Hidden Cardiotoxicity of Rofecoxib Can be Revealed in Experimental Models of Ischemia/Reperfusion

Cardiac adverse effects are among the leading causes of the discontinuation of clinical trials and the withdrawal of drugs from the market. The novel concept of 'hidden cardiotoxicity' is defined as cardiotoxicity of a drug that manifests in the diseased (e.g. ischemic/reperfused), but not in the healthy heart or as a drug-induced deterioration of cardiac stress adaptation (e.g. ischemic conditioning). Here, we aimed to test if the cardiotoxicity of a selective COX-2 inhibitor rofecoxib that was revealed during its clinical use, i.e., increased occurrence of proarrhythmic and thrombotic events, could have been revealed in early phases of drug development by using preclinical models of ischemia/reperfusion (I/R) injury. Rats that were treated with rofecoxib or vehicle for four weeks were subjected to 30 min. coronary artery occlusion and 120 min. reperfusion with or without cardioprotection that is induced by ischemic preconditioning (IPC). Rofecoxib increased overall the arrhythmias including ventricular fibrillation (VF) during I/R. The proarrhythmic effect of rofecoxib during I/R was not observed in the IPC group. Rofecoxib prolonged the action potential duration (APD) in isolated papillary muscles, which was not seen in the simulated IPC group. Interestingly, while showing hidden cardiotoxicity manifested as a proarrhythmic effect during I/R, rofecoxib decreased the infarct size and increased the survival of adult rat cardiac myocytes that were subjected to simulated I/R injury. This is the first demonstration that rofecoxib increased acute mortality due to its proarrhythmic effect via increased APD during I/R. Rofecoxib did not interfere with the cardiprotective effect of IPC; moreover, IPC was able to protect against rofecoxib-induced hidden cardiotoxicity. These results show that cardiac safety testing with simple preclinical models of I/R injury uncovers hidden cardiotoxicity of rofecoxib and might reveal the hidden cardiotoxicity of other drugs

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments

BACKGROUND: In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined. METHODS: Ion currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 mug/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts. RESULTS: ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 muM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 muM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts. CONCLUSIONS: The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in <i>In Vitro</i> and <i>In Vivo</i> Experiments - Fig 12

<p><b>Panel A</b>. Effect of ORM-10962 (0.3 mg/kg) in ischemia-reperfusion induced ventricular arrhythmias during reperfusion after 6 min coronary artery ligation in anesthetized (pentobarbitone, 60 mg/kg <i>i</i>.<i>p</i>.) rats. Time to the development of ventricular arrhythmias was measured on the ECG. The ordinates show the numbering of the animals. There was no significant change in the arrhythmias following administration of ORM-10962. <b>Panel B.</b> Effect of ORM-10962 (1 μM) in global ischemia-reperfusion induced ventricular fibrillation in isolated guinea pig hearts. ORM-10962 did not influence significantly the duration of ventricular fibrillation.</p

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in <i>In Vitro</i> and <i>In Vivo</i> Experiments - Fig 10

<p><b>Panel A.</b> The effect of 1 μM ORM-10962 on the delayed afterdepolarization (DAD) amplitude in dog right ventricular Purkinje fibres. DAD was evoked by a 40 stimulus train with a stimulation cycle length of 400 ms in the presence of 150 nM digoxin. Trace <b>a</b> is a control recording, trace <b>b</b> indicates the induction of DAD by 150 nM digoxin, and trace <b>c</b> demonstrates that 1 μM ORM-10962 almost completely abolished DAD. <b>Panel B.</b> Effect of ORM-10962 (1 μM) on digoxin-induced automaticity in dog right ventricular Purkinje fibres. Trace <b>a</b> is a control recording. Spontaneous activity was recorded after a 40 stimulus train with a stimulation cycle length of 400 ms in the presence of 150 nM digoxin (trace <b>b</b>). Application of 1 μM ORM-10962 in the presence of digoxin abolished the spontaneous activity (trace <b>c</b>).</p

Effect of ORM-10962 (0.3 mg/kg) on ouabain (10 μg/kg/min i.<i>v</i>.) induced arrhythmias in anesthetized (pentobarbitone, 45 mg/kg <i>i</i>.<i>p</i>.) guinea-pigs.

<p><b>Panel A</b>. ORM-10962 was applied as a pre-treatment 10 min before starting permanent ouabain infusion. Mean time to the development of ventricular arrhythmias is indicated on the Figure. <b>Panel B.</b> ORM-10962 was administered after the termination of 16 min ouabain infusion. In both <b>Panels</b> the ordinate indicates the numbering of the animals. There was a considerable antiarrhythmic effect; the duration of arrhythmic periods was shorter under the influence of ORM-10962.</p

Lack of effect of ORM-10962 on the inward rectifier potassium and transient outward potassium currents (I_K1 and I_to) in dog ventricular myocytes.

<p><b>Panels A and B.</b> Original I_K1 (top) and I_to (bottom) current traces (left panels) recorded under control conditions and in the presence of 1 μM ORM-10962, and the corresponding I-V current-voltage relationships (right panels) before and after application of 1 μM ORM-10962, respectively. The insets in the I-V diagrams show the voltage protocol applied during measurements. Values represent means ± SEM, n = 8 and 10, respectively.</p

Effect of ORM-10962 (0.3 mg/kg) on ventricular arrhythmias induced by reperfusion after 6 min coronary artery ligation in anesthetized (60 mg/kg <i>i</i>.<i>p</i>.) rats.

<p>Effect of ORM-10962 (0.3 mg/kg) on ventricular arrhythmias induced by reperfusion after 6 min coronary artery ligation in anesthetized (60 mg/kg <i>i</i>.<i>p</i>.) rats.</p

Suspension of ouabain induced arrhythmias by ORM-10962, administered after the termination of ouabain infusion (10 μg/kg/min for 16 min) in anesthetized guinea pigs.

<p>Suspension of ouabain induced arrhythmias by ORM-10962, administered after the termination of ouabain infusion (10 μg/kg/min for 16 min) in anesthetized guinea pigs.</p