Automated high throughput human CYP isoform activity assay using SPE-LC/MS method: application in CYP inhibition evaluation

被引：59

作者：

Yin, H ^{[1
]}

Racha, J ^{[1
]}

Li, SY ^{[1
]}

Olejnik, N ^{[1
]}

Satoh, H ^{[1
]}

Moore, D ^{[1
]}

机构：

[1] Hoffmann La Roche Inc, Drug Metab & Pharmacokinet, Nutley, NJ 07110 USA

来源：

XENOBIOTICA | 2000年 / 30卷 / 02期

关键词：

D O I：

10.1080/004982500237749

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on K-m values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 (and 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC(50)values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.

引用

页码：141 / 154

页数：14

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