Detect structural cardiotoxicity of novel therapeutics using Cyprotex’s spontaneously beating tri-cultured cardiac 3D microtissue high content screening (HCS) assay.
Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.
Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens.
6Nam KH, Smith AS, Lone S, Kwon S and Kim DH (2015) Biomimetic 3D Tissue Models for Advanced High-Throughput Drug Screening. J Lab Autom; In press
|Microtissue||Human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM’s), cardiac endothelial cells and cardiac fibroblasts|
|Analysis Platform||Confocal Cellomics ArrayScan® XTI (Thermo Scientific)|
|Test Article Concentration||8 point dose response curve with top concentration based on 100x Cmax or solubility limit
3 replicates per concentration
|Test Article Requirements||50 µL of a DMSO solution at a concentration of 200x top concentration (top concentration = 100x Cmax) or equivalent amount in solid compound|
|Time Points||72 hours (others available on request)|
|Quality Controls||Negative control: 0.5% DMSO (vehicle)
Positive controls: Sunitinib (Ca2+ homeostasis) and dasatinib (mitochondrial membrane potential)
|Data Delivery||Minimum effective concentration (MEC) and AC50 value for each measured parameter (microtissue count, microtissue size, DNA structure, calcium homeostasis (Ca2+), mitochondrial mass (Mito Mass), mitochondrial membrane potential (MMP) and cellular ATP content)|
|Compound||Cmax (µM)||In vivo toxicity||hESC-CM prediction (Pointon et al, 2013)||H9c2 monolayer||H9c2 MTs||Tri-culture cardiac MTs||Most Sensitive Feature|
|Dasatinib7||0.72||Structural cardiotoxin||Positive structural cardiotoxin||0.529||NR||2.08||MMP|
|Cyclophosphamide13||153.20||Negative structural cardiotoxins||NR||NR||30.8||Mito Mass|
|Buspirone HCl||0.03||NR||0.237||NR||Microtissue size|
All reference compound toxicities were correctly predicted in the spontaneously beating cardiac tri-culture 3D microtissue model including isoproterenol (MEC 2.1 µM, calcium dyshomeostasis (Table 1 and Figure 2)) and cyclophosphamide (MEC 30.8 µM, mitochondrial mass (Table 1)) which previously went undetected by Pointon et al (2013)5 and Cyprotex’s in-house H9c2 data.
Control compound sunitinib displays cytosolic calcium increase (calcium dyshomeostasis) followed by gross cytotoxicity (microtissue loss) (Figure 2a) while control compound dasatinib displays mitochondrial membrane potential loss without gross cytotoxicity (microtissue loss) (Figure 2b). The combination of an in vitro 3D model that better recapitulates the in vivo cellular physiology of the myocardium with a multiparametric HCS and cytotoxicity assay presents a viable screening strategy for the accurate detection of novel therapeutics that cause drug induced structural cardiovascular toxicity early in drug development.
1 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
2 Brutsaert DL (2003). Cardiac endothelial-myocardial signaling: Its role in cardiac growth, contractile performance, and rhythmicity. Phys Revs 83; 59-115.
3 Souders CA et al., (2009). Cardiac fibroblast:the renaissance cell. Circ Res 105; 1164-1176.
4 Mikaelian I et al., (2010). Primary endothelial damage is the mechanism of cardiotoxicity of tubulin-binding drugs. Tox Sci 117(1); 144-151
5 Pointon A et al., (2013) Phenotypic profiling of structural cardiotoxins in vitro reveals dependency on multiple mechanisms of toxicity. Tox Sci 132(2); 317-326
6 Nam KH et al., (2015) Biomimetic 3D tissue models for advanced high-throughput drug screening. J Lab Autom; In press
7 Force T et al., (2007). Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. Nat Rev Cancer 7; 332-344
8 Minotti G et al., (2004). Doxorubicin cardiotoxicity and the control of iron metabolism: quinone-dependent and independent mechanisms. Methods Enzymol 378; 340–361
9 Schimmel KJ et al., (2004). Cardiotoxicity of cytotoxic drugs. Cancer Treat Rev 30(2); 181–191
10Anderlini P et al., (1995). Idarubicin cardiotoxicity: a retrospective study in acute myeloid leukemia and myelodysplasia. J Clin Oncol 13(11); 2827-2834
11Kerkelä R et al., (2006). Cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Nat Med 12(8); 908-916
12 Chu TF et al., (2007). Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib. Lancet 370; 2011-2019
13 Floyd JD et al., (2005). Cardiotoxicity of cancer therapy. J Clin Oncol 23(30); 7685-7696
14 Zhang J et al., (2008). Isoproterenol-induced cardiotoxicity in Sprague-Dawley rats: correlation of reversible and irreversible myocardial injury with release of cardiac troponin T and roles of iNOS in myocardial injury. Toxicol Pathol 36(2); 277-278