人肝脏微粒体在体外对丝裂霉素C的代谢

Aim To provide the profiles of metabolism of mitomycin C(MMC)by human liver microsomes in vitro,MMC was incubated with human liver microsomes,then the supernatant component was isolated and detected by HPLC.Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol(DIC)on metabolism of MMC.Standard,reaction,background control(microsomes was inactivated),negative control(no NADPH),and inhibitor group(adding DIC)were assigned,the results were analyzed by Graphpad Prism 4.0 software.Reaction group compared with background control and negative control groups,3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes.Their retention times were 10.0,14.0,14.8 min(named as M1,M2,M3),respectively.Their formation was kept as Sigmoidal dose-response and their K_m were 0.52(95% CI,0.40-0.67)mmol·L-1,0.81(95% CI,0.59-1.10)mmol·L-1,0.54(95% CI,0.41-0.71)mmol·L-1,respectively.The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC.DIC can inhibit formation of M2,it’s dose-effect fitted to Sigmoidal curve and it’s IC_ 50 was 59.68(95% CI,40.66-87.61)μmol·L-1,which indicated DT-diaphorase could take part in the formation of M2.MMC can be metabolized by human liver microsomes in vitro,and at least three metabolites of MMC could be isolated by HPLC in the experiment,further study showed DT-diaphorase participated in the formation of M2.

人肝微粒体CYP亚型在新型吲哚醌类化合物629代谢中的作用

目的研究人肝微粒体重组体系中不同CYP亚型在丝裂霉素C(MMC)的衍生物5-氮丙啶-3-羟甲基-1-甲基吲哚-4,7-二酮[5-(aziridin-1-yl)-3-hydroxymethyl-1-methylin-dole-4,7-dione,简称629]代谢中的作用。方法不同浓度629与人肝脏微粒体共孵育,给予细胞不同CYP亚型特异性抑制剂的处理,用高压液相色谱法(high pressure liquid chro-matography,HPLC)分离、检测629的消失情况。结果HPLC检测到629在肝脏微粒体中的代谢遵循酶动力学剂量效应关系,Km值为336μmol·L-1。P450酶系中CYP1A2、CYP2B6和CYP2A6被抑制后,可影响629的代谢(P<0.05),并且3者中CYP1A2的影响较大,但三者间差异无显著性(P>0.05);而CYP3A4、CYP2C19、CYP2C9、CYP2E1和CYP2D6对629的代谢无影响(P>0.05)。结论新型吲哚醌类生物还原物629可在肝脏代谢,其中CYP1A2、CYP2B6和CYP2A6可参与629的代谢,这为新型生物还原活性物设计、开发及临床上的合理用药具有重大的意义。

中药降脂方对肝细胞色素P450同工酶3A4的抑制作用

目的:分析中药复方降脂方对人体主要药物代谢酶——肝细胞色素P450(Cytchrome P450,CYP)同工酶(3A4)的抑制作用。方法:选择不同浓度的中药健脾泄浊降脂方,与人肝微粒体预孵育15 min后,加入CYP 3A4的底物睾酮及还原型辅酶烟酰胺腺嘌呤二核苷酸(NADPH),于37℃共同孵育30 min,之后加入0.5 ml预冷的甲醇终止反应。以紫外247 nm为基础的HPLC的方法检测探针底物睾酮的代谢产物6-β-羟基睾酮以判断CYP3A4的活性,确立中药降脂方对CYP 3A4的抑制作用。结果:降脂方全方及其拆方1(健脾化浊、活血化瘀)和拆方2(平补肝肾、活血通络)与人肝微粒体共同孵育后对CYP 3A4均有抑制作用,其中拆方1与拆方2的作用更明显,三方的IC50分别为0.024 64、0.008 96和0.007 69;而且随着中药复方浓度的升高,酶活性逐渐下降。结论:中药复方降脂方及其拆方对CYP 3A4酶均有抑制作用,且存在浓度效应关系,其中拆方作用较复方更明显。