氟喹诺酮类药物对大鼠肝外组织药物代谢酶的影响

目的:探讨氟喹诺酮类药物(FQs)对大鼠肺、脑、肾、小肠中细胞色素P450(CYP)含量、氨基比林N- 脱甲基酶(AMND)、红霉素N-脱甲基酶(ERND)活性的影响。方法:体外实验中环丙沙星、妥苏沙星和司帕沙星的药物终浓度均为1 mmol·L-1;体内实验按1 mmol·kg-1剂量灌胃,qd×7。制备大鼠肺、脑、肾、小肠S9,分光光度法测定CYP含量、AMND及ERND活性变化。结果:大鼠肺、肾组织CYP含量低;3种药物可不同程度地抑制各组织AMND活性(P<0．05)。肾和小肠中可测到ERND活性,而肺、脑中未测到;给药后肾、小肠 ERND活性有降低趋势,但大多无显著差异。结论:FQs对肝外组织CYP含量无显著影响,对AMND活性有抑制作用,对ERND有抑制趋势。     

CYP2C19基因型和CYP2C9对人肝微粒体中氟西汀N-去甲基代谢的影响(英文)

目的:本实验旨在研究CYP2C19基因型人肝微粒体中氟西汀N-去甲基代谢的酶促动力学特点并鉴定参与此代谢途径的细胞色素P-450酶。方法:测定基因型CYP2C19肝微粒体中去甲氟西汀形成的酶促动力学。鉴定氟西汀N-去甲基酶活性与细胞色素P-450 2C9,2C19,1A2和2D6酶活性的相关性,同时应用各种细胞色素P-450酶的选择性抑制剂和化学探针进行抑制实验,从而确定参与氟西汀N-去甲基代谢的细胞色素P-450酶。结果:去甲氟西汀生成的酶促动力学数据符合单酶模型,并具有Michaelis-Menten动力学特征。当底物浓度为氟西汀25μmol/L和100μmol/L时,去甲氟西汀(N-FLU)的生成率分别与甲磺丁脲3-羟化酶活性显著相关(r_1=0.821,P_1=0.001;r_2=0.668,P_2=0.013),当底物浓度为氟西汀100μmol/L时,N-FLU的生成率与S-美芬妥因4’-羟化酶活性显著相关(r=0.717,P=0.006)。PM肝微粒中磺胺苯吡唑和醋竹桃霉素对氟西汀N-去甲基代谢的抑制作用显著大于EM(73％vs 45％,P<0.01)。结论:在生理底物浓度下,CYP2C9是催化人肝微粒体中氟西汀N-去甲基代谢的主要CYP-450酶;而高底物浓度时,以CYP2C19的作用为主。     

人肝微粒体中尼莫地平代谢的酶动力学及其抑制(英文)

目的:在体外研究人肝微粒体中尼莫地平代谢的酶动力学及选择性的细胞色素P-450(CYP450)酶抑制剂对尼莫地平代谢的影响。方法:采用人肝微粒体研究尼莫地平脱氢酶的代谢动力学,运用Eadie-Hof-stee方程估算其动力学参数。在体外运用CYP450酶的选择性抑制剂探讨其对尼莫地平代谢的影响及人肝微粒体中参与尼莫地平二氢吡啶环脱氢代谢的CYP450酶。结果:尼莫地平在人肝微粒体中的代谢存在较大的个体差异,尼莫地平脱氢酶的K_m为(36±11)μmol,其V_m为(17±7)μmol·g~(-1)·min~(-1)。酮康唑和三乙酰竹桃霉素竞争性地抑制尼莫地平二氢吡啶环脱氢代谢,其K_i值分别为0.59和122.2μmol。非那西丁、奎尼丁、DDC、Sul和Tra对尼莫地平二氢吡啶环脱氢代谢没有明显的影响。结论:尼莫地平在体内的药物动力学个体差异与其在肝中的代谢存在多态性有关。CYP3A参与了尼莫地平二氢吡啶环脱氢代谢,CYP3A的抑制剂可能会与尼莫地平发生代谢相互作用。     

氟喹诺酮类药的药理特点

氟喹诺酮类药物是20世纪70年代后期发展起来的第三代喹诺酮类抗生素,在前两代的基础上,在C-6位上接上了氟原子,使该类药物抗菌谱更广,抗菌活性增强,交叉耐药现象更少,耐药性降低,口服吸收更好,体内广泛更分布,组织穿透力增强,半衰期增长,使用更方便。     

氟喹诺酮类抗菌药的不良反应

喹诺酮类尤其是氟喹诺酮类以其抗菌谱广，抗菌作用强，口服吸收好等优点，广泛应用于临床，但该类药物的不良反应逐渐被发现并引起医患重视，本文对氟喹诺酮类的有害药物反应综述。     

利福喷丁对小鼠肝微粒体药酶的诱导作用

连续给小鼠口服利福喷丁40mg/kg或20mg/kg14日后,用药鼠的戊巴比妥钠催眠时间显著缩短,利福喷丁血浓下降,鼠肝重增加,而SGPT活性无改变。鼠肝细胞中细胞色素P-450和细胞色素B_b含量明显增加,表明利福喷丁有诱导小鼠肝药酶的作用。     

西尼地平在人肝微粒体内代谢及代谢抑制

目的：在体外研究西尼地平在人肝微粒体内的代谢及选择性细胞色素P-450（CYP450）酶抑制剂对其代谢的影响。方法：在体外用人肝微粒体研究西尼地平的代谢,并用CYP450酶的选择性抑制剂探讨其对西尼地平代谢的影响及人肝微粒体中参与西尼地平二氢吡啶环脱氢代谢的CYP450酶。结果：西尼地平在人肝微粒体内被迅速代谢物M1,二氢吡啶环侧链脱甲基代谢物M2,二氢吡嘧环脱氢及其侧链脱甲基代谢物M3,酮康唑竞争性地抑制西尼地平二氢吡啶环的脱氢代谢,同时降低西尼地平的代谢速率,而其它抑制剂,奎尼丁,α-Naphthoflavone,diethyldithiocarbamate,sulfaphenazole和tra-nylcypromine对西尼地平二氢吡啶环的脱氢代谢没有明显的影响。结论：西尼地平在人肝微粒体内被迅速代谢,其二氢吡啶环的脱氢代谢是其代谢的关键性的步骤,CYP3A作为主要的CYP酶参与了西尼地平二氢吡啶环的脱氢代谢,CYP3A的抑制剂可能会与西尼地平发生代谢相互作用。     

环丙沙星对人肝微粒体药物酶活性的影响

目的：观察环丙沙星对人肝微粒体药物代谢酶活性的影响。方法：用正常人新鲜肝组织低温匀浆,低温超速离心分离肝微粒体,测定肝微粒体细胞色素P450（CP450）的含量。以环丙沙星为处理因素,作肝微粒体药物代谢活性测定的体外试验。反应体系中微粒体蛋白的终浓度为1．0g/L,环丙沙星的终浓度为400mg/L。结果：环丙沙星对人肝微粒体药物代谢酶的活性抑制有选择性,对不同酶其抑制强度不同。对多种酶的抑制强弱顺序为：戊巴比妥侧链羟化酶＞苯并芘羟化酶＞乙基吗啡N－脱甲基酶, 其抑制率分别为0．34、0．30、和0．18。结论：环丙沙星对人肝微粒体多种药物代谢酶活性有明显的抑制作用。提示对肝功异常者、使用戊巴比妥钠麻醉的病人、经常服用吗啡类药物及高苯比芘含量环境工作人员该类药物应慎用。     

卤酚化合物LM49对大鼠肝微粒体细胞色素P450的影响

目的研究LM49对大鼠肝微粒体蛋白及细胞色素P450含量的影响。方法将大鼠分成空白对照组、溶剂对照组、阳性对照组(苯巴比妥组、地塞米松组、β-萘黄酮组)、LM49低、中、高剂量组。给药后采用超速离心法制备大鼠肝微粒体;BCA法测定大鼠肝微粒体蛋白浓度;Omura and Sato法测定大鼠肝微粒体细胞色素P450的含量。结果 给予不同剂量的LM49后,大鼠肝微粒体的蛋白及细胞色素P450含量均明显降低。低、中、高剂量组与对照组比较差异有统计学意义(P<0.05)。结论 LM49对大鼠肝微粒体细胞色素P450具有一定的抑制作用,可能引起肝药酶对某些药物代谢的改变。     

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.     

Drug-metabolizing activity of human and rat liver,lung, kidney and intestine slices

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V_max in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.     

体外人肝微粒体中阿米替林N-去甲基代谢的酶促动力学分析和抑制

采用6例人的肝微粒体应用酶促动力学分析和抑制研究阐明参与阿米替林（AT）N-去甲基代谢的CYP450的种类及性质. 去甲替林（NT）生成的酶促动力学数据符合一两酶模型，其中高亲和力酶具有Michaelis-Menten 动力学特征，而低亲和力酶则具有底物别构激活的特性. 当AT为2 μmol·L^-1时S-美芬妥英和呋喃茶碱均可使NT生成被抑制达50%左右；较高浓度的酮康唑也可使NT生成明显受到抑制，但醋竹桃霉素几乎对此没有影响; 而当AT为100 μmol·L^-1 时，酮康唑和醋竹桃霉素均是NT生成的强抑制剂. 结果提示：当底物的浓度较低时，CYP1A2和CYP2C19是催化AT体外人肝微粒体中N-去甲基代谢的主要CYP450酶类；当底物浓度较高时，由于底物别构激活的特性，CYP3A4逐渐占据主导地位.     

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。     

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。     

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.     

Enzyme kinetic analysis and inhibition of amitriptyline N-demethylation in human liver microsomes in vitro

采用６例人的肝微粒体应用酶促动力学分析和抑制研究阐明参与阿米替林（ＡＴ）Ｎ－去甲基代谢的ＣＹＰ４５０的种类及性质．去甲替林（ＮＴ）生成的酶促动力学数据符合一两酶模型，其中高亲和力酶具有Ｍｉｃｈａｅｌｉｓ－Ｍｅｎｔｅｎ动力学特征，而低亲和力酶则具有底物别构激活的特性．当ＡＴ为２μｍｏｌ·Ｌ－１时Ｓ－美芬妥英和呋喃茶碱均可使ＮＴ生成被抑制达５０％左右；较高浓度的酮康唑也可使ＮＴ生成明显受到抑制，但醋竹桃霉素几乎对此没有影响；而当ＡＴ为１００μｍｏｌ·Ｌ－１时，酮康唑和醋竹桃霉素均是ＮＴ生成的强抑制剂．结果提示：当底物的浓度较低时，ＣＹＰ１Ａ２和ＣＹＰ２Ｃ１９是催化ＡＴ体外人肝微粒体中Ｎ－去甲基代谢的主要ＣＹＰ４５０酶类；当底物浓度较高时，由于底物别构激活的特性，ＣＹＰ３Ａ４逐渐占据主导地位．     

Effect of single‐walled carbon nanotubes on cytochrome P450 activity in human liver microsomes in vitro

Single‐walled carbon nanotubes (SWCNTs) are made from a rolled single sheet of graphene with a diameter in the nanometer range. SWCNTs are potential carriers for drug delivery systems because antibodies or drugs can be loaded on their surface; however, their effect on the activities of cytochrome P450 (CYP) remains unclear. The aim of this study was to investigate the effect of two kinds of SWCNTs with different lengths (FH‐P‐ and SO‐SWCNTs) on human CYP activity. In addition, other nano‐sized carbon materials, such as carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ were also evaluated to compare their effects on CYP activities. Ten CYP substrates (phenacetin, coumarin, bupropion, paclitaxel, tolbutamide, S‐mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone) were used. Testosterone 6β‐hydroxylation and midazolam 1′‐hydroxylation, which are catalysed by both CYP3A4 and CYP3A5 in liver microsomes, were decreased by 25% and 45%, respectively, in the presence of 0.1 mg/ml SO‐SWCNT. Dextromethorphan O‐demethylation, which is catalysed mainly by CYP2D6, was decreased by 40% in the presence of SO‐SWCNT. Other CYP activities, however, were not attenuated by SO‐SWCNT. FH‐P‐SWCNT, carbon black, fullerene‐C₆₀, and fullerene‐C₇₀ at 0.1 mg/ml had no effect on CYP activities. The K_i values for testosterone 6β‐hydroxylation, midazolam 1′‐hydroxylation, and dextromethorphan O‐demethylation in liver microsomes were 136, 34, and 56 μg/ml, respectively. SO‐SWCNT was determined to be a competitive inhibitor of CYP3A4, CYP3A5, and CYP2D6. These results suggest that the effect of SO‐SWCNT differs among CYP isoforms, and that the inhibition potency depends on the physicochemical properties of the nanocarbons.