The best time to discover that a drug has cardiovascular liabilities is early in the drug research process. Assays that identify toxicities in preclinical phases can help.

The cardiovascular system is a vital part of the human body. It is responsible for important tasks such as supplying nutrients, removing waste, distributing hormones and regulatory substances, maintaining osmotic balance and homeostasis. It is also one of the more delicate systems in the human body and can be impacted by both cardiovascular and non-cardiovascular drugs. As a result, drug development programs are complex and often require multidisciplinary research when assessing potential cardiovascular toxicity.

Cardiac Potential

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Figure 1. Schematic representation of an electric cardiac cell action potential (A) detailing the different ionic current components (Na: sodium, Ca: calcium and Kr: hERG-related potassium) and its translation into the body-surface electrocardiogram (B) where the major wave complexes are identified (PQRST). (Source: Charles River Laboratories)

QT liability
The cardiac system has a number of potential toxicity targets, including heart rate, conductivity, and contractility, all of which are important areas of research. One of the main issues for drug development is the interruption of the heart’s electrical cycle due to blocking the human ether-à-go-go related gene (hERG) channel (Figure 1A), which is responsible for fluxing potassium out of the cell while the heart is repolarizing. If the hERG channel is blocked, QT-interval prolongation (Figure 1B)—an increase in the amount of time the heart muscles and myocytes need to prepare for the next heart beat—can occur leading to a fatal arrhythmia called torsade de pointes.

Many non-cardiovascular medications, including antibiotics, antihistamines, and antipsychotics, can contribute to hERG-channel blockage and prolong the user’s QT interval. This problem has resulted in a large number of drugs being pulled from the market, caused prescription restrictions, labeling predicaments, approval delays and refusals, and wasted resources. Because of this, potential QT liability should be addressed early and monitored throughout the drug development process.

Early discovery screening
A number of different in vitro, ex vivo, and in vivo strategies can be conducted early in the discovery screening process to determine if a compound has a QT liability. These assays (See Table 1.) can provide data to identify potential toxicities early in the development process and allow researchers to focus their efforts and resources on compounds without dangerous liabilities.

In vitro
These assays are conducted in cells transformed to over-express the hERG protein. The voltage patch-clamp technique is currently the gold standard for detecting hERG blockers. This test measures current through a single, specific channel, in this case, the hERG channel. Researchers can then determine the concentration of the drug that reduces current by 50 percent (IC50). The lower the IC50 the higher the likelihood the drug will be a hERG blocker.

As accurate as the voltage patch-clamp technique is, it is not a high-throughput test and more efficiency may be needed to test a large number of compounds. The more labor-intensive an assay, the more resources are required. Several other assays offer higher throughput than the patch-clamp test, including the radiolabeled hERG-binding, rubidium-flux, and membrane-potential assays.

The radiolabeled hERG-binding assay determines the concentration of a test compound that can compete off a radioligand from a hERG channel. A number of compounds are known to actively bind to the hERG channel and can be used as controls to determine if a test article can compete off of those bound ligands.

The rubidium flux assay uses rubidium, a naturally occurring substance within a cell, as a surrogate for potassium. External and internal amounts of rubidium are measured in a preloaded cell where a test article and potassium chloride have been added to determine how well the hERG channels are performing.

The membrane-potential assay involves loading cells with a voltage-sensitive dye and the test article. Potassium chloride is then added to induce cell depolarization. The emission from the dye will be modified if there is hERG blocking, and it can then be quantified accordingly.

Assay  Throughput Specialized equipment  Sensitivity  Reliability  Specificity  Predictive Value for QT prolongation 
in vitro
Membrane Potential Dyes +++ Yes/No Very Low Low Poor Poor
Rubidium Efflux +++ Yes Moderate High Good Good
Radioligand Binding ++ No High High Good Good
Patch Clamp ++ Yes High High Good Good
ex vivo
Purkinje Fibers ++ Yes High High Moderate Good
Isolated Heart + Yes High High Moderate Good
in vivo
Transgenic Mice + Yes/No Low Moderate Low Poor

The results of both the hERG-binding and rubidium-flux assays correlate well to the gold standard of the voltage patch-clamp. While hERG binding correlates the closest, rubidium flux offers a higher throughput since it takes less equipment and time to set up and perform. The membrane-potential assay, on the other hand, does not correlate well and typically should not be used for selecting compounds.

Ex vivo
A number of different ion channels must interact together to make a heart beat properly, and a compound could affect one or more of those channels. The benefits gained from ex vivo methods is that they offer a larger picture of cardiac processes and allow researchers to better identify potential toxicities.

Ex vivo methods of testing include the use of Purkinje-fiber and isolated-heart methods and allow the recording of action potential durations. Purkinje fiber uses whole tissue and offers a better picture than in vitro methods for possible mixed-channel effects. The isolated-heart method involves the use of a whole heart and can identify channel effects as well as hemodynamic alterations.


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In vivo
In vivo assays offer an even broader picture of the cardiovascular system, showing not just voltage and electrocardiography (ECG) changes, but also pressure changes. The cardiovascular system is directed by a wide variety of receptors and modulators to maintain homeostasis, so studying the whole heart in the context of the whole body can offer an enormous amount of valuable information. Of course, in vivo methods are more expensive and have a lower throughput than in vitro methods. Because rodents don’t naturally express the hERG gene, several murine models with human transgenes have been developed. Despite this, the mouse heart’s electrophysiological singularities mean it is not a suitable model for drug-induced cardiac arrhythmias.

The advantages of early discovery screening include minimizing the risks of investing in unsafe compounds and bringing a safer drug forward with fewer liabilities. These are particularly important points in programs where compounds can be sent back to the chemist for modification. After all, a single, small, chemical change to the structure of a compound can make the difference between a rejected drug and a drug brought successfully and safely to market.

In addition to determining lead compounds for development, discovery screening allows for the collection of data that can be invaluable as the program moves into the regulated preclinical package. The results of discovery assays can aid in setting doses for toxicology and safety pharmacology studies, and identification of key time periods for the assessment of potential toxicities.

Preclinical safety
Once a lead compound is identified, the next step is the preclinical safety package, which includes toxicology and safety pharmacology studies. Safety pharmacology is required by the FDA and other regulatory bodies to assess cardiovascular, respiratory, and central nervous system (CNS) safety. The cardiovascular study is typically conducted as a stand-alone, but may be included in toxicology studies, as is the case in the development of most large molecule biologics programs.

One guideline of particular importance in safety pharmacology is ICH Guideline S7B: The Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals. It is specifically focused on potential effects on QT interval and hERG-channel blocking and outlines the minimum requirements for testing that need to be performed in the preclinical safety package.

A common mistake is to limit cardiovascular safety assessments to heart rate, blood pressure, and ECG. However, this methodology is often not enough. In some situations the participant’s heart rate, blood pressure, and ECG will not be affected by a drug, but their left ventricular pressure will be. Left ventricular pressure is an important indicator of the health of cardiac muscle and the heart overall. Measuring this can help discover a severe cardiovascular liability that would otherwise have been missed.

Identifying potential cardiovascular liabilities is an important part of the drug discovery and development process. A number of assays can be used to identify potential toxicities early in the drug discovery process, thereby minimizing the amount resources expended on compounds with cardiovascular issues. When a strategy of early screening is employed, there are many opportunities to assess the risks and benefits of a particular compound and minimize unexpected findings during the preclinical development program. The full complement of data generated as a compound moves toward the clinic is relevant in determining its overall safety.

About the Authors
Alain Stricker-Krongrad has more than a decade of experience as a preclinical pharmacologist with expertise in cardiovascular therapies. He is an adjunct professor of pharmaceutics at the Massachusetts College of Pharmacy. Stephen Wilson has more than eight years experience in cardiovascular pharmacology and safety pharmacology testing and is the author of more than 200 preclinical testing reports.

This article was published in Drug Discovery & Development magazine: Vol. 12, No. 6, June, 2009, pp. 26-28.