Predicting QT prolongation in humans during early drug development using hERG inhibition and an anaesthetized guinea-pig model

Abstract

Background and purpose: Drug-induced prolongation of the QT interval can lead to torsade de pointes, a life-threatening ventricular arrhythmia. Finding appropriate assays from among the plethora of options available to predict reliably this serious adverse effect in humans remains a challenging issue for the discovery and development of drugs. The purpose of the present study was to develop and verify a reliable and relatively simple approach for assessing, during preclinical development, the propensity of drugs to prolong the QT interval in humans.

Experimental approach: Sixteen marketed drugs from various pharmacological classes with a known incidence -- or lack thereof -- of QT prolongation in humans were examined in hERG (human ether a-go-go-related gene) patch-clamp assay and an anaesthetized guinea-pig assay for QT prolongation using specific protocols. Drug concentrations in perfusates from hERG assays and plasma samples from guinea-pigs were determined using liquid chromatography-mass spectrometry.

Key results: Various pharmacological agents that inhibit hERG currents prolong the QT interval in anaesthetized guinea-pigs in a manner similar to that seen in humans and at comparable drug exposures. Several compounds not associated with QT prolongation in humans failed to prolong the QT interval in this model.

Conclusions and implications: Analysis of hERG inhibitory potency in conjunction with drug exposures and QT interval measurements in anaesthetized guinea-pigs can reliably predict, during preclinical drug development, the risk of human QT prolongation. A strategy is proposed for mitigating the risk of QT prolongation of new chemical entities during early lead optimization.

Figure 1

Effects of verapamil on hERG (human ether a-go-go-related gene) potassium currents. (a) Stably transfected Chinese hamster ovary (CHO)-K1 cells were held at −80 mV, then stepped to +20 mV for 400 ms followed by a second pulse to −40 mV for 400 ms to elicit large, slowly deactivating hERG tail currents. This pulse protocol was repeated every 10 sec while the cells were exposed to verapamil at different concentrations. (b) The percent inhibition at each dose was calculated (n=4–8 cells for each concentration), and the concentration–response curve was then fitted to a Hill equation as described in the Methods. In this example, the IC₅₀ for verapamil inhibition of hERG was 0.94 μM. The IC₅₀ values for the other drugs evaluated in this study are listed in Table 2.

Figure 2

Free safety margins were calculated by dividing the hERG (human ether a-go-go-related gene) IC₅₀ values (Table 2) by the therapeutic free C_max plasma concentrations calculated using the total C_max and the protein binding from Table 1. Group A (open circles, ○) contains eight drugs that induce QTc (heart rate corrected QT interval of the ECG) prolongation with TdP incidents reported. The three drugs in group B (plus symbols, +) cause QTc prolongation but have not been reported to cause TdP. Neither QTc prolongation nor TdP incidents were found for the five drugs in group C (multiplication symbols, × ). The dashed horizontal lines represent free safety margins of 30 and 300.

Figure 3

Example of a drug that prolongs the QT interval (moxifloxacin) (a) versus another that does not (amoxicillin) (b). Baseline-corrected QTc (heart rate corrected QT interval of the ECG) time courses (left panel) and averaged responses (right panel) in each case are depicted. Each of the drug doses was administered to anaesthetized guinea-pigs on a milligram per kilogram basis by slow intravenous infusion over 10 min (horizontal bars). For each animal, baseline-corrected QTc interval was calculated by comparing the mean QTc interval during baseline (that is, pre-infusion) with the mean QTc interval post-infusion (that is, from 10.5 to 30 min after the start of the 10-min infusion period). Baseline-corrected QTc is plotted as a function of the dose in the right panel graphs. The mean values are connected by lines and the error bars represent the standard error of the mean. The vehicle data correspond to the 0 mg kg⁻¹ dose. Sixteen drugs were evaluated in this model, and the data are summarized in Figure 4.

Figure 4

Summary of QTc (heart rate corrected QT interval of the ECG) expressed as differences from vehicle. The y axis shows the mean difference from vehicle (of baseline-corrected QTc) for each drug and the low (L), medium (M) and high (H) doses (Table 3) that were tested in the anaesthetized guinea-pig model. QTc values that are significantly higher than vehicle (P<0.05) are indicated by asterisks. Every drug in groups A and B showed at least one significant increase over vehicle, but none of the drugs in group C had any significant increases over vehicle.

Figure 5

A testing strategy incorporating the hERG (human ether a-go-go-related gene) electrophysiology and anaesthetized guinea-pig QTc (heart rate corrected QT interval of the ECG) assays. The solid lines represent the standard progression path, whereas the dashed lines represent exceptions on a case-by-case basis. Compounds are initially evaluated in a hERG electrophysiology assay conducted using the defined protocol described in Methods. Compounds with hERG IC₅₀ values of >10 μM are usually progressed, whereas compounds with IC₅₀ values of <1 μM are usually withheld. Compounds with hERG IC₅₀ values between 1 and 10 μM are evaluated for their potential to prolong the QTc in a specific anaesthetized guinea-pig model (see Methods for protocol details). If a compound is found to prolong QTc in this model, an in vivo safety margin (SMiv) is calculated by dividing the peak drug concentration obtained from an anaesthetized guinea-pig study (where the QTc increased by more than 5%) by the peak drug concentration obtained during an efficacy study in an appropriate animal model. If the SMiv is greater than 30, the compound is further progressed; if the SMiv is less than 30, the compound is put on ‘hold', and research continues for an alternative clinical candidate.