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Assessment of Drug-Induced Cardiotoxicity in Zebrafish
Louis J. D’Amico1, Wen Lin Seng1, Yi Yang2, Willi Suter2, and Patricia McGrath1
1Phylonix Pharmaceuticals, Inc., Cambridge, Massachusetts, U.S.A.
2Novartis Pharmaceuticals, East Hanover, New Jersey, U.S.A.
Abstract
The goal of this study was to validate use of zebrafish for Drug
Cardiotoxic manifestations in humans
Cardiotoxicity in Zebrafish
Effect of Terfenadine on Heart Rate
Effect of Clozapine on Heart Rate
assessing drug-induced cardiotoxicity. We treated 2-day Heart rate
Rhythmicity
Morphology
Circulation
zebrafish with varying concentrations (0.01 µM-1000 µM) of 24 Doxorubicin
Cardiomyopathy, decreased heart rate, contractility validation drugs, including: doxorubicin, 5-fluorouracil, cyclophosphamide, mitoxantrone, terfenadine, clomipramine, thioridazine, gentamicin, tetracycline, amantadine, disopyramide, 5-fluorouracil
lidocaine, metoprolol, mexiletine, phenytoin, procainamide, propafenone, clozapine, erythromycin, quinidine, astemizole, Cyclophosphamide
Cardiac myocyte death, decreased heart rate, amiodarone, verapamil, haloperidol, and clomipramine. Heart Concentration (µ M)
Concentration (µ M)
rate, rhythmicity, circulation, and morphology were easily Mitoxantrone
Acute heart failure, decreased left ventricular assessed in the transparent animals. Drugs that elicited ejection fraction (LVEF), decreased heart rate. Effect of Haloperidol on Heart Rate
Effect of Tetracycline on Heart Rate
cardiomyopathy, arrhythmia, negative inotropic effects, or QT Terfenadine
prolongation in humans including: doxorubicin, 5-fluorouracil, terfenadine, and lidocaine also caused bradycardia, AV block, and slow circulation in zebrafish. Severe impairment of cardiac Clomipramine
function after treatment with terfenadine and clomipramine resulted in the formation of pericardial edema, hemorrhage, Thioridazine
Negative inotropic effect, QT prolongation, PR bradycardia, as well as death at higher concentrations. QT- prolonging drugs such as terfenadine, quinidine, and thioridazine Gentamicin
Rare clinical finding: Negative inotropic effect induced acute atrioventricular block (AV block); A:V ratios ranging Concentration (µ M)
Concentration (µ M)
from 2/1 to 8/1 were observed. Gentamicin, amantadine and Tetracycline
Rare clinical finding; Negative inotropic effect tetracycline, which rarely induce cardiotoxicity in normal clinical use, did not cause significant cardiotoxicity in zebrafish. Results Effect of Amiodarone on A:V Ratio
Effect of Clomipramine on A:V Ratio
for drug effects on zebrafish heart rate show 100% concordance Amantadine
with results in mammals, which according to ECVAM standards, is excellent. This data supports use of zebrafish as a predictive Disopyramide
animal model for assessing drug-induced cardiotoxicity. Lidocaine
Introduction
Metoprolol
Concentration (µ M)
Concentration (µ M)
Cardiotoxicity: Cardiotoxicity is a major problem with hundreds of
Mexiletine
pharmaceutical agents, industrial chemicals and naturally Examples of dose-response and A:V ratio analysis.
occurring products. In the pharmaceutical sector, several Phenytoin
recorded heart rates of zebrafish treated with 1% DMSO (carrier control). A decrease in compounds have been shown to lengthen cardiac repolarization, ventricular rate is observed when zebrafish are treated with terfenadine, clozapine, and leading to arrhythmia and its clinical manifestation, Torsades de haloperidol, while tetracycline has no significant effect on heart arte (note log scale). Data pointes. Previous research has shown that compounds capable Procainamide
are reported as mean ± SD, and P<0.05 was used as the significance level. One-way of inducing repolarization abnormalities cause bradycardia in ANOVA with a post hoc Dunnett’s test was used to identify concentrations that significantly zebrafish. In current research, we look at the effect of 24 drugs on Propafenone
altered heart rate, indicated with an “*”. The bar graphs demonstrate a dose dependent
increase in A:V block. Amiodarone treatment resulted in a ~2:1 A:V ratio at 3.21 and Clozapine
6.42µM, and increased at higher concentrations. Conversely, Clomipramine, despite The Zebrafish Heart: The zebrafish (Danio rerio) is a useful
causing a dose-dependent decrease in heart rate, does not generate an AV block.
animal model system for studying cardiovascular development, Erythromycin
genetics, and cardiotoxicity. Zebrafish use gills for respiration and Drug Name
Known cardiotoxicity in Cardiotoxicity observed
Correlation of
have a single-loop circulatory system. The heart consists of two humans?
in zebrafish?
cardiotoxicity between
chambers: an atrium that receives blood and a ventricle that Quinidine
Arrhythmia; bradycardia; abnormal prolongation of humans and zebrafish?
pumps blood to the body. Both the mammalian and zebrafish heart share the development of: specialized chambers, outflow Astemizole
tracts to an intricate vasculature, valves to ensure directionality, specialized endothelial cells (endocardium) to drive a high- Amiodarone
Arrhythmia; bradycardia; SA node dysfunction pressure system, and an electrical system to regulate rhythm. There is inflow of blood from a major vein to an atrium, the blood Verapamil
moves to a muscular ventricle for delivery to the aorta, valves are
present to direct blood flow, and the heartbeat is associated with
pacemaker activity. The underlying development, patterning,
Haloperidol
QT prolongation; ventricular arrhytmias; genes, functions, as well as disease characteristics are similar to humans, making zebrafish a valuable animal model for studying Summary of drug-induced cardiotoxicity in zebrafish. 24 compounds were tested for cardiotoxicity in 2dpf zebrafish embryos. The most common form(s) of cardiotoxicity observed in humans are
B Zebrafish
listed for each drug, and the corresponding effects observed in zebrafish are noted. Gentamycin, tetracycline, and amantadine were predicted to show no signs of cardiotoxicity in zebrafish based on findings in humans. Several compounds known to have adverse cardiotoxic effects in humans caused a decrease in heart rate in zebrafish.
Atrioventricular block was observed at higher concentrations of a number of compounds. Defects in cardiovascular morphology were primarily noted by an increase in pericardial edema, though instances of hemorrhage, as well as swelling of the atrium and ventricle were also noted. Some compounds caused a corresponding decrease in circulation, particularly at higher concentrations. Circulation and Morphology Assessment
Comparison of human and zebrafish ECG. ECG pattern in
human (A) and adult zebrafish (B). The zebrafish and human
heart share strikingly similar patterns of electrical activity, including distinct P, QRS, and T waves.
Correlation of human and zebrafish cardiotoxicity.
human and zebrafish cardiotoxicity demonstrates the utility of the zebrafish model as a tool Heart Rate Assessment: 2 days post fertilization (dpf) zebrafish were
for assessing drug-induced cardiotoxicity.
incubated with drugs for 4 hours at 28°C. After incubation, zebrafish (N=10) were visualized on a Zeiss Stemi-1000 dissecting scope, and the number of ventricular contractions in a 30 second period was counted Conclusions
manually. The number of contractions was multiplied by 2 to calculate Examples of Malformations Observed in Cardiotoxicity Assessment. A & B) Images of 3dpf zebrafish treated for 24h with 1% DMSO (2.5x and 8x, respectively). No abnormalities are observed.
the heart rate, reported in beats per minute (BPM).
C) 2.5x image of a drug-treated 3pdf zebrafish. The white arrow points to the heart, where blood can clearly be seen in the chambers. Ventricular contractions have stopped, allowing blood to
Drugs known to cause cardiotoxic adverse side effects in humans show similar effects in accumulate in both chambers, and pool just posterior to the atrium (surrounding the yolk). Secondary toxicity has caused necrosis of the musculature along the tail, and caused curvature of the body. Atrial:Ventricular Ratio: Following ventricular measurement, atrial
contractions were counted using the same methods described above.
D) A milder drug-induced toxicity example at 8.0x magnification. The white arrow points to the pericardium, which is clearly enlarged. Otherwise the zebrafish exhibits no evidence of cardiotoxicity.
Zebrafish assays are rapid, quantitative, and reproducible. Only small amounts of drug Cardiac Morphology and Circulation: 2dpf zebrafish were incubated
with a single concentration of drug for 24h at 28°C. Following treatment, zebrafish (N=10) were observed for circulation defects, gross Zebrafish in vivo cardiotoxicity assays provide useful information that supplements morphological defects, edema, and thrombosis.

Source: http://www.phylonix.com/ACtoxPosterHandout.pdf