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Background Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of SK-channels that is currently undergoing clinical trials for treatment of AF. Here we present preclinical data from conscious pigs with persistent AF treated with AP30663. Purpose To examine the pharmakokintetics (PK) of AP30663 in anaesthetized pigs and to test whether AP30663 could cardiovert AF that was resistant to treatment by 4 mg/kg vernakalant in pigs. Methods A total of 12 Danish landrace pigs (gilts) were used for the experiments (2 for PK and 10 for cardioversion of AF). Ten conscious pigs with implanted neurostimulators were tachypaced in the right atrium for 17±5 days until persistent AF that did not respond to treatment with 4 mg/kg vernakalant was obtained. After 3±2 days of vernakalant-resistant AF, the pigs received an infusion of 20 mg/kg AP30663 over 60 minutes. During the infusion the pigs remained conscious and the ECG was monitored. If AF reverted within this period, burst pacing with 50 Hz was applied thrice. Cardioversion was considered successful if sustained AF was reverted. Protection against reinduction of AF was considered successful if no episodes of AF lasting for more than 10 minutes could be reinduced by burst pacing. Results Six out of ten pigs with vernakalant-resistant AF cardioverted during infusion of 20 mg/kg AP30663 over 60 minutes. Four out of six pigs were protected against re-induction of AF by burst pacing. The average time to cardioversion was 29±18 minutes corresponding to calculated free plasma concentrations of 1.0–1.4 μM AP30663. In all the conscious pigs AP30663 was well tolerated with no adverse events. The maximal plasma concentration (Cmax) of AP30663 observed at the end of infusion was 4532±844 ng/ml corresponding to an unbound concentration of 1.54±0.29 μM. The plasma concentrations during infusion were described well by first-order kinetics with a half-time of 5.2 minutes, whereas the plasma concentrations after infusion followed Michaeilis-Menten kinetics with a fast half-life of 6.8 minutes and a slow half-life of 93.7 minutes. Thus, it seems that there is a fast distribution to other tissues and an elimination half-life of approximately 90 minutes. The plasma concentration during infusion reached 90% of the steady state concentration after 17 minutes. Conclusion In an advanced pig model of persistent AF where clinically relevant doses of vernakalant could no longer convert the AF to SR, AP30663 able to convert the pigs to sinus rhythm and protect against reinduction of AF. No signs of adverse events were observed during or after infusion of AP30663. The SK channel inhibitor AP30663 had a fast distribution and an elimination half-life of approximately 90 minutes. The results support the development of AP30663 for treatment of AF in man.

Background Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). AP30663 is a small molecule inhibitor of SK-channels that is effective in converting vernakalant-resistant AF in tachy-paced pigs. Detailed understanding of the molecular mechanism of AP30663 is important for the development of SK channel inhibition for use in man. Purpose To establish the electrophysiological profile, mechanism of action and efficacy in prolonging atrial refractoriness ex vivo of AP30663. Methods AP30663 potency and mechanism of action were established by whole cell and inside-out patch clamp recordings of expressed SK channels. The ion channel selectivity profile of AP30663 was investigated on heterologous expressed channels. Effects of AP30663 or vehicle (DMSO) on atrial refractoriness (AERP) and ventricular repolarization (QTcB) were investigated on isolated perfused guinea pig hearts. Results AP30663 was found to be a selective negative allosteric modulator of SK channels (IC50=0.77±x0.13 μM) with no or minor effects on a panel of other cardiac ion channels, including hERG/KV11.1, (IKr), KV7.1/KCNE1 (IKs), KV4.3/KChiP2 (Ito), Kir2.1 (IK1), Kir3.1/Kir3.4 (IKACh), KV1.5 (IKur), NaV1.5 (INa) and CaV1.2 (ICa). AP30663 inhibited the SK-channel by right-shifting the calcium activation curve of the SK-channel (the EC50 of Ca2+ increased from 0.43±0.02 μM (control, n=6) to 1.37±0.05 μM (in the presence of 7μM AP30663, n=6). In isolated guinea pig hearts, administration of vehicle had no effect on AERP or QTcB. AP30663 significantly prolonged the AERP in 3 μM (to 131±6% of baseline) and 10 μM (to 165±3% of baseline) without any effects on the QTcB. Conclusion AP30663 is a selective negative allosteric modulator of SK channels, acting by means of shifting the calcium dependence of SK-channel activation. AP30663 prolonged atrial refractoriness without affecting the QT-interval in isolated perfused heart preparations. These properties support continued development of AP30663 for treatment of AF in man. Acknowledgement/Funding Innovation Fund Denmark, Wellcome Trust

Background Hypokalemia is commonly encountered in the clinic. Hypokalemia reduces the cardiac repolarization reserve and causes increases in intracellular calcium. This prolongs the QT-interval and increases the risk of ventricular arrhythmia; a risk that can be further exacerbated by concomitant administration of classical class 3 anti-arrhythmic agents. Small conductance Ca2+-activated K+-channels (SK-channels) are a promising new atrial selective target for treatment of atrial fibrillation (AF). Under physiological conditions SK channels play an insignificant role in ventricular repolarization. However, this might change under hypokalemia because of concomitant increases in intracellur calcium. Purpose To study the effects of SK channel inhibition with the tool compound AP14145 or ICA under hypokalemic conditions as compared to the class 3 anti-arrhythmic agent dofetilide and time matched controls (TMC). Methods Guinea pig hearts were isolated and retrogradely perfused with normokalemic (4.5 mM K+) Krebs-Henseleit solution, followed by perfusion with drug or vehicle control. The perfusion was then changed to hypokalemic solution (2.5 mM K+) in presence of drug for 20 min, followed by 20 min perfusion with normokalemic solution in presence of drug. A total of 24 animals were included in the study and randomly assigned to 4 groups: ICA, AP14145, dofetilide or TMC. QT-interval, ventricular effective refractory period, extra systoles and incidence of ventricular tachycardia (VT) or fibrillation (VF) were recorded for each perfusion period. Results Hypokalemia caused a small increase in QT-interval. Application of SK channel inhibitors did not cause further changes, whereas dofetilide prolonged QT compared to hypokalemia alone. During hypokalemia 3 out of 6 hearts in the TMC group developed VF (one spontaneously, two following S1S2 stimulation) whereas 4 out of 6 hearts developed VF in the dofetilide group (one spontaneously, three following S1S2 stimulation). In comparison only 1 heart out of 6 developed VF when treated with the SK channel inhibitor ICA (spontaneously) or AP14145 (following S1S2 stimulation). Conclusion Hypokalemia was associated with an increased risk of VF, an effect that was exaggerated by co-administration of dofetilide. In comparison, the structurally and functionally different SK channel inhibitors, ICA and AP14145, protected the heart from hypokalemia induced VF. These results with the tool compound AP14145 support that SK inhibition may be associated with a better safety and tolerability profile than dofetilide. Acknowledgement/Funding Innovation Fund Denmark

Background and purpose Prolongation of cardiac action potentials is considered antiarrhythmic in the atria but can be proarrhytmic in ventricles if the current carried by Kv11.1-channels (IKr) is inhibited. The current mediated by KCa2-channels, IKCa, is considered a promising new target for treatment of atrial fibrillation. Selective inhibitors of IKr— (dofetilide) and I-KCa (AP14145) were used to compare the effects on ventricular and atrial repolarisation. Ondansetron which has been reported to be a potent blocker of both IKr and IKCa was included to examine its potential atrial antiarrhythmic properties. Methods The expression of KCa2- and Kv11.1-channels in the guinea pig heart was investigated using qPCR. Whole-cell patch clamp technique was used to investigate the effects of dofetilide, AP14145, and ondansetron on IKCa and/or IKr. The effect of dofetilide, AP14145, and ondansetron on atrial and ventricular repolarisation was investigated in isolated hearts. A novel atrial paced in vivo guinea pig model was further validated using AP14145 and dofetilide. Results AP14145 increased AERP (29 ms ex vivo and 38 ms in vivo) without prolonging QTcB both ex vivo and in vivo. In contrast, dofetilide increased QTcB (41 ms) and, to a lesser extent, AERP (16 ms) in isolated hearts and prolonged QTcB (61ms) with no effects on AERP in the in vivo guinea pig model. Ondansetron did not inhibit IKCa, but did inhibit IKr in vitro. Ondansetron prolonged ventricular (25 ms), but not atrial repolarisation ex vivo. Conclusion IKCa inhibition by AP14145 selectively increased atrial repolarisation whereas IKr inhibition by dofetilide and ondansetron increases ventricular repolarisation to a larger extent than atrial repolarisation. Data support that IKCa inhibition may be of value in treating atrial fibrillation without causing adverse effects in the ventricles. Acknowledgement/Funding Innovation Fund Denmark and Wellcome Trust