ACA-24-3995.Highlighted: Determination of Lopinavir and Ritonavir in hERG solution to Support In Vitro hERG Block Potency Assessment Using LC-MS/MS: The Challenge of Poor Drug Solubility

Abstract

Background

ICH-S7B recommends assessing hERG channel block using patch clamp method to understand the risk of clinical QT_C prolongation and the associated rare but potentially fatal ventricular tachyarrhythmia, Torsade de Pointes. Drug concentrations exposed to the cells during hERG assays may differ from the intended concentrations due to either human errors or drug properties leading to drug loss. Concentration verification is challenging for drugs with limited solubility, samples may contain solutes and insoluble particles that may solubilize during sample analysis and resulting in inaccurate measurements of cell exposure. Lopinavir and ritonavir (LOP, RIT) are poorly soluble in water and their solubilities in hERG recording solution are unknown but expected to be limited. This study describes a procedure to prepare drug solutions for hERG assays, sample collection, and analysis.

Results

Drug solutions were prepared by prolonged stirring followed by filtration to remove insoluble precipitates, as verified using the light scattering method. LC-MS/MS methods for determination of LOP and RIT in the hERG recording solution were fully validated over a concentration range of 10-500 nM. Analytes were stable in hERG solution for 3 freeze/thaw cycles, 6 hours at room temperature (RT), and 99 days at -80 °C in low-binding tubes. The developed methods were used to assess cell exposure and hERG block potency, the IC₅₀ (half-maximal inhibitory concentration) value was 5.1 μM for LOP and 14.3 μM for RIT.

Significance

The established protocol can be generalizable to other drugs with limited solubility for hERG assays and other in vitro assays to allow accurate assessment of the concentration-response relationship.