measurement-heart-contractility-isolated-adult-human-primary

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...

AnaBios Corporation

Biological differences in sensory processing between human and model organisms may present significant obstacles to translational approaches in treating chronic pain. To better understand the physiology of human sensory neurons, we performed whole-cell patch-clamp recordings from 141 human dorsal root ganglion (hDRG) neurons from 5 young adult donors without chronic pain. Nearly all small-diameter hDRG neurons (<50 μm) displayed an inflection on the descending slope of the action potential, a defining feature of rodent nociceptive neurons. A high proportion of hDRG neurons were responsive to the algogens allyl isothiocyanate (AITC) and ATP, as well as the pruritogens histamine and chloroquine. We show that a subset of hDRG neurons responded to the inflammatory compounds bradykinin and prostaglandin E2 with action potential discharge and show evidence of sensitization including lower rheobase. Compared to electrically evoked action potentials, chemically induced action potentials were triggered from less depolarized thresholds and showed distinct afterhyperpolarization kinetics. These data indicate that most small/medium hDRG neurons can be classified as nociceptors, that they respond directly to compounds that produce pain and itch, and that they can be activated and sensitized by inflammatory mediators. The use of hDRG neurons as preclinical vehicles for target validation is discussed.

Human sensory neurons: Membrane properties and sensitization by inflammatory mediators.

While current S7B/E14 guidelines have succeeded in protecting patients from QT-prolonging drugs, the absence of a predictive paradigm identifying pro-arrhythmic risks has limited the development of valuable drug programs. We investigated if a human ex-vivo action potential (AP)-based model could provide a more predictive approach for assessing pro-arrhythmic risk in man. Human ventricular trabeculae from ethically consented organ donors were used to evaluate the effects of dofetilide, d,l-sotalol, quinidine, paracetamol and verapamil on AP duration (APD) and recognized pro-arrhythmia predictors (short-term variability of APD at 90% repolarization (STV(APD90)), triangulation (ADP90-APD30) and incidence of early afterdepolarizations at 1 and 2Hz to quantitatively identify the pro-arrhythmic risk. Each drug was blinded and tested separately with 3 concentrations in triplicate trabeculae from 5 hearts, with one vehicle time control per heart. Electrophysiological stability of the model was not affected by sequential applications of vehicle (0.1% dimethyl sulfoxide). Paracetamol and verapamil did not significantly alter anyone of the AP parameters and were classified as devoid of pro-arrhythmic risk. Dofetilide, d,l-sotalol and quinidine exhibited an increase in the manifestation of pro-arrhythmia markers. The model provided quantitative and actionable activity flags and the relatively low total variability in tissue response allowed for the identification of pro-arrhythmic signals. Power analysis indicated that a total of 6 trabeculae derived from 2 hearts are sufficient to identify drug-induced pro-arrhythmia. Thus, the human ex-vivo AP-based model provides an integrative translational assay assisting in shaping clinical development plans that could be used in conjunction with the new CiPA-proposed approach. 

Human ex-vivo action potential model for pro-arrhythmia risk assessment.

Cardiac safety remains the leading cause of drug development discontinuation. We developed a human cardiomyocyte-based model that has the potential to provide a predictive preclinical approach for simultaneously predicting drug-induced inotropic and pro-arrhythmia risk.  Adult human primary cardiomyocytes from ethically consented organ donors were used to measure contractility transients. We used measures of changes in contractility parameters as markers to infer both drug-induced inotropic effect (sarcomere shortening) and pro-arrhythmia (aftercontraction, AC); contractility escape (CE); time to 90% relaxation (TR90). We addressed the clinical relevance of this approach by evaluating the effects of 23 torsadogenic and 10 non-torsadogenic drugs. Each drug was tested separately at four multiples of the free effective therapeutic plasma concentration (fETPC).  Human cardiomyocyte-based model differentiated between torsadogenic and non-torsadogenic drugs. For example, dofetilide, a torsadogenic drug, caused ACs and increased TR90 starting at 10-fold the fETPC, while CE events were observed at the highest multiple of fETPC (100-fold). Verapamil, a non-torsadogenic drug, did not change TR90 and induced no AC or CE up to the highest multiple of fETPCs tested in this study (222-fold). When drug pro-arrhythmic activity was evaluated at 10-fold of the fETPC, AC parameter had excellent assay sensitivity and specificity values of 96 and 100%, respectively. This high predictivity supports the translational safety potential of this preparation and of the selected marker. The data demonstrate that human cardiomyocytes could also identify drugs associated with inotropic effects. hERG channel blockers, like dofetilide, had no effects on sarcomere shortening, while multi-ion channel blockers, like verapamil, inhibited sarcomere shortening.  Isolated adult human primary cardiomyocytes can simultaneously predict risks associated with inotropic activity and pro-arrhythmia and may enable the generation of reliable and predictive data for assessing human cardiotoxicity at an early stage in drug discovery.

Adult Human Primary Cardiomyocyte-Based Model for the Simultaneous Prediction of Drug-Induced Inotropic and Pro-arrhythmia Risk.

To assess drug-induced pro-arrhythmic risk, especially Torsades de Pointe (TdP), new models have been proposed, such as  modeling of ventricular action potential (AP) and stem cell-derived cardiomyocytes (SC-CMs). Previously we evaluated the electrophysiological profile of 15 reference drugs in hESC-CMs and hiPSC-CMs for their effects on intracellular AP and extracellular field potential, respectively. Our findings indicated that SC-CMs exhibited immature phenotype and had the propensity to generate false positives in predicting TdP risk. To expand our knowledge with mature human cardiac tissues for drug-induced pro-arrhythmic risk assessment, human ventricular trabeculae (hVT) from ethically consented organ donors were used to evaluate the effects of the same 15 drugs (8 torsadogenic, 5 non-torsadogenic, and 2 discovery molecules) on AP parameters at 1 and 2 Hz. Each drug was tested blindly with 4 concentrations in duplicate trabeculae from 2 hearts. To identify the pro-arrhythmic risk of each drug, a pro-arrhythmic score was calculated as the weighted sum of percent drug-induced changes compared to baseline in various AP parameters, including AP duration and recognized pro-arrhythmia predictors such as triangulation, beat-to-beat variability and incidence of early-afterdepolarizations, at each concentration. In addition, to understand the translation of this preclinical hVT AP-based model to clinical studies, a ratio that relates each testing concentration to the human therapeutic unbound Cmax (Cmax) was calculated. At a ratio of 10, for the 8 torsadogenic drugs, 7 were correctly identified by the pro-arrhythmic score; 1 was mislabeled. For the 5 non-torsadogenic drugs, 4 were correctly identified as safe; 1 was mislabeled. Calculation of sensitivity, specificity, positive predictive value, and negative predictive value indicated excellent performance. For example, at a ratio of 10, scores for sensitivity, specificity, positive predictive value and negative predictive values were 0.88, 0.8, 0.88 and 0.8, respectively. Thus, the hVT AP-based model combined with the integrated analysis of pro-arrhythmic score can differentiate between torsadogenic and non-torsadogenic drugs, and has a greater predictive performance when compared to human SC-CM models.

Action Potential Recording and Pro-arrhythmia Risk Analysis in Human Ventricular Trabeculae.

Effects of non-cardiac drugs on cardiac contractility can lead to serious adverse events. Furthermore, programs aimed at treating heart failure have had limited success and this therapeutic area remains a major unmet medical need. The challenges in assessing drug effect on cardiac contractility point to the fundamental translational value of the current preclinical models. Therefore, we sought to develop an adult human primary cardiomyocyte contractility model that has the potential to provide a predictive preclinical approach for simultaneously predicting drug-induced inotropic effect (sarcomere shortening) and generating multi-parameter data to profile different mechanisms of action based on cluster analysis of a set of 12 contractility parameters. We report that 17 positive and 9 negative inotropes covering diverse mechanisms of action exerted concentration-dependent increases and decreases in sarcomere shortening, respectively. Interestingly, the multiparametric readout allowed for the differentiation of inotropes operating via distinct mechanisms. Hierarchical clustering of contractility transient parameters, coupled with principal component analysis, enabled the classification of subsets of both positive as well as negative inotropes, in a mechanism-related mode. Thus, human cardiomyocyte contractility model could accurately facilitate informed mechanistic-based decision making, risk management and discovery of molecules with the most desirable pharmacological profile for the correction of heart failure.

Multiparametric Mechanistic Profiling of Inotropic Drugs in Adult Human Primary Cardiomyocytes.

The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor.

Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity.

Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences.

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AnaBios Corporation

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes