Improving assessment of cardiovascular arrhythmic safety of new pharmacologic agents

Fourteen drugs have been removed from the market worldwide due to an increased risk of torsade de pointes (TdP), a potentially fatal ventricular arrhythmia. Almost all of the removed drugs that have been linked to an increased risk for TdP have been shown to block the human ether-à-go-go-related gene (hERG) potassium channel. In addition, block of the hERG potassium channel results in a prolongation of the duration of ventricular repolarization measured as the QT interval on the electrocardiogram (ECG). Therefore, almost all new drugs must be studied in a thorough QT (TQT) study to determine if they have the potential to prolong the heart rate corrected QT interval (QTc). The TQT study is an expensive study that in addition to including a negative control (placebo), also includes a positive pharmacologic control to ensure assay sensitivity and proper study conduct. Not all drugs that block the hERG potassium channel and prolong the QTc interval have been linked with a risk for TdP likely due to additional inward current block. For example block of the late sodium (amiodarone, ranolazine) or L-type calcium (verapamil). In addition, not every study is able to detect the QTc prolongation associated with the positive pharmacological control. It is unknown which factors have a greater influence on study quality and the ability to demonstrate assay sensitivity. TQT studies from the Food and Drug Administration (FDA) ECGWarehouse were used to investigate factors of assay sensitivity and how they relate to ECG quality metrics, as well as new ECG biomarkers that could complement the QTc interval and increase specificity of the TQT study. In addition, two prospective clinical trials were conducted to evaluate the performance of the new ECG biomarkers. The first clinical trial focuses on comparing selective hERG potassium channel blockers to multichannel blockers. The goal of the second clinical trial is to evaluate if selective late sodium or L-type calcium channel blockers could reduce drug-induced QTc prolongation. Finally, data from the first clinical trial was used to study dynamic ECG biomarkers. The retrospective analysis of TQT studies showed that the most influential factors of assay sensitivity is reader variability and stability of heart rate. The latter being driven in part by study conduct. In addition, the retrospective analysis suggested that by breaking down the QTc interval into QRS, J-Tpeakc and Tpeak-Tend intervals that it is possible to detect the presence of inward current block (late sodium or L-type calcium), that can reduce the risk for TdP. In two prospective clinical studies the proposed ECG biomarkers were shown to be able to detect the presence of inward current block. Moreover, the second clinical trial showed that a selective late sodium current blocker (mexiletine or lidoacaine) shortens dofetilide-induced QTc prolongation. Lastly, using ECG measurements from periods of postural maneuvers and light exercises it was possible to detect the presence of reverse use dependence and increased instability of the QT interval (dynamic ECG biomarkers) associated with hERG potassium channel block. No changes in the dynamic ECG biomarkers was observed with ranolazine, and only small changes was observed with verapamil. Overall, the findings in this thesis show that by ensuring consistently measured QT intervals and maximizing heart rate stability the ability to detect the QTc interval prolongation associated with the positive control is improved. Ensuring consistent QT measurements also results in improved quality of QTc measurements, quantified using QTc quality metrics. In addition, the use of the J-Tpeakc and Tpeak-Tend intervals allows for discrimination between drugs that are selective hERG potassium channel blockers, and are associated with a high risk for TdP, and multichannel blockers with a low risk for TdP

Assessing effect of beat detector on detection dependent signal quality indices

Patient monitoring algorithms which use multimodal physiological waveforms are needed to reduce alarm fatigue by alarming only for physiologic events and not signal artifacts. When combining information from multiple ECG signals, computational approaches that automatically identify artifacts in ECG signals play an important role. Signal quality indices (SQIs) have been derived which can differentiate between ECG signal artifacts and normal QRS morphology. Some of these SQIs are derived using beat detections and might be affected by the beat detector used. Using ECG signals from the PhysioNet/Computing in Cardiology Challenge 2015 training set, we studied the effect of beat detector on previously reported ECG SQIs derived using beat detections. We found that, while being affected by the beat detector, some of these SQIs can predict beat detector failure. Using beat detector specific SQIs can improve the designs of robust monitoring algorithms

ECGlib: Library for Processing Electrocardiograms

Abstract To facilitate evaluation of ECGs in the FDA ECG Warehouse, public databases and clinical trials we have developed a C++ library for processing ECGs (ECGlib). ECGlib has a modular design, and is capable of handling files stored in many different formats, e.g. ISHNE, Physionet and FDA HL7. Moreover, ECGlib provides functions to do standard ECG signal processing, such as noise removal, QRS detection, classification, median beat creation and ECG waveform delineation. The performance of the different components of ECGlib has been evaluated using publicly available databases from Physionet (MIT-BIH and QTDB). The performance of ECGlib processing methodologies is comparable to state-of-the-art methods. We have also developed a MATLAB/Octave interface for ECGlib and are working on an interface for Python, R and Julia. Lastly, ECGlib comes with a set of command line tools that utilize parallel processing to quickly enable researchers to process large databases. We believe frameworks such as the one described can be used to facilitate research of ECG signals and we are working on making the library publicly available under an open source license

Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted

Effects of Salicornia-Based Skin Cream Application on Healthy Humans’ Experimental Model of Pain and Itching

Halophyte plants are salt-tolerant and are acclimated for growth in saline soils such as along coastal areas. Among the halophytes, the Salicornia species have been used as both folk medicine and functional food for many years due to their high levels of bioactive compounds with supposed anti-inflammatory and antioxidative effects. However, the properties of Salicornia bioactive extracts on pain and itching still remain unclear. In this study, 30 healthy volunteers were randomized to treatments with 10% Salicornia-based cream or placebo cream for 24 or 48 h. On day 0, and 24 or 48 h post cream application, cold/heat detection and pain thresholds, mechanical pain thresholds and sensitivity, trans-epidermal water loss, histamine- and cowhage-evoked itch, and micro-vascular reactivity (neurogenic inflammation) were assessed to evaluate the analgesic, anti-pruritogenic and vasomotor effects. Skin permeability was reduced in the Salicornia-treated area for 48 h compared with 24 h application (p-value < 0.05). After 48 h of application, a decrease in mechanical-evoked itching (hyperkinesis) compared with 24 h treatment (p-value < 0.05) and increased warm detection and heat pain thresholds (p-value < 0.05) was found. Histamine-induced neurogenic inflammation showed a significant reduction in the cream-treated areas after 48 h compared with 24 h (p-value < 0.05). The results of this study indicate the overall inhibitory effect of Salicornia on hyperkinesis (mechanically evoked itch), the analgesic effect on thermal sensation, and modulation of the skin barrier architecture. Further studies are needed for the assessment of the long-term effects

Targeting TGF-ß in the Central Nervous System: Assessment of Cynomolgus Monkey—Toxicity and Pharmacokinetics for an LNA-Antisense Oligonucleotide

Increasingly antisense oligonucleotides (ASOs) are developed for potential treatment of CNS disorders, and due to the inability to cross the blood brain barrier, they require direct administration into the cerebrospinal fluid (CSF). In this regard, intrathecal (i.th.) administration in cynomolgus monkeys (Macaca fascicularis) is a well-established approach for preclinical safety studies. Here, we present an innovative preclinical approach that is intended to support rapid entry into clinical development with ASOs targeting the CNS. The preclinical approach comprises one non-GLP study in 26 non-human primates, followed by a pivotal GLP repeated dose toxicity study in the same species. No pivotal rodent studies were conducted, and regulatory guidance to initiate this study was met by in vitro work. The non-GLP study consists of three separate phases: Phase A determines toxicity after i.th. administrations with five escalating dose levels in a single male and female animal, respectively. Dosing is conducted on days 1, 8, 15, 22, and 29 and the experiment is terminated 36 days after start of the study. The second phase (Phase B) investigates pharmacokinetics over a 2- or 4-week period at two dose levels following single administrations in eight (8) animals (4 females, 4 males). Finally, a third phase (Phase C) investigates toxicity and pharmacokinetics after repeated (9×) dosing over a 13-week period at two dose levels in sixteen (8 females, 8 males) animals. In each phase, clinical observations and physical/neurological parameters are investigated directly pre-dose, 4 h and 24 h post-dose, respectively. In all phases, CSF and blood samples are taken pre-dose and after each dosing, for determination of test article concentration, biomarkers of tolerability and biomarkers of pharmacology. In all phases, tissue samples from the liver, kidney, spinal cord, and brain are collected for determination of NVP-13 tissue concentrations. The above concept has successfully supported first-in-human clinical trials. The entire non-GLP program is completed within less than six months and requires fewer animals in comparison to the conduct of three independent studies

Safe and Effective Cynomolgus Monkey GLP—Tox Study with Repetitive Intrathecal Application of a TGFBR2 Targeting LNA-Gapmer Antisense Oligonucleotide as Treatment Candidate for Neurodegenerative Disorders

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFβ system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide “NVP-13”, targeting TGFBR2, effectively reduced its expression and lowered TGFβ signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13