Inhibition of coumarin 7-hydroxylase activity in human liver microsomes
Inhibition of coumarin 7-hydroxylase activity in human liver microsomes
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DOI：
10.1006/abbi.1997.9964
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发表时间：
1997-05-01
影响因子：
3.9
通讯作者：
Parkinson, A
中科院分区：
文献类型：
--
作者：
Draper, AJ;Madan, A;Parkinson, A
研究了9种有机溶剂和47种常用的P450底物和抑制剂对人肝微粒体香豆素7-羟化酶（CYP 2A 6）活性的影响。在检查的9种有机溶剂（终浓度1%，v/v）中，只有甲醇不抑制人肝微粒体对香豆素（0.5 - 50 μ M）的7-羟基化。结构上与香豆素相关的二氧六环和四氢呋喃是最具抑制性的溶剂。虽然香豆素7-羟基化的速率在9份人肝微粒体和cDNA表达的CYP 2A 6样品中变化很大（Vmax = 179至2470 pmol/mg蛋白/min），但香豆素7-羟基化的Km相当恒定（范围为0.50至0.70 μ M）。根据Ki> 200 μ M的定义，以下化学品对CYP 2A 6几乎没有抑制作用或没有抑制作用：咖啡因、氯唑沙宗、西咪替丁、美沙芬、地西泮、双氯芬酸、红霉素、乙炔基睾酮、乙炔睾酮、氟康唑、呋拉茶碱、糠醛、己巴比妥、伊曲康唑、美芬妥英、甲巯咪唑、甲硝唑、柚皮素、柚皮苷、硝苯地平、诺氟沙星、炔诺孕酮、奥芬那君、奎尼丁、罂粟碱、非那西丁，乙胺嘧啶、雷尼替丁、螺内酯、磺胺苯吡唑、磺酰草胺、睾酮、甲苯磺丁脲、三乙酰竹桃霉素和华法林。换句话说，当香豆素的浓度等于K-m（0.50 μ M）时，这些化学品在终浓度为100 μ M时不能抑制CYP 2A 6。以下化学品被归类为CYP 2A 6的强抑制剂（定义为Ki < 200 μ M）：克霉唑、二乙基二硫代氨基甲酸酯、椭圆藤碱、酮康唑、8-甲氧基哌庚烯、4-甲基吡唑、甲吡酮、咪康唑、α-萘甲酮、尼古丁、对硝基苯酚和反苯环丙胺。每种化学物质抑制香豆素7-羟基化的效力与研究的人肝微粒体样品无关。香豆素7-羟化酶最有效的抑制剂之一是8-甲氧基补骨脂素（methoxsalen），它被确定为CYP 2A 6的基于机制的抑制剂（自杀底物）（k（失活）0.5 min（-1））。除8-甲氧基补骨脂素外，人肝微粒体和NADPH与上述抑制剂预孵育不会增加其抑制CYP 2A 6的能力。CYP 2A 6最有效的竞争性抑制剂是反苯环丙胺（Ki = 0.04 μ M）。几种强烈抑制CYP 2A 6的化学物质，如酮康唑和反苯环丙胺，通常用于选择性抑制CYP 2A 6以外的人P450酶。本研究的结果强调了对常用P450抑制剂特异性进行系统评价的必要性。(C)北京：科学出版社.
Nine organic solvents and 47 commonly used P450 substrates and inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents examined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 mu M) by human liver microsomes. Dioxane and tetrahydrofuran, which are structurally related to coumarin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of human liver microsomes and cDNA-expressed CYP2A6 (V-max = 179 to 2470 pmol/mg protein/min), the K-m for coumarin 7-hydroxylation was fairly constant (ranging from 0.50 to 0.70 mu M). The following chemicals caused little or no inhibition of CYP2A6 as defined by a K-i > 200 mu M: caffeine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofenac, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, furafylline, furfural, hexobarbital, itraconazole, mephenytoin, methimazole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, norgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethamine, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testosterone, tolbutamide, troleandomycin, and warfarin. In other words, these chemicals, at a final concentration of 100 mu M, failed to inhibit CYP2A6 when the concentration of coumarin was equal to K-m (0.50 mu M). The following chemicals were classified as strong inhibitors of CYP2A6 (defined by K-i < 200 mu M): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyrapone, miconazole, alpha-naphthoflavone, nicotine, p-nitrophenol, and tranylcypromine. The potency with which each chemical inhibited the 7-hydroxylation of coumarin was independent of which sample of human liver microsomes was studied. One of the most potent inhibitors of coumarin 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determined to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (k(inactivation) 0.5 min(-1)). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementioned inhibitors did not increase their ability to inhibit CYP2A6. The most potent competitive inhibitor of CYP2A6 was tranylcypromine (K-i = 0.04 mu M). Several of the chemicals that strongly inhibited CYP2A6, such as ketoconazole and tranylcypromine, are often used with the intention of selectively inhibiting human P450 enzymes other than CYP2A6. The results of this study underscore the need for a systematic evaluation of the specificity of commonly used P450 inhibitors. (C) 1997 Academic Press.