灯盏花素注射液对大鼠体外肝微粒体细胞色素P450酶活性的影响

摘 要 目的:研究灯盏花素注射液对大鼠肝微粒体CYP1A2、CYP2C9、CYP2D6、CYP2E1和CYP3A1/2 五个亚型酶活性的影响。方法: 采用大鼠肝微粒体体外孵育法,选用非那西丁（CYP1A2）、甲苯磺丁脲（CYP2C9）、右美沙芬（CYP2D6）、氯唑沙宗（CYP2E1）和睾酮（CYP3A1/2）作为5个亚型酶的探针药物,孵育系统中加入不同浓度的灯盏花素注射液,用HPLC法测定5个亚型酶探针药物代谢产物的生成量,比较空白对照组与不同浓度灯盏花素注射液给药组探针药物的活性,反映灯盏花素注射液对5个亚型酶活性的影响。结果:大鼠肝微粒体体外孵育系统中,灯盏花素注射液对大鼠CYP3A1/2的IC₅₀为29.40μg·ml^-1,K_i为37.78 μg·ml^-1;对CYP1A2、CYP2C9、CYP2D6和CYP2E1的IC₅₀＞200 μg·ml^-1。结论:灯盏花素注射液对大鼠体外肝微粒体CYP3A1/2有弱的抑制作用,对CYP1A2、CYP2C9、CYP2D6和CYP2E1活性无明显影响。     

混合探针底物法同时预测细胞色素P450酶5种亚型的抑制作用

本研究建立了混合探针底物法同时预测细胞色素P450(cytochrome P-450，CYP450)酶5种亚型的抑制作用。将CYP450酶5种亚型的特异性探针底物非那西丁(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、奥美拉唑(CYP2C19)及咪达唑仑(CYP3A4)同时与人肝微粒体在体外进行孵化反应，采用液相色谱-串联质谱分析方法同时测定5个特异性底物及其生成的对应的5种代谢产物(对乙酰氨基酚、右啡烷、4-羟基甲苯磺丁脲、5-羟基奥美拉唑和1′-羟基咪达唑仑)。并选择5种细胞色素P450酶的特异性抑制剂α-萘黄酮(CYP1A2)、奎尼丁(CYP2D6)、磺胺苯吡唑(CYP2C9)、氟康唑(CYP2C19)和酮康唑(CYP3A4)加入到其所对应酶的单个探针底物及混合探针底物的反应体系中，测定生成的代谢物，计算相应IC₅₀值，对方法进行验证。5种特异性的抑制剂与混合探针底物反应后所得的IC₅₀值和与单个探针底物反应后所得的IC₅₀值具有很好的一致性且与文献报道的基本一致。本研究建立的混合探针底物法可以用于快速高通量地同时预测化合物对CYP450酶5种亚型活性的抑制作用。     

探针药物法评价厚朴酚、和厚朴酚对大鼠肝微粒体中CYP450酶的抑制作用

目的 研究厚朴中的主要成分厚朴酚、和厚朴酚对CYP450酶的7种亚型酶活性的影响,预测可能的药物间相互作用,为中药的临床应用提供理论依据。方法 采用Cocktail探针方法,将厚朴酚、和厚朴酚和CYP450酶7种亚型的特异性探针底物：甲苯磺丁脲（CYP2C）、香豆素（CYP2A6）、右美沙芬（CYP2D6）、氯唑沙宗（CYP2E1）、睾酮（CYP3A）、非那西丁（CYP1A2）、安非他酮（CYP 2B6）与大鼠肝微粒体进行孵化反应,结合UPLC-MS/MS的多反应监测技术,测定对应的7种代谢产物（4-羟基甲苯磺丁脲、7-羟基香豆素、右啡烷、6-羟基氯唑沙宗、6β-羟基睾酮、对乙酰氨基酚和羟基丙酮）的峰面积,通过与对照组比较,确定厚朴酚、和厚朴酚对以上7种酶活性的影响,计算相应的IC₅₀,评价是否有抑制作用。结果 厚朴酚、和厚朴酚对大鼠肝微粒体中的4种亚型酶（CYP2C、CYP2D6、CYP2E1和CYP2B6）活性均有抑制作用,且随化合物浓度和预温育时间增加而增加机械抑制;另外3种亚型酶（CYP2A6、CYP3A4和CYP1A2）则不呈现此抑制规律。结论 厚朴在与以上4种酶（CYP2C、CYP2D6、CYP2E1、CYP2B6）代谢的药物联合用药时,易产生药物相互作用,抑制药物代谢。本研究也为中药厚朴的临床应用以及新药研发提供重要的数据支持。     

注射用益气复脉(冻干)对人肝微粒体CYP450各亚型酶活性的影响

目的 研究注射用益气复脉（冻干,YQFM）对人肝微粒体的细胞色素P450亚型酶CYP1A2、CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6和CYP3A4活性的影响。方法 差速离心法制备人肝微粒体,体外人肝微粒体、YQFM （6.5、32.5、65.0、325.0、650.0、3 250.0和6 500.0 μg/mL）和7种底物探针在还原型辅酶β-NADPH的作用下,37℃水浴孵育30 min,同时设置阴性对照（空白缓冲液）和阳性对照（各亚酶的选择性抑制剂）组;应用液相色谱-串联质谱（LC-MS/MS）法测定探针底物的代谢产物生成量,酶活性以相对阴性对照的百分比表示。结果 与阴性对照组比较,32.5、65.0、325.0、650.0、3 250.0浓度的YQFM对CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6和CYP3A4活性均不发挥抑制作用;半数抑制浓度（IC₅₀）均大于6 500 μg/mL。结论 YQFM在临床使用过程中发生药物相互作用的概率较小。     

艾瑞昔布对CYP2C9酶活性及mRNA和蛋白表达的影响

目的:评价艾瑞昔布对CYP2C9酶活性及mRNA和蛋白表达的影响。方法:采用超高效液相色谱(UPLC),色谱柱：Acquity UPLC BEH-C₁₈柱(2.1 mm×50 mm, 1.7μm);流动相为水-乙腈(76∶24,V/V);流速0.4 mL·min^-1;检测波长235 nm;柱温30℃;进样量2μL;内标为卡马西平。选择甲苯磺丁脲为CYP2C9酶特异性探针药物,与艾瑞昔布在肝微粒体中共同孵育,UPLC测定代谢产物4-羟基甲苯磺丁脲的生成量,考察艾瑞昔布对CYP2C9酶活性的影响。Wistar大鼠给予艾瑞昔布,RT-PCR和Western Blot测定肝组织中CYP2C9酶活性及mRNA和蛋白表达。结果:随着艾瑞昔布浓度的升高,肝微粒体孵育体系中4-羟基甲苯磺丁脲生成量降低,艾瑞昔布对CYP2C9活性呈剂量依赖性抑制作用,IC₅₀值为74.77μmol·L^-1;随着给药时间延长,大鼠肝脏CYP2C9酶的mRNA和蛋白表达减少(P<0.05)。结论:艾瑞昔布可降低CYP2C9酶的mR...     

苦碟子注射液对大鼠肝微粒体CYP450酶5个亚型的体外抑制作用

目的研究苦碟子注射液对大鼠肝微粒体CYP450酶的体外抑制作用。方法制备大鼠肝微粒体。将苦碟子注射液分别与混合探针药物非那西丁、甲苯磺丁脲、奥美拉唑、睾酮和氯唑沙宗共同孵育,UPLC-MS/MS法检测各探针药物的代谢物。结果在测定浓度范围内,苦碟子注射液对CYP2E1、CYP2C9和CYP2C19的活性抑制率小于50%;对CYP1A2、CYP3A4的IC_(50)值分别为12.68%、8.11%,远高于临床日用药浓度0.20%~0.80%。结论在正常剂量下,苦碟子注射液对大鼠CYP2E1、CYP2C9、CYP2C19几乎不显示抑制作用,对CYP1A2、CYP3A4几乎没有抑制作用。     

甲基原薯蓣皂苷对人肝微粒体中7种CYP450酶活性的影响

目的研究甲基原薯蓣皂苷对CYP450酶的7种亚型酶活性的影响。方法将MPD和CYP450酶7种亚型的特异性探针底物咖啡因(CYP1A2)、右美沙芬(CYP2D6)、甲苯磺丁脲(CYP2C9)、s-美芬妥因(CYP2C19)、氯唑沙宗(CYP2E1)、香豆素(CYP2A6)及咪达唑仑(CYP3A4)与人肝微粒体进行孵化反应,采用HPLC和LC-MS/MS法测定对应的7种代谢产物(1,7-二甲基黄嘌呤、去甲右美沙芬、4-羟基甲苯磺丁脲、4-羟基美芬妥因、6-羟基氯唑沙宗、7-羟基香豆素和1-羟基咪达唑仑)的浓度,通过与对照组比较,确定MPD对以上7种酶活性的影响。结果MPD在1~10μmol.L-1时对7种酶均无明显抑制作用,在100μmol.L-1时对CYP2D6有抑制趋势,但对其他6种酶无抑制作用,均无统计学意义(P>0.05)。结论MPD在与以上6种酶(CYP1A2、CYP2E1、CYP2C19、CYP3A4、CYP2C9和CYP2A6)代谢的药物联合用药时,发生药物相互作用的可能性较小。     

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.     

Polymorphism of CYP2D6, CYP2C19, CYP2C9 and CYP2C8 in the Faroese population

Objective The purpose of the study was to study the distribution of poor and extensive metabolizers of CYP2C19 and CYP2D6 and to genotype for CYP2C8 and CYP2C9 among 312 randomly selected Faroese.Methods and results The participants were phenotyped for CYP2D6 with the use of sparteine. The distribution of the sparteine metabolic ratio (sparteine/didehydrosparteines) was bimodal, and 14.5% (n=44; 95% CI: 10.7–18.9%) of the subjects were phenotyped as poor metabolizers. The frequency of poor metabolizers was higher (P=0.0002; ² test) among the Faroese than in other European populations (7.4%). Genotype analyses for the CYP2D6*3, *4, *6 and *9 alleles were performed using real-time polymerase chain reaction (PCR) (TaqMan, Foster City, CA, USA), and we found 14.6% (n = 45) (95% CI: 10.8–19.0%) with deficient CYP2D6 genes (*3/*4, *4/*4, *4/*6, *6/*6) in the Faroese population. The subjects were phenotyped for CYP2C19 with the use of mephenytoin and 10 subjects, i.e., 3.2% (95% CI: 1.6–5.9%) were phenotyped as poor metabolizers. Genotype analysis for the CYP2C19*2 and *3 alleles was performed by means of PCR analysis, and 2.9% (n=9) (95% CI: 1.3–5.4%) of the Faroese were found to have a deficient CYP2C19 gene all explained by the CYP2C19*2/*2 genotype. The allele frequencies of the CYP2C9*2 and CYP2C9*3 alleles were 8.8% (95% CI: 6.7–11.4%) and 5.3% (95% CI: 3.7–7.4%), respectively, while the CYP2C8*3 allele frequency was 6.9% (95% CI: 5.0–9.2%). Real-time PCR (TaqMan) was used for both CYP2C9 and CYP2C8 genotype analyses.Conclusion The frequency of CYP2D6 poor metabolizers is twofold higher among the Faroese population than other Caucasians, while the frequencies of Faroese subjects with decreased CYP2C19, CYP2C8 and CYP2C9 enzyme activity are the same as seen in other Caucasian populations. A possible consequence might be a higher incidence of side effects among Faroese patients taking pharmaceuticals that are CYP2D6 substrates.     

Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC₅₀ value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC₅₀ values greater than 250 μg/ml. Hence there appears to be little likelihood of drug–herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.     

Detection of human CYP2C8, CYP2C9, and CYP2J2 in cardiovascular tissues.

The cytochrome P450 (P450) enzymes CYP2C8, CYP2C9, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids, which are known to be vital in regulation of vascular tone and cardiovascular homeostasis. Because there is limited information regarding the relative expression of these P450 enzymes in cardiovascular tissues, this study examined the expression of CYP2C8, CYP2C9, and CYP2J2 mRNA and protein in human heart, aorta, and coronary artery samples by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. CYP2J2 and CYP2C9 mRNA levels were highly variable in human hearts, whereas CYP2C8 mRNA was present in lower abundance. CYP2J2 mRNA was approximately 10(3) times higher than CYP2C9 or CYP2C8 in human heart. However, CYP2C9 mRNA was more abundant than CYP2J2 or CYP2C8 in one ischemic heart. In human aorta, mean CYP2C9 mRNA levels were approximately 50 times higher than that of CYP2J2 and 5-fold higher than that of CYP2C8. In human coronary artery, mean values for CYP2C9 mRNA were approximately 2-fold higher than that of CYP2J2 mRNA and 6-fold higher than that of CYP2C8 mRNA. Immunoblotting results show relatively high levels of CYP2J2 and CYP2C8 protein in human hearts, which was confirmed by immunohistochemistry. CYP2C9 protein was also detected at high levels in one ischemic heart by immunoblotting. CYP2C9 was present at higher levels than CYPJ2 in aorta and coronary artery, whereas CYP2C8 protein was below the limits of detection. The expression of CYP2J2 and CYP2C8 in human heart, and CYPC9 and CYP2J2 in aorta and coronary artery is consistent with a physiological role for these enzymes in these tissues.     

Investigation of enzyme selectivity in the human CYP2C subfamily: homology modelling of CYP2C8, CYP2C9 and CYP2C19 from the CYP2C5 crystallographic template

Homology modelling of human CYP2C subfamily enzymes, CYP2C8, CYP2C9 and CYP2C19, based on the rabbit CYP2C5 crystal structure template is reported. The relatively high sequence homologies (75-80%) between the rabbit CYP2C5 and human CYP2C subfamily enzymes tend to indicate that the resulting structures should prove adequate models of these major catalysts of human drug metabolism. Selective substrates of all three human CYP2C enzymes are found to fit closely within the putative active sites in a manner which is consistent with site-directed mutagenesis experiments and known positions of substrate metabolism. The specific interactions between substrates and enzymes can be used to rationalize the variation in substrate binding affinity and generate QSAR models for both inhibition and metabolism via CYP2C family enzymes, yielding a generally good agreement with experimental binding data obtained from Km values, with correlation coefficients (R values) of between 0.97 and 0.99 depending on the QSAR equation produced.     

Effects of polymorphisms in CYP2D6, CYP2C9, and CYP2C19 on trimipramine pharmacokinetics

Little is known about the impact of cytochrome P450 polymorphisms on the metabolism of trimipramine, which is still widely used as antidepressant due to its positive effect on sleep patterns. A single oral dose of 75 mg trimipramine was given to 42 healthy volunteers selected according to their CYP2D6, CYP2C19, and CYP2C9 genotypes. The reference group included 8 subjects with homozygous active wild-type genotypes of all 3 enzymes (EM). This group was compared with 7 intermediate (IM) with 1 and 7 poor metabolizers (PM) with zero active alleles of CYP2D6 and CYP2C19, respectively, and with 4 subjects with the genotype CYP2C9*3/*3. Pharmacokinetics of trimipramine and its demethylated metabolite strongly depended on the CYP2D6 genotype. Median oral clearance of trimipramine was 276 L/h (range 180-444) in the reference group but only 36 L/h (range 24-48) in CYP2D6 PMs (P < 0.001). These differences could only be explained by an effect of CYP genotypes on both parameters, systemic clearance and bioavailability, the latter being at least 3-fold higher in CYP2D6 PMs than in the reference group. The desmethyltrimipramine area under the concentration-time curve was 40-fold greater in CYP2D6 PMs than in the reference group (1.7 vs. 0.04 mg/L x h in EMs), but below the quantification limit in most carriers of deficiencies of CYP2C19 or CYP2C9. This indicates that both CYP2C enzymes contribute to the demethylation of desmethyltrimipramine and CYP2D6 to further metabolism.     

中国汉族和维吾尔族药物代谢酶CYP3A4、CYP2C9、CYP2C19、CYP2D6基因多态性分析

目的对中国汉族、维吾尔族健康人群CYP3A4、CYP2C9、CYP2C19、CYP2D6进行基因多态性分析,并对汉族和维吾尔族人群等位基因频率和基因型频率进行比较。方法聚合酶链反应一限制性片段长度多态性(PCR-RFLP)法对CYP3A4、CYP2C9、CYP2C19、CYP2D6进行分型。结果汉族、维吾尔族健康人群CYP3A4*5等位基因频率为0,CYP3A4*18等位基因频率分别为0.183 8、0.140 2;CYP2C9*2等位基因频率分别为0.011 0、0.095 8,CYP2C9*13等位基因频率分别为0、0.002 3;CYP2C19*2等位基因频率分别为0.386 0、0.324 8,CYP2C19*3等位基因频率分别为0.051 5、0.021 0;CYP2D6*10等位基因频率分别为0.573 5、0.224 3。结论本研究在汉族、维吾尔族健康人群中未发现CYP3A4*5等位基因。汉族、维吾尔族健康人群CYP3A4*18、CYP2C9*13、CYP2C19*2、CYP2C19*3等位基因频率差异均无统计学意义。维吾尔族健康人群CYP2C9*2等位基因频率远高于汉族(P<0.01);CYP2D6*10等位基因频率远低于汉族(P<0.01),存在民族差异。     

Polymorphisms of drug-metabolizing enzymes CYP2C9, CYP2C19, CYP2D6, CYP1A1, NAT2 and of P-glycoprotein in a Russian population

Objective The frequency of functionally important mutations and alleles of genes coding for xenobiotic metabolizing enzymes shows a wide ethnic variation. However, little is known of the frequency distribution of the major allelic variants in the Russian population.Methods Using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) genotyping assays and the real-time PCR with fluorescent probes, the frequencies of functionally important variants of the cytochromes P₄₅₀ (CYP) 2C9, 2C19, 2D6, 1A1 as well as arylamine N-acetyltransferase 2 (NAT2) and P-glycoprotein (MDR1) were determined in a sample of 290 Russian volunteers derived from Voronezh area.Results CYP2C9*2 and *3 alleles were found with allelic frequencies of 10.5% and 6.7%, respectively. The novel intron-2 T>C mutation at exon 2 +73 bp occurred in 24.8% of alleles. CYP2C19*2 and *3 alleles occurred in 11.4% and 0.3%, respectively. Six persons (2.1%) carried two of these CYP2C19 alleles responsible for poor metabolizing activity. Of all subjects, 5.9% were CYP2D6 poor metabolizers, whereas 3.4% were addressed to ultra-rapid metabolizers (CYP2D6*1×2/*1). The CYP1A1*2A allele was found in 4.7%, *2B in 5.0%, *4 in 2.6%, and the 5-mutations –3219C>T, –3229G>A, and the novel –4335G>A in 6.0%, 2.9% and 26.0% of alleles, respectively. Genotyping of eight different single nucleotide polymorphisms in the NAT2 gene provided in 58.0% a genotype associated with slow acetylation. The MDR1 triple variants G2677T and G2677A in exon 21 had an allelic frequency of 41.9% and 3.3%, respectively, and the variant C3435T in exon 26 one of 54.3%. Frequencies of functionally important haplotypes were calculated.Conclusion The overview of allele distribution of important xenobiotic-metabolizing enzymes among a Russian population shows similarity to other Caucasians. The data will be useful for clinical pharmacokinetic investigations and for drug dosage recommendations in the Russian population.     

Amiodarone N-deethylation by CYP2C8 and its variants,CYP2C8*3 and CYP2C8 P404A

Amiodarone is a potent Class III antiarrhythmic drug. The N-deethylation of amiodarone to desethylamiodarone is known to be catalyzed by cytochrome P450 (CYP) 2C8. In the present study, amiodarone N-deethylation by the CYP2C8s, CYP2C8*1 (wild-type), CYP2C8*3, and CYP2C8 P404A (Pro404Ala substitution in exon 8), was investigated by their transient expression in Hep G2 cells. The expression levels of CYP2C8*1 and CYP2C8*3 were similar, whereas the level of CYP2C8 P404A was 55.6% of that of CYP2C8*1. The kinetic parameters of amiodarone N-deethylation were obtained by means of Lineweaver-Burk analysis. The intrinsic clearance (Vmax/Km, per mg of microsomal protein) of amiodarone by CYP2C8 P404A but not CYP2C8*3 was significantly (48.7%) less than that of CYP2C8*1. These results suggest that CYP2C8 P404A but not CYP2C8*3 is less effective in the N-deethylation of amiodarone.