Our data provide validation of the arterially perfused rabbit ventricular wedge preparation as a preclinical experimental model for assessment of drugs with TdP liability. The relative risk of 13 drugs with and without known TdP liability was evaluated using a quantitative approach in which all investigators and technicians were blinded to the names, concentrations and molecular weights of the drugs. The results point to the isolated arterially perfused rabbit ventricular wedge preparation as a preclinical model with a high sensitivity and specificity for identifying drugs with a potential for causing TdP.
The incidence of drug-induced TdP ranges from <0.1% for noncardiac agents such as terfenadine or moxifloxacin up to 3–8% for some antiarrhythmic drugs, such as quinidine.21–24
Drugs with a relatively low TdP liability may be difficult to identify without a preclinical model of high sensitivity designed to avoid false negatives.2,8
Indeed, regulatory agencies have emphasized the importance of a high sensitivity in preclinical testing.25
The sensitivity of a preclinical test is determined, in part, by the species selected. Among those available for electrophysiological study, the rabbit is well-suited for simulating humans with a markedly reduced repolarization reserve due to its intrinsically weak IKs
throughout the ventricular endocardium.26
As a consequence, the entire endocardium in the adult rabbit exhibits characteristics of M cells, which are known to be exquisitely sensitive to QT prolonging drugs, owing in part to the weaker IKs
in this cell type. This characteristic of the rabbit ventricle, particularly in female gender, renders it exceptionally sensitive even to weak QT prolonging agents.2,7,8,27
In the present study, a positive signal for QT prolongation was observed for all drugs with a known TdP risk, even for drugs that tested negative in other preclinical models. For example, terfenadine, a non-cardiac drug with a TdP risk in humans, caused little QT or action potential prolongation in conscious dogs,28
or in isolated canine Purkinje fibers,29
porcine myocardium or Purkinje fibers,30
but prolonged QT interval by more than 20% in the rabbit left ventricular wedge preparation and increased dispersion of repolarization by nearly 50%.
Electrophysiological stability of a preclinical model is another important factor that influences the predictive value of preclinical testing. Electrophysiological instability reduces the signal to noise ratio of the test, and may produce false negatives as well as false positives. Because the ventricular wedge preparation is arterially-perfused via the native coronary artery, the preparation remains electrically stable for at least 4 hours.17,31,32
In our hands, the QT interval of the rabbit left ventricular wedge preparation changes less than 2% over a period of four hours after the initial one hour equilibration period. Thus, the wedge preparation permits a temporal window sufficient for studying multiple concentrations of a drug, with a long exposure at each level.
Although regulatory agencies have expressed particular interest in the sensitivity of the preclinical test, specificity is of equal importance to the pharmaceutical industry in order to avoid false positives that could unwarrantedly remove promising drugs from their pipeline. While measurements of the QT interval or of hERG inhibition can detect drugs with a TdP liability with great sensitivity, this approach often lacks specificity. hERG inhibition alone in many cases does not translate directly into TdP risk, since drugs like verapamil, ranolazine, sodium pentobarbital and fluoxetine, possess hERG-blocking activities at clinically relevant concentrations, but are not associated with TdP.3,4
Agents like amiodarone, which also blocks IKr
produce marked QT prolongation but rarely cause TdP even in patients who have previously developed TdP as a complication of other QT prolonging agents.33
/QT ratio and the incidence of EADs and EAD- induced triggered activity are additional parameters used in the present study to assess the relative risk for drug-induced TdP. These parameters increase both the sensitivity and specificity of the rabbit ventricular wedge preparation because they provide unique signals specifically related to the development of TdP.7–9
Spatial dispersion of repolarization and EAD-induced triggered activity are well recognized as the substrate and trigger for the development of TdP.9
Interestingly, the Tp-e
/QT ratio may serve as an important parameter useful not only in differentiating between potent and weak IKr
blockers but also in distinguishing pure IKr
blockade from combined inhibition of IKr
and inward currents. Compounds with a measurable incidence of TdP risk such as cisapride, clarithromycin, sparfloxacin, erythromycin, dofetilide and sotalol amplify transmural dispersion of repolarization by preferentially prolonging sub- and endocardial action potential duration due to their effect on IKr
and therefore markedly increase the Tp-e
/QT ratio (, and ).2,7
Some agents such as terfenadine at lower dose cause only a modest increase of QT interval, but a significant increase in the Tp-e
/QT ratio (20.3 ± 2.9%), identifying it as a drug with significant TdP liability. It is noteworthy that previous preclinical models have failed to identify terfenadine as having a TdP risk. On the other hand, although azithromycin significantly prolongs both of the QT and Tp-e
intervals at higher doses, the Tp-e
/QT ratio remains relatively unchanged probably due to the fact that azithromycin inhibits inward sodium current at the same dose range (our unpublished data). This is probably one of the reasons why azithromycin carries a much smaller TdP risk than erythromycin clarithromycin and sparfloxacin. Similarly, cisapride at higher doses, which inhibits inward currents,34
markedly reduces the Tp-e
/QT ratio, and abolishes EADs and R-on-T ectopic beats despite the fact both the QT and Tp-e
intervals remain prolonged.
The safety margin, clinical pharmacokinetic profile and pharmacodynamics of each compound must all be taken into consideration in the final analysis. For example, fexofenadine causes a significant increase in the QT and Tp-e
intervals and received a significant TdP score at doses greater than 100 fold its free TPC ( and ). This is consistent with its clinical outcome, which is that the drug has a very low risk of TdP.35
Moxifloxacin, a fluoroquinolone antibiotic that modestly prolongs the QT interval in humans, received a small but significant TdP score at concentrations ranging from 5 to 50 fold its free TPC (). These results are consistent with clinical outcome data demonstrating a low incidence of TdP.
The isolated Langendorff-perfused rabbit heart preparation as a preclinical model to identify drugs with TdP liability was recently validated in blinded assessments, and it exhibited a high sensitivity.36,37
The isolated arterially-perfused rabbit ventricular wedge preparation differs from the Langendorff-perfused rabbit heart preparation with regard to two critically important aspects. First, the rabbit ventricular wedge preparation is electrically stable for more than 4 hours, as demonstrated by us and others.17
In contrast, the Langendorff-perfused rabbit heart preparation deteriorates faster, showing QT interval abbreviation of >3.5% (11 ms) over a one hour perfusion period.36,38
Drug testing in the latter must therefore be completed within one to two hours, necessitating relatively short (10 min) exposure to each drug concentration.36,37
This is an important limitation in the case of drugs that have a delayed time-course of action due to slow intracellular accumulation.39
Secondly, studies involving the wedge preparation permit measurement of three core parameters specifically related to the development of TdP: the QT interval or APD, TDR or Tp-e
and EAD-induced triggered activity, in addition to TdP. Relative TdP risk can therefore be scored semi-quantitatively. Based on preclinical tests involving measurement of monophasic action potentials in the Landgendorff preparation, Hondeghem et al. introduced the interesting concept of TRIaD (e.g. triangulation, reverse use dependence and instability) for the assessment of drug-induced TdP risk, which still remains to be fully validated. Interpretation of the data is difficult at times. For example, amiodarone, a QT prolonging agent, which rarely causes TdP in humans, produces triangulation, reverse use dependence and instability.37
In summary, our blinded study validates the isolated arterially perfused rabbit ventricular wedge preparation as a promising preclinical model for the assessment of drug-induced proarrhythmia. This approach can not only distinguish between TdP positive and negative agents but can also scale the relative risk of each compound from integrated information of QT prolongation, transmural dispersion and EAD activity.
Limitations of the study
Analysis of the effluent collected from each preparation yielded drug concentrations consistent with the prepared stock solution with one exception. The concentrations of thioridazine were significantly lower than that in the corresponding stock solutions, suggesting the possibility that the compound might have been partially adsorbed to glassware and the perfusion tubing. This may be the reason why thioridazine, which is associated with a fairly high TdP risk,3
received TdP score that may potentially underestimate its potential risk.