Gastrointestinal-tract models and techniques for use in safety pharmacology

Introduction: The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (I_Kr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [³H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value.Methods: To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [³H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K⁺ channels. The object of these experiments was to (1) compare binding K_i values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K⁺ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing K_i values from binding studies at 5 and 60 mM [K⁺]_o to functional IC₅₀ values for hERG current block using 56 structurally diverse drugs.Results: We found (a) comparable K_i values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K⁺]_o lowered the K_d and increased the B_max for [³H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding K_i values and functional IC₅₀ values for hERG current block.Discussion: In conclusion, increasing K⁺ concentrations results in an increase in both [³H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between K_i values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K⁺]_o within assays. There is good correlation between binding K_i values and functional (whole-cell patch clamp) IC₅₀ values at both 5 and 60 mM K⁺ concentrations (R² values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [³H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K⁺ channel.