Patient safety relies on numerous early warning systems in drug development, designed to catch harmful effects long before candidates reach human trials. One of the most important tests is the hERG (human Ether-a-go-go) assay, which can predict cardiotoxic liabilities in vitro. They prevent costly late-stage failures and withdrawals and, more importantly, protect patients.
The hERG gene encodes a potassium ion channel that helps reset the electrical activity of heart cells between beats. If a drug is found to block this channel excessively, the heart may not function properly, which will be indicated by QT interval prolongation on an ECG. Prolonged QT increases can cause sudden cardiac death, and in the past, otherwise effective drugs have been withdrawn from the market because they blocked hERG.
In February 2022, the pharmaceutical industry shifted its approach to hERGs following the adoption of new international guidelines, ICH E14/S7B Q&As. The new regulation instructed drug developers to use a hERG safety margin threshold to assess drug safety in laboratory tests. However, there was a catch. Even when organizations followed the same protocols, the exact results of hERG tests varied from one lab to another. This made it essential for each lab to establish its own internal threshold using reference drugs, rather than relying on those published elsewhere.
WuXi AppTec put these rules into practice to establish its own hERG safety margin threshold, and with the following tips, laboratories can ensure they’re following the best practice considerations.
1. Define Your Own hERG Safety Margin Threshold
Developers previously relied on rules of thumb; however, the new guidelines now require sponsors to use their own specific cut-off points for hERG safety. Assay results can vary between labs for several reasons, including differences in patch-clamp equipment, and other subtle factors.
WuXi AppTec determined an internal hERG safety margin threshold of about 57 using the methods documented below. However, while that works for our setup, it cannot be replicated in other laboratories. To establish a reliable calibration dataset, labs can follow some of our best practices.
2. Calibrate hERG Safety Margin with Reference Drugs
To define a valid threshold, developers must anchor their assays to drugs with known clinical QT effects. ICH E14/S7B Q&As calls on labs to use a panel of well-characterized compounds to establish assay sensitivity.
To establish an internal hERG safety margin, WuXi AppTec chose moxifloxacin, dofetilide, ondansetron, and cisapride. Each drug has a well-documented relationship between dose, hERG block, and QT prolongation, and produces clear, dose-dependent reductions in hERG current. The four reference drugs were tested two to five times in accordance with best practices in two laboratories.
3. Stick to Best Practices in hERG Assay Design
Regulators provide guidance on expectations for temperature, voltage protocol, and the use of controls for assays that determine thresholds. Our scientists followed the recommended voltage protocol command, with a stimulation frequency of 0.2 Hz. We ran our assays at near-physiological temperature of 35-37°C, and the full blocker 1 µM E-4031. Assays were conducted at two laboratories, both utilizing a manual patch-clamp system.
This level of rigor is required for any laboratory hoping to determine and use its own hERG threshold.
4. Use Robust Data
A single IC₅₀ value from a lone experiment cannot support regulatory decisions. Guidance calls for repeated experiments, pooled data, and robust analysis. To accomplish this, our team gathered 13 IC₅₀ values for the four reference drugs. Data across drugs, laboratories, and several repeated experiments were pooled using a random-effect meta-analysis, using the metafor package in R software.
Log-normal distributions were assumed for both plasma concentration and hERG IC50, and our researchers reported their margins with confidence intervals. This robust approach ensures that developers can present properly defined margins to regulators.
5. Integrate the hERG Safety Margin Into the Bigger Picture
hERG is no longer assessed in isolation. The new guidance allows high-quality nonclinical “double negative” results, meaning a negative hERG finding paired with negative in vivo QT studies. This can reduce or even replace thorough QT trials in humans.
“These Q&As focus on connecting the ICH S7B and E14 Guidelines for specific scenarios where the nonclinical data are informative in clinical study implementation and evaluation…to decrease the need for clinical Thorough QT (TQT) studies,” ICH E14/S7B Q&As states.
WuXi AppTec was able to demonstrate that its internal threshold produced results that were consistent with ICH reference datasets, showing regulators that the methodology was reliable.
A Final Word
Cardiac safety is an imperative element of drug development. The 2022 ICH Guidance drastically altered the mindset surrounding hERG assays, and developers should ensure that each laboratory used establishes its own hERG safety margin threshold rather than relying on a published one. By calibrating assays with the correct drugs, validating results under best practice considerations, and analyzing them with modern statistical methods, labs can ensure their thresholds are accurate.
For sponsors seeking to alleviate clinical burden, streamline development, and ensure patient safety, collaborating with a lab partner that possesses the expertise and know-how to navigate these regulatory requirements will ease their path to market.
