右旋黄皮酰胺在大鼠肝微粒体中的代谢转化

目的：研究黄皮酰胺的主要代谢途径,为进一步研究黄皮酰胺代谢的立体选择性打下基础。方法：用大鼠肝微粒体体外温孵法对右旋黄皮酰胺((+)-clausenamide)进行温孵,用硅胶柱色谱、制备TLC分离纯化代谢产物并通过光谱分析鉴定其结构。结果：分离得到5个代谢产物CM1,CM3,CM4,CM5及CM6,其结构分别鉴定为6-羟基,4-羟基,4,6-二羟基,4-苯环邻位羟基,4,7-苯环间位-二羟基黄皮酰胺。结论：黄皮酰胺的代谢主要发生羟化或双羟化,CM3是其主要代谢产物,量较少的CM4,CM6为其进一步代谢产生的双羟基代谢产物;另2个代谢产物CM1,CM5产生的量也较少;CM2未分离得到,但通过HPLC分析知其为右旋黄皮酰胺的微量代谢产物。     

左旋黄皮酰胺在大鼠体内的排泄

左旋黄皮酰胺[（-）-clausenamide]是从芸香科黄皮属植物黄皮[Clausena lansium（lour） sheels]叶的水浸膏中分离得到的有效成分，经不对称合成和拆分制备而得。药效学研究表明，左旋黄皮酰胺促进突触体谷氨酸释放，增加大鼠脑皮层厚度和海马CAL区突触数及NMDA受体密度，提高小鼠脑皮层和海马的胆碱乙酰转移酶活性，对抗樟柳碱引起的乙酰胆碱含量降低。这些结果表明左旋黄皮酰胺具有较好的促智和神经保护作用以及潜在的抗老年痴呆作用；右旋体作用不明显，且有较强的毒性。     

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。     

水黄皮素在大鼠肝微粒体中的代谢研究

目的 研究水黄皮素在大鼠肝微粒体中的代谢稳定性,确定水黄皮素的代谢酶表型.方法 将水黄皮素与大鼠肝微粒体于37℃孵育,应用UPLC-MS/MS法检测孵育液中水黄皮素的含量,考察水黄皮素的代谢稳定性.将水黄皮素与各细胞色素P450(CYP)酶中的各同工酶CYP2E1、2C19、1A2、2D6、2C9和3A4的特异性抑制剂共孵育,确定其代谢酶表型.结果 在大鼠肝微粒体中,水黄皮素的t1/2=1.97 h、Cl =0.70 mL·h-1 ·mg-1,CYP1 A2是其主要代谢酶.结论 水黄皮素在大鼠肝微粒体中是由多个CYP同工酶介导代谢的,其中,CYP1 A2酶的介导作用最强.     

反相高效液相色谱法测定大鼠血浆中左旋黄皮酰胺及其主要代谢产物和药代动力学

目的　探讨左旋黄皮酰胺［（-）clau］及其主要代谢产物6-OH-clau在大鼠体内的药代动力学。方法　建立了RP-HPLC-UV法。固定相为Kromasil-100 C₁₈（5 μm）色谱柱,流动相为乙腈-甲醇-水（21∶16.5∶62.5), DM-9384作内标,氯仿作提取溶剂。结果　测得（-）clau的回收率为96.91%～105.74%,日内、日间RSD低于7%,最低检测浓度为24 ng.mL^-1,（-）clau和6-OH-clau分别在0.047～968 μg.mL^-1和0.049～200 μg.mL^-1,线性关系良好(γ=0.999); （-）clau和6-OH-clau血浆浓度-时间曲线符合二室开放模型,同时求得两者的药代动力学参数。结论　数据表明（-）clau在大鼠血浆中的分布、代谢转化和消除均较快。     

HPLC/MS法研究左旋黄皮酰胺及其代谢物在Beagle犬血浆中的药代动力学HPLC/MS法研究左旋黄皮酰胺及其代谢物在Beagle犬血浆中的药代动力学

目的用HPLC/MS法研究左旋黄皮酰胺［(-)-clau］及其代谢物6-羟基-黄皮酰胺(6-OH-clau)在Beagle犬血浆中的药代动力学过程。方法Beagle犬灌胃左旋黄皮酰胺30 mg·kg^-1，采集静脉血样，血浆经乙酸乙酯萃取分离后，用HPLC/MS选择性正离子检测内标(格列吡嗪，［M+H］⁺，m/z 446)法测定左旋黄皮酰胺(［M+H］⁺，m/z 298)及6-羟基-黄皮酰胺(［M+H-H₂O］⁺，m/z 296)的浓度，以甲醇-水-冰醋酸(60∶40∶0.8)为流动相，流速1.0 mL·min^-1。用3P97软件计算药代动力学参数。结果左旋黄皮酰胺和6-羟基-黄皮酰胺分别在1.0～200 ng·mL^-1和0.2～40.0 ng·mL^-1线性关系良好(r>0.999)，萃取回收率均大于85%。原药及其代谢物的体内过程均符合二室模型；左旋黄皮酰胺及6-羟基-黄皮酰胺的C_max分别为(21±10) ng·mL^-1和(3.9±2.2) ng·mL^-1；T_max分别为(0.8±0.5) h和(1.3±0.5) h；T_1/2α分别为(0.9±0.6) h和(1.4±0.6) h；T_1/2β分别为(19±23) h和(13±12) h；AUC_{0-24 h}分别为(69±14) h·ng·mL^-1和(12±7) h·ng·mL^-1。结论Beagle犬灌胃左旋黄皮酰胺后迅速吸收，血药浓度一相消除很快，但末端消除较慢；其代谢物6-羟基-黄皮酰胺血药浓度经时过程与左旋黄皮酰胺相似，但血药浓度相对较小。     

米氮平在鼠肝微粒体中的体外代谢研究

目的:通过不同诱导剂预处理鼠肝,研究米氮平在肝微粒体中的代谢主要受何种酶影响,同时研究米氮平对介导其自身代谢的P450酶亚型是否有影响,为米氮平的临床合理应用提供科学依据。方法:将米氮平与不同诱导剂诱导的鼠肝微粒体进行体外孵育代谢,以乙腈中止反应,样品用25%氨水碱化后以环己烷提取。用RPHPLC测定剩余的米氮平含量,流动相为甲醇水(含10mmol·L-1NH4AC,5mmol·L-1SDS,pH3.5)6238(VV),检测波长为307nm。结果:本文测定方法回收率高,日内和日间精密度均良好,符合生物样本检测要求。苯巴比妥诱导的鼠肝微粒体对米氮平的代谢具有明显的催化作用,利福平也有一定的催化能力,米氮平诱导的鼠肝微粒体与对照组对米氮平的代谢无明显差异。结论:由苯巴比妥诱导的P450酶亚型(主要为细胞色素P4503A4)和利福平诱导的P450酶亚型(主要为细胞色素P4502C92C19,同时也对P4503A4有一定的诱导作用)在米氮平的体外代谢中起着重要作用;而米氮平诱导组对于米氮平代谢无明显影响,预示米氮平对介导其自身代谢的P450酶亚型无明显的诱导或抑制作用。     

HPLC—MS法测定血浆中左旋黄皮酰胺的浓度

目的:建立测定血浆中左旋黄皮酰胺浓度的 LC-MS 方法。方法:血浆样品经醋酸乙酯提取处理后,以2%冰醋酸-甲醇(45∶55)为流动相,在1.0 mL·min~(-1)的流速下,用 Shimadzu ODS 色谱柱(150mm×4.6mm,5μm)分离,样品经电喷雾离子化后,通过 HP 1100 HPLC-MS 质谱仪液相色谱串联质谱法,以格列吡嗪为内标测定左旋黄皮酰胺的浓度。结果:血浆中左旋黄皮酰胺的线性范围为0.01-10μg·mL~(-1),最低定量浓度为10ng·mL~(-1),准确度在80%-90%之间,日内、日间精密度(RSD)在±10%之内。结论:该方法高效、准确,可用于左旋黄皮酰胺在体内的药代动力学研究及血浆中左旋黄皮酰胺的含量测定以控制质量。     

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。     

MN9202在Beagle犬肝微粒体酶中的代谢动力学

目的研究MN9202在Beagle犬肝微粒体酶中的代谢。方法差速离心法制备Beagle犬肝微粒体酶，0.4 μmol·L^-1的MN9202与1.0 g·L^-1的肝微粒体酶在37 ℃水浴中孵育30 min，加入0.5 mL碱化液终止反应，然后采用RP-HPLC法测定孵育液中MN9202原形药物的浓度。根据所测浓度与反应速度做Lineweave-Brurk双倒数曲线，推导出药物的米氏常数K_m和最大反应速度V_max,并计算机体内在清除率。同时观察不同浓度和不同种类的人肝微粒体酶(CYP450)抑制剂对MN9202代谢的影响。结果MN9202在Beagle犬肝微粒体酶中的K_m为(22.6±8.0) μmol·L^-1；V_max为(0.54±0.17) μmol·g^-1·min^-1；CL_int为(0.024 2±0.000 9) L·g^-1·min^-1。醋竹桃霉素(Tro)和酮康唑(Ket)能够显著抑制MN9202的代谢；反苯环丙胺(Tra)对MN9202的代谢也有一定的抑制作用，而其他CYP450抑制剂对MN9202的代谢无明显影响。结论CYP3A和CYP2C19参与了MN9202的代谢，人CYP3A和CYP2C19的抑制剂可能使MN9202的代谢受到抑制，造成药物的药效或毒性的增加。     

左旋和右旋吡喹酮在人和大鼠肝微粒体内的代谢

左旋吡喹酮[(-)PQT]在人和大鼠肝微粒体中人谢生成产物[M];在[M]色谱峰处,右旋吡喹酮[(+)PQT]无明显代谢物生成.人肝微粒体代谢(?)-PQT生成MI的K_m和V_(max)分别为58±s13μmol·L~1和1.3±s 0.6 nmol·mg~1·min~1·RF.在人肝微粒体中,(?)与(+)PQT原药消除的K_m和V_(max)比值分别为0.86±s 0.28和1.5±s 0.5;(-)-和(+)PQT人肝内在清除率分别为1.3±s 0.5和0.7±s0.3 ml·h~1·mg~1,(-)-/(+)PQT的比值平均为1.8±s 0.5(-)与(-)-PQT在大鼠肝微粒体中消除速率的比值为1.83±s 0.27,结果说明PQT对映异构体在人和大鼠肝微粒体内代谢表现较严格的立体选择性     

Selective metabolism of E-3,4-bis(4-ethylphenyl)hex-3-ene in rat liver microsomes

The synthetic stilbene derivative E-3,4-bis(4-ethylphenyl)hex-3-ene (E-DE-BPH) has been proposed as a potential anticancer drug with a new mode of action. We report here on the in vitro metabolism of E-DE-BPH in liver microsomes of rats and pigs. The formation of five metabolites, which could be separated on a reverse-phase HPLC column with UV detection, was observed in microsomal incubations. To facilitate the structural identification of these metabolites, two different deuterium-labeled forms of E-DE-BPH were synthesized. By comparing the mass spectra obtained for the metabolites of unlabeled E-DE-BPH and of the two deuterated forms, it could be demonstrated that E-DE-BPH was oxidized by liver microsomes exclusively at the benzylic positions of the molecule. The major metabolite was identified as E-3-(4-(1-hydroxyethyl)phenyl)-4-(4-ethylphenyl)hex-3-ene. Four minor metabolites were formed from the major metabolite, either by hydroxylation at the other benzylic position to yield a bishydroxylated metabolite, or by oxidation of the hydroxyl group to form E-3-(4-acetylphenyl)-4-(4-ethylphenyl)hex-3-ene. The latter compound was also obtained by chemical oxidation of the monohydroxylated metabolite of E-DE-BPH. Since no products containing hydroxyl groups at the aromatic rings or at other aliphatic sites of the molecule were detected, a surprisingly selective oxidative metabolism of E-DE-BPH appears to occur with rat and pig liver microsomes.     

Biotransformation of letrozole in rat liver microsomes: effects of gender and tamoxifen

The in vitro metabolic kinetics of letrozole were investigated by incubating letrozole (10-500 microM) in female or male rat liver microsomes to assess the effect of gender and to predict the in vivo biotransformation characteristics of letrozole in rats. The effects of tamoxifen (TAM) on the metabolic kinetics of letrozole were also examined by incubating letrozole in female rat liver microsomes in the presence or absence of TAM. The effects of chronic pretreatment of female rats with TAM (0.5, 1.0, 5.0 mg/kg/day, i.p. for 7 consecutive days) on liver microsomal protein content and metabolic activity were also examined. The formation rate of the carbinol metabolite of letrozole, CGP44 645, was significantly higher (p<0.05) in male rat liver microsomes in comparison to female. The V(max)/K(m) ratio for letrozole metabolism in female rat liver microsomes did not change significantly (p>0.05) in the presence of TAM. After chronic pretreatment of female rats with TAM (up to a dose of 1.0mg/kg/day), the hepatic microsomal protein content was significantly increased but the formation rate of CGP44 645, when normalized for protein content, did not change significantly. These results suggest that there is a marked gender difference in letrozole metabolism in rats. It also appears that acute treatment of female rat liver microsomes with TAM produces negligible inhibitory effect on the CYP mediated metabolic clearance of letrozole. However, chronic pretreatment of female rats with TAM appear to induce CYPs, but does not significantly impact the metabolic activities of the enzymes associated with the formation of the carbinol metabolite of letrozole.     

Comparative metabolism of benzo(a)pyrene by ovarian microsomes of various species

Knowledge of the ability of the female reproductive system to metabolize polycyclic aromatic hydrocarbons (PAHs) is critical to the diagnosis and management of female infertility and for risk assessment purposes. The PAHs are a family of widespread pollutants that are released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, PAHs become activated to reactive metabolites that interfere with target organ function and as a consequence cause toxicity. The extent of susceptibility to PAH exposure may depend on the ability of animals to metabolize these chemicals. The present study has been undertaken to assess whether any differences exist among mammals in the metabolism of benzo(a)pyrene (BaP), a prototypical PAH compound. Microsomes isolated from the liver and ovaries of rats, mice, goats, sheep, pigs, and cows were incubated with 5 μM BaP. Postincubation, samples were extracted with ethyl acetate and analyzed for BaP/metabolites by reverse‐phase HPLC with fluorescence detection. The rate of metabolism (pmol of metabolite/min/mg protein) was found to be more in liver than in ovary in all the species studied (P < 0.05). The differences in metabolite concentrations were statistically significant (P < 0.0001) among the various species in both organs studied. Multiple species comparison also revealed that the differences were statistically significant (P < 0.001) between rodents (rat and mouse) and higher mammals (ewe, sow, and cow). Even among the higher mammals, in a majority of the cases, the differences in metabolite concentrations were significantly different (P < 0.001) both in ovary and liver. The BaP metabolites identified were 4,5‐diol; 7,8‐diol; 9,10‐diol; 3‐hydroxy BaP; and 9‐hydroxy BaP. The rodent microsomes produced considerably higher proportion of BaP 4,5‐diol and 9,10‐diol than did cow, sow, goat, and sheep. However, microsomes from higher mammals converted a greater proportion of BaP to 3‐hydroxy and 9‐hydroxy BaP, the detoxification products of BaP. Overall, our results revealed a great variation among species to metabolize BaP. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Irreversible binding of 3-14C-antipyrine to hepatic protein in vivo and in metabolizing liver microsomes

Summary After i.p. injection of 3-¹⁴C-antipyrine (10 mole=1.9 mg with 10 Ci per 10 g of body weight) to mice radioactivity was irreversibly bound to liver proteins. The irreversible binding reached maximal values of 0.15 nmole/mg protein in liver microsomes after 30–60 min.During 60 min incubation with liver microsomes of mice and rabbits (phenobarbital pretreated) and a NADPH-regenerating system 3-¹⁴C-antipyrine was irreversibly bound to microsomal protein at a rate of 1.5 nmole/mg protein (mouse) and 3 nmole/mg protein (rabbit).In identical incubates with rabbit liver microsomes the 4-hydroxylation of antipyrine was 24 nmole/mg protein in 60 min and formaldehyde production from antipyrine 3 nmole/mg protein in 60 min.In incubates with rabbit liver microsomes the binding rate was 80–90% inhibited by 1mM metyrapone, SKF 525-A and trichloropropene epoxide respectively; 4-hydroxylation was 70–80% inhibited by the same substances. In the presence of 1 mM GSH, cysteine or ethylene diamine binding was 30–40% inhibited, whereas 4-hydroxylation showed no inhibition.Some of the results were presented at the 17th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, March 1976     

山冈橐吾碱在雌性大鼠肝微粒体内的代谢(英文)

研究了山冈橐吾碱 (clivorine)在雌性大鼠肝微粒体内的代谢 .山冈橐吾碱在雌性大鼠肝微粒体内的主要代谢物为两个非吡咯代谢物M1和M2 .与雄性大鼠不同 ,生成肝毒性的吡咯代谢物为其次要的代谢途径 .文献报道山冈橐吾碱在雄性大鼠肝微粒体内的主要代谢方式是形成相应的吡咯代谢物 .这提示山冈橐吾碱在雌雄大鼠肝微粒体内的主要代谢方式不同 .CYP4 5 0特异性抑制剂黄胺苯吡唑(CYP2C) ,毛果芸香碱 (CYP2A1) ,二乙基二硫代氨基甲酸钠 (CYP2E1)和酮康唑 (CYP3A)对M1和M2的形成无明显的影响 .黄素单氧化酶的特异性抑制剂甲巯咪唑可以显著地抑制M2 的形成 ,但对M1的形成无明显的抑制作用 ,且M1在肝微粒体中的形成为NADPH非依赖性 ,上述结果提示参与M1和M2代谢的酶分别为肝微粒体中的水解酶和黄素单氧化酶 .另一方面 ,毛果芸香碱 ,黄胺苯吡唑和二乙基二硫代氨基甲酸钠对山冈橐吾碱的吡咯代谢物的形成无明显的影响 ,而CYP3A的特异性抑制剂酮康唑可以显著地抑制吡咯代谢物的生成 ,且山冈橐吾碱在重组的大鼠肝CYP2C12 ,CYP2E1温孵液中无代谢 ,而在重组的大鼠肝CYP3A1和CYP3A2的温孵液中山冈橐吾碱被代谢成相应的吡咯代谢物 .这提示CYP3A作为主要的CYP4 5 0酶参与了山冈橐吾碱的肝毒性吡咯代谢物的形成 .山冈橐吾碱?     

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

AIM

The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin.

METHODS

The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4′-hydroxylation (S)-mephenytoin 4′-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1′-hydroxylation/testosterone 6β-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences.

RESULTS

The samples of the two ethnic groups were not significantly different in cytochrome-b₅ concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A.

CONCLUSIONS

These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.