Time for a Fully Integrated Nonclinical-Clinical Risk Assessment to Streamline QT Prolongation Liability Determinations: A Pharma Industry Perspective.

Defining an appropriate and efficient assessment of drug-induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc-prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14-based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B-based "double-negative" nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high-dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double-negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Rapid cooling of the heart with total liquid ventilation prevents transmural myocardial infarction following prolonged ischemia in rabbits.

STUDY AIM: Total liquid ventilation (TLV) with cooled perfluorocarbons has been demonstrated to induce an ultrafast cardioprotective cooling in rabbits. However, it remains unknown whether this technically challenging strategy would be actually more potent than a conventional external cooling after a prolonged ischemia inducing transmural myocardial infarction. METHODS: Anesthetized rabbits were randomly submitted to 60min of coronary artery occlusion (CAO) under normothermic conditions (Control group, n=7) or with cooling started at the 5th min of CAO (target left atrial temperature: 32 degrees C). Cooling procedures were either external cooling using cold blankets (EC group, n=7) or ultrafast cooling initiated by 20min of TLV (TLV group, n=6). An additional group underwent a similar ultrafast cooling protocol started at the 20th min of CAO (TLV(delayed) group, n=6). After reperfusion, all hypothermic animals were rewarmed and infarct size was assessed after 4h. RESULTS: In the EC group, the target temperature was reached only at 60min of CAO whereas this time-interval was dramatically reduced to 15 and 25min of CAO in TLV and TLV(delayed), respectively. Infarct sizes were significantly reduced in TLV and TLV(delayed) but not in EC groups as compared to Control (45+/-18%, 58+/-5%, 78+/-10% and 82+/-7% of the risk zone, respectively). Similar significant differences were observed for the sizes of the no-reflow zones (15+/-9%, 23+/-8%, 49+/-11% and 58+/-13% of the risk zone, respectively). CONCLUSION: Cooling induced by TLV afforded a potent cardioprotection and prevented transmural infarction following prolonged and severe ischemia, even when started later than a surface cooling in rabbits.

Chronic heart rate reduction with ivabradine improves systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term increase in FKBP12/12.6 expression.

AIMS: To investigate the adaptations of left ventricular function and calcium handling to chronic heart rate reduction with ivabradine in the reperfused heart. METHODS AND RESULTS: Rabbits underwent 20 min coronary artery occlusion followed by 3 weeks of reperfusion. Throughout reperfusion, rabbits received ivabradine (10 mg/kg/day) or vehicle (control). Ivabradine reduced heart rate by about 20% and improved both ejection fraction (+35%) and systolic displacement (+26%) after 3 weeks of treatment. Interestingly, this was associated with a two-fold increase expression of FKBP12/12.6. There was no difference in the expressions of phospholamban, SERCA2a, calsequestrin, ryanodine, phospho-ryanodine, and Na(2+)/Ca(2+) exchanger in the two groups. Infarct scar and vascular density were similar in both groups. Administration of a single intravenous bolus of ivabradine (1 mg/kg) in control rabbits at 3 weeks of reperfusion also significantly improved acutely ejection fraction and systolic displacement. CONCLUSION: Chronic heart rate reduction protects the myocardium against ventricular dysfunction induced by myocardial ischaemia followed by 3 weeks of reperfusion. Beyond pure heart rate reduction, ivabradine improves global and regional systolic function of the reperfused heart through a dual mechanism involving a direct mechanical effect and a long-term adaptation in calcium handling, as supported by the increase in FKBP12/12.6 expression.

Relation of the ischaemic substrate to left ventricular remodelling by cardiac magnetic resonance at 1.5 T in rabbits.

OBJECTIVES: Contrast-enhanced cardiac magnetic resonance (CMR) for infarct sizing has been validated in large animals, but studies and follow-up are restricted. We sought to (1) validate CMR for assessment of myocardial area at risk (MAR) and infarct size (IS) in a rabbit model of reperfused myocardial infarction (MI); (2) analyse the relation between ischaemic substrates and subsequent left ventricular (LV) remodelling. METHODS: Experimental reperfused acute MI was induced in 16 rabbits. Ten animals underwent cross-registered cine and contrast-enhanced CMR and histopathology at day 3 for assessment of MAR and IS (group 1). The remaining six rabbits underwent serial CMR for the study of LV remodelling (group 2). RESULTS: In group 1, mean IS was 12.7 +/- 6.4% and 12.7 +/- 6.9% of total LV myocardial mass on CMR (late-enhancement technique) and histopathology (P = 0.52; r = 0.93). No significant difference occurred between CMR and histopathology for the calculation of MAR and IS/MAR ratio (P = 0.18 and P = 0.17), whereas correlations were strong (r = 0.92 and r = 0.95). In group 2, mean LV end-diastolic, end-systolic volumes and LV mass were significantly increased at 3 weeks compared with measurements at day 3 (P < 0.01). Significant correlations between initial IS and the increase in LV end-diastolic volume (r = 0.66) and the increase in LV mass (r = 0.48) were observed, as well as correlations between initial MAR and the increase in LV end-diastolic volume (r = 0.70) and the increase in LV mass (r = 0.37). CONCLUSIONS: Comprehensive CMR provides accurate assessment of IS and MAR in reperfused rabbit MI. Infarct size is closely related to LV remodelling. Through the infarct size/MAR ratio, this approach has great potential for assessing interventions aimed at cardioprotection.

[Heart rate reduction: beneficial effects in heart failure and post-infarcted myocardium]. / La réduction de la fréquence cardiaque: intérêt dans l'insuffisance cardiaque et le post infarctus.

Heart rate (HR) is an independent predicting factor of cardiovascular events in patients with advanced heart failure. Clinical trials conducted with beta-blockers have demonstrated that reduction of HR is a fundamental mechanism to explain the reduction of morbidity-mortality in such a disease. Trials conducted with ivabradine, the first selective inhibitor of the I(f) current in cardiac pacemaker cells, have also clearly confirmed that reduction of HR lead to powerful and sustained favourable effects in patients with cardiac failure and post-infarcted myocardial diseases. These effects are not only due to the reduction of the risk factors associated with myocardial ischemia but also to the reduction of cardiac and perivascular fibrosis associated with the remodelling process at the ventricular level. Although the mechanisms associated with reduction of HR following administration of beta-blockers and ivabradine are to a large extent similar, others like their effects on post-systolic contraction are totally opposed. So, new clinical trials are necessary to know whether it is possible to substitute these drugs or conversely to associate them for an optimal therapeutic strategy in patients with advanced heart failure and post-infarcted myocardium.

The ceiling effect of pharmacological postconditioning with the phytoestrogen genistein is reversed by the GSK3beta inhibitor SB 216763 [3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] through mitochondrial ATP-dependent potassium channel opening.

In the present study, we investigated the efficacy of pharmacological postconditioning induced by 17beta-estradiol and the phytoestrogen, genistein, against myocardial infarction induced by increasing durations of coronary artery occlusion (CAO). Anesthetized rabbits underwent either 20-min (protocol A) or 30-min (protocol B) CAO, followed by 4 h of coronary artery reperfusion (CAR). Before CAR, they randomly received an intravenous injection of either vehicle (control), 100 or 1000 microg/kg genistein (Geni(100) and Geni(1000), respectively), or 100 microg/kg 17beta-estradiol (17beta-E(100)). In protocol A, infarct size was significantly reduced in Geni(100) (n = 6), Geni(1000) (n = 6), and 17beta-E(100) (n = 6) versus control (n = 9) (6 +/- 2, 15 +/- 4, and 11 +/- 3 versus 35 +/- 5%, respectively). In protocol B, none of these drugs reduced infarct size versus control. Western blots demonstrated an increase of Akt phosphorylation in the Geni(100) and 17beta-E(100) hearts submitted to 20-min CAO but not to 30-min CAO. The selective GSK3beta inhibitor SB 216763 (0.2 mg/kg) [3-(2,4)-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] did not exhibit cardioprotection at this dose, but its administration restored the cardioprotective effect of genistein and 17beta-estradiol with 30-min CAO. Administration of 5-hydroxydecanoate (5 mg/kg) abolished the cardioprotective effects of Geni(100) and 17beta-E(100) alone with 20-min CAO and also those observed when combined to SB 216763 with 30-min CAO. Thus, pharmacological postconditioning with genistein and 17beta-estradiol is limited by a "ceiling effect of protection" along with a loss of Akt phosphorylation. However, this ceiling effect is reversed by concomitant inhibition of GSK3beta by SB 216763 through opening of mitochondrial ATP-dependent potassium channels.

Rapid cooling preserves the ischaemic myocardium against mitochondrial damage and left ventricular dysfunction.

AIMS: We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion. METHODS AND RESULTS: Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV(15') groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32-34 degrees C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (15.1 +/- 3.3%, 16.4 +/- 2.3%, 1.8 +/- 0.6%, and 1.1 +/- 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (4 +/- 1%, 11 +/- 5%, 39 +/- 2%, and 42 +/- 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 +/- 12 vs. 86 +/- 12 microM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control. CONCLUSION: Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.

Making the heart resistant to infarction: how can we further decrease infarct size?

Acute myocardial infarction (AMI) following coronary artery occlusion is a common cause of mortality and morbidity world-wide. Patients currently receive reperfusion therapy as the only anti-infarct intervention. A number of agents have been evaluated to further improve myocardial salvage, but until recently, none has demonstrated clear efficacy in clinical trials. A new target of cardioprotection, the Reperfusion Injury Salvage Kinase (RISK) pathway, has been proposed. These kinases are involved in mediating the cardioprotection of myocardial preconditioning and postconditioning induced by short non-lethal cycles of ischemia/reperfusion performed before (preconditioning) or just after (postconditioning) a lethal ischemic insult. Many pharmacological interventions are now available that protect the heart by activating the RISK pathway at the time of reperfusion. The present review will examine the efficacy of several strategies that have been proposed to protect the acutely ischemic myocardium including (1) those intended to directly alter adverse reperfusion events (e.g., calcium overload and free radical attack), (2) those based on activation of the RISK pathway including postconditioning, and (3) myocardial cooling.

Differential effects of postconditioning on myocardial stunning and infarction: a study in conscious dogs and anesthetized rabbits.

Postconditioning, i.e., brief intermittent episodes of myocardial ischemia-reperfusion performed at the onset of reperfusion, reduces infarct size after prolonged ischemia. Our goal was to determine whether postconditioning is protective against myocardial stunning. Accordingly, conscious chronically instrumented dogs (sonomicrometry, coronary balloon occluder) were subjected to a control sequence (10 min coronary artery occlusion, CAO, followed by coronary artery reperfusion, CAR) and a week apart to postconditioning with four cycles of brief CAR and CAO performed at completion of the 10 min CAO. Three postconditioning protocols were investigated, i.e., 15 s CAR/15 s CAO (n=5), 30 s CAR/30 s CAO (n=7), and 1 min CAR/1 min CAO (n=6). Left ventricular wall thickening was abolished during CAO and similarly reduced during subsequent stunning in control and postconditioning sequences (e.g., at 1 h CAR, 33+/-4 vs. 34+/-4%, 30+/-4 vs. 30+/-4%, and 33+/-4 vs. 32+/-4% for 15 s postconditioning, 30 s postconditioning, and 1 min postconditioning vs. corresponding control, respectively). We confirmed this result in anesthetized rabbits by demonstrating that shortening of left ventricular segment length was similarly depressed after 10 min CAO in control and postconditioning sequences (4 cycles of 30 s CAR/30 s CAO). In additional rabbits, the same postconditioning protocol significantly reduced infarct size after 30 min CAO and 3 h CAR (39+/-7%, n=6 vs. 56+/-4%, n=7 of the area at risk in postconditioning vs. control, respectively). Thus, contrasting to its beneficial effects on myocardial infarction, postconditioning does not protect against myocardial stunning in dogs and rabbits. Conversely, additional episodes of ischemia-reperfusion with postconditioning do not worsen myocardial stunning.