microsomal metabolites of XN were tentatively identified as 1) 5"-isopropyl-5"-hydroxydihydrofurano[2",3":3',4']-2',4-dihydroxy-6'-methoxychalcone; 2) 5"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":3',4']-2',4-dihydroxy-6'-methoxychalcone; and 3) a derivative of XN with an additional hydroxyl function at the B ring. The nonpolar XN metabolite was identified as dehydrocycloxanthohumol.