The potential for CYP2D6 inhibition screening using a novel scintillation proximity assay-based approach

High throughput inhibition screens for human cytochrome P450s (CYPs) are be ing used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enab led the development of a homogeneous high throughput assay for cytochrome P 450 2D6 (CYP2D6) inhibition screen using [O-methyl-C-14]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [C-14]HCHO, on SPA beads. Enzyme kinetics parameters V-max and app arent K-m, determined using pooled human liver microsomes and microsomes fr om baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 re ductase, were 245 pmol [C-14]HCHO/min/mg protein and 11 muM, and 27 pmol [C -14]HCHO/min/pmol and 1.6 muM, respectively. In incubations containing eith er pooled microsomes or recombinant CYP2D6, [C-14]dextromethorphan O-demeth ylase activity was inhibited in the presence of quinidine (IC50 = 1.0 muM a nd 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC50 > 25 muM). In agreement, a selective CY P2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [ C-14]dextromethorphan O-demethylase activity in human liver microsomes, whe reas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibi tory effect. SPA-based [C-14]dextromethorphan O-demethylase activity was al so shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase meas ured by high-performance liquid chromatography in a bank of human liver mic rosomes (N = 15 different organ donors). In a series of known CYP2D6 inhibi tors/substrates, the SPA-based assay resolved potent inhibitors (IC50 <2 <m u>M) from weak inhibitors (IC50 greater than or equal to 20 muM). It is con cluded that the SPA-based assay described herein is suitable for CYP2D6 inh ibition screening using either native human liver microsomes or cDNA-expres sed CYP2D6.