Electrophysiological evaluation of novel f-channels blockers.

Purpose - In the sino-atrial node a major role in rhythm generation and regulation is played by f-channels. They mediate a sodium-potassium inward current (If) that is activated upon hyperpolarization and are encoded by Hyperpolarization-activated Cyclic Nucleotide-gated sodium-potassium channel family genes (HCN1-4). At ventricular level If is overexpressed in cardiac diseases, where it may contribute to the increased propensity for arrhythmias. Selective f-channel blockers have a potential therapeutic use as bradycardic and antiarrhythmic agents. Zatebradine and ivabradine act as f-channel blockers but they show no or partial cardiac selectivity, both of them blocking the neuronal HCN isoforms. As a consequence of this, there is an unmet need to develop new blockers selective for the mammalian sino-atrial channel that is largely determined by HCN4 isoform. Methods - On the basis of zatebradine structure different analogues (C1-C5)were synthesized. By using the patch-clamp technique the effect on If was measured on isolated HEK293 cells expressing mHCN1, mHCN2 and hHCN4 and on native guinea-pig and rabbit sino-atrial cells. Results- At 10 μM concentration all compounds reduced maximal If amplitude; however, potencies and selectivity (defined by EC50) differed considerably. Ivabradine, taken as reference compound, showed no isoform selectivity; C1 and C4 were more potent on HCN1 (2.31±0.4 and 0.60±0.07 μM respectively) and C2 was more potent on HCN4 (9.74±7.7μM). C3 was more effective on HCN1 and HCN4isofomsand C5, had low activity on all isoforms. If current-voltage curves revealed that blockade was concentration-dependent, did not reverse upon drug removal and did not change current properties. Finally, data obtained in SAN cells suggest that effects on native If resemble those obtained on HCN4 isoform, in accordance with the hypothesis that HCN4 has a major contribution in SAN cells. Conclusions - Present results indicate that drug interaction with different HCN isoforms has diverse structural requirements. Current investigations are aimed to characterize the pharmacological profile of the new f-channel blocker and improve their isoform selectivity.