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八种抑制性单克隆抗体确定了个体细胞色素P-450在人肝微粒体中地西泮、7-乙氧基香豆素和丙咪嗪代谢中的作用。

Eight inhibitory monoclonal antibodies define the role of individual P-450s in human liver microsomal diazepam, 7-ethoxycoumarin, and imipramine metabolism.

作者信息

Yang T J, Krausz K W, Sai Y, Gonzalez F J, Gelboin H V

机构信息

Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

出版信息

Drug Metab Dispos. 1999 Jan;27(1):102-9.

PMID:9884317
Abstract

Eight inhibitory monoclonal antibodies (MAbs) individually specific to human cytochrome P-450 (P-450) 1A1, 1A2, 2A6, 2B6, 2C subfamily (2C8, 2C9, 2C18 and 2C19), 2D6, 2E1, and 3A4/5 were used to define the role of single P-450s in the metabolism of diazepam (DZ), 7-ethoxycoumarin (7-EC), and imipramine (IMI) in human liver microsomes (HLM). The MAbs were added combinatorially to six HLM samples. With DZ as a substrate, more than 80% of temazepam (TMZ) formation was inhibited in all six samples by the addition of MAb to 3A4/5, indicating an 80% contribution of 3A4/5 to TMZ formation. Nordiazepam formation was inhibited with MAbs to 2B6 (6-23%), 2C subfamily (12-61%) and 3A4/5 (14-45%). The MAbs to 1A1, 1A2, 2A6, 2D6, and 2E1 did not inhibit TMZ or nordiazepam formation; this indicates their noninvolvement in DZ metabolism. The MAb-defined P-450 contribution to 7-EC Odeethylation in six HLM samples was 17 to 60% for 2E1, 15 to 46% for 2A6, and 5 to 22% for 1A2, reflecting the role and variation of each P-450 in this activity. MAbs to 1A1, the 2C subfamily, 2D6, and 3A4/5 did not affect 7-EC metabolism in the HLM samples. IMI is metabolized mainly to 2-hydroxyimipramine by expressed 2C19 and 2D6, and desipramine (DIM) by expressed 1A2, 2C18, 2C19 and 2D6. Expressed 1A1, 2C9, and 3A4 showed low activities for the formation of DIM. Of six HLM samples, five showed IMI hydroxylation activity (0.35-2.6 nmol/min/nmol P-450) while one (HL43) lacked hydroxylation activity. All six HLM samples showed N-deethylation activity (0.74-1.4 nmol/min/nmol P-450). The MAb-determined contribution of 2D6 and 2C19 to 2-hydroxyimipramine formation ranged from 47 to 90% and from 0 to 49%, respectively, while HL43 did not show 2-hydroxylation. The role of P-450s involved in DIM formation varied for 2C19 (13-50%), 1A2 (23-41%), and 3A4 (8-26%). These studies demonstrate a system for identifying the quantitative metabolic role of single P-450s and their interindividual variability in a tissue containing multiple P-450s. The system using inhibitory MAbs is simple, precise, and applicable to any P-450-mediated catalytic activity including that for drugs, carcinogens, mutagens, toxic chemicals and endobiotics.

摘要

使用了八种分别对人细胞色素P - 450(P - 450)1A1、1A2、2A6、2B6、2C亚家族(2C8、2C9、2C18和2C19)、2D6、2E1以及3A4/5具有特异性的抑制性单克隆抗体(MAb),来确定单个P - 450在人肝微粒体(HLM)中地西泮(DZ)、7 - 乙氧基香豆素(7 - EC)和丙咪嗪(IMI)代谢中的作用。将这些单克隆抗体组合添加到六个HLM样本中。以DZ为底物,通过添加针对3A4/5的单克隆抗体,在所有六个样本中超过80%的替马西泮(TMZ)形成受到抑制,表明3A4/5对TMZ形成的贡献为80%。去甲地西泮的形成受到针对2B6(6 - 23%)、2C亚家族(12 - 61%)和3A4/5(14 - 45%)的单克隆抗体的抑制。针对1A1、1A2、2A6、2D6和2E1的单克隆抗体未抑制TMZ或去甲地西泮的形成;这表明它们不参与DZ的代谢。在六个HLM样本中,单克隆抗体确定的P - 450对7 - EC O - 去乙基化的贡献,2E1为17%至60%,2A6为15%至46%,1A2为5%至22%,反映了每种P - 450在该活性中的作用和变化。针对1A1、2C亚家族、2D6和3A4/5的单克隆抗体不影响HLM样本中7 - EC的代谢。IMI主要通过表达的2C19和2D6代谢为2 - 羟基丙咪嗪,通过表达的1A2、2C18、2C19和2D6代谢为去甲丙咪嗪(DIM)。表达的1A1、2C9和3A4对DIM的形成显示出较低的活性。在六个HLM样本中,五个显示出IMI羟基化活性(0.35 - 2.6 nmol/分钟/ nmol P - 450),而一个(HL43)缺乏羟基化活性。所有六个HLM样本均显示出N - 去乙基化活性(0.74 - 1.4 nmol/分钟/ nmol P - 450)。单克隆抗体确定的2D6和2C19对2 - 羟基丙咪嗪形成的贡献分别为47%至90%和0至49%,而HL43未显示出2 - 羟基化。参与DIM形成的P - 450的作用在2C19(13 - 50%)、1A2(23 - 41%)和3A4(8 - 26%)中有所不同。这些研究证明了一种用于确定单个P - 450的定量代谢作用及其在含有多种P - 450的组织中的个体间变异性的系统。使用抑制性单克隆抗体的该系统简单、精确,并且适用于任何P - 450介导的催化活性,包括对药物、致癌物、诱变剂、有毒化学物质和内源性物质的催化活性。

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