The Potential for CYP2D6 Inhibition Screening Using a Novel Scintillation Proximity Assay-Based Approach

Abstract

High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.

Current methods of cytochrome P450 analysis

“…Данные методы основаны на использовании субстратов, меченных 3 Н или 14 С, которые селективно окисляются исследуемыми изоформами P450 [65][66][67]. После разделения пробы методом ВЭЖХ концентрацию метаболита измеряется с использованием радиохимического детектора.…”

“…Существуют версии методик, которые не требуют хроматографического разделения и реализованы в виде содержащих сцинтиллянт шариков, импрегнированных полиэтиленимином, которые связывают 14 С-формальдегид. Образование 14 С-формальдегида приводит к возбуждению сцинтиллянта, что вызывает свечение [67]. Данные методы предложены авторами для оценки степени ингибирования цитохромов Р450 новыми лекарственными препаратами, их преимуществом является использование любых субстратов, включая эндогенные соединения для оценки активности.…”

A High-Throughput Method for Enzyme Kinetic Studies

“…These values are in general agreement with the values reported in the literature. [7][8][9] These examples demonstrate the utility of the system for detailed kinetic studies, for example, estimation of inhibition constants, or to screen a large number of compounds by a highthroughput method made possible by the feasibility for frequent sampling required for kinetic work. One of the major features that confer flexibility to the system is the ability to have compounds and reagents in a well format rather than in reservoirs.…”