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Cardiotox Screen: Cardiac Safety Liability Assessment

Our novel Cardiotox Screen assay utilizes a combination of cellular assays comprising multi-parameter phenotypic profiling techniques to assess both the functional and structural cardiotoxicity potential of novel compounds from a single plate based assay.

Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Background Information

  • Cardiotoxicity is a major cause of drug attrition during pre-clinical and clinical drug development.1
  • Drugs can exhibit functional changes defined as an acute alteration in the mechanical function of the myocardium or structural in nature as defined by morphological damage to cardiomyocytes and/or loss of viability.
  • In recent years in vitro strategies have been developed to allow the high throughput assessment of functional cardiomyocyte changes through fast kinetic monitoring of calcium transients, while structural morphology can be monitored in a high throughput manner using high content imaging (HCI) combined with biochemical intracellular ATP assessment.
  • Pointon et al., 2013 highlighted calcium homeostasis, mitochondrial function and ATP content as key endpoints for the in vitro detection of structural cardiotoxicity.2
  • Functional cardiotoxins can alter contraction frequency (chronotrophy), force (inotropy) or pattern (arrhythmia) creating disturbances in calcium transient patterns within contracting cardiomyocytes.
  • Fast kinetic fluorescent reading of cardiomyocyte calcium transients has been shown to detect atypical patterns and changes in cell-beating rate caused by hERG, Ca2+ and Na+ channel blockers.3
  • Cardiotoxins can also indirectly affect expression of ion channels, which alongside morphological changes require a longer period of compound exposure.
  • Our novel Cardiotox Screen panel assesses both structural and functional cardiotoxicity endpoints from a single cell population by utilizing our proprietary software to detect and analyze individual calcium transients alongside high content image analysis of cellular morphology and biochemical cytotoxicity assessment.
  • The full Cardiotox Screen panel comprises acute and pretreated (24h) exposure periods to capture cardiotoxins with time-dependent effects and is used in the assessment of cardiac safety liability in drug discovery.
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.

1 Sirenko O et al, (2012) J Biomol Screen 18(1); 39-53

Protocol

Cardiotox Screen protocol

Cell Line Induced pluripotent stem cell (iPSC) derived cardiomyocytes
Analysis Platform Cytation 3 Cell Imaging Multi-Mode reader and Cellomics ArrayScan® XTI or CX7 (Thermo Scientific)
Test Compound Concentrations 8 point dose response curve with top concentration based on 100x Cmax or solubility limit. 3 replicates per concentration*
Compound Requirements 150 μL of a DMSO* solution to achieve 200 x top concentration maintained at 0.5% DMSO or equivalent amount in solid compound
Time Points Acute and 24 hr pre-incubation*
Quality Controls Negative control: 0.5% DMSO (vehicle)*
Positive control: 2 appropriate compounds
Data Delivery Minimum effective concentration (MEC) and AC50 value for each measured parameter; peak count, amplitude, frequency, full peak width, full width half maximum (FWHM), full rise time, rise time from 10%, full decay time, decay time to 10%, peak width at 10%, peak spacing (below 10%), cell count, nuclear size, DNA structure (DNA), calcium homeostasis (Ca2+) mitochondrial mass (Mito Mass), mitochondrial membrane potential (MMP) and cellular ATP content (ATP)*

* other options available on request.

Data

Data from Cyprotex's Cardiotox Screen

 
Figure 1
Representative calcium transients for iPSC-derived cardiomyocytes treated with (A) vehicle, (B) isoproterenol, and (C) diltiazem.
 
Figure 2
Representative dose response curves for iPSC-derived cardiomyocytes treated with isoproterenol and diltiazem.

Calcium transient profiling allows the detection of functional cardiotoxicity; isoproterenol increases calcium transient peak frequency thus displaying positive chronotropy (MEC; 0.01 µM) while diltiazem decreases amplitude thus displaying negative inotropy (MEC; 0.02 µM).
 
Figure 3
Representative high content images of iPSC-derived cardiomyocytes stained with EarlyTox calcium dye (green), TMRE (red) and Hoechst (blue).
 
Figure 4
Representative dose response curves for (A) FWHM, (B) calcium homeostasis & (C) cellular ATP for dual cardiotoxin sunitinib. Representative dose response curves for (D) peak width at 10%, (E) DNA structure, and (F) cell count for dual cardiotoxin doxorubicin.

By combining the calcium transient profiling assay with a downstream high content screening and cellular ATP assay we can detect signs of morphological changes; sunitinib is a dual toxicity compound (functional and structural cardiotoxin) which exhibits an increase in calcium transient peak width at half maximum (FWHM) (MEC; 3.5 µM), alongside morphological calcium changes and decreased cellular ATP (MEC; 1.4 µM and 60.0 µM, respectively) correlating to its known effects in vivo.4

Doxorubicin, also a dual cardiotoxin, displays an increase in calcium transient peak width at 10% (MEC; 62.7 µM) correlating to its in vivo negative inotropy findings alongside a reduction in DNA structure (MEC; 1.4 µM) and cell count (MEC; 13.7 µM) correlating to its known DNA intercalation mechanism.5
 
Table 1
Detection of functional, structural and cardiac risk with 14 reference compounds using Cardiotox Screen; categorized according to in vivo literature with normalization to total plasma Cmax.

Utilizing a high content multi-time point approach (acute and 24 hour pre-incubation) with therapeutic index normalization to cut offs approaching human relevant total plasma Cmax, Cardiotox Screen is able to predict functional, structural and overall cardiotoxicity risk with sensitivities of 89%, 83% and 100%, specificities of 100%, 88% and 100%, and accuracies of 93%, 86% and 100%, respectively.

By combining the in vitro assessment of both structural and functional mechanisms of cardiotoxicity in a single plate based assay, Cardiotox Screen is able to accurately predict cardiotoxicity liabilities early in preclinical screening and can improve the in vitro to in vivo translation and risk assessment of novel compounds.

References

1 Sirenko O et al. (2012) Multiparameter in vitro assessment of compound effects on cardiomyocyte physiology using iPSC cells. J Biomol Screen 18(1): 39-53
2 Pointon A et al (2013) Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity. Toxicol Sci 132(2): 317-326
3 Laverty HG et al. (2011) How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines? Br J Pharmacol 163(4): 675-693
4 Cross MJ et al. (2015) Physiological, pharmacological and toxicological considerations of drug-induced structural cardiac injury. Br J Pharmacol 172(4): 957-974
5 Ravenscroft S et al. (2016) Cardiac non-myocyte cells show enhanced pharmacological function suggestive of contractile maturity in stem cell derived cardiomyocyte microtissues. Toxicol Sci 152(1): 99-112

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