CYP2C19基因型和CYP2C9对人肝微粒体中氟西汀N-去甲基代谢的影响(英文)

目的:本实验旨在研究CYP2C19基因型人肝微粒体中氟西汀N-去甲基代谢的酶促动力学特点并鉴定参与此代谢途径的细胞色素P-450酶。方法:测定基因型CYP2C19肝微粒体中去甲氟西汀形成的酶促动力学。鉴定氟西汀N-去甲基酶活性与细胞色素P-450 2C9,2C19,1A2和2D6酶活性的相关性,同时应用各种细胞色素P-450酶的选择性抑制剂和化学探针进行抑制实验,从而确定参与氟西汀N-去甲基代谢的细胞色素P-450酶。结果:去甲氟西汀生成的酶促动力学数据符合单酶模型,并具有Michaelis-Menten动力学特征。当底物浓度为氟西汀25μmol/L和100μmol/L时,去甲氟西汀(N-FLU)的生成率分别与甲磺丁脲3-羟化酶活性显著相关(r_1=0.821,P_1=0.001;r_2=0.668,P_2=0.013),当底物浓度为氟西汀100μmol/L时,N-FLU的生成率与S-美芬妥因4’-羟化酶活性显著相关(r=0.717,P=0.006)。PM肝微粒中磺胺苯吡唑和醋竹桃霉素对氟西汀N-去甲基代谢的抑制作用显著大于EM(73％vs 45％,P<0.01)。结论:在生理底物浓度下,CYP2C9是催化人肝微粒体中氟西汀N-去甲基代谢的主要CYP-450酶;而高底物浓度时,以CYP2C19的作用为主。

Effects of CYP2C19 genotype and CYP2C9 on fluoxetine N-demethylation in human liver microsomes

AIM: The present study was designed to define the kinetic behavior of fluoxetine N-demethylation in human liver microsomes and to identify the isoforms of cytochrome P-450 (CYP) involved in this metabolic pathway. METHODS: The kinetics of Ne formation of norfluoxetine was determined in human liver microsomes from six genotyped CYP2C19 extensive metabolizers (EM). The correlation studies between the fluoxetine N-demethylase activity and various CYP enzyme activities were performed. Selective inhibitors or chemical probes of various cytochrome P-450 isoforms were also employed. RESULTS: The kinetics of norfluoxetine formation in all liver microsomes were fitted by a single-enzyme Michaelis-Menten equation (mean Km=32 micromol/L+/-7 micromol/L). Significant correlations were found between N-demethylation of fluoxetine at both 25 micromol/L and 100 micromol/L and 3-hydroxylation of tolbutamide at 250 micromol/L (r1=0.821, P1=0.001; r2=0.668, P2=0.013), respectively, and S-mephenytoin 4'-hydroxylase activity (r=0.717, P=0.006) at high substrate concentration of 100 micromol/L. S-mephenytoin (SMP) (a CYP2C19 substrate) at high concentration and sulfaphenazole (SUL) (a selective inhibitor of CYP2C9) substantially inhibited norfluoxetine formation. The reaction was minimally inhibited by coincubation with chemical probe, inhibitor of CYP3A4 (triacetyloleandomycin, TAO). The inhibition of fluoxetine N-demethylation at high substrate concentration (100 micromol/L) was greater in PM livers than in EM livers (73 % vs 45 %, P < 0.01) when the microsomes were precoincubated with SUL plus TAO. CONCLUSION: Cytochrome P-450 CYP2C9 is likely to be a major CYP isoform catalyzing fluoxetine N-demethylation in human liver microsomes at a substrate concentration close to the therapeutic level, while polymorphic CYP2C19 may play a more important role in this metabolic pathway at high substrate concentration.