He K, Iyer K R, Hayes R N, Sinz M W, Woolf T F, Hollenberg P F
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Chem Res Toxicol. 1998 Apr;11(4):252-9. doi: 10.1021/tx970192k.
Grapefruit juice has been found to significantly increase oral bioavailability of several drugs metabolized by cytochrome P450 3A4 (P450 3A4) through inhibiting the enzymatic activity and decreasing the content of intestinal P450 3A4. HPLC/MS/MS and HPLC/UV analyses of ethyl acetate extracts from grapefruit juice revealed the presence of several furanocoumarins of which bergamottin (BG) is the major one. BG was shown to inactivate P450 3A4 in a reconstituted system consisting of purified P450 3A4, NADPH-cytochrome P450 reductase, cytochrome b5, and phospholipids. Inactivation was time- and concentration-dependent and required metabolism of BG. The loss of catalytic activity exhibited pseudo-first-order kinetics. The values of kinactivation and KI calculated from the inactivation studies were 0.3 min-1 and 7.7 microM, respectively. While approximately 70% of the erythromycin N-demethylation activity was lost during incubation with BG in the reconstituted system, P450 3A4 retained more than 90% of the heme as determined either by UV-visible spectroscopy or by HPLC. However, approximately 50% of the apoP450 in the BG-inactivated P450 3A4 incubation mixture could not be recovered from a reverse-phase HPLC column when compared with the -NADPH control. The mechanism of the inactivation appears to involve modification of the apoP450 in the active site of the enzyme instead of heme adduct formation or heme fragmentation. These results indicate that BG, the primary furanocoumarin extracted from grapefruit juice, is a mechanism-based inactivator of P450 3A4. BG was also found to inhibit the activities of P450s 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes.
已发现葡萄柚汁可通过抑制细胞色素P450 3A4（P450 3A4）的酶活性并降低肠道P450 3A4的含量，显著提高几种经P450 3A4代谢的药物的口服生物利用度。对葡萄柚汁乙酸乙酯提取物的HPLC/MS/MS和HPLC/UV分析显示存在几种呋喃香豆素，其中佛手柑内酯（BG）是主要成分。在由纯化的P450 3A4、NADPH-细胞色素P450还原酶、细胞色素b5和磷脂组成的重组系统中，BG可使P450 3A4失活。失活具有时间和浓度依赖性，且需要BG的代谢。催化活性的丧失表现为假一级动力学。从失活研究计算得到的kinactivation和KI值分别为0.3 min-1和7.7 microM。在重组系统中与BG孵育期间，约70%的红霉素N-脱甲基活性丧失，而通过紫外可见光谱或HPLC测定，P450 3A4保留了超过90%的血红素。然而，与-NADPH对照相比，在BG失活的P450 3A4孵育混合物中，约50%的脱辅基P450无法从反相HPLC柱上回收。失活机制似乎涉及酶活性位点中脱辅基P450的修饰，而非血红素加合物形成或血红素断裂。这些结果表明，从葡萄柚汁中提取的主要呋喃香豆素BG是P450 3A4的一种基于机制的失活剂。还发现BG可抑制人肝微粒体中P450s 1A2、2A6、2C9、2C19、2D6、2E1和3A4的活性。
