延胡索与白芷组分配伍对延胡索乙素在大鼠肝微粒体中酶促反应动力学的影响

目的研究延胡索总生物碱(TA)中延胡索乙素(TET)在肝微粒体中的酶促反应动力学,并比较白芷有效组分白芷香豆素(Cou)、挥发油(VO)与TA配伍后对TET酶促反应动力学的影响。方法超速离心法制备大鼠肝微粒体,采用高效液相色谱法测定孵育液中TET原形药物的浓度。比较TA、TA-Cou、TA-VO、TA-Cou-VO配伍各组中TET的酶促反应动力学,推导出药物米氏常数(Km)和最大反应速度(Vmax);并计算TET肝内清除率(CLint)。结果 TA配伍组中的TET在大鼠肝微粒体内代谢反应的Vmax、Km和CLint分别为0.12μmol/(L.min.mg)、5.40μmol/L、0.022 L/(min.mg);TA-Cou组分别为0.27μmol/(L.min.mg)、40.18μmol/L、0.006 L/(min.mg);TA-VO组分别为0.57μmol/(L.min.mg)、22.60μmol/L、0.025 L/(min.mg);TA-Cou-VO组分别为0.84μmol/(L.min.mg)、23.25μmol/L、0.036 L/(min.mg)。结论 TA配伍白芷有效组分可降低TA中TET在肝内的CLint。     

In vitro inhibition of Huanglian [Rhizoma coptidis (L.)] and its six active alkaloids on six cytochrome P450 isoforms in human liver microsomes

Huanglian (Rhizoma Coptidis) as a popular herb has been used for the treatment of various diseases such as diarrhea, eye inflammation and women's abdominal ailments. Alkaloids are considered to be responsible for its pharmacological effects. In this investigation, Huanglian and its six alkaloids (coptisine, epiberberine, berberine, jateorrhizine, palmatine and magnoflorine) were systematically evaluated for their inhibition of six cytochrome P450 isoforms (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) in human liver microsomes by the LC‐MS/MS method. Huanglian showed the strongest inhibition of CYP2D6, followed by CYP1A2 and CYP3A4_T. The IC₅₀ values were 5.8 µg/mL, 36.8 µg/mL and 59.2 µg/mL, respectively. Of the constituents tested, coptisine and epiberberine showed strong inhibition of CYP2D6 with IC₅₀ values of 4.4 µm and 7.7 µm ; berberine, jateorrhizine and palmatine showed weak inhibition of CYP2D6 with IC₅₀ values of 45.5 µm , 49.4 µm and 92.6 µm , respectively; jateorrhizine showed moderate inhibition of CYP3A4_T with an IC₅₀ value of 13.3 µm ; coptisine showed weak inhibition of CYP1A2 with an IC₅₀ value of 37.3 µm . In addition, activation was observed in coptisine/CYP2C9 and palmatine/CYP2C9/CYP2C19. Other CYP450 isoforms were not affected markedly by the six alkaloids. In conclusion, Huanglian showed in vitro inhibition of CYP2D6, the inhibition might be contributed mostly by protoberberine alkaloids, especially coptisine and epiberberine. Herb–drug interactions may occur through the CYP2D6 inhibition. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes

The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.     

Mechanisms underlying the inhibition of the cytochrome P450 system by copper ions

Copper toxicity has been associated to the capacity of free copper ions to catalyze the production of superoxide anion and hydroxyl radical, reactive species that modify the structure and/or function of biomolecules. In addition, nonspecific Cu²⁺‐binding to thiol enzymes, which modifies their catalytic activities, has been reported. Cytochrome P450 (CYP450) monooxygenase is a thiol protein that binds substrates in the first and limiting step of CYP450 system catalytic cycle, necessary for the metabolism of lipophilic xenobiotics. Therefore, copper ions have the potential to oxidize and bind to cysteinyl residues of this monooxygenase, altering the CYP450 system activity. To test this postulate, we studied the effect of Cu²⁺ alone and Cu²⁺/ascorbate in rat liver microsomes, to independently evaluate its nonspecific binding and its pro‐oxidant effects, respectively. We assessed these effects on the absorbance spectrum of the monooxygenase, as a measure of structural damage, and p‐nitroanisole O‐demethylating activity of CYP450 system, as a marker of functional impairment. Data obtained indicate that Cu²⁺ could both oxidize and bind to some amino acid residues of the CYP450 monooxygenase but not to its heme group. The differences observed between the effects of Cu²⁺ and Cu²⁺/ascorbate show that both mechanisms are involved in the catalytic activity inhibition of CYP450 system by copper ions. The significance of these findings on the pharmacokinetics and pharmacodynamics of drugs is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

藁本内酯在大鼠肝微粒体中代谢的酶动力学

研究体外大鼠肝微粒体藁本内酯代谢的酶动力学及选择性CYP450酶抑制剂对其代谢的影响。建立测定肝微粒体孵育液中藁本内酯含量的LC-MS法,以尼群地平为内标,二者的定量离子m/z分别选择173和315。考察确定最佳温孵条件,进行藁本内酯代谢的酶促反应动力学研究,通过特异性抑制试验,探讨参与藁本内酯代谢的主要同工酶。结果显示,酮康唑、甲氧苄啶、α-萘黄酮显著抑制藁本内酯的体外代谢,而奥美拉唑、4-甲基吡唑、奎尼丁对其体外代谢影响不大。可见CYP3A4、CYP2C9和CYP1A2是参与藁本内酯代谢的主要代谢酶,CYP2C19、CYP2E1和CYP2D6没有明显参与催化其代谢。

     

Limited influence of UGT1A1*28 and no effect of UGT2B7*2 polymorphisms on UGT1A1 or UGT2B7 activities and protein expression in human liver microsomes

AIMS: UGT1A1 and UGT2B7 are enzymes that commonly contribute to drug glucuronidation. Since genetic factors have been suggested to contribute to variability in activities and expression levels of these enzymes, a quantitative assessment of the influence of the major genotypes (UGT1A1*28 or UGT2B7*2) on enzyme activities was conducted. METHODS: Using a bank of microsomal samples from 59 human livers, the effect of UGT1A1*28 or UGT2B7*2 polymorphisms were investigated on rates of estradiol 3-glucuronidation (a marker of UGT1A1 enzyme activity) or zidovudine glucuronidation (a marker of UGT2B7 enzyme activity) and levels of immunoreactive protein for each enzyme. Glucuronidation rates for both enzymes were measured at K(m)/S(50) and 10 times K(m)/S(50) concentrations. RESULTS: UGT1A1 and UGT2B7 enzyme activities varied up to 16-fold and sixfold, respectively. Rates at K(m)/S(50) concentration closely correlated with rates at 10 times K(m)/S(50) concentration for both enzymes (but not at 1/10th K(m) for UGT2B7). Enzyme activities correlated with relative levels of immunoreactive protein for UGT1A1 and UGT2B7. Furthermore, rates of zidovudine glucuronidation correlated well with rates of glucuronidation of the UGT2B7 substrate gemcabene, but did not correlate with UGT1A1 enzyme activities. For the UGT1A1*28 polymorphism, consistent with levels of UGT1A1 immunoreactive protein, mean UGT1A1 activity was 2.5- and 3.2-fold lower for TA(6)/TA(7) (P < 0.05) and TA(7)/TA(7) (P < 0.001) genotypes in comparison with the TA(6)/TA(6) genotype. CONCLUSIONS: Relative to the observed 16-fold variability in UGT1A1 activity, these data indicate only a partial (approximately 40%) contribution of the UGT1A1*28 polymorphism to variability of interindividual differences in UGT1A1 enzyme activity. For the UGT2B7*2 polymorphism, genotype had no influence on immunoreactive UGT2B7 protein or the rate of 3'-azido-3'-deoxythymidine glucuronidation.     

Quantitation of UGT1A1 in human liver microsomes using stable isotope-labelled peptides and mass spectrometry based proteomic approaches

1.?UDP-glucuronosyltransferases (UGTs) are a group of drug-metabolizing enzymes that catalyse the conjugation of endogeonous compounds and xenobiotics to yield hydrophilic glucuronides which subsequently undergo excretion. This report describes an approach for the identification and accurate quantitation of human UGT1A1 in complex biological matrices using liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) analysis of protein digests.

2.?A stable isotope-labelled (SIL) peptide of a unique peptide spanning residues 54–69 in exon 1 of the human UGT1A1 protein with the sequence RIYLSADPALVVIEHG was synthesized. The peptide sequence synthesized was in the reverse order of the human peptide with the stable isotope-labels in the amino acid arginine (¹³C₆¹⁵N₄) resulting in an increase in the mass of the SIL peptide of 10 amu, from 1753 to 1763. The SIL peptide was quantitated by injecting increasing concentrations of the peptide into the LC-MS to obtain a standard curve.

3.?The labelled peptide along with precursor ion monitoring was used to quantify the levels of UGT1A1 in commercial recombinant preparations (supersomes) and individual human liver microsomal samples and pooled human liver micrsomes obtained from BD Biosciences.

4.?Glucuronidation activity studies were performed, which demonstrated a positive correlation between enzyme activity levels and the UGT1A1 content in the liver microsomes obtained from individual human donors.     

磷,砷,四氯化碳肝损伤线粒体和微粒体乳酸脱氢酶同工酶的改变

本文对P4,NaAsO_2,CCl_4亚慢性肝损伤大鼠肝细胞线粒体,微粒体LDH同工酶进行聚丙烯酰胺凝胶电泳,光密度扫描定量,对其标志酶组化染色,微机图象定量分析,结果表明,三组动物肝小叶线粒体标志酶SDH活性均有下降,其中以CCl_4组SDH下降较迟.NaAsO_2组染毒第8wk可见线粒体LDH_5升高,染毒12wk LDH_(2,3)下降,LDH_5升高与对照组比较无显著性差异,但仍高于P_4组,各组间微粒体LDH同工酶和其标志酶G-6-P酶活性变化存在差异;P_4组微粒体LDH同工酶改变明显且较其他两组早。     

人肝微粒体中西尼地平及其代谢物的HPLC测定法及代谢动力学

目的　建立人肝微粒体中西尼地平及其脱氢代谢物的 HPLC测定法 ,并用本法研究西尼地平在人肝微粒体中代谢的动力学。方法　色谱柱为 Hypersil C1 8柱 (2 5 0 mm× 4.6mm ID,5μm) ,流动相为乙腈 -甲醇 -0 .0 1 mol/ L四丁基溴化铵溶液 (5 5∶ 5∶ 40 ,V/ V/ V) ,柱温 5 0℃ ,检测波长 UV 2 3 8nm,样品用乙醚 -正己烷 (1∶ 1 ,V/ V)提取。用人肝微粒体研究西尼地平的代谢。结果　本法的回收率为 90 .62 %～ 1 0 9.90 % ,西尼地平和其脱氢代谢物的日间、日内 RSD分别为 4.70 %～ 8.3 5 %和 3 .88%～ 8.1 1 % ,西尼地平及其脱氢代谢物的浓度分别在 0 .77μg/ ml～ 98.2 4μg/ ml和 0 .1 3 μg/ ml～ 5 .3 6μg/ ml范围内与峰面积呈良好的线性相关性。在温孵时间为1 0～ 60 min,微粒体蛋白浓度为 1 mg/ ml时 ,西尼地平呈线性消除 ,而其脱氢代谢物呈线性增加。结论　西尼地平在人肝微粒体内被迅速代谢 ,人肝 P45 0酶参与了西尼地平的脱氢氧化     

戊二醛交联法固定鼠肝微粒体

报告了用戊二醛交联法固定鼠肝微粒体并以ＮＡＤＰＨ－细胞色素Ｃ－还原酶为标志酶的初步研究结果。固定后酶的耐热及耐碱性均有提高，但催化活性尚不能令人满意。