人肝细胞色素P450 2C8/9、2E1比活性测定

目的　建立甲苯磺丁脲羟化酶 (CYP2C8/ 9)和氯羟苯口恶唑 6 羟化酶 (CYP2E1)比活性的测定方法 ,为深入研究CYP45 0在药物代谢中的作用奠定基础。方法　从成人肝细胞中提取微粒体 ,测定其蛋白含量 ,以甲苯磺丁脲、氯羟苯口恶唑为底物 ,用HPLC以梯度洗脱法测定其代谢产物羟基甲苯磺丁脲及 6 羟基氯羟苯 口恶唑生成量 ,据此计算人肝细胞色素P45 0 (CYP45 0 )同工酶甲苯磺丁脲羟化酶 (CYP2C8/9)和氯羟苯 口恶唑 6 羟化酶 (CYP2E1)比活性。结果　于不同时间反复测定的CYP2C8/ 9、CYP2E1比活性无差异。结论　CYP2C8/ 9、CYP2E1的测定方法较为简单、稳定、重复性好 ,可用于新药筛选、安全性评价及肝脏病理学、毒理学研究。     

人肝细胞色素P450 2C8/9、2E1比活性测定

目的建立甲苯磺丁脲羟化酶(CYP2C8/9)和氯羟苯口恶唑6-羟化酶(CYP2E1)比活性的测定方法,为深入研究CYP450在药物代谢中的作用奠定基础.方法从成人肝细胞中提取微粒体,测定其蛋白含量,以甲苯磺丁脲、氯羟苯口恶唑为底物,用HPLC以梯度洗脱法测定其代谢产物羟基甲苯磺丁脲及6-羟基氯羟苯口恶唑生成量,据此计算人肝细胞色素P450(CYP450)同工酶甲苯磺丁脲羟化酶(CYP2C8/9)和氯羟苯口恶唑6-羟化酶(CYP2E1)比活性.结果于不同时间反复测定的CYP2C8/9、CYP2E1比活性无差异.结论 CYP2C8/9、CYP2E1的测定方法较为简单、稳定、重复性好,可用于新药筛选、安全性评价及肝脏病理学、毒理学研究.     

细胞色素P450 2C9酶活性测定探针的分析

P450 2C9是人体中重要的药物代谢酶.P450 2C9基因编码区的多态性造成氨基酸序列的变化,主要包括P450 2C9*2和P450 2C9*3两种突变体,变异纯合子对P450 2C9底物的代谢能力明显降低.目前常用的P450 2C9的探药包括甲苯磺丁脲、华法令和洛沙坦等,各种探药在检测方法、不良反应和灵敏度方面各有优缺点.     

人肝细胞色素P450含量及其同工酶1A1,2A6活性的测定

从成人肝细胞中提取微粒体测定其蛋白浓度、细胞色素Ｐ４５０（ＣＰＹ４５０）的总量,并建立了通过测定代产物异恶唑和香豆素生成量确定ＣＹＰ４５０同工酶ＣＹＰ１Ａ１、ＣＹＰ２Ａ６活性的测定方法。结果表明：该测定方法简单、稳定、重复性好,人肝微粒体于－８０℃保存６个月人活性无明显影响。     

中国健康人群P450 2C9基因多态性与甲苯磺丁脲代谢的关系

目的 研究甲苯磺丁脲人体代谢过程与中国健康志愿者P450 2C9基因多态性的关系。方法 不同P450 2C9＊1／＊1（n=53）、＊1／＊3（n=9）及＊3／＊3（n=1）基因型的健康志愿者单次口服甲苯磺丁脲500mg后，用HPLC方法测定血和尿中的甲苯磺丁脲及其代谢产物浓度，研究甲苯磺丁脲的药代过程。结果 P450 2C9＊1／＊3与P450 2C9＊3／＊3基因型的受试者与P450 2C9＊1／＊1基因型相比，AUC（0-∞）增加20％和116％，t1/2增加60％和813％，CL0为68％和11％，CLform仅为39％和3％。结论 P450 2C9＊1／＊3对甲苯磺丁脲的代谢活性较P4502C9＊1／＊1显著降低，P4502C9＊3／＊342谢活性更低，并呈现基因剂量关系。     

肝细胞微粒体的制备和细胞色素P450氧化酶活性测定

目的：为测定人肝细胞微粒体细胞色素Ｐ４５０氧化酶的活性。方法：用差速离心法制备３例人肝细胞微粒体。结果：细胞色素Ｐ４５０的含量为０．５２３±０．００５ｎｍｏｌ·ｍｇ－１；细胞色素ｂ５为０．２８５±０．０２５ｎｍｏｌ·ｍｇ－１；氨基比林Ｎ－脱甲基酶的活力为０．５±０．６ｎｍｏｌ·ｍｇ－１；乙基吗啡Ｎ－脱甲基酶活力为０．９８±０．０８ｎｍｏｌ·ｍｇ－１。结论：Ｐ４５０酶活性影响因素较多，个体差异大。临床用药时应考虑患者的个体情况。     

细胞色素P450 CYP2C9基因多态性对甲苯磺丁脲代谢动力学的影响

目的研究细胞色素P450 CYP2C9基因多态性对甲苯磺丁脲代谢动力学的影响。方法用基因芯片对137名健康志愿者进行CYP2C9基因多态性检测，将受试者分为CYP2C9野生型、杂合突变型和纯合突变型3组，用高效液相色谱法检测甲苯磺丁脲在受试者体内的药物代谢动力学参数，统计分析各组间药代动力学性质差异。结果在137名受试者中发现了9个CYP2C9*1/*3杂合型突变体和1个CYP2C9*3/*3纯合型突变体，其余为野生型个体。将9名CYP2C9*1/*3，1名CYP2C9*3/*3以及随机抽取的10名野生型个体分组，以甲苯磺丁脲为探药进行药物代谢动力学研究。结果在杂合型突变个体组以及纯合型突变个体组中，甲苯磺丁脲的代谢率显著低于对照的野生型个体组。结论CYP2C9基因多态性对甲苯磺丁脲代谢具有显著影响并呈基因剂量效应，检测突变型个体对指导临床合理用药和个体化医疗具有重要意义。     

吴茱萸次碱在人肝微粒体中对细胞色素P450酶的抑制作用

目的研究吴茱萸次碱(WZY)在人肝微粒体中对细胞色素P450酶的抑制作用。方法对照组和抑制组酶活性均用探针药测定,探针药物及其代谢产物用HPLC进行检测。用代谢产物与母药比值来表达酶的活性。结果加入50μmol·L-1吴茱萸次碱组CYP1A2,CYP2C19,CYP2E1和CYP2D6的活性显著降低,CYP3A4和CYP2C9活性无显著变化。结论吴茱萸次碱对CYP1A2,CYP2C19,CYP2E1和CYP2D6的活性有显著抑制作用,而对CYP2C9和CYP3A4的活性无显著影响。     

酒精性肝损伤大鼠细胞色素P450 CYP2E1和细胞色素P450 CYP3A的代谢活性

目的观察酒精性肝损伤对大鼠细胞色素P450CYP3A(CYP3A)和细胞色素P450CYP2E1(CYP2E1)代谢活性的影响。方法采用ig给予白酒制备大鼠酒精性肝损伤模型,检测血清中谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,采用HE染色法光镜下观测酒精对肝脏损伤程度。大鼠ip给予CYP3A探针药物咪达唑仑10mg·kg-1或ig给予CYP2E1探针药物氯唑沙宗50mg·kg-1后,采用高效液相色谱法测定不同时间点大鼠血浆中咪达唑仑和氯唑沙宗的血药浓度,并应用3P87软件计算其药代动力学参数,以考察CYP2E1和CYP3A的代谢活性的变化。大鼠ig给予氯唑沙宗80mg·kg-1后,热板方法测定大鼠添足次数和添足反射潜伏期。结果酒精性肝损伤可致大鼠肝小叶结构不清,肝索排列紊乱,肝细胞体积增大,呈弥漫性中度水变性,肝窦受压,大部分肝细胞胞浆内见大小不等的脂肪空泡;与正常对照组相比,酒精性肝损伤组大鼠GPT和GOT活性分别增加了16.0%和20.0%(P<0.05,P<0.01)。酒精性肝损伤致大鼠CYP2E1对探针药物氯唑沙宗的代谢活性增强,AUC,t1/2和cmax分别降低了38.0%,30.5%和35.0%(P<0.05);酒精肝损伤组大鼠氯唑沙宗镇痛效果明显降低;酒精性肝损伤致大鼠CYP3A对探针药物咪达唑仑的代谢活性增强,AUC,t1/2和cmax分别降低了122.6%,54.9%和56.9%(P<0.01,P<0.05)。结论酒精性肝损伤可使大鼠CYP2E1和CYP3A代谢活性增强。     

中国人肝微粒体中细胞色素P450酶含量和活性的个体间差异(英文)

目的:比较不同中国人肝微粒体中几种重要细胞色素P450(CYP)的酶含量和活性。方法:运用West-ern斑点分析和光密度扫描,对17个汉族、17个壮族和8个苗族受试者肝微粒体中的细胞色素P4501A2(CYP1A2)、2C9及3A4进行定量;非那西丁、甲磺丁脲、异喹胍和奥美拉唑分别用于体外测量CYP1A2、2C9、2D6及3A4的活性。结果:CYP1A2、2C9及3A4的含量和活性具有很大的个体间变异,另外CYP2D6的活性在各样本间也有很大差异;CYP3A4(32％)是中国人肝微粒体中含量最丰富的CYP,CYP2C9(19％)和CYP1A2(16％)的含量也很可观;除了CYP1A2的含量和活性具有一定的种族和性别差异外,未发现其它CYP具有种族和性别差异;CYP1A2、2C9和3A4的酶蛋白含量分别和它们的活性具有很好的相关性。结论:我们的结果为在中国人中进行药物代谢研究提供了非常有价值的信息。     

Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes

Cytochrome P450 (CYP) substrates that yield fluorescent metabolites were used for rapid screening of drug metabolism activities of 13 recombinant human cytochromes P450, human liver microsomes and human hepatocytes. Reproducible results were obtained using a fluorescent plate reader (CytoFluor) more expediently than those generated using conventional HPLC methods. Typically, results for 96 samples were obtained with the plate reader in less than 10 min as opposed to 15-35 min/sample required by conventional HPLC. The fluorescent substrates used to measure CYP activities were as follows: 3-cyano-7-ethoxycoumarin (CEC) for CYP1A1, CYP1A2, CYP2C9 and CYP2C19; 7-ethoxyresorufin (7-ER) for CYP1A1, CYP1A2 and CYP1B1; 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC) for CYP2D6; dibenzylfluorescein (DBF) for CYP3A4, CYP3A5 and CYP2C8; 7-methoxy-4-trifluoromethylcoumarin (7-MFC) for CYP2E1, CYP2B6 and CYP2C18; and coumarin for CYP2A6. The chemical inhibition and correlation data indicated that the following substrates can be used as specific functional probes for individual cytochrome P450 present in human liver microsomes: coumarin for CYP2A6 (r=0.82), AMMC for CYP2D6 (r=0.83) and DBF for CYP3A4 (r=0.92). The fluorescent plate reader was found to be useful for the rapid assessment of CYP activities (positive control) in both intact cells and subcellular fractions.     

丹参注射液对人肝微粒体酶CYP2C9,CYP2C19,CYP2D6体外抑制作用的研究

目的 评价丹参注射液在体外对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6活性的影响。方法 将丹参注射液与CYP450酶3种亚型(CYP2C9、CYP2C19、CYP2D6)的特异性探针底物甲苯磺丁脲、奥美拉唑、右美沙芬与大鼠肝微粒体进行孵育,采用LC-MS/MS法测定对应3种代谢产物4-羟基甲苯磺丁脲、5-羟基奥美拉唑、右啡烷的浓度,求算出IC₅₀。结果 丹参注射液对CYP2C9、CYP2C19和CYP2D6的IC₅₀值均>50 μg/mL。结论 丹参注射液对人肝微粒体酶CYP2C9、CYP2C19、CYP2D6没有抑制作用。     

Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9

1.?Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated.

2.?We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4′-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC₅₀) 1.3 and 16.1?μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively.

3.?Glycyrol decreased CYP2C9-catalyzed diclofenac 4′-hydroxylation activity with IC₅₀ values of 0.67?μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs.     

Inhibition of cytochrome P450 2E1 by propofol in human and porcine liver microsomes

While almost anesthetics are metabolized by the cytochrome P450 (CYP) 3A4, some major volatile ones such as halothane and sevoflurane are metabolized by CYP2E1 in humans. To determine whether 2,6-diisopropylphenol (propofol), a widely used intravenous anesthetic agent, known to inhibit CYP3A4 and CYP1A2, also inhibits CYP2E1, 6-OH hydroxylation of chlorzoxazone, a prototypical CYP2E1 substrate, was estimated using two pools of human microsomes and one pool of porcine microsomes from seven livers. Basal human enzyme activities were characterized by a V(max) of 1426+/-230 and 288+/-29 pmol min(-1)mg(-1) protein and a K(m) of 122+/-47 and 149+/-42 microM, while the corresponding porcine activities were associated with a V(max) of 352+/-42 pmol min(-1)mg(-1) protein and a K(m) of 167+/-38 microM. A competitive inhibition of CYP2E1 by propofol was observed with low inhibition constants in the therapeutic range in both porcine (19 microM) and human (48 microM) liver microsomes. These in vitro results suggest that propofol could have a protective effect on toxic metabolite activation of compounds catalyzed by CYP2E1.     

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。     

Involvement of multiple human cytochromes P450 in the liver microsomal metabolism of astemizole and a comparison with terfenadine

AIMS: The aims of the present study were to investigate the metabolism of astemizole in human liver microsomes, to assess possible pharmacokinetic drug-interactions with astemizole and to compare its metabolism with terfenadine, a typical H1 receptor antagonist known to be metabolized predominantly by CYP3A4. METHODS: Astemizole or terfenadine were incubated with human liver microsomes or recombinant cytochromes P450 in the absence or presence of chemical inhibitors and antibodies. RESULTS: Troleandomycin, a CYP3A4 inhibitor, markedly reduced the oxidation of terfenadine (26% of controls) in human liver microsomes, but showed only a marginal inhibition on the oxidation of astemizole (81% of controls). Three metabolites of astemizole were detected in a liver microsomal system, i.e. desmethylastemizole (DES-AST), 6-hydroxyastemizole (6OH-AST) and norastemizole (NOR-AST) at the ratio of 7.4 : 2.8 : 1. Experiments with recombinant P450s and antibodies indicate a negligible role for CYP3A4 on the main metabolic route of astemizole, i.e. formation of DES-AST, although CYP3A4 may mediate the relatively minor metabolic routes to 6OH-AST and NOR-AST. Recombinant CYP2D6 catalysed the formation of 6OH-AST and DES-AST. Studies with human liver microsomes, however, suggest a major role for a mono P450 in DES-AST formation. CONCLUSIONS: In contrast to terfenadine, a minor role for CYP3A4 and involvement of multiple P450 isozymes are suggested in the metabolism of astemizole. These differences in P450 isozymes involved in the metabolism of astemizole and terfenadine may associate with distinct pharmacokinetic influences observed with coadministration of drugs metabolized by CYP3A4.