西洋参总皂苷对大鼠肝微粒体CYP1A2活性的影响

目的考察西洋参总皂苷对大鼠肝微粒体CYP1A2活性的影响。方法将大鼠随机分为空白对照组(空白组)、西洋参总皂苷低剂量组[低剂量组,50 mg/(kg·d)]、西洋参总皂苷中剂量组[中剂量组,100 mg/(kg·d)]、西洋参总皂苷高剂量组[高剂量组,200 mg/(kg·d)]和β-萘黄酮阳性药对照组[阳性药组,80 mg/(kg·d)],预处理11 d后,观察大鼠肝微粒体CYP1A2酶活性、CYP1A2 mRNA表达和CYP1A2酶蛋白表达情况,研究CYP1A2特异性探针底物非那西丁在预处理大鼠体内的药动学变化。结果预处理后,大鼠肝微粒体CYP1A2活性、mRNA和蛋白表达随西洋参总皂苷剂量升高有增加趋势,非那西丁在预处理大鼠体内代谢速率加快。结论西洋参总皂苷对大鼠肝微粒体CYP1A2活性有一定诱导作用。     

高效液相色谱法检测人肝微粒体CYP3A活性

目的：测定普罗帕酮和N 去丙基普罗帕酮的比值以表达人肝微粒体中CYP3A的活性。方法：以1 mg·mL 1微粒体蛋白浓度37℃孵育普罗帕酮1 h，以HPLC测定普罗帕酮和N 去丙基普罗帕酮的含量。结果： 普罗帕酮和N 去丙基普罗帕酮的线性方程分别为Y＝0.452 0X＋0.003 029(r＝0.999 7)；Y＝1.137 3X＋0.012 740(r＝0.998 5)。结论：N 去丙基普罗帕酮与普罗帕酮的最大比值为1.54，CYP3A具有饱和性。     

高效液相色谱法测定大鼠肝微粒体CYP3A酶的活性

目的 以咪达唑仑为探针药用HPLC法测定大鼠肝微粒体CYP3A酶活性.方法 用ZORBAX SB-C18色谱柱分离;流动相为乙腈-水-0.1％三氟乙酸;流速为1 mL· min-1;柱温40℃;检测波长为230 nm.肝微粒体加入咪达唑仑孵育5 min后,用乙腈终止反应,加入内标溶液50 μL,混匀后离心10 min,取上清进行HPLC分析;计算Km和Vmax.结果 1-羟基咪达唑仑浓度在0.06～3.00 mg· L-1内线性关系良好;日内、日间精密度均＜10％,回收率＞75％.咪达唑仑羟化反应的Km =7.32μmol· L-,Vmax=0.59 nmol·min-1·mg-1 pro.结论 本方法稳定可靠,能准确反映CYP3A酶的活性.     

大鼠肝微粒体中CYP2C9酶的活性及动力学研究

目的 以双氯芬酸为探针药,建立HPLC测定大鼠肝微粒体CYP2C9酶活性的方法,并对其进行动力学考察。方法 采用Agilent ZORBAX SB-C18色谱柱;流动相为乙腈-水-0.1%三氟乙酸,梯度洗脱;流速为1 mL·min^-1;柱温30 ℃;检测波长为278 nm。大鼠肝微粒体加入双氯芬酸钠孵育30 min后,用盐酸和乙酸乙酯终止反应,加入内标地西泮,涡旋后高速离心,取上层有机相吹干复溶进样检测;以Lineweaver-Burk作图计算Km和Vmax。结果 双氯芬酸、4-羟基双氯芬酸和内标分离良好且无内源性干扰。4-羟基双氯芬酸浓度在0.05-10 μmol·L^-1内线性关系良好（r=0.999 8）,定量下限为0.05 μmol·L^-1;日内、日间精密度均〈10%,回收率〉75%。动力学考察表明选择盐酸和乙酸乙酯作为终止试剂效果良好,测得大鼠肝微粒体中双氯芬酸羟化反应的Km为26.87 μmol·L^-1,Vmax为2.359 nmol·min^-1·mg^-1 pro。结论 该方法稳定,结果能准确反映CYP2C9酶的活性,可用于相关动力学研究。     

高效液相色谱法测定氟康唑血药浓度

氟康唑（Ｆｌｕｃｏｎａｚｏｌｅ）为新型三唑类抗真菌药物。本文采用高效液相色谱法测定其血药浓度，ＯＤＳ色谱柱（１５０×４．６ｍｍ，１０μｍ），流动相为乙腈－０．０５ｍｏｌ／Ｌ，磷酸二氢钾溶液（２３∶７７，Ｖ／Ｖ），非那西丁为内标，标准曲线回归方程为Ｙ＝０．２５９５Ｘ－０．０１１９８，ｒ＝０．９９９９，３种浓度平均回收率１０１．５８％。并进行了１０名健康志愿者的血药浓度测定。     

非那西丁探针法动态观测非酒精性脂肪肝大鼠发病进程中的CYP1A2活性

目的:建立探针法动态观测非酒精性脂肪肝大鼠发病进程中细胞色素(CY)P450酶系的活性。方法:制备非酒精性脂肪肝大鼠模型,分别于造模后第0、7、9、11、13、15、17周给予探针药物非那西丁并收集尿液,检测其中非那西丁代谢产物对乙酰氨基酚的各代谢指标以考察CYP1A2的活性变化情况,同时设立正常大鼠组为对照。含量检测采用高效液相色谱法,色谱柱为HypersilODS,流动相为甲醇-水(12.5∶87.5),流速为1.0 mL.min-1,检测波长为254 nm。结果:对乙酰氨基酚的单位体重代谢量在第9周时达到最高,明显高于第0周和正常组((15.02±3.12)、(10.03±3.60)、(9.96±4.00)mg.kg-1),随后又逐渐降低,即CYP1A2的活性先升高后降低。结论:建立的探针法简便可靠,适用于大鼠尿中对乙酰氨基酚的测定以及CYP1A2活性的研究。     

反相高效液相色谱法测定阿霉素的血药浓度

本文采用反相ＨＰＬＣ法测定阿霉素的血药浓度，血浆样品用甲醇－氯仿（１：４）在ｐＨ９．０的条件下提取后进样，５ｍｍｏｌ／Ｌ磷酸－异丙醇－甲醇－乙腈（４５：３５：１０：１０，ｐＨ２．９）为流动相，荧光检测器λｅｘ＝４５０ｎｍ，λｅｍ＝５３０ｎｍ。最低检测浓度１０ｎｇ／ｍｌ，线性范围在３０－１５００ｎｇ／ｍｌ内ｒ＝０。９９８７，日内ＲＳＤ为１．５％－２．４％，日间ＲＳＤ为１．８％－３．７％。并对７例肺癌     

反相高效液相色谱法测定鼠肝微粒体中依普黄酮及其在代谢研究中的应用

目的　建立鼠肝微粒体中依普黄酮的反相高效液相色谱测定法,以研究依普黄酮的体外代谢。方法　本法中依普黄酮与鼠肝微粒体共孵育之后用氯仿提取,采用地非三唑为内标,以Nova parkC₁₈柱为分析柱,乙腈0.1%醋酸溶液(60∶40)为流动相,流速1.0mL·min^-1,紫外检测波长为250nm。结果　依普黄酮在1～100μg·mL^-1内线性关系良好(r=0 .9998)。检测限为0.02μg·mL^-1(S/N≥3) ,定量限为0.1μg·mL^-1(RSD<5.0% ,S/N=8,n=3)。方法回收率达96 .90 %～112 .8% ,日内,日间RSD分别<8.0%和<10% (n=5)。结论　此法简便,准确,可用于依普黄酮的体外代谢研究     

反相高效液相色谱法测定鼠肝微粒体中依普黄酮及其在代谢研究中的应用

目的　建立鼠肝微粒体中依普黄酮的反相高效液相色谱测定法 ,以研究依普黄酮的体外代谢。方法　本法中依普黄酮与鼠肝微粒体共孵育之后用氯仿提取 ,采用地非三唑为内标 ,以Nova parkC18柱为分析柱 ,乙腈 0 .1%醋酸溶液 ( 6 0∶4 0 )为流动相 ,流速 1.0mL·min-1,紫外检测波长为 2 5 0nm。结果　依普黄酮在 1～ 10 0 μg·mL-1内线性关系良好 (r =0 .9998)。检测限为 0 .0 2 μg·mL-1(S/N≥ 3) ,定量限为 0 .1μg·mL-1(RSD <5 .0 % ,S/N =8,n =3)。方法回收率达 96 .90 %～ 112 .8% ,日内 ,日间RSD分别 <8.0 %和 <10 % (n =5 )。结论　此法简便 ,准确 ,可用于依普黄酮的体外代谢研究     

HPLC法测定大鼠UGT1A6的酶活性及体外动力学分析

目的:建立灵敏、稳定的高效液相色谱法,以对硝基酚为探针底物,评价体外大鼠肝微粒体中UGT1A6酶活性。方法:色谱柱为Agilent Zorbax SB-C18柱(250 mm×4.6 mm,5μm),流动相为20 mmol.L-1磷酸二氢钾缓冲液-甲醇(50∶50,V/V),流速0.8 mL.min-1,柱温30℃,检测波长280 nm。对硝基酚与大鼠肝微粒体在37℃共同孵育一段时间后,加入冰乙腈终止反应,于4℃下12000 r.min-1离心15 min后,吸取上清进行HPLC分析,以双倒数作图法计算酶动力学参数。结果:对硝基酚保留时间为8.95 min,线性范围为0.5～100μmol.L-1,回归方程为Y=5973.12X+571.58(r=0.9999),最低检测限(LOD)为0.1μmol.L-1(S/N≥3),最低定量限(LLOQ)为0.5μmol.L-1,回收率为104.5%～105.5%,日内、日间RSD均小于10%,温孵体系中其他内源性物质不干扰测定;计算酶动力学参数Km为24.63μmol.L-1,Vmax为18.87 nmol.min-1.mg.protein-1。结论:该方法灵敏度高、稳定性好,适合体外对硝基酚的测定,可用于大鼠体外UGT1A6酶活性的评价及酶动力学研究。     

尼扎替丁对大鼠CYP1A2亚型的影响

目的通过尼扎替丁的大鼠体内、外实验,观察尼扎替丁对大鼠CYP1A2亚型的影响。方法通过HPLC法测定全血中咖啡因的代谢率,观测尼扎替丁对大鼠CYP1A2活性的影响;通过W estern b lot法测定尼扎替丁对大鼠肝微粒体CYP1A2蛋白表达的调控;通过HPLC法测定肝微粒体重组系统对乙酰氨基酚的含量,确定尼扎替丁对大鼠肝微粒体CYP1A2亚型的作用。结果实验组中给予大鼠不同浓度的尼扎替丁(14、27、54 mg.kg-1),其咖啡因代谢率为29.6%±12.5%、32.4%±13.4%、37.5%±15.0%,对照组为26.9%±11.9%,各剂量组及对照组间差异均无显著性(P>0.05);实验各剂量组与对照组的CYP1A2蛋白表达差异无显著性;肝微粒体体外重组系统中,实验组各浓度尼扎替丁对CYP1A2没有抑制作用,CYP1A2的活性>100%;对照组α-萘黄酮有明显的抑制作用,IC50=0.0306μmol.L-1。结论体内、外实验结果均表明尼扎替丁对大鼠CYP1A2没有抑制作用。     

茵栀黄注射液等28种中药制剂对大鼠体外CYP1A2的作用

目的:观察茵栀黄注射液等28种中药制剂对大鼠CYP1A2的作用。方法:以非那西丁为探针,通过研究其体外转化率的变化评价受试中药制剂对大鼠CYP1A2的影响。结果:清开灵注射液、茵栀黄注射液、穿心莲注射液使非那西丁的体外转化率分别为(255.87±32.33)pmol.min-1.mg-1、(204.01±43.60)pmol.min-1.mg-1、(220.15±48.39)pmol.min-1.mg-1,显著低于对照组(432.68±69.71)pmol.min-1.mg-1(P<0.05)。3种药物的IC50分别为3.55,2.43,2.49 mL.(100 mL)-1。结论:清开灵注射液、茵栀黄注射液、穿心莲注射液体外对大鼠CYP1A2有抑制作用,且呈浓度依赖性。     

Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes

The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the V_max/K_m values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25?nM^?1?min^?1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a V_max value of 6.96?nmol?min^?1?nmol^?1 P450 and apparent K_m value of 0.09?mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the K_m values for the formation of fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03?mM, and V_max values were 2.94, 6.1 and 13.8?nmol?min^?1?nmol^?1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.     

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。     

大鼠肝细胞色素P4503A活性测定及其孵育条件的优化

目的:测定大鼠肝微粒体中细胞色素P4503A(CYP3A)的活性,并对其体外孵育条件进行优化。方法:采用1’-羟基咪哒唑仑的生成量表示肝中CYP3A的活性,高效液相色谱法测定肝微粒体中1’-羟基咪哒唑仑的浓度,用单因素试验法对其体外孵育条件进行优化,用线性Lineweaver-Burk双倒数作图法研究肝CYP3A酶促动力学。结果:1’-羟基咪哒唑仑在18.20~1 820.00μg.L-1范围内线性关系良好;体外优化的孵育条件为5μmol.L-1咪哒唑仑、0.102mg肝微粒体、孵育15min;肝CYP3A酶促动力学参数Km为1.67μmol.L-1,Vmax为95.15pmol.min-1.mg-1。结论:HPLC法操作简便、灵敏、快速,适用于肝微粒体中1’-羟基咪哒唑仑的浓度测定,肝CYP3A孵育条件及其酶促动力学研究为研究经CYP3A代谢的药物相互作用及其他物质对CYP3A酶的影响提供理论依据。     

Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes

Nicotine C-oxidation by recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes was investigated using a convenient high-performance liquid chromatographic method. Experiments with recombinant human P450 enzymes in baculovirus systems, which co-express human nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-P450 reductase, revealed that CYP2A6 had the highest nicotine C-oxidation activities followed by CYP2B6 and CYP2D6; the K _m values by these three P450 enzymes were determined to be 11.0, 105, and 132 μM, respectively, and the V _max values to be 11.0, 8.2, and 8.6 nmol/min per nmol P450, respectively. CYP2E1, 2C19, 1A2, 2C8, 3A4, 2C9, and 1A1 catalysed nicotine C-oxidation only at high (500 μM) substrate concentration. CYP1B1, 2C18, 3A5, and 4A11 had no measurable activities even at 500 μM nicotine. In liver microsomes of 16 human samples, nicotine C-oxidation activities were correlated with CYP2A6 contents at 10 μM substrate concentration, whereas such correlation coefficients were decreased when the substrate concentration was increased to 500 μM. Contribution of CYP2B6 (as well as CYP2A6) was demonstrated by experiments with the effects of orphenadrine (and also coumarin and anti-CYP2A6) on the nicotine C-oxidation activities by human liver microsomes at 500 μM nicotine. CYP2D6 was found to have minor roles since quinidine did not inhibit microsomal nicotine C-oxidation at both 10 and 500 μM substrate concentrations. These results support the view that CYP2A6 has major roles for nicotine C-oxidation at lower substrate concentration and both CYP2A6 and 2B6 play roles at higher substrate concentrations in human liver microsomes. Received: 27 October 1998 / Accepted: 11 January 1999     

Inhibitory effects of antiarrhythmic drugs on phenacetin O-deethylation catalysed by human CYP1A2

Aims The aim of the study was to clarify whether the pharmacokinetic interaction between theophylline and mexiletine is mediated by inhibition of CYP1A2 and to assess the possible interaction potential of other antiarrhythmic drugs with drugs metabolized by CYP1A2.
Methods The inhibitory effects of mexiletine and 10 antiarrhythmic drugs on phenacetin O -deethylation, a marker reaction of CYP1A2, were studied using human liver microsomes and cDNA-expressed CYP1A2.
Results Propafenone and mexiletine inhibited phenacetin O -deethylation with I C ₅₀ values of 29 and 37  μm, respectively. Disopyramide, procainamide and pilsicainide produced negligible inhibition of phenacetin O -deethylation (I C ₅₀>1  mm ). Amiodarone, bepridil, aprindine, lignocaine, flecainide and quinidine inhibited phenacetin O -deethylation in a concentration-dependent manner, although the inhibitory effects were relatively weak with I C ₅₀ values ranging from 86 to 704  μm. Propafenone and mexiletine selectively abolished the high-affinity component of phenacetin O -deethylation in human liver microsomes. In addition, propafenone and mexiletine inhibited phenacetin O -deethylation catalysed by cDNA-expressed CYP1A2.
Conclusions These data suggest that, among the antiarrhythmic drugs studied, propafenone and mexiletine are relatively potent inhibitors of CYP1A2, which may cause a drug-drug interaction with drugs metabolized by CYP1A2.     

五味子甲素对大鼠肝微粒体CYP3A活性的影响

目的：通过体外药物代谢实验探讨五味子甲素对CYP3A活性的影响。方法：以大鼠肝微粒体为载体,选取咪达唑仑（MDZ）作为药物“探针”,建立高效液相色谱（HPLC）检测方法,体外给药测定五味子甲素对MDZ的IC50值以及相关酶动力学参数。结果：孵育体系内源性物质不干扰测定,方法快捷、稳定、灵敏度高。在肝微粒中,五味子甲素对MDZ的IC50浓度为6．26μmol／mL,相关酶动力学参数：Km=15．77μmol／L,Ki=5．50μmol／mL。结论：五味子甲素对大鼠肝微粒体CYP3A活性具有抑制作用,其抑制作用为混合型,即：非竞争与反竞争抑制。