以普罗帕酮为探针测定人肝微粒体中CYP2D6的活性

目的:测定普罗帕酮(PPF)和5-羟基普罗帕酮(5-OHP)的比值以表达人肝微粒体中CYP2D6的活性.方法:以1g·L-1微粒体蛋白浓度37℃孵育PPF 1 h,以HPLC测定PPF和5-OHP的含量.结果:PPF和5-OHP的线性方程分别为Y=0.452 0 C 0.003 0,r=0.999 7;Y=0.749 4 C-0.020 5,r=0.997 5.结论:人肝微粒体CYP2D6酶的活性可以通过测定5-OHP与PPF的比值进行预测.     

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.     

高效液相色谱法测定鼠肝微粒体中CYP1A2酶的活性及动力学考察

目的:建立以非那西丁为探针的高效液相色谱-紫外检测的实验方法,测定大鼠肝微粒体中CYP1A2酶活性并对其进行动力学考察。方法:采用Shimadzu Shim-Pack VP-ODS柱(150 mm×4.6 mm,5μm),流动相为:100 mmol·L-1磷酸二氢钠缓冲液(pH 4.3)和乙腈,梯度洗脱,流速为1.0 mL·min-1,柱温为室温,检测波长245 nm。非那西丁与大鼠肝微粒体在37℃温孵60 min,加入冰甲醇终止,12000 r.min-1离心10 min,取上清进行HPLC分析,以Lineweaver-Burk作图计算Vmax与Km值。结果:非那西丁、对乙酰氨基酚及其内标间乙酰氨基酚三者分离良好且无内源性干扰。对乙酰氨基酚最低检测限50 nmol·L-1,线性范围0.1～10μmol·L-1。日内日间精密度均小于10%。回收率大于75%。动力学考察表明选择甲醇作为终止试剂效果较好,非那西丁在0.2 mg·mL-1大鼠肝微粒体体系中孵育60 min,测得动力学参数Vmax为0.21 nmol·min-1.mgprotein-1,Km为20.39μmol·L-1。结论:该方法稳定,结果能准确地反映CYP1A2酶的活性,可以用于相关动力学研究。     

建立用大鼠肝微粒体快速考察药物体外CYP3A1活性抑制的方法

目的研究酮康唑对大鼠肝微粒体细胞色素P450同工酶3A1（CYP3A1）活性的作用,建立一种用大鼠肝微粒体快速考察药物体外CYP3A1活性抑制的方法。方法采集大鼠肝微粒体,随机分为对照组和药物处理组。酮康唑药物处理组分别加入不同浓度的酮康唑,对照组只加入培养液,混匀后放入37℃培养箱中孵育15 min,然后加入CYP 3A1底物睾酮,继续孵育10 min,最后加入内标物氢化可的松。用HPLC法测定睾酮经大鼠肝微粒体温孵后生成6-β羟基睾酮的量,代表CYP3A1的活性。结果各个处理组与对照组的比较差异均有显著性差异（P〈0.05）;提示在一定浓度范围内,大鼠同工酶CYP3A1的相对活性百分比随着酮康唑浓度的增加而逐渐减低,各处理组与对照组比较差异均有显著性意义（P〈0.5）。半数抑制浓度（IC50）值为3.25μg/ml。结论酮康唑对大鼠肝微粒体细胞色素P450同工酶3A1的活性有强抑制作用,建立的通过大鼠肝微粒体快速考察药物体外CYP3A1探针抑制的方法重复性和稳定性均良好,为评价新药对大鼠肝微粒体CYP3A1的活性的体外作用提供可靠的检测手段。     

建立用大鼠肝微粒体快速考察药物体外CYP3A1活性抑制的方法

目的 研究酮康唑对大鼠肝微粒体细胞色素P450同工酶3A1( CYP3A1)活性的作用,建立一种用大鼠肝微粒体快速考察药物体外CYP3A1活性抑制的方法.方法 采集大鼠肝微粒体,随机分为对照组和药物处理组.酮康唑药物处理组分别加入不同浓度的酮康唑,对照组只加入培养液,混匀后放入37℃培养箱中孵育15 min,然后加入CYP 3A1底物睾酮,继续孵育10 min,最后加入内标物氢化可的松.用HPLC法测定睾酮经大鼠肝微粒体温孵后生成6-β羟基睾酮的量,代表CYP3A1的活性.结果 各个处理组与对照组的比较差异均有显著性差异(P＜0.05);提示在一定浓度范围内,大鼠同工酶CYP3A1的相对活性百分比随着酮康唑浓度的增加而逐渐减低,各处理组与对照组比较差异均有显著性意义(P＜0.5).半数抑制浓度( IC50)值为3.25 μg/ml.结论 酮康唑对大鼠肝微粒体细胞色素P450同工酶3A1的活性有强抑制作用,建立的通过大鼠肝微粒体快速考察药物体外CYP3A1探针抑制的方法重复性和稳定性均良好,为评价新药对大鼠肝微粒体CYP3A1的活性的体外作用提供可靠的检测手段.     

反相高效液相色谱法测定人肝微粒体中帕洛诺司琼的含量

目的:为了研究帕洛诺司琼的体外代谢,建立人肝微粒体中帕洛诺司琼的反相高效液相色谱测定法。方法:帕洛诺司琼与人肝微粒体共孵育之后,以Nova-park C18(5μm,200×4.6mm)柱为分析柱,甲醇- 0.01 mol/L磷酸二氢钾缓冲盐(80:20,v/v)为流动相,流速1.0mL/min,紫外检测波长为240nm。结果:帕洛诺司琼在5-100μmol/L范围内线性关系良好(r=0.999 8)。检测限为0.2μmol/L(S/N≥3),定量限为1.0μmol/L(RSD=4.81%,n=3)。方法回收率为96.0%-103.0%,日内、日间RSD分别<7.0%和<10% (n=5)。结论:此法简便,准确,可用于研究帕洛诺司琼的体外代谢。     

重组肝CYP3A4与CYP3A29细胞微粒体药物代谢动力学比较

目的 比较巴马小型猪CYP3A29和人CYP3 A4稳定表达重组肝癌细胞株微粒体的药物代谢特征,在分子水平为巴马小型猪作为临床前药物代谢实验动物提供科学依据.方法 以CYP3A特异性代谢底物硝苯地平、睾酮及其抑制药酮康唑为探针药物,将探针药物与重组CYP3A4、CYP3A29细胞微粒体在优化的微粒体浓度、药物浓度、孵育时间等条件下进行体外孵育,高效液相色谱法检测其药物代谢(抑制)动力学参数,并将二者进行比较分析.结果 巴马小型猪CYP3A29与CYP3A4在硝苯地平和睾酮代谢差异无统计学意义(P＞0.05).酮康唑的抑制活性为:当代谢底物为硝苯地平时,酮康唑对CYP3A29和CYP3A4的半数抑制浓度分别为0.090,0.132 μmol·L-1(P＜0.05);当代谢底物为睾酮时,酮康唑对CYP3A29和CYP3A4的半数抑制浓度分别为0.056,0.032 μmol·L-1(P＜0.05).结论 重组CYP3A29与CYP3 A4细胞微粒体对硝苯地平和睾酮活性差异不显著;酮康唑对重组CYP3A29与CYP3A4细胞微粒体硝苯地平和睾酮代谢活性有差异.     

大鼠肝微粒体法测定3种中药对CYP3A4 亚型的作用

［摘要］目的建立肝微粒体测定法,观察中药制剂对CYP3A4 亚型的作用。方法取大鼠肝脏,制备肝微粒体,分别优化肝微粒体体外温孵系统的反应时间、蛋白浓度及探针药物咪达唑仑的浓度;高效液相色谱 质谱联用（HPLC MS MS）法测定大鼠肝微粒体中咪达唑仑的浓度,计算咪达唑仑的活性;在肝微粒体体外温孵系统中分别加入不同浓度的血脂康胶囊、通心络胶囊、枣仁安神胶囊内容物及对照药物酮康唑,测定其半数抑制浓度（IC50）及抑制常数（Ki）。结果肝微粒体体外温孵系统的反应条件为0.4 g&;#8226;L 1大鼠肝微粒体,4 μmol&;#8226;L 1咪达唑仑溶液,37 ℃温育5 min。酮康唑、血脂康胶囊、通心络胶囊的IC50分别为（3.4±0.2）,（25.1±0.3）,（56.2±0.3） mg&;#8226;L 1,枣仁安神胶囊的IC50﹥150 mg&;#8226;L 1;酮康唑、血脂康胶囊、通心络胶囊的Ki分别为（1.7±0.1）,（25.0±0.2）,（50.0±0.3） mg&;#8226;L 1。结论建立了中药制剂对CYP3A4亚型作用的大鼠肝微粒体研究模型。血脂康胶囊和通心络胶囊对大鼠CYP3A4亚型有较弱的抑制作用。     

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。     

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。     

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。     

The involvement of CYP1A2 and CYP3A4 in the metabolism of clozapine

Aims Clozapine (CLZ), an atypical neuroleptic with a high risk of causing agranulocytosis, is metabolized in the liver to desmethylclozapine (DCLZ) and clozapine N-oxide (CLZ-NO). This study investigated the involvement of different CYP isoforms in the formation of these two metabolites. Methods Human liver microsomal incubations, chemical inhibitors, specific antibodies, and different cytochrome P450 expression systems were used. Results K_m and V_max values determined in human liver microsomes were lower for the demethylation (61±21 μm, 159±42 pmol min⁻¹ mg protein⁻¹ mean±s.d.; n=4), than for the N-oxidation of CLZ (308±1.5 μm, 456±167pmol min⁻¹ mg protein⁻¹; n=3). Formation of DCLZ was inhibited by fluvoxamine (53±28% at 10 μm ), triacetyloleandomycin (33±15% at 10 μm ), and ketoconazole (51±28% at 2 μm ) and by antibodies against CYP1A2 and CYP3A4. CLZ-NO formation was inhibited by triacetyloleandomycin (34±16% at 10 μm ) and ketoconazole (51±13% at 2 μm ), and by antibodies against CYP3A4. There was a significant correlation between CYP3A content and DCLZ formation in microsomes from 15 human livers (r=0.67; P=0.04). A high but not significant correlation coefficient was found for CYP3A content and CLZ-NO formation (r=0.59; P=0.09). Using expression systems it was shown that CYP1A2 and CYP3A4 formed DCLZ and CLZ-NO. K_m and V_max values were lower in the CYP1A2 expression system compared to CYP3A4 for both metabolic reactions. Conclusions It is concluded that CYP1A2 and CYP3A4 are involved in the demethylation of CLZ and CYP3A4 in the N-oxidation of CLZ. Close monitoring of CLZ plasma levels is recommended in patients treated at the same time with other drugs affecting these two enzymes.     

Ilaprazole,a new proton pump inhibitor,is primarily metabolized to ilaprazole sulfone by CYP3A4 and 3A5

1. Ilaprazole is a new proton pump inhibitor, designed for treatment of gastric ulcers, and developed by Il-Yang Pharmaceutical Co (Seoul, Korea). It is extensively metabolised to the major metabolite ilaprazole sulfone.

2. In the present study, several in vitro approaches were used to identify the cytochrome P450 (CYP) enzymes responsible for ilaprazole sulfone formation. Concentrations of ilaprazole sulfone were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

3. Incubation of ilaprazole with cDNA-expressed recombinant CYPs indicated that CYP3A was the major enzyme that catalyses ilaprozole to ilaprazole sulfone. This reaction was inhibited significantly by ketoconazole, a CYP3A inhibitor, and azamulin, a mechanism-based inhibitor of CYP3A, while no substantial effect was observed using selective inhibitors for eight other P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1).

4. In addition, the formation of ilaprazole sulfone correlated well with CYP3A-catalysed testosterone 6β-hydroxylation and midazolam 1′-hydroxylation in 20 different human liver microsome panels. The intrinsic clearance of the formation of ilaprazole sulfone by CYP3A4 was 16-fold higher than that by CYP3A5.

5. Collectively, these results indicate that the formation of the major metabolite of ilaprazole, ilaprazole sulfone, is predominantly catalysed by CYP3A4/5.

     

CYP3A4 and CYP3A5 catalyse the conversion of the N-methyl-D-aspartate (NMDA) antagonist CJ-036878 to two novel dimers

CJ-036878, N-(3-phenethoxybenzyl)-4-hydroxybenzamide, was developed as an antagonist of the N-methyl-D-aspartate receptor NR2B subunit. Two dimeric metabolites, CJ-047710 and CJ-047713, were identified from the incubation mixture with CJ-036878 in human liver microsomes (HLM). The identification of the enzymes involved in the formation of these dimeric metabolites was investigated in the current study. Inhibition of the formation of CJ-047710 and CJ-047713 in pooled HLM by 1-aminobenztriazole, SKF-525A, and ketoconazole were observed. Ketoconazole played a significant role in inhibiting formation of these two metabolites in a concentration-dependent manner. Recombinant CYP3A4 and CYP3A5 exhibited a markedly high activity toward the formation of CJ-047710 and CJ-047713 from CJ-036878, but the contribution of other CYP enzymes to these formations was at a very low level or negligible. The formation of CJ-047710 and CJ-047713 in pooled HLM, CYP3A4, and CYP3A5 showed sigmoid characteristics. S₅₀ values for CJ-047710 and CJ-047713 formation in HLM were almost equivalent with those for CYP3A4 and CYP3A5. For the CYP3A enzymes, maximal clearance due to auto-activation values for CJ-047710 and CJ-047713 formation catalysed by CYP3A5 were 3.6- and 3.1-fold higher than those catalysed by CYP3A4. This is the first report that shows both CYP3A4 and CYP3A5 simultaneously contribute to dimerization through oxidative C-C and C-O coupling reactions.     

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.     

三七总皂苷主要成分对人肝微粒体CYP3A4酶的抑制作用研究

目的：研究三七总皂苷主要成分人参皂苷Rg1、人参皂苷Rb1和三七皂苷R1对人肝微粒体CYP3A4酶的体外抑制作用.方法：采用人肝微粒体体外孵育法,在孵育体系中分别加入不同浓度的人参皂苷Rg1、人参皂苷Rb1和三七皂苷R1与探针底物睾酮共孵育,用LC-MS /MS 法测定代谢产物6β-羟基睾酮的生成量反映CYP3A4酶活性的影响.结果：人参皂苷Rg1在2～1 000 μmol·L-1的测试浓度范围内对CYP3A4未见剂量相关的抑制作用;人参皂苷Rb1在2～200 μmol·L-1的测试浓度范围内对CYP3A4未见剂量相关的抑制作用,在1 000 μmol·L-1的测试浓度下对CYP3A4具有轻微抑制作用;三七皂苷R1在2～1 000 μmol·L-1的测试浓度范围内对CYP3A4的IC50为126 μmol·L-1（＞100 μmol·L-1）.结论：三七总皂苷3个主要有效成分单体对CYP3A4酶的体外抑制作用不同,人参皂苷Rg1无抑制作用,人参皂苷Rb1高浓度下有轻微抑制作用,三七皂苷R1有弱抑制作用,三七总皂苷制剂与CYP3A4酶代谢相关的药物之间产生相互作用的可能性低.     

HPLC法测定大鼠肝微粒体中睾酮的含量

目的:建立以高效液相色谱法测定大鼠肝微粒体中细胞色素P4503A4(CYP3A4)探针底物睾酮含量的方法。方法:色谱柱为Ultimate-XB C18,流动相为水-乙腈(50∶50),内标为地西泮,流速为1.0mL·min-1,检测波长为245nm,柱温为30℃,进样量为20μL。结果:睾酮生物样品检测浓度的线性范围为10.0～200.0μmo·lL-1(r=0.9995);低、中、高浓度的平均方法回收率分别为86.15%、103.10%、97.48%,平均相对回收率为111.15%、107.24%、100.78%,日内RSD分别为3.92%、2.46%、2.82%,日间RSD分别为2.98%、1.34%、2.41%。结论:本方法简便快速、灵敏度高、重复性好,可用于大鼠肝微粒体中CYP3A4活性的测定及相关研究应用。     

胡柚提取物抑制硝苯地平肝微粒体酶代谢的研究

目的:探讨胡柚不同部位提取物对体外小鼠肝微粒体酶代谢活性的抑制效应。方法:在小鼠肝微粒体孵育体系中加入硝苯地平和胡柚不同部位的提取物后,应用HPLC法测定体系中硝苯地平的剩余含量。结果:硝苯地平的标准曲线方程为Y=2.136×10⁴X+2.334×10⁴（r=0.999 3）,线性范围:0.15～240 mg·L^-1。含有相同浓度硝苯地平及3种胡柚提取物的小鼠肝微粒体孵育体系中,柚汁、果肉、柚皮中剩余硝苯地平浓度分别为8.802,7.497,4.227mg·L^-1。结论:胡柚不同部位提取物对小鼠肝微粒体酶活性有抑制作用,尤其是柚汁和果肉部分活性较强。