2023 FDA Science Forum
Determination of drugs with poor solubility in hERG external solution by LC-MS/MS to support hERG Potency Assessment
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Contributing OfficeCenter for Drug Evaluation and Research
Abstract
ICH S7B recommends assessing hERG channel block using patch clamp method to understand the risk of clinical QTC prolongation and the rare but potentially fatal ventricular tachyarrhythmia, Torsade de Pointes. Because drug concentrations exposed to the recorded cells can differ from nominal due to non-specific binding, instability, and experimenter-induced errors, ICH S7B and ICH E14/S7B Q&As recommend verifying drug concentrations exposed to the recorded cells to improve the accuracy of the hERG block potency assessment.
An additional challenge is quantifying concentrations of drugs that are poorly soluble in the solutions used to assess hERG block: samples may contain solutes and insoluble drug particles that are solubilized during sample processing resulting in inaccurate measurements of cell exposure. Lopinavir and ritonavir (LOP, RIT) are poorly soluble in water. Their solubilities in hERG solution are unknown but expected to be limited. To prepare and collect samples that reflect just free molecules thus required special solution handling procedure. Drug solutions were prepared by prolonged stirring for 1.0 hour followed by filtration to remove precipitates. This was verified using light scattering method. Sensitive LC-MS/MS methods for LOP and RIT have been developed and validated over a concentration range of 10-500 nM, target analytes and stable labeled internal standards were isolated on a Phenomenex Kinetex XB-C18; 2.1x50 mm; 1.7 µm column, using 10 mM Ammonium Format with 0.1% FA in water as mobile phase A, and 0.11% FA in acetonitrile aas mobile phase B. Analytes were stable for 3 freeze/thaw cycles, 6 hours at RT, 99 days at -80 ◦C in low-binding tubes. The developed methods were used to measure concentrations of LOP and RIT exposed to the recorded cells during real patch clamp experiments to calculate drug potencies; IC50 was found to be 5.1 µM and 12.6 µM, for LOP and RIT, respectively. The established protocol can be generalized to drugs with solubility issues and provide an example for conducting hERG assays to ensure that concentrations measured from drug solution samples reflect solubilized molecules that can interact with hERG channels.
