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,K1=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。     

体外探讨五味子乙素对大鼠肝微粒体CYP3A的影响及其作用机制

目的通过体外药物代谢实验探讨五味子乙素对CYP3A活性的影响,并探讨其作用机制。方法以大鼠肝微粒体为载体,选取咪达唑仑为药物"探针",建立高效液相色谱法(HPLC)检测五味子乙素对肝微粒体代谢咪达唑仑的影响,体外给药测定其IC50值以及相关酶动力学参数,并推测其可能的作用机制。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。结果显示五味子乙素呈剂量依赖性抑制体外肝微粒体咪达唑仑代谢,其IC50为5.50g.L-1,据此计算其Ki为4.32mol·L-1。结论五味子乙素体外抑制大鼠肝微粒体CYP3A活性,其作用机制为可逆性抑制,属于非竞争性抑制类型。     

五味子及木脂素成分对肝CYP3A酶活性的影响

目的研究五味子和五味子科8种木脂素成分对大鼠肝CYP3A1／2活性和人肝CYP3A4活性的影响。方法在体外大鼠及健康人肝微粒体孵育体系中加入底物睾酮和不同浓度的五味子或木脂素成分，甲醇终止反应，用高效液相色谱仪检测睾酮的羟化代谢产物68羟基睾酮的生成量反映CYP3A酶活性。人原代肝细胞和五味子水提物共同培养3d后同法测定CYP3A酶活性的变化。结果大鼠肝微粒体孵育体系中，五味子水提物和醇提物对CYP3A1／2酶活性有浓度依赖性抑制作用，IC50分别为487．8和175．7mg·L^-1，五味子酯甲、南五味子素、异南五味子素、内南五味子素和angeloyl binankadsurina A的IC50分别为3．77、6．18、20．67、16．43和23．07μmol·L^-1；人肝微粒体孵育体系中，五味子水提物抑制CYP3A4酶活性的IC50为317．3mg·L^-1，8种木脂素成分对酶活性的抑制作用与大鼠微粒体结果相似；人原代肝细胞培养中，五味子水提物（1500mg·L^-1）浓度可以抑制50％以上的CYP3A4酶活性。结论体外给药五味子和五种木脂素成分对大鼠肝CYV3A1／2活性和人肝CYP3A4活性有浓度依赖性抑制作用。     

20味中成药对5个人肝微粒体酶活性的影响

目的：考察六神丸等20个常用中成药对CYP1A2、CYP2C9、CYP2C19、CYP2D6和CYP3A4这5个人肝微粒体中主要的药物代谢酶的活性是否有影响。方法：采用人肝微粒体孵育法,分别采用非那西丁、甲苯磺丁脲、美芬妥因、氢溴酸右美沙芬和咪达唑仑作为5个酶的探针药物,采用液相色谱－质谱联用法测定了探针药物代谢产物的浓度,比较分别加入待测药物和空白缓冲液时代谢产物的初始生成速度。结果：六神丸对CYP1A2有明显抑制作用,IC50为28．75μg／mL;肝苏颗粒对CYP2C19有明显抑制作用,IC50为29．80μg／mL;丹参片对CYP1A2有明显抑制作用,IC50为48．55μg／mL;贝羚胶囊对CYP1A2有明显抑制作用,IC50为17．45μg／mL;复方夏天无片对CYP2D6有明显抑制作用,IC50为40．26μg／mL;丹参酮胶囊对CYP1A2、CYP2C9、CYP2C19、CYP2D6和CY173A4均有明显抑制作用,IC50分别为7．04、13．97、55．77、33．90μg／mL和34．57μg／mL;通风定胶囊对CYP2D6有明显抑制作用,IC铀为37．56μg／mL结论：所测定的多数中成药对药物代谢酶有明显的抑制作用,这些药物与药物代谢酶的底物药物联用发生药物相互作用的可能性较大,临床联合用药须谨慎。     

Cocktail 探针药物法评价辣椒素对大鼠CYP450亚型的影响

目的：采用cocktail 探针药物法研究辣椒素在体外对大鼠肝微粒体CYP 450四种亚型的影响。方法：分为试验组和对照组,试验组采用大鼠肝微粒体孵育体系、探针药物和系列浓度的辣椒素（对照组仅加入缓冲液）共同孵育20 min,反应终止后用HPLC方法检测代谢产物的生成量以代表酶的活性。采用Graphpad prism 5.0计算辣椒素对各亚型的IC50值。将辣椒素与大鼠肝微粒体分别预孵育0,5,10,15,20,30 min,计算不同预孵育时间下各亚型的相对活性百分比。结果：辣椒素对大鼠肝微粒体CYP1A2、CYP2C11、CYP2E1和CYP3A2的IC50值分别为36.21,17.19,51.64,18.86μmol·L-1。预孵育没有增强辣椒素对CYP450的抑制作用。结论：辣椒素在体外对大鼠肝微粒CYP1A2、CYP2C11、CYP2E1和CYP3A2具有抑制作用,且未呈现出预孵育时间依赖性。     

血塞通注射液对鼠肝CYP3A体外抑制作用研究

目的：研究血塞通注射液对大鼠肝微粒体CYP3A酶活性的体外抑制作用。方法：在大鼠肝微粒体体外孵育体系中加入CYP3A酶的底物睾酮和不同体积的血塞通注射液,用高效液相色谱法测定睾酮的羟化代谢产物6β-羟基睾酮的生成量反映CYP3A酶的活性。酮康唑为阳性对照药。结果：在大鼠肝微粒体体外孵育体系中,血塞通注射液对CYP3A酶的IC50和Ki值分别为血塞通注射液原液的0．87％和0．43％。结论：血塞通注射液在体外对大鼠肝微粒体CYP3A酶活性有抑制作用,符合混合型抑制模型。     

抗肿瘤活性物CH330331在大鼠肝微粒体代谢及对药酶影响作用的研究

目的：体外研究大鼠肝微粒体中CH330331代谢的酶促动力学,并利用＂Cocktail＂探针药物模型,研究CH330331对主要CYP450亚型的体外抑制作用。方法：优化CH330331在大鼠肝微粒体中孵育的条件,并进行酶促动力学研究;探讨体外＂Cocktail＂探针药物模型的组成,并研究CH330331对CYP450亚型的体外抑制作用。结果：CH330331代谢的酶促动力学参数：最大反应速率（Vmax）为2.08μmol/（min.mgpro-tein）,米氏常数（Km）为18.96μmol/L。CH330331对大鼠CYP1A2、CYP2C9和CYP2D6有弱抑制作用,对大鼠CYP2C19、CYP2E1和CYP3A4没有抑制作用。结论：临床使用中CH330331可以增加主要通过CYPCYP1A2,CYP2C9和CYP2D6代谢的药物浓度。     

五味子甲素对CYP3A活性的影响及其体内外相关性研究

目的:通过"探针"法探讨五味子甲素体内、外对大鼠肝微粒体CYP3A活性的影响及其相关性。方法:选取咪达唑仑作为CYP3A探针,用高效液相色谱法测定五味子甲素体外孵育给药和体内灌胃给药后大鼠肝微粒体酶CYP3A活性,通过药动学参数K_m确定其体内、外作用的相关性。结果:体外试验结果表明,五味子甲素非竞争性抑制咪达唑仑的代谢,其K_i为5.5μmol·L^-1,体内试验亦证实了五味子甲素能显著抑制肝微粒体CYP3A酶活性（P<0.01）并呈剂量依赖性,其体内给药的K_i值为30.67 mg·kg^-1。体内外K_m值十分接近。结论:五味子甲素在体内和体外均可非竞争性抑制大鼠肝微粒体酶CYP3A活性,其对CYP3A影响的体内、体外结果具有良好的相关性。     

刺五加注射液对大鼠肝CYP3A活性的影响

目的:考察刺五加注射液对大鼠肝细胞色素P4503A活性的影响。方法:制备大鼠空白肝微粒体,加入刺五加注射液孵育,HPLC法测定咪达唑仑浓度,计算咪达唑仑代谢速率评价刺五加注射液体外对大鼠CYP3A酶的影响。大鼠随机分为对照组和实验组,分别尾静脉注射生理盐水和刺五加注射液,连续7 d,制备肝微粒体,进行孵育反应,评价刺五加注射液体内对大鼠CYP3A的影响。结果:刺五加注射液体外对大鼠CYP3A酶具有抑制作用,测得刺五加注射液抑制CYP3A酶的IC50为5.00 mL/100mL。大鼠连续7 d静脉注射刺五加注射液,使探针药物咪达唑仑的代谢减少了21.11%。结论:刺五加注射液对大鼠CYP3A的活性有抑制作用。     

灯盏细辛注射液对大鼠肝微粒体CYP3A的抑制作用

目的：研究灯盏细辛注射液对大鼠肝微粒体CYP3A酶活性的抑制作用。方法：在体外大鼠肝微粒体孵育体系中加入底物睾酮和不同体积的灯盏细辛注射液,用高效液相色谱法测定睾酮的羟化代谢产物6β-羟基睾酮的生成量反映CYP3A酶的活性。酮康唑用作阳性对照药。结果：在体外孵育体系中,灯盏细辛注射液对大鼠肝微粒体CYP3A酶活性有抑制作用,IC50和Ki值分别为0.40%和0.24%（V/V）。结论：体外给药灯盏细辛注射液对大鼠肝微粒体CYP3A酶活性有抑制作用,符合混合型抑制模型。     

In vivo inhibition of CYP3A-mediated midazolam metabolism by anchusan in rats

Cytochrome P450 (CYP)-mediated drug interactions caused by Kampo medicine have not been investigated sufficiently. The current study was conducted to reveal the effect of anchusan, a commonly used Kampo formula for gastrointestinal disease, on CYP3A-mediated drug metabolism in rats. The pharmacokinetics of midazolam (MDZ) was investigated after the single or one-week administration of anchusan (500 mg/kg) to evaluate its inhibitory and inducible effect on CYP3A, respectively. MDZ was administrated 16 h after the last anchsan treatment in the multiple dose study, while their intervals were 2 or 16 h in the single dose study. Unexpectedly, the multiple-pretreatment of anchusan increased the AUC of MDZ by 2.4-fold rather than decreasing it, and the CYP3A contents and activities were unchanged in hepatic and intestinal microsomes of these rats. In contrast, no significant inhibitory effects on MDZ metabolism were observed by the single anchusan pretreatment. In vitro study showed that the preincubation of anchusan and some of its component extracts with rat liver microsomes reduced CYP3A activity in a time- and NADPH-dependent manner. These results suggested that anchusan increased the serum MDZ concentration in rats, at least in part, by the time-dependent inhibition of CYP3A.     

Investigation of drug-drug interaction via mechanism-based inhibition of cytochrome P450 3A by macrolides in dexamethasone-treated female rats

The in vitro and in vivo inhibition of cytochrome P450 (CYP) 3A with mechanism-based inhibition (MBI) by macrolides was investigated using dexamethasone-treated female rats (DEX-female rats). In the in vitro CYP inhibition studies using erythromycin (ERM) and clarithromycin (CAM), similar inhibition responses were observed between human and DEX-female rat liver microsomes, however, there were fewer effects in intact male rats. The ex vivo study showed that midazolam (MDZ) metabolism in liver microsomes of DEX-female rats was reduced by ERM administration and the inhibitory effect was increased with increasing ERM doses, indicating that metabolite intermediate complex formation caused irreversible inhibition of CYP3A activity in DEX-female rats as well as in humans. In the in vivo studies, ERM and CAM significantly increased the area under the plasma concentration-time curve of MDZ and decreased the total clearance in DEX-female rats. It was concluded that the DDIs via MBI of CYP3A following macrolide administration in humans could be reproduced in female rats, suggesting that DEX-female rats can serve as an in vivo model for assessing this DDI in humans.     

CYP3A5-mediated metabolism of midazolam in recombinant systems is highly sensitive to NADPH-cytochrome P450 reductase activity

Data from in vitro drug metabolism studies with recombinant enzyme systems are frequently used to predict human drug metabolism in vivo. However, for the CYP3A probe substrate midazolam (MDZ), considerable variability in enzyme kinetic parameters has been observed in different in vitro studies. The aim of this study was to explore the effect of varying activities of the electron donor NADPH-cytochrome P450 reductase (CPR) on CYP3A5-mediated metabolism of MDZ. Microsomes with similar levels of CYP3A5 but 12-fold difference in CPR activity showed a 30-fold difference in intrinsic clearance for the formation of 1'-OH-MDZ. Significantly higher K(m) and lower V(max) for the formation of 1'-OH-MDZ were found in microsomes with low CPR activity compared with microsomes with higher CPR activity (P?=?0.024 and 0.001). In the microsomes with lowest CPR activity, the formation of 1'-OH-MDZ displayed Michaelis-Menten kinetics, whereas substrate inhibition was observed in the two preparations with higher CPR activity. The present study shows that the CPR activity in different recombinant enzyme preparations is crucial for in vitro CYP3A5-mediated clearance of MDZ. This suggests that the CPR activity of enzyme preparations could be an important factor for the ability of in vitro data to predict human drug metabolism in vivo.     

Evaluation of animal models for intestinal first-pass metabolism of drug candidates to be metabolized by CYP3A enzymes via in vivo and in vitro oxidation of midazolam and triazolam

Abstract

1. To search an appropriate evaluation methodology for the intestinal first-pass metabolism of new drug candidates, grapefruit juice (GFJ)- and vehicle (tap water)-pretreated mice or rats were orally administered midazolam (MDZ) or triazolam (TRZ), and blood levels of the parent compounds and their metabolites were measured by liquid chromatography/MS/MS. A significant effect of GFJ to elevate the blood levels was observed only for TRZ in mice.

2. In vitro experiments using mouse, rat and human intestinal and hepatic microsomal fractions demonstrated that GFJ suppressed the intestinal microsomal oxidation of MDZ and especially TRZ. Substrate inhibition by MDZ caused reduction in 1′-hydroxylation but not 4-hydroxylation in both intestinal and hepatic microsomal fractions. The kinetic profiles of MDZ oxidation and the substrate inhibition in mouse intestinal and hepatic microsomal fractions were very similar to those in human microsomes but were different from those in rat microsomes. Furthermore, MDZ caused mechanism-based inactivation of cytochrome P450 3A-dependent TRZ 1′-hydroxylation in mouse, rat and human intestinal microsomes with similar potencies.

3. These results are useful information in the analysis of data obtained in mouse and rat for the evaluation of first-pass effects of drug candidates to be metabolized by CYP3A enzymes.     

染料木黄酮在雌雄大鼠肝微粒体中的代谢差异

目的研究染料木黄酮在♀、♂大鼠肝微粒体中的代谢差异。方法制备♀、♂大鼠肝微粒体,确定染料木黄酮代谢的酶动力学条件,分别用CYP1A2抗体和选择性CYP1A2抑制剂呋喃茶碱与大鼠肝微粒体和染料木黄酮共同温孵,测定染料木黄酮在♀、♂大鼠肝微粒体中的代谢速率,评价♀、♂大鼠CYP1A2的相对百分比活性。结果在CYP1A2抗体浓度为1∶400,孵育时间为30 m in条件下,♂大鼠肝微粒体代谢染料木黄酮的相对代谢率为(20.95±2.13)%,♀动物为(13.73±1.26)%。在选择性CYP1A2抑制剂呋喃茶碱浓度为3.125μmol.L-1,孵育时间为30 m in条件下,♂动物为(58.02±3.35)%,而♀大鼠肝微粒体代谢染料木黄酮的相对代谢率为(43.82±2.65)%,两者之间差异有显著性(P<0.01)。结论染料木黄酮在♂大鼠肝微粒体中代谢较♀大鼠快,提示♂大鼠肝微粒体CYP1A2酶活性高于♀大鼠。     

探针药物法测定CYP450 3A的活性

目的：以睾酮为探针药物采用高效液相色谱法测定GYP4503A的活性。方法：采用大鼠肝微粒体体外代谢模型,以有机溶剂乙腈终止反应并沉淀蛋白,上清液进高效液相色谱分析。根据Lineweaver-Burk双倒数作图法方程式计算酶动力学参数。结果：在蛋白浓度为1．0mg&#183;mL^-1,反应时间为5min时酶促反应近似直线进行,测得酶动力学参数Km值为38．84μmol&#183;L^-1,Vmax值为1．83μmol&#183;L^-1&#183;min^-1&#183;mg^-1 protein。结论：本方法简便、有效,适合实验室用于测定CYP4503A的活性,为进一步研究药物相互作用奠定了基础。     

Flavonoids diosmetin and luteolin inhibit midazolam metabolism by human liver microsomes and recombinant CYP 3A4 and CYP3A5 enzymes

We evaluated the effects of increasing concentrations of the flavonoids salvigenin, diosmetin and luteolin on the in vitro metabolism of midazolam (MDZ), a probe substrate for cytochrome P450 (CYP) 3A enzymes, which is converted into 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) by human liver microsomes. Salvigenin had only a modest effect on MDZ metabolism, whereas diosmetin and luteolin inhibited in a concentration-dependent manner the formation of both 1'-OH-MDZ and 4-OH-MDZ, with apparent K(i) values in the 30-50mumol range. Both diosmetin and luteolin decreased 1'-OH-MDZ formation by human recombinant CYP3A4, but not CYP3A5, whereas they decreased 4-OH-MDZ formation by both recombinant enzymes. To assess whether any relationship exists between the physico-chemical characteristics of flavones and their effects on MDZ metabolism, we tested the effects of three other flavones (flavone, tangeretin, chrysin) on MDZ metabolism by human liver microsomes. Whereas flavones possessing more than two hydroxyl groups (luteolin, diosmetin) inhibited MDZ biotransformation, flavones lacking hydroxyl groups in their A and B rings (flavone, tangeretin) stimulated MDZ metabolism. We also found close relationships between the maximum stimulatory or inhibitory effects of flavones on 1'-OH-MDZ and 4-OH-MDZ formation rates and their log of octanol/water partition coefficients (logP) or their total number of hydroxyl groups. The results of the study may be of clinical relevance since they suggest that luteolin and diosmetin may cause pharmacokinetic interactions with co-administered drugs metabolized via CYP3A.