The limitations of current preclinical drug testing systems used by pharmaceutical companies and other drug developers contribute to the high failure rate of drug candidates. Unexpected cardiotoxicity is one of the top two major safety-related reasons for failure of both drugs and drug candidates. Incorporating human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) assays early in preclinical development offers the potential to improve clinical predictability, decrease rescue and development costs, and avoid adverse patient effects, late-stage clinical termination, and product recall from the market.
We produce fully-functional hPSC-CMs at a high level of purity (>95%), without genetic modification or cell selection. This is important because genetic modification and cell selection skew the resulting cell population and may distort the clinical predictivity of the assay. In addition to expressing all of the key ion channels and various cardiomyocytic markers of the human heart, our hPSC-CMs, which we call VSTA-CMs™, function reliably in all cardiac toxicity assays relevant to cardiac drug effects developed and tested to date.
Utilizing VSTA-CMs™, our CardioSafe 3D assay system can screen for both cardiomyocyte cytotoxicity and arrhythmogenesis (or development of irregular beating patterns). We believe CardioSafe 3D is sensitive, stable, reproducible and capable of generating data enabling a more accurate prediction of the in vivo cardiac effects of new small molecule drug candidates, including drug rescue candidates and drug rescue variants, than is possible with existing preclinical testing systems, including the FDA-required in vitro hERG assay.
We have developed and validated two customized and functional components of our CardioSafe 3D bioassay system to assess multiple different categories of cardiac toxicities. The first consists of a suite of fluorescence or luminescence based high-throughput hPSC-CM assays. These CardioSafe 3D assays measure drug-induced cardiomyopathy, including the following:
- mitochondrial membrane depolarization;
- oxidative stress; and
- energy metabolism disruption.
These CardioSafe 3D assays were correlated with reported clinical results involving reference compounds known to be cardiotoxic in humans and compounds known to be safe in humans. These reference compounds were representative of eight different drug classes, including:
- Ion channel blockers: amiodarone, nifedipine
- hERG trafficking blockers: pentamidine, amoxapine
- α-1 adrenoreceptors: doxazosin
- Protein and DNA synthesis inhibitors: emetine
- DNA intercalating agents: doxorubicin
- Antibiotics: ampicillin, cefazolin
- NSAID: aspirin
- Kinase inhibitors: staurosporine
This suite of CardioSafe 3D assays provided measurement of cardiac drug effects with high sensitivity that are consistent with the expected cardiac responses to each of these compounds. Based on our results, we believe our CardioSafe 3D assays provide valuable tools for both assessing the effects of pharmaceutical compounds on cardiac cytotoxicity and for elucidating the specific mechanisms of cardiac toxicity, thereby laying a solid foundation for our drug rescue programs.
The other component of our CardioSafe 3D assay system is a sensitive and reliable medium throughput multi-electrode array (MEA) assay developed to predict drug-induced alterations of electrophysiological function of the human heart. We have validated this key component of our CardioSafe 3D bioassay system with numerous drugs, each with reported toxic or non-toxic cardiac effects in humans.
Our CardioSafe 3D MEA assay was reproducible and consistent with the known human cardiac effects of all compounds studied, based on the mechanisms of action and dosage of the compounds. For instance, by using CardioSafe 3D, we were able to distinguish between the cardiac effects of terfenadine (SeldaneTM), withdrawn by the FDA due to cardiotoxicity, and the cardiac effects of the close structurally related fexofenadine (AllegraTM), the non-cardiotoxic chemical variant of terfenadine, which remains on the market. Our validation data suggest that CardioSafe 3D provides valuable tools for in vitro preclinical cardiac safety screening, which we believe will contribute to the efficient and rapid identification of novel, safer Drug Rescue Variants in our drug rescue programs.
To further evaluate the potential of CardioSafe 3D to predict cardiac toxicity of new drug candidates, including Drug Rescue Variants, we have assessed cardiac effects induced by small molecule kinase inhibitors (KIs), which belong to a new category of drugs that have revolutionized cancer therapy due to decreased systemic toxicity and an increased tumor cell specific effect compared to classic cancer drugs. Since 1998, the FDA has approved thirty small molecule KIs for cancer therapy. However, many of these FDA-approved KIs have been implicated in causing serious adverse cardiac events in patients which were not identified during drug development.
In our KI-induced cardiotoxicity study, we evaluated nineteen well-known anti-cancer KIs with CardioSafe 3D, some of which are FDA-approved and have been documented as cardiotoxic. This important validation set of anti-cancer KI compounds is as follows:
- Inhibitors to growth factor receptors: sunitinib, axitinib, imatinib, dasatinib, sorafenib, erlotinib, lapatinib, tyrphostin AG1478)
- Inhibitors to the mTOR pathway: everolimus, temsirolimus
- Inhibitors to cell cycle regulators: tozasertib, barasertib, alvocidib
- Inhibitors to the PI3K pathway : perifosine, LY294002, XL765
- Inhibitors to the MEK pathway: PD325901, AZD6264
- Inhibitors to the JAK and other pathways: lestaurtinib
CardioSafe 3D successfully detected cardiotoxicity induced by each of the representative compounds, cardiotoxicity associated with clinical adverse cardiac events, in each of the foregoing six different KI categories. CardioSafe 3D is able to distinguish between cardiotoxic and safe compounds, and even as between those compounds which inhibit the same kinase pathways. For instance, both sunitinib and axitinib inhibit VEGFR, PDGFR and c-Kit pathways, and CardioSafe 3D indicated that sunitinib is cardiotoxic and axitinib is safe, which is consistent with the reported clinical outcomes.
Furthermore, the CardioSafe 3D profile of each KI provided clues to the potential mechanism(s) causing cardiotoxicity. For example, cardiotoxicity induced by perifosine showed apoptotic responses at lower concentrations, while imatinib was most active in the oxidative stress assays. In addition, no cardiac toxicity or alteration in electrophysiology was detected with drugs that do not have a cardiac liability, emphasizing the specificity of CardioSafe 3D. Having information on the pathways associated with the cardiotoxic effects of compounds provides important advantages for novel medicinal chemistry approaches and compound modifications for our CardioSafe 3D drug rescue programs.
CardioSafe 3D enables the sensitive measurement of drug effects with results that are consistent with known clinical responses to the compounds. For example, our data indicated that sunitinib and dasatinib caused QT prolongation, arrhythmia, and/or altered contraction rates in hPSC-CMs, which are consistent with clinical observations.
We believe our CardioSafe 3D validation data demonstrate that CardioSafe 3D will improve clinical predictivity as an in vitro cardiac safety assay, helping not only to identify potential cardiac toxicities early in development, but also to discover important potential mechanisms of cardiotoxicity. We believe the results of our CardioSafe 3D validation studies indicate that CardioSafe 3D may be effectively used to identify novel drug candidates, including Drug Rescue Variants, with reduced heart toxicity. By providing more accurate and timely indications of alterations in electrophysiological activity, as well as potential cardiac cytotoxicity of new drug candidates, than live animal models or cellular assay systems currently used by pharmaceutical companies, including the FDA-required in vitro hERG assay, we believe the results of our CardioSafe 3D validation studies support our opportunities to leverage substantial prior investment by pharmaceutical companies and others in drug discovery and efficacy validation of drug candidates with established therapeutic and commercial potential that have been terminated prior to FDA approval due to unexpected heart toxicity concerns.