Tjia J F, Colbert J, Back D J
Department of Pharmacology and Therapeutics, University of Liverpool, United Kingdom.
J Pharmacol Exp Ther. 1996 Mar;276(3):912-7.
In this paper we describe the kinetics of formation of 1-methylxanthine (1-MX), 3-methylxanthine (3-MX) and 1,3-dimethyluric acid (1,3-DMU) from theophylline in human liver microsomal incubations and use the selective inhibitor approach to define the role of the individual cytochrome P450s (CYP) in each pathway. A biphasic model fitted the data best for the formation of each metabolite. The high-affinity site Km and Vmax values were: 1-MX, Km = 0.29 +/- 0.21 mM, Vmax = 5.92 +/- 3.74 pmol.mg(-1).min(-1) (mean +/- S.D.; n = 4); 3-MX, Km = 0.28 +/- 0.08 mM, Vmax = 3.32 +/- 2.19 pmol.mg(-1).min(-1); 1,3-DMU,Km = 0.31 +/- 0.14 mM, Vmax = 43.3 +/- 9.3 pmol.mg(-1).min(-1). The relative contribution of the high- and the low-affinity enzymes in 1,3-DMU formation was calculated based on the enzyme kinetic parameters. To characterize the high-affinity site, a range of CYP isozyme substrates and inhibitors were incubated with 100 microM theophylline. The CYP1A2 inhibitors furafylline, ellipticine and alpha-naphthoflavone were potent inhibitors of both 1-MX and 3-MX formation with more that 80% of N-demethylase activities inhibited below a concentration of 5 microM. These compounds also markedly inhibited 1,3-DMU formation. Enzyme kinetic and selective inhibition data indicated that about 80% of 1,3-DMU formation was catalyzed by the high-affinity isoform (CYP1A2) at a theophylline concentration of 100 microM. To investigate the role of other isoforms in 8-hydroxylation, experiments were performed involving incubation with a combination of inhibitors. It is evident that in addition to CYP1A2, CYP2E1 has a minor role om 8-hydroxylation. This based on the fact that 80% inhibition was seen on preincubation with furafylline and about 90% inhibition on preincubation with furafylline plus diethyldithiocarbamate. Low concentrations of ketoconazole (selective for CYP3A4) only produced marginal inhibition of 1,3-DMU and, therefore, CYP3A4 is only of minor significance in this reaction. Human B-lymphoblastoid cell lines expressing CYP1A2 catalyzed theophylline metabolism with formation of 1-MX, 3-MX and 1,3-MDU. CYP2E1 cells also catalyzed formation of 1,3-DMU. The CYP3A4 cell line did not catalyze theophylline metabolism.
在本文中，我们描述了在人肝微粒体孵育体系中，茶碱形成1-甲基黄嘌呤（1-MX）、3-甲基黄嘌呤（3-MX）和1,3-二甲基尿酸（1,3-DMU）的动力学过程，并采用选择性抑制剂法来确定各细胞色素P450（CYP）在每条途径中的作用。双相模型最适合各代谢产物形成的数据。高亲和力位点的Km和Vmax值分别为：1-MX，Km = 0.29 ± 0.21 mM，Vmax = 5.92 ± 3.74 pmol·mg⁻¹·min⁻¹（平均值 ± 标准差；n = 4）；3-MX，Km = 0.28 ± 0.08 mM，Vmax = 3.32 ± 2.19 pmol·mg⁻¹·min⁻¹；1,3-DMU，Km = 0.31 ± 0.14 mM，Vmax = 43.3 ± 9.3 pmol·mg⁻¹·min⁻¹。根据酶动力学参数计算了1,3-DMU形成过程中高亲和力和低亲和力酶的相对贡献。为了表征高亲和力位点，将一系列CYP同工酶底物和抑制剂与100 μM茶碱一起孵育。CYP1A2抑制剂呋拉茶碱、玫瑰树碱和α-萘黄酮是1-MX和3-MX形成的有效抑制剂，在浓度低于5 μM时，超过80%的N-脱甲基酶活性被抑制。这些化合物也显著抑制1,3-DMU的形成。酶动力学和选择性抑制数据表明，在茶碱浓度为100 μM时，约80%的1,3-DMU形成是由高亲和力同工型（CYP1A2）催化的。为了研究其他同工型在8-羟基化中的作用，进行了涉及与抑制剂组合孵育的实验。很明显，除CYP1A2外，CYP2E1在8-羟基化中起次要作用。这是基于以下事实：用呋拉茶碱预孵育时可观察到80%的抑制，用呋拉茶碱加二乙基二硫代氨基甲酸盐预孵育时抑制率约为90%。低浓度的酮康唑（对CYP3A4有选择性）仅对1,3-DMU产生轻微抑制，因此，CYP3A4在该反应中仅具有次要意义。表达CYP1A2的人B淋巴细胞系催化茶碱代谢生成1-MX、3-MX和1,3-MDU。CYP2E1细胞也催化1,3-DMU的形成。CYP3A4细胞系不催化茶碱代谢。
