新型抗慢性肾功能不全药硝克柳胺在大鼠肝微粒体和胞浆的代谢

目的：研究硝克柳胺在大鼠肝微粒体和胞浆中的代谢动力学及参与硝克柳胺体外代谢的药物代谢酶鉴定。方法：硝克柳胺分别与大鼠肝微粒体和胞浆温孵一定时间后，应用HPLC方法测定硝克柳胺含量，计算体外代谢速率和动力学参数（Kin，Vmax）。在微粒体或胞浆中加入选择性CYP抑制剂、硝基还原酶抑制剂，鉴定参与硝克柳胺代谢的药酶类型。结果：硝克柳胺在肝微粒体代谢是依赖NADPH／NADH的酶促反应，缺氧代谢高于有氧代谢，微粒体代谢明显快于胞浆。CYP2C6特异性抑制剂西米替丁、NADPH-细胞色素C还原酶特异性抑制剂2‘-单磷酸腺苷（2’-AMP）和细胞色素b5还原酶特异性抑制剂6-丙基-2-硫脲嘧啶（PTU）可明显抑制肝微粒体中硝克柳胺的代谢，其它CYP特异性抑制剂对硝克柳胺的代谢无明显影响。NAD（P）H脱氢酶特异性抑制剂双香豆素和黄嘌呤氧化酶特异性抑制剂别嘌呤醇也可显著抑制肝胞浆中硝克柳胺的代谢。结论：硝克柳胺的体外代谢是依赖多个药物代谢酶参与的酶促反应，包括微粒体NADPH-细胞色素C还原酶、细胞色素b5还原酶和CYP2C6以及胞浆NAD（P）H脱氢酶和黄嘌呤氧化酶。     

去氢厄弗酚在小鼠肝微粒体中代谢产物及CYP450酶亚型的鉴定

目的建立去氢厄弗酚(DHE)小鼠体外肝微粒体孵育方法,鉴定DHE在小鼠肝微粒体中的代谢产物及参与DHE代谢的CYP450酶亚型。方法采用UPLC-Q-TOF-MS/MS分析鉴定DHE在体外肝微粒体共温孵后的代谢产物,筛选7种CYP450酶亚型,并通过特异性化学抑制剂法,鉴别参与DHE代谢的主要CYP450酶亚型。结果在体外肝微粒体共温孵后,检测到4个代谢产物;所筛选的7种CYP450酶亚型中,CYP1A2、CYP2C8和CYP2D2对DHE体外肝微粒体代谢的参与度较高。结论在肝脏中,有多种代谢酶亚型参与DHE的代谢,表明DHE在临床上不易与其他药物产生相互作用。     

丝裂霉素C在体外和体内对大鼠肝脏CYP2D1/2,CYP2C11和CYP1A2活性的影响

目的研究丝裂霉素C(MMC)在体外和体内对大鼠肝脏CYP2D1/2,CYP2C11和CYP1A2活性的影响。方法用诱导剂和抑制剂分别在体内和体外调节大鼠肝脏P450同工酶活性，并用HPLC检测3种同工酶各自底物的特定代谢产物，以计算同工酶活性。结果在体外, MMC可以使地塞米松诱导的大鼠肝脏微粒体CYP2D1/2,CYP2C11和CYP1A2活性分别抑制(19±6)%(P<0.05),(85±10)％(P<0.01)和(36±6)%(P<0.05)，并使β-萘黄酮诱导的CYP1A2活性降低(58±6)%(P<0.01)。在体内，以20% LD₅₀的剂量连续3 d或6 d腹腔注射MMC 对大鼠肝脏CYP2D1/2，CYP2C11和CYP1A2活性的影响无统计学差异。结论在体外MMC可以抑制大鼠肝微粒体CYP2D1/2，CYP2C11和CYP1A2的活性，但在体内对这3种细胞色素P450同工酶活性的影响无统计学差异。     

硝克柳胺在大鼠和人肝脏的体外代谢研究

研究硝克柳胺在大鼠肝微粒体和胞浆中的代谢动力学，鉴定硝克柳胺在大鼠和人肝微粒体中的主要代谢产物及参与代谢的药物代谢酶。采用高效液相色谱-紫外检测(HPLC-UV)方法测定大鼠肝微粒体和胞浆中硝克柳胺浓度，应用选择性抑制剂鉴定参与硝克柳胺代谢的药物代谢酶类型，采用液相色谱-串联质谱联用(LC-MS/MS)法分离鉴定硝克柳胺在大鼠和人肝微粒体中的主要代谢产物。硝克柳胺在大鼠和人肝微粒体的主要代谢产物(M₁)为硝基还原产物［3-(3′-羧基-4′-羟基苯胺羰基)-6-氨基-7-羟基-8-甲基香豆素］，大鼠体内(血浆、尿液、胆汁及肝组织)主要代谢产物与M₁一致。硝克柳胺的体外代谢是依赖多个药物代谢酶参与的酶促反应，包括微粒体CYP450还原酶、细胞色素b₅还原酶和CYP2C6以及胞浆NAD(P)H脱氢酶和黄嘌呤氧化酶。     

肝微粒体体外温孵法与基因重组P450酶系在药物体外肝代谢研究中的应用进展

目的 介绍肝微粒体体外温孵法与基因重组P450酶系在药物体外肝代谢中应用进展。方法 根据近几年的文献资料进行分析、综合、归纳，分别按肝微粒体体外温孵法与基因重组P450酶系进行介绍。结果与结论 两种方法在药物体外代谢研究上既有统一又可互补，二者相结合可更有效的应用于药物的高通量筛选、代谢物种选择及代谢物生成等研究领域。     

力达霉素的体外代谢

旨在研究力达霉素在血浆和肝微粒体中的体外代谢性质,指导临床合理用药。选择HPLC-MS/MS测定方法,通过测定力达霉素的活性成分,考察力达霉素在大鼠、比格犬、猕猴和人血浆及肝微粒体中的代谢稳定性以及在人肝微粒体中对细胞色素P450(cytochrome P450,CYP450)各亚型酶的抑制作用。结果表明,力达霉素在4个种属血浆中均有代谢,其代谢速率为大鼠>比格犬>人>猕猴;在4个种属肝微粒体中,只有在猕猴肝微粒体中代谢;在浓度为0.000 5～10 ng·mL-1时,对人肝微粒体中细胞色素P450各亚型酶几乎无抑制作用。可见力达霉素在人体内的代谢性质与比格犬体内较相似,而且临床上当力达霉素与通过CYP450酶代谢的药物合用时,不会导致这些药物的代谢减慢。     

TM208在大鼠肝微粒体中代谢产物与细胞色素P450亚型代谢酶鉴别研究

在大鼠肝微粒体的体外代谢中研究作用于TM208的细胞色素P450亚型代谢酶。以不含细胞色素P450化学抑制剂的样品为对照，研究不同细胞色素P450亚型选择性化学抑制剂对TM208代谢转化率的影响。CYP2D和CYP2B的选择性抑制剂对TM208的代谢表现出浓度依赖性较强抑制作用，CYP1A的选择性抑制剂对TM208的代谢表现出一定抑制作用。CYP3A的选择性抑制剂对TM208的代谢没有表现出明显的抑制作用。TM208在人鼠肝微粒体体外代谢中主要通过CYP2D和CYP2B两种细胞色素P450亚型代谢酶参与代谢。     

药物非临床安全性评价中细胞色素P450s酶的研究方法

目前,在药物安全性评价中越来越关注细胞色素P450s酶系统在药物代谢、药物血浆暴露量、药物相互拮抗和诱导等方面的作用。现常用的细胞色素P450s研究方法有以下几方面。1以肝脏为基础的体外代谢的研究1.1肝微粒体外代谢法由制备的肝微粒体在模拟生理温度及环境条件下与药物进行     

UPLC-MS/MS方法研究pongachin在肝微粒体中的代谢特征

目的:采用不同种属肝微粒体,研究黄酮类化合物pongachin的体外代谢特性,明确参与pongachin代谢的CYP酶亚型。方法:选择UPLC-MS/MS测定方法,通过测定pongachin的剩余浓度,考察pongachin在人、大鼠和猴3个种属肝微粒体中的代谢稳定性,以及在大鼠肝微粒体中的代谢表型。结果:pongachin在人、大鼠和猴肝微粒体中t1/2分别为36.86、30.13和20.50 min;其清除率为人大鼠猴。在大鼠肝微粒体中,CYP2E1、CYP2C、CYP1A2对pongachin的代谢有较强抑制作用。结论:pongachin在人和大鼠肝微粒体中代谢稳定性较好(30 min),且在人和大鼠肝微粒体中代谢相似,在临床上与其他抑制CYP2E1、CYP2C、CYP1A2代谢的药物合用时应注意药物间的相互作用。     

肉豆蔻木脂素的体外代谢初步研究

目的建立诱导的大鼠肝微粒体药物代谢酶体外转化模型,评价肉豆蔻木脂素的体外代谢情况,为规模化制备肉豆蔻木脂素的代谢产物提供方法.方法将肉豆蔻木脂素与苯巴比妥诱导的大鼠肝微粒体药物代谢酶共温孵,用高效液相色谱法检测肉豆蔻木脂素及其代谢产物.结果肉豆蔻木脂素在苯巴比妥诱导的大鼠肝微粒体药物代谢酶作用下,可以被代谢,并且发现了7个代谢产物.结论建立的肝微粒体药物代谢酶模型可靠有效,可用于肉豆蔻中肉豆蔻木脂素的体外代谢研究.     

五味子醇甲在大鼠肝微粒体内的代谢动力学和性别差异

体外研究五味子醇甲(schizandrin，SZ)在大鼠肝微粒体内的代谢动力学和性别差异。制备正常雌、雄大鼠肝微粒体，与SZ共同温孵，以高效液相色谱法测定SZ及其代谢产物。SZ在雄鼠肝微粒体内代谢反应的最大速率V_max、米氏常数K_m和清除率Cl_int分别为(21.88±2.30) μmol·L^-1·min^-1·mg^-1(protein)，(389.00±46.26) μmol·L^-1和(0.056 3±0.000 7) min·mg^-1(protein)；在雌鼠肝微粒体内代谢反应的最大速率V_max、米氏常数K_m和清除率Clint_{分别为(0.61±0.07) μmol·L^-1·min^-1·mg^-1(protein)，(72.64±13.61) μmol·L^-1和(0.008 4±0.000 8) min·mg^-1(protein)，雌、雄鼠肝微粒体内SZ的主要代谢物不同，分别为7,8-顺二羟基五味子醇甲(M1)和7,8-顺二羟基-2-去甲基五味子醇甲(M2b)。酮康唑、奎尼丁和奥芬得林对SZ的在雌、雄大鼠肝微粒体内代谢均有不同程度的抑制作用，西咪替丁对其在雄鼠肝微粒体内的代谢也有一定的抑制作用。SZ在雌、雄大鼠肝微粒体中代谢动力学及代谢产物存在明显的性别差异，这种差异可能主要是由CYP3A和CYP2C11在大鼠肝微粒体内的性别差异引起的。}     

HPLC法测定利多卡因及其代谢产物估算人肝微粒体CYP3A的活性

目的　以利多卡因 (LDC)与其代谢产物的比值估算人肝微粒体中CYP3A的活性。方法　以 1 0g·L- 1微粒体蛋白浓度 3 7℃孵育利多卡因 60min ,以HPLC测定利多卡因及其代谢产物单乙基甘氨二甲基苯酰胺 (MEGX)和甘氨二甲基苯酰胺 (GX)的含量。结果　LDC、MEGX和GX的标准曲线方程分别为 ^Y =0 2 93 4X -0 0 0 5661(r =0 9997)、^Y =0 7913X -0 0 0 8916(r =0 9993 )和 ^Y =0 6799X -0 0 0 7770 (r =0 9985)。体外孵育的最佳条件为 2 0mg·L- 1的LDC在浓度为 1 0 g·L- 1的微粒体中 ,孵育 60min ,代谢产物与利多卡因的平均比值为 3 2 8。结论　(MEGX +GX) /LDC可用来估算人肝微粒体CYP3A的活性。     

Biotransformation of 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075), a novel antimicrotubule agent, by mouse, rat, dog, and human liver microsomes.

6-Methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole (BPR0L075) is a novel synthetic indole compound with microtubule binding activity. Incubation of BPR0L075 with mouse, rat, dog, and human liver microsomes in the presence of NADPH resulted in the formation of six metabolites. Liquid chromatography-tandem mass spectrometry and comparison with the synthetic reference standards identified two metabolites (M1 and M5) as the products derived from hydroxylation on the indole moiety of the molecule. M3 was also identified as a product derived from hydroxylation, but the structure of this metabolite was not identified because of the lack of a reference standard. M2, M4, and M6 were identified as the products derived from O-demethylation. M2, 6-desmethyl-BPR0L075, was the major metabolite formed by the liver microsomes of the four species. No qualitative species difference in the metabolism of BPR0L075 was observed. There was quantitative species difference in the metabolism of BPR0L075 among the four species. Whereas mouse and rat liver microsomes metabolized BPR0L075 predominantly via O-demethylation, dog liver microsomes metabolized BPR0L075 by O-demethylation and hydroxylation to about the same extent. The rank order of intrinsic clearance rates for the conversion of BPR0L075 to 6-desmethyl-BPR0L075 was mouse > rat > human > dog. Incubation of BPR0L075 with baculovirus-insect cell-expressed human cytochrome P450 (P450) isozymes showed that CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the O-demethylation and hydroxylation of BPR0L075 but to a different degree. Among the six P450 isozymes tested, CYP1A2 and 2D6 were most active on catalyzing the metabolism of BPR0L075. CYP1A2 catalyzed mainly the formation of M1, M2, and M3. M2 was the predominant metabolite formed by CYP2D6.     

甲基莲心碱在大鼠肝脏中的代谢产物及其途径

大鼠灌胃给予甲基莲心碱 20 mg·kg^-1，采用液相色谱-串联质谱联用法对大鼠肝脏中的代谢产物进行分析；并建立肝微粒体温浴及NADPH再生体系，采用高效液相色谱-紫外检测法研究CYP450亚型的特异性抑制剂对甲基莲心碱体外代谢的影响。在正离子检测方式下，除甲基莲心碱外共检测到4种代谢产物M₁、M₂(主要代谢产物)、M₃和M₄。其中，M₂和M₄通过与对照品的色谱和质谱比对，确认为莲心碱和异莲心碱，而M₁ 和M₃可能为去甲基莲心碱和去甲基异莲心碱。CYP3A1的特异性抑制剂酮康唑和CYP2D1的特异性抑制剂奎尼丁均可抑制甲基莲心碱在肝微粒体温孵液中的代谢，其主要代谢产物莲心碱的生成抑制率分别为25.7%和80.5%。因此提示，甲基莲心碱在肝脏中的主要代谢途径是苄基和喹啉环上的甲氧基脱甲基化，其主要代谢物为莲心碱，CYP2D1和CYP3A1均参与了其生物转化。     

人肝微粒体CYP亚型在新型吲哚醌类化合物629代谢中的作用

目的研究人肝微粒体重组体系中不同CYP亚型在丝裂霉素C(MMC)的衍生物5-氮丙啶-3-羟甲基-1-甲基吲哚-4,7-二酮[5-(aziridin-1-yl)-3-hydroxymethyl-1-methylin-dole-4,7-dione,简称629]代谢中的作用。方法不同浓度629与人肝脏微粒体共孵育,给予细胞不同CYP亚型特异性抑制剂的处理,用高压液相色谱法(high pressure liquid chro-matography,HPLC)分离、检测629的消失情况。结果HPLC检测到629在肝脏微粒体中的代谢遵循酶动力学剂量效应关系,Km值为336μmol·L-1。P450酶系中CYP1A2、CYP2B6和CYP2A6被抑制后,可影响629的代谢(P<0.05),并且3者中CYP1A2的影响较大,但三者间差异无显著性(P>0.05);而CYP3A4、CYP2C19、CYP2C9、CYP2E1和CYP2D6对629的代谢无影响(P>0.05)。结论新型吲哚醌类生物还原物629可在肝脏代谢,其中CYP1A2、CYP2B6和CYP2A6可参与629的代谢,这为新型生物还原活性物设计、开发及临床上的合理用药具有重大的意义。     

Inhibitory effect of omeprazole on the metabolism of midazolam in vitro

To examine the effect of omeprazole on the hepatic drug metabolizing enzyme system microsomes from rat and human liver samples were incubated with midazolam (CAS 59467-70-8) in the absence and presence of various concentrations of omeprazole (CAS 73590-58-6), its sulfone metabolite and for comparison also with cimetidine. In the extracted incubation mixtures unchanged midazolam, a-OH-midazolam, 4-OH-midazolam and di-OH-midazolam were analyzed by HPLC. In both species omeprazole (and its sulfone) inhibited the formation of all three oxidized metabolites of midazolam and the corresponding IC50-values (range 0.2-1.3 mmol/l for rat microsomes and 0.2-1.5 mmol/l for human microsomes) were comparable to cimetidine (range 0.05 to 3.8 mmol/l). These results indicate that the oxidative metabolism of midazolam can be inhibited in vitro by omeprazole (and/or its sulfone metabolite) and this interaction should be considered if both drugs are administered concomitantly in man.     

In vitro metabolism of a triclyclic alkaloid (M445526) in human liver microsomes and hepatocytes

The in vitro metabolism of M445,526 (ZD6,126 phenol) was investigated by incubating [(14)C]-M445,526 at a concentration of 10 microg ml(-1) with human hepatic microsomes (4 mg ml(-1)) or human hepatocytes (2 x 10(6) cells ml(-1)) for up to 180 min. Following incubation with microsomes and hepatocytes, up to 78% and 40% of [(14)C]-M445,526 was metabolized after 180 and 120 min, respectively. High-performance liquid chromatography (HPLC) with radiochemical detection confirmed extensive metabolism of [(14)C]-M445,526 by microsomes and hepatocytes. Mass spectrometry and (1)H-NMR spectroscopy enabled structural identification of up to eight metabolites. Human liver microsomes formed one major (O-desmethyl) and three minor (a further O-desmethyl and two different hydroxylated) phase I metabolites. Human hepatocytes produced one major metabolite, a sulphate conjugate of the major O-desmethyl metabolite formed by microsomes. Four minor metabolites were also formed, primarily by O-demethylation with subsequent glucuronidation. Taken collectively, [(14)C]-M445,526 underwent extensive in vitro metabolism by human liver fractions. These data were confirmed by subsequent human in vivo studies.     

Determination of microsome and hepatocyte scaling factors for in vitro/in vivo extrapolation in the rat and dog

1. In vivo clearance predictions from in vitro assays require scaling factors to relate the concentrations of hepatocytes or microsomal protein to the intact liver. 2. The aims were to measure the variability in scaling factors for Wistar rat and beagle dog for which the literature is particularly scarce and determine any sex differences. 3. Scaling factors were determined by comparing the cytochrome P450 (P450) content in hepatocytes or microsomes against the P450 content of fresh liver homogenate. The use of fresh homogenate is recommended as freezing can increase contamination and affect the P450 assay. 4. Mean(geo) hepatic microsomal concentrations in Wistar rats were 61 mg g(-1) liver (95% confidence interval (CI); 47-75 mg g(-1) liver) and in beagle dogs 55 mg g(-1) liver (95% CI = 48-62 mg g(-1) liver). Mean(geo) hepatocellularity was 163 x 10(6) cells g(-1) liver for Wistar rats (95% CI = 127-199 x 10(6) cells g(-1) liver) and 169 x 10(6) cells g(-1) liver (95% CI = 131-207 x 10(6) cells g(-1) liver) for beagle dogs. The data generated in this study indicate a consistency in scaling factors between rat and dog. No sex differences were observed.     

The in vitro biosynthesis and stability measurement with agyl-glycuronide isoformes of the main metabolite of ipriflavone.

The formation and stability of 1-beta-glucuronide conjugate of the main metabolite of ipriflavone [7-(1-carboxy-ethoxy)-isoflavone] (CI)--were studied by using liver microsomes, hepatocytes, and isolated perfused liver of untreated and 3-methylcholanthrene (MC) treated dog and rat, and human liver microsomes. MC treatment enhanced the rate of conjugation twice as much as that of the control in the microsomes of both dogs and rats. Conjugation of CI by microsomes results in two metabolites, both sensitive to pH and temperature. Other two glucuronide forms appeared in experiments with hepatocytes and perfused liver. Mass spectrometry supported. The conclusion, assumption that both metabolites produced by microsomes are glucuronide conjugate isoforms of CI, and that they could be distinguished according to the intensity of peaks on FAB-MIKE spectra. The beta-glucuronidase enzyme hydrolysed only the 1-beta-glucuronide isomer, the other, migrated form remained unchanged. D-saccharic-acid-1,4-lactone, a specific inhibitor of beta-glucuronidase enzyme, decreased the rate of enzymatic cleavage. Standard curves of CI were prepared by HPLC, and 1-beta-CI-glucuronide was quantified according to the amount of CI formed by hydrolysis. The stability of conjugates greatly depends on pH and temperature, and the rate of degradation and isomerization is sensitive to the value of both. Lowering the pH from 7.4 to 5.0 and the temperature from 37 degrees C to 18 degrees C increased the stability of glucuronides. Increasing the pH to 12.0 results in very rapid acyl migration and hydrolysis.