Effects of Cardiotoxins from Naja oxiana Cobra Venom on Rat Heart Muscle and Aorta: A Comparative Study of Toxin-Induced Contraction Mechanisms.

Cardiotoxins (CaTxs) are a group of snake toxins that affect the cardiovascular system (CVS). Two types (S and P) of CaTxs are known, but the exact differences in the effects of these types on CVS have not been thoroughly studied. We investigated cellular mechanisms of action on CVS for Naja oxiana cobra CaTxs CTX-1 (S-type) and CTX-2 (P-type) focusing on the papillary muscle (PM) contractility and contraction of aortic rings (AR) supplemented by pharmacological analysis. It was found that CTX-1 and CTX-2 exerted dose-dependent effects manifested in PM contracture and AR contraction. CTX-2 impaired functions of PM and AR more strongly than CTX-1. Effects of CaTxs on PM were significantly reduced by nifedipine, an L-type Ca2+ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na+/Ca2+ exchange. Furthermore, 2-aminoethoxydiphenyl borate, an inhibitor of store-operated calcium entry, partially restored PM contractility damaged by CaTxs. The CaTx influence on AR contracture was significantly reduced by nifedipine and KB-R7943. The involvement of reverse-mode Na+/Ca2+ exchange in the effect of CaTxs on the rat aorta was shown for the first time. The results obtained indicate that CaTx effects on CVS are mainly associated with disturbance of transporting systems responsible for the Ca2+ influx.

Development of a Next-Generation Biosensing Platform for Simultaneous Detection of Mechano- and Electrophysiology of the Drug-Induced Cardiomyocytes.

Detection of adverse effects of cardiac toxicity at an early stage by in vitro methods is crucial for the preclinical drug screening. Over the years, several kinds of biosensing platforms have been proposed by the scientific society for the detection of cardiac toxicity. However, the proposed tissue platforms have been optimized to measure either mechanophysiology or electrophysiology of the cardiomyocytes but not both. Herein, we demonstrate in detail our successful attempt toward developing a novel "multifunctional microphysiological system" also known as "organs-on-chips" to measure simultaneously the mechanical and electrical characteristics of cardiomyocytes in vitro. The proposed device can rapidly recognize drug-induced cardiovascular toxicity in real time, which is one of the most significant factors for drug discovery and postmarketing surveillance. We confirm that the proposed sensor delivers the direct relationship between the contraction force and cell impedance of cardiomyocytes under the influence of different cardiovascular drugs such as verapamil, astemizole, and lidocaine. The obtained assay results provide a great potential for a deep understanding of the drug effects on the cardiomyocytes in vitro.

Action potential-based MEA platform for in vitro screening of drug-induced cardiotoxicity using human iPSCs and rat neonatal myocytes.

Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10-7 to 2.10-5M) and verapamil (7 concentrations from 10-11 to 10-5M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10-6M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10-6M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10-6M; and (3) suppressing spontaneous activity with EC50 of 3.8·10-6M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10-8M and prolonged the APD with EC50 of 2.5·10-8M. Verapamil reduced rates of fast depolarization and repolarization with IC50s of 1.8 and 2.2·10-7M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety.

Cardiac voltage-gated ion channels in safety pharmacology: Review of the landscape leading to the CiPA initiative.

Voltage gated ion channels are central in defining the fundamental properties of the ventricular cardiac action potential (AP), and are also involved in the development of drug-induced arrhythmias. Many drugs can inhibit cardiac ion currents, including the Na+ current (INa), L-type Ca2+ current (Ica-L), and K+ currents (Ito, IK1, IKs, and IKr), and thereby affect AP properties in a manner that can trigger or sustain cardiac arrhythmias. Since publication of ICH E14 and S7B over a decade ago, there has been a focus on drug effects on QT prolongation clinically, and on the rapidly activating delayed rectifier current (IKr), nonclinically, for evaluation of proarrhythmic risk. This focus on QT interval prolongation and a single ionic current likely impacted negatively some drugs that lack proarrhythmic liability in humans. To rectify this issue, the Comprehensive in vitro proarrhythmia assay (CiPA) initiative has been proposed to integrate drug effects on multiple cardiac ionic currents with in silico modelling of human ventricular action potentials, and in vitro data obtained from human stem cell-derived ventricular cardiomyocytes to estimate proarrhythmic risk of new drugs with improved accuracy. In this review, we present the physiological functions and the molecular basis of major cardiac ion channels that contribute to the ventricle AP, and discuss the CiPA paradigm in drug development.

CSAHi study: Detection of drug-induced ion channel/receptor responses, QT prolongation, and arrhythmia using multi-electrode arrays in combination with human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: The use of multi-electrode arrays (MEA) in combination with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides a promising method to predict comprehensive cardiotoxicity, including drug-induced QT prolongation and arrhythmia. We previously demonstrated that MEA in combination with hiPSC-CMs could provide a generalizable platform by using 7 reference drugs at 10 testing facilities. Using this approach, we evaluated responses to reference drugs that modulate a range of cardiac ion currents and have a range of arrhythmogenic effects. METHODS: We used the MEA system (MED64) and commercially available hiPSC-CMs (iCell cardiomyocytes) to evaluate drug effects on the beat rate, field potential duration (FPD), FPD corrected by Fridericia's formula (FPDc), and the incidence of arrhythmia-like waveforms. RESULTS: This assay detected the repolarization effects of Bay K8644, mibefradil, NS1643, levcromakalim, and ouabain; and the chronotropic effects of isoproterenol, ZD7288, and BaCl2. Chronotropy was also affected by K+ and Ca2+ current modulation. This system detected repolarization delays and the arrhythmogenic effects of quinidine, cisapride, thioridazine, astemizole, bepridil, and pimozide more sensitively than the established guinea pig papillary muscle action potential assay. It also predicted clinical QT prolongation by drugs with multiple ion channel effects (fluoxetine, amiodarone, tolterodine, vanoxerine, alfuzosin, and ranolazine). DISCUSSION: MEA in combination with hiPSC-CMs may provide a powerful method to detect various cardiac electrophysiological effects, QT prolongation, and arrhythmia during drug discovery. However, the data require careful interpretation to predict chronotropic effects and arrhythmogenic effects of candidate drugs with multiple ion channel effects.

Dietary phytoestrogens present in soy dramatically increase cardiotoxicity in male mice receiving a chemotherapeutic tyrosine kinase inhibitor.

Use of soy supplements to inhibit cancer cell growth is increasing among patients due to the perception that phytoestrogens in soy inhibit carcinogenesis via induction of apoptosis. Genistein, the most prevalent phytoestrogen in soy, is a potent endocrine disruptor and tyrosine kinase inhibitor (TKI) that causes apoptosis in many cells types. Chemotherapeutic TKIs limit cancer cell growth via the same mechanisms. However, TKIs such as Sunitinib cause cardiotoxicity in a significant number of patients. Molecular interactions between Sunitinib and dietary TKIs like genistein have not been examined in cardiomyocytes. Significant lethality occurred in mice treated with Sunitinib and fed a phytoestrogen-supplemented diet. Isolated cardiomyocytes co-treated with genistein and Sunitinib exhibited additive inhibition of signaling molecules important for normal cardiac function and increased apoptosis compared with Sunitinib alone. Thus, dietary soy supplementation should be avoided during administration of Sunitinib due to exacerbated cardiotoxicity, despite evidence for positive effects in cancer.

Toward functional screening of cardioactive and cardiotoxic drugs with zebrafish in vivo using pseudodynamic three-dimensional imaging.

Given the high mortality in patients with cardiovascular diseases and the life-threatening consequences of drugs with unforeseen adverse effects on hearts, a critical evaluation of the pharmacological response of cardiovascular function on model animals is important especially in the early stages of drug development. We report a proof-of-principle study to demonstrate the utility of zebrafish as an analytical platform to predict the cardiac response of new drugs or chemicals on human beings. With pseudodynamic 3D imaging, we derive individual parameters that are central to the cardiac function of zebrafish, including the ventricular stroke volume, ejection fraction, cardiac output, heart rate, diastolic filling function, and ventricular mass. We evaluate both inotropic and chronotropic responses of the heart of zebrafish treated with drugs that are commonly prescribed and possess varied known cardiac activities. We reveal deranged cardiac function of a zebrafish model of cardiomyopathy induced with a cardiotoxic drug. The cardiac function of zebrafish exhibits a pharmacological response similar to that of human beings. We compare also cardiac parameters obtained in this work with those derived with conventional 2D approximation and show that the latter tends to overestimate the cardiac parameters and produces results of greater variation. In view of the growing interest of using zebrafish in both fundamental and translational biomedical research, we envisage that our approach should benefit not only contemporary pharmaceutical development but also exploratory research such as gene, stem cell, or regenerative therapies targeting congenital or acquired heart diseases.

Administration of chromium(III) and manganese(II) as a potential protective approach against daunorubicin-induced cardiotoxicity: in vitro and in vivo experimental evidence.

Daunorubicin (DNR) is a widely used antitumor drug, but its application is limited because of its cardiotoxic side effects. The present study was designed to investigate the interaction between DNR and cardiac myosin (CM) in the presence of chromium(III) (Cr(3+)) and manganese(II) (Mn(2+)) using fluorescence spectrometry under simulative physiological conditions with the aim of exploring the influence of metal ion on DNR-CM complex and finding out an aggressive approach to abrogate of DNR-induced cardiotoxicity. In detail, the quenching and binding constant of ternary system, including metal ion, DNR, and CM, were measured and compared with the DNR-CM. The data from in vitro experiments indicate that the presence of Cr(3+) or Mn(2+) distinctly decreased the binding force between DNR and CM, and alleviated the cardiac toxicity caused by DNR. In addition, the variations in mice body weight and myocardial enzyme level were examined by in vivo experiments. Animals receiving Cr(3+) or Mn(2+) supplementation of DNR showed preservation of the normal pattern of the heart, especially 2.0 mg Cr(3+)/kg body wt or 50.0 mg Mn(2+)/kg body wt exhibited an obviously protective effect accompanied with body weight raise when compared with the mice treated with DNR alone, decreased the ratio of heart to body weight (BW) and the ratio of left ventricular mass to BW to the normal levels, and inhibited the leak of myocardial enzyme caused by DNR. As a result, this study suggests that pretreatment of lower dose of Cr(3+) (2 mg/kg wt) and moderate dose of Mn(2+) (50 mg/kg wt) might be useful and play an important role in ameliorating the cardiotoxicity of DNR treatment in cancer patients.

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca(2+) flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration-response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive "cardiosafety" ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development.

Multiparameter in vitro assessment of compound effects on cardiomyocyte physiology using iPSC cells.

A large percentage of drugs fail in clinical studies due to cardiac toxicity; thus, development of sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocytes is extremely important for drug development. Human cardiomyocytes derived from stem cell sources offer more clinically relevant cell-based models than those presently available. Human-induced pluripotent stem cell-derived cardiomyocytes are especially attractive because they express ion channels and demonstrate spontaneous mechanical and electrical activity similar to adult cardiomyocytes. Here we demonstrate techniques for measuring the impact of pharmacologic compounds on the beating rate of cardiomyocytes with ImageXpress Micro and FLIPR Tetra systems. The assays employ calcium-sensitive dyes to monitor changes in Ca(2+) fluxes synchronous with cell beating, which allows monitoring of the beat rate, amplitude, and other parameters. We demonstrate here that the system is able to detect concentration-dependent atypical patterns caused by hERG inhibitors and other ion channel blockers. We also show that both positive and negative chronotropic effects on cardiac rate can be observed and IC(50) values determined. This methodology is well suited for safety testing and can be used to estimate efficacy and dosing of drug candidates prior to clinical studies.

Evaluation of drugs with specific organ toxicities in organ-specific cell lines.

Safety attrition of drugs during preclinical development as well as in late-stage clinical trials continues to be a challenge for the pharmaceutical industry for patient welfare and financial reasons. Hepatic, cardiac, and nephrotoxicity remain the main reasons for compound termination. In recent years, efforts have been made to identify such liabilities earlier in the drug development process, through utilization of in silico and cytotoxicity models. Several publications have aimed to predict specific organ toxicities. For example, two large-scale evaluations of hepatotoxic compounds have been conducted. In contrast, only small cardiotoxic and nephrotoxic compound sets have been evaluated. Here, we investigated the utility of hepatic-, cardiac-, and kidney-derived cell lines to (1) accurately predict cytotoxicity and (2) to accurately predict specific organ toxicities. We tested 273 hepatotoxic, 191 cardiotoxic, and 85 nephrotoxic compounds in HepG2 (hepatocellular carcinoma), H9c2 (embryonic myocardium), and NRK-52E (kidney proximal tubule) cells for their cytotoxicity. We found that the majority of compounds, regardless of their designated organ toxicities, had similar effects in all three cell lines. Only approximately 5% of compounds showed differential toxicity responses in the cell lines with no obvious correlation to the known in vivo organ toxicity. Our results suggest that from a general screening perspective, different cell lines have relatively equal value in assessing general cytotoxicity and that specific organ toxicity cannot be accurately predicted using such a simple approach. Select organ toxicity potentially results from compound accumulation in a particular tissue, cell types within organs, metabolism, and off-target effects. Our analysis, however, demonstrates that the prediction can be improved significantly when human C(max) values are incorporated.

Oxidative stress and myocardial gene alterations associated with Doxorubicin-induced cardiotoxicity in rats persist for 2 months after treatment cessation.

The molecular mechanisms underlying doxorubicin (DOX)-induced cardiomyopathy include alterations in cardiomyocytes' oxidative stress status and in gene expression. Although such alterations have been reported during in vivo DOX treatment of animals, it remains to be clarified whether they persist after treatment cessation. To address this question, rats were injected with either saline (1 ml/kg/day i.p; control) or DOX (1 mg/kg/day i.p.) for 10 days, and 70 days later cardiac functional parameters were evaluated in vivo by left ventricular catheterization. Hearts were also harvested for histological analyses as well as measurements of oxidative stress parameters by various techniques and gene expression by quantitative polymerase chain reaction of markers of cardiac pathological remodeling, namely atrial natriuretic factor, myosin heavy chain ß, vascular endothelial growth factor A (VEGF-A), and sarcoplasmic reticulum Ca(+2) ATPase. Compared with controls, DOX-treated rats displayed marked alterations in most parameters even 2 months after cessation of treatment. These included 1) lower left ventricular contractility (+dP/dt), 2) increased levels of plasma and myocardial oxidative stress markers, namely thiobarbituric acid reactive substances or dihydroethidium fluorescence, and 3) markedly altered transcript levels for all measured markers of cardiac remodeling, except VEGF-A. These changes correlated significantly with +dP/dt values assessed in the two groups of animals. In conclusion, this study demonstrated that as many as 2 months after cessation of DOX treatment cardiac alterations persisted, reflecting increased oxidative stress and pathological remodeling, the latter being linked to the development of contractile dysfunction.

A twenty-eight-day mechanistic time course study in the rhesus monkey with hepatitis C virus protease inhibitor BILN 2061.

BILN 2061 is a potent, reversible inhibitor of hepatitis C virus NS3/NS4A serine protease. Early clinical proof of principle with the drug was offset by the results of subsequent safety studies in Rhesus monkeys revealing cardiotoxicity that featured myocardial vacuolation corresponding to mitochondrial swelling. Here we describe an investigation into the nature, onset, and reversibility of the lesion, and an assessment of potentially predictive biomarkers for the change. Rhesus monkeys were orally administered 1,000 mg/kg/day BILN 2061 and either necropsied after one, three, fourteen, or twenty-eight doses or afforded a ten-week recovery period. The results of electrocardiographic and plasma troponin I and T measurements were unaffected by BILN 2061, but cardiac myocytic vacuolation, correlated with mitochondrial swelling, was observed after three or more doses. Echocardiographic traces obtained after twenty-eight consecutive days of dosing revealed two animals with diminished left ventricular cardiac ejection fraction. One animal was immediately necropsied and exhibited marked cardiotoxicity. The other was afforded a ten-week treatment-free period during which the left ventricular ejection fraction returned to normal. All recovery animal hearts were microscopically and ultrastructurally normal. High-dose BILN 2061 cardiotoxicity in Rhesus monkeys appeared early in the treatment regimen and exhibited reversibility. A reliable biomarker has yet to be identified.

Preclinical evaluation of potential nilotinib cardiotoxicity.

In vitro, concentrations ≥ 10 µM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.27 µM) did not induce overt cardiovascular pathologies or heart failure in vitro or in vivo under study conditions.

hERG in vitro interchange factors--development and verification.

Literature data analysis shows that the hERG interactions experiments carried out in different conditions with use of different in vitro systems for the same substance can result with different IC50 values. One of the initial components of the proposed drug development supporting system is a set of extrapolation factors enabling the unrestricted choice of some elements of the experimental procedure without resultin significant depreciation. Therefore the main objective of this work was to develop the extrapolation factors allowing inter-system (HEK, CHO, XO) and inter-temperature (room and physiological) IC50 values comparison based on the collected and analyzed hERG IC50 data. The efficiency of the obtained factors was then verified in comparison with the native HEK IC50 values at the physiological temperature. Low values of all the error estimates for the proposed factors evidence their good predictive value which, in turn, ground their application during drug candidates' cardiotoxic risk evaluation or further in silico modeling. Utilization of the proposed factors during drug development process allows a more flexible choice of experimental model to exploit in the electrophysiological investigations, as well as makes it possible to experiment in ambient temperature, which is more convenient. The factors also facilitate data comparisons and allow one to draw reasonable conclusions for further extrapolation.