Project stages

Stage 1/2022 (Reporting period: 01.06.2022 – 31.12.2022). Proarrhythmogenic risk testing and analysis of BBB transport mechanisms of cenobamate (CBM) – part I

Summary: In Stage 1/2022 of the PCE 39/2022 project, the in vitro electrophysiology and fluorescent immunolabeling protocols were standardized. The effects of cenobamate (CBM) at low physiological concentrations (10 and 20 μM) on cardiac ion channels and on the blood-brain barrier were analyzed. The proarrhythmogenic effect of CBM was tested by voltage-clamp mode patch-clamp recordings in the whole-cell configuration on the HEK293T cell line transfected with hERG1 human cardiac ion channels. Preliminary results indicated that 20 μM CBM does not inhibit hERG1, but alters hERG1 channel activation and inactivation kinetics. The effect of 10 μM CBM on human cerebral microvasculature endothelial cells HBEC5i was also tested by analyzing the expression changes of tight junction proteins and efflux pumps by fluorescent immunolabeling followed by confocal microscopy analysis. Preliminary results indicated a decrease in ZO-1 and claudin-5 expression and an increase in P-gp expression after exposure of endothelial cells to cenobamate, 10 μM for 24 hours.

Stage 2/2023 (Reporting period: 01.01.2023 – 31.12.2023). Proarrhythmogenic risk testing and analysis of BBB transport mechanisms of CBM (part II) and FFA

Summary: Cenobamate applied at concentrations in the clinically relevant range (mean Cmax values ​​of 45.5 µg/ml=169.98 µM were obtained in human clinical trials by administering 400 mg/day p.o. for 14 consecutive days – study YKP3089C018) led to the significant inhibition of cardiac ion channels Nav1.5 and Cav1.2, which may explain the shortening effects dose-dependent QTc interval. Computer simulations with an O’Hara-Rudy 2011 model supplemented with pharmacological data, particularly related to the state-dependent blockade of Nav1.5, reproduced the effect of dose-dependent shortening of the duration of action potentials, with relevant effects also appearing at 0.5 x Max. These blocking effects, especially on Nav1.5, although generally considered to exert antiarrhythmic effects by decreasing the velocity of propagation of depolarization in the myocardium, may trigger reentrant arrhythmias in pathological conditions such as myocardial ischemia or other structural heart diseases. Both drugs permeabilize the endothelial monolayer, and decrease the expression level of ZO-1, claudin-5, and P-gp expression, which may contribute to the establishment of drug resistance at the BBB level. Considering the effects of the two drugs on human cardiac ion channels and endothelial cells in the human microvasculature, the guidelines for clinical use should be revised.