THE IMPACT OF DRUG-INDUCED QT INTERVAL PROLONGATION ON DRUG DISCOVERY AND DEVELOPMENT

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1 THE IMPACT OF DRUG-INDUCED QT INTERVAL PROLONGATION ON DRUG DISCOVERY AND DEVELOPMENT Bernard Fermini and Anthony A. Fossa During the past decade, a number of non-cardiovascular drugs have had their label revised or have been withdrawn from the market because of unexpected post-marketing reports of sudden cardiac death associated with a prolongation of the QT interval, and an increased propensity to develop a ventricular tachyarrhythmia called Torsades de Pointes. Although a direct link between QT interval prolongation and arrhythmogenesis is still unclear, QT prolongation is now the subject of increased regulatory review and is considered a significant risk factor for predicting human safety of New Chemical Entities. Consequently, pharmaceutical companies are striving to improve the drug discovery and development process to identify, as early as possible, the risk of novel agents, or their metabolites, of causing QT interval prolongation and to make appropriate go/no-go decisions or modify their development programme accordingly. QT INTERVAL On the electrocardiogram, this measures the total time of ventricular depolarization and repolarization, roughly demonstrating the duration of the action potential. It is measured from the onset of the Q wave to the end of the T wave. ELECTROCARDIOGRAM (ECG). A graphic record of the heart s electrical currents. Pfizer Global Research and Development, Eastern Point Road MS 4083, Groton, Connecticut 06340, USA. Correspondence to B. F. bernard_fermini@ groton.pfizer.com doi: /nrd1108 The duration of the QT INTERVAL on the ELECTROCARDIOGRAM (ECG) is defined as the period between the beginning of the QRS COMPLEX and the end of the T wave. When corrected for individual heart rate, the QT interval is defined as corrected QT, or QTc (BOX 1). It is a reflection of ventricular ACTION POTENTIAL duration (APD) (FIG. 1), and represents the time during which the ventricles DEPOLARIZE, and REPOLARIZE. Numerous overlapping ionic currents contribute to determine the morphology and duration of ventricular APD. Rapid entry of sodium ions through selective sodium channels initiates the depolarization of the ventricles (phase 0) (FIG. 1), followed by a rapid repolarization through transiently activating and inactivating outward potassium channels (I to, phase 1). This is followed by a plateau phase (phase 2), which is mainly determined by the entry of calcium ions through L-type calcium channels. Repolarization, or phase 3, results from the inactivation of calcium channels, and the increase in net outward potassium currents carried mainly by the slow (I Ks ) and rapid (I Kr ) components of the delayed rectifier potassium channels. Inwardly-rectifying potassium channels (I K1 ) contribute to phase 3 repolarization and to the maintenance of the resting membrane potential (phase 4). In humans, I Kr seems to play a significant role in determining ventricular APD and repolarization, as congenital mutations of this channel are associated with a decrease in current amplitude and prolongation of the QT interval 1. A reduction of outward currents and/or an increase of inward currents will prolong APD, and might increase the propensity of developing early after-depolarizations (EADs) (FIG. 2). When generated in the presence of transmural heterogeneity in ventricular repolarization, EADs are believed to contribute to the generation of extrasystoles that can trigger TORSADES DE POINTES 2 (TdP), a potentially life-threatening ventricular tachyarrhythmia characterized by a distinct twisting morphology of the QRS COMPLEX around the isoelectric baseline (FIG. 3). Between June 1990 and March 2001, eight non-cardiovascular pharmaceuticals were removed from the market in the United States and elsewhere because of their propensity to delay cardiac repolarization, prolong the QT interval on the ECG, and cause TdP. At present, more than 50 non-cardiac drugs have been reported to significantly prolong the corrected QT interval and/or NATURE REVIEWS DRUG DISCOVERY VOLUME 2 JUNE

2 Box 1 QT Interval and correction formulas QRS COMPLEX A graphic representation of part of the cardiac cycle, specifically the heart s electrical impulse as it passes through the ventricles as seen on an electrocardiogram. ACTION POTENTIAL Change in voltage generated across the membrane of a nerve or muscle cell when the cell is activated by electrical, chemical or mechanical stimuli. The evaluation of the effect of a drug on the QT interval is a fundamental component of the required safety database, and the results of these analyses must be submitted in support of any new drug application. The determinant that mostly influences the QT interval is the heart rate (HR). As the QT interval has an inverse relationship to HR, the measured QT intervals are generally corrected for HR (QTc), or more precisely the preceding the RR interval (a measure of the time duration between two consecutive R waves on the QRS complex of the electrocardiogram. Accurate measurement and interpretation of the QT interval is complicated by the dynamic nature of this QT/RR interval relationship, which fluctuates constantly with HR changes, and is influenced by multiple, often related, factors such as age, gender, plasma electrolytes, diurnal fluctuations, physiological or pathological state and autonomic tone. To correct QT interval data for these changes in HR, correction formulas were initially proposed by both Bazett and Fridericia back in These mathematical functions attempt to normalize observed QT intervals for an RR interval of 1000 ms (60 beats per minute (bpm)) into comparable referenced QTc values. In clinical practice, the formulas of Bazett and Fridericia are the most widely used. In general, Bazett s over-corrects at elevated heart rates (that is, the corrected QT interval is greater than the measured QT interval), and under-corrects at heart rates below 60 bpm. So, many mathematical functions (both linear and non-linear regression analysis) have been derived over the years to more accurately correct the QT/RR interval for individuals or populations, under different physiological conditions. Even so, these approaches still allow for errors in the determination of the actual QT interval if physiological HR changes occur outside of the baseline QT/RR interval range used to define the correction formula. This latter point is particularly problematic when studying the effect of a drug on the QT interval and is often neglected. For instance, drugs that do not affect ventricular repolarization or the QT interval directly, but rather change the HR of patients treated by virtue of their therapeutic efficacy (for example, alleviating infections, improving tissue oxygenation and reducing hypertension), could erroneously be considered to alter the QTc because of the lack of overlap in the range of RR intervals defined at baseline, when compared with drug treatment. A thorough statistical evaluation of both baseline and post-drug QT/RR interval datasets across the entire range of the heart rates (especially those not overlapping) might be essential to insure the validity of the correction factor used for QTc determinations. So, it is necessary to select and define the most appropriate method for accurately and reliably measuring QT interval changes produced by medicinal products to avoid unwarranted regulatory concerns or underestimation of their potential to prolong the QT interval resulting from the use of inappropriately applied formulas for rate correction. DEPOLARIZATION A decrease in the electrical potential across a membrane, such as when the inside of a cell becomes less negative. REPOLARIZATION Recovery of the resting potential. TORSADES DE POINTES A form of polymorphic ventricular tachycardia characterized by QRS complexes that gradually rotate around the isoelectric baseline. Torsades de Pointes is associated with a long QT interval on the ECG and an absolute or relative bradycardia. ARRHYTHMIA A generalized term used to denote disturbances in heart rhythm. DYSRHYTHMIA Any abnormality in the rate, regularity or sequence of heart activity. ANTI-ARRHYTHMIC An agent that has the ability to decrease the incidence of arrhythmias. CHANNEL BLOCKER A compound that decreases the ability of charged atoms to pass through ion channels, thereby inhibiting the electrical activity of the cell. induce TdP 3 (TABLE 1). The potential for non-cardiac drugs to alter cardiac repolarization by prolonging the QT interval and to induce potentially fatal ARRHYTHMIAS, such as TdP, is now the second leading cause for withdrawing approved drugs from the market 4. Because of this, any evidence that a new drug prolongs the QTc interval is a significant development concern for pharmaceutical companies, as it raises the apprehension of TdP, and its subsequent regulatory approvability. Furthermore, the focus of interpretation of QTc prolongation by non-cardiovascular drugs by worldwide regulatory agencies has changed from one of noted side effect to that of life-threatening fatal outcome. Yet, although QTc prolongation can lead to serious cardiac DYSRHYTHMIA, the incidence of potentially lethal TdP due to treatment with compounds known to prolong the QT interval is relatively low. Using the antihistamine terfenadine (Seldane) as an example, approximately 429 serious cardiovascular events, including 98 deaths, were reported from the time of its introduction in the United States in 1985, until about 1996 (REF. 5). Terfenadine was withdrawn from the US market in By then, it had become the tenth most prescribed medication in the United States. A retrospective analysis showed that the number of adverse reactions with terfenadine was fewer than 0.25 per million defined daily doses sold 5. Overall, the background incidence rate of TdP differs depending on the population being examined. The most described incidence is with ANTI-ARRHYTH- MIC drugs, where it extends up to 8% for the class IA anti-arrhythmic quinidine 6. In addition, there is currently no conclusive evidence to show that drug-induced prolongation of APD or QT interval inevitably lead to TdP. The link between QT interval prolongation and TdP is seemingly very complex and affected by a host of influencing factors 7, including age, electrolyte imbalance (hypokalemia, hypomagnesemia, hypocalcemia), gender, disease states (cardiomyopathy, myocardial ischaemia and infarction, hypertension, hypothyroidism, diabetes, renal or hepatic dysfunction) and concomitant medications. Furthermore, not all drugs that prolong APD or the QT interval carry the same potential to induce TdP. For example, the calcium-channel BLOCKER verapamil, which is used for the treatment of hypertension, has been shown to prolong the QT interval in a manner that is linearly correlated to its plasma concentration 8, but there are few described cases of verapamil-induced TdP 9. The risk of arrhythmia seems to increase with increasing QTc. Data on QTc intervals of cases of TdP on a number of cardiac and non-cardiac drugs indicate that a QTc interval of >500 ms is considered a significant risk of induction of an arrhythmia Furthermore, available data suggest that in individual subjects, an increase of 60 ms in peak or maximum QTc interval over baseline is also predictive of a potential risk 12. As stated in a recent draft concept paper issued by the FDA s Center for Drug Evaluation and Research (CDER) and Health Canada s Therapeutic Products Directorate (see below), the FDA regards a mean 20 ms prolongation as worrisome. Approval of non anti-arrhythmic drugs with this liability would probably be denied, unless they offer very 440 JUNE 2003 VOLUME 2

3 ARRHYTHMOGENESIS Having the tendency to increase the incidence of arrhythmias. P Phase 0 (Depolarization) Sodium current (I Na ) Phase 4 (Resting membrane potential) Potassium current (I K1 ) clinically meaningful benefits. Compounds associated with a mean ms prolongation would face an uphill battle, and would need to show important therapeutic roles. A prolongation of the mean QT/QTc interval by 5 10 ms would result in increased scrutiny of the benefit and risks. Regulatory agencies are concerned that, for the majority of the QT prolonging drugs, their ARRHYTHMOGENIC potential was only recognized during later post-marketing surveillance and/or many months/ years after they were approved and used in the clinic. As a result, regulatory agencies will continue to rely on QT interval prolongation as a surrogate indication for TdP. Evolution of regulatory guidelines Beginning in the late 1980s, spontaneous case reports of cardiotoxicity related to use of the non-sedating H 1 -antihistamine terfenadine began to appear in the literature, at first following overdose 13, but then also with concurrent administration of ketoconazole 14. Terfenadine was shortly thereafter shown to cause TdP 14 and sudden death 15 by prolonging cardiac repolarization following inhibition of potassium currents 11 and, more specifically, the delayed rectifier potassium channel I Kr 16.Interestingly, terfenadine is metabolized rapidly by the isozyme P450 3A4 to its pharmacologically active carboxylic metabolite, and its plasma concentration does R Q S QRS interval Phase 1 (Rapid repolarization) Potassium current (I to ) QT interval Action potential duration T 1 mm 40 ms Phase 2 (Plateau) Calcium current (I Ca ) Phase 3 (Repolarization) Potassium currents (I Ks, I Kr ) Figure 1 Temporal correlation between action potential duration and the QT interval on the surface ECG. The surface electrocardiogram (ECG), which provides information on the electrical events occurring within the heart, is obtained by placing electrodes on the surface of the body. Typically, the P wave reflects atrial depolarization, the QRS complex reflects ventricular depolarization and the T wave is indicative of ventricular repolarization. The QRS complex is produced by the upstroke (phase 0) of the action potential. The isoelectric S T segment corresponds to the plateau (phase 2), whereas the T wave is indicative of ventricular repolarization (phase 3). The resting membrane potential corresponds to phase 4. The duration of the QT interval on the ECG is defined as the duration between the beginning of the QRS complex and the end of the T wave. It is a reflection of ventricular action potential duration, and represents the time during which the ventricles depolarize, and repolarize. Numerous overlapping ionic currents contribute to determining the morphology and duration of the ventricular action potential duration (see text). not reach detectable levels in the absence of any concurrent metabolic inhibitors. Prospective studies confirmed that its cardiac toxicity was associated with interactions with agents that impaired its metabolism (for example, ketoconazole, intraconazole, erythromycin, clarithromycin, troeandromycin, and grapefruit juice) or with its use in patients with significant liver disease. Following several modifications of its prescribing information, terfenadine was quickly replaced on the market by its carboxylic acid metabolite, fexofenadine, which did not block potassium channels but retained the same therapeutic benefits as the parent drug. Reports of similar effects with other non-cardiovascular agents began to be recognized and the benefits versus the risks associated with the use of some drugs were questioned 17.At that time, no regulatory guidance to the pharmaceutical industry existed for studying drugs that could affect cardiac repolarization. Then in 1997, the European Agency for the Evaluation of Medicinal Products (EMEA) was the first to issue a points to consider document from the Committee for Proprietary Medicinal Products (CPMP). This document outlined a series of experimental non-clinical and clinical models for assessing the potential for QT prolongation by non-cardiovascular agents 18. The non-clinical approaches emphasized in vitro electrophysiological studies examining action potentials in isolated cardiac tissues, such as Purkinje fibres and papillary muscle. The in vivo approaches focused on large animal assessment effects on blood pressure, heart rate, and more robust measurements of electrocardiogram intervals, including descriptions of morphology changes in T wave. Specific methods and magnitude of changes were noted in the document, opening the debate on the scientific basis and relevance of all these recommendations. During the past two years, regulatory scrutiny has continued to increase, with Health Canada issuing a detailed draft guidance document entitled Assessment of the QT Prolongation Potential of Non-anti-arrhythmic Drugs 19. This document lists all the current non-clinical and clinical methodologies to be used in screening new chemical entities (NCE) and recommends termination of development of NCEs if positive findings occur. More recently, a preliminary concept paper for clinical testing entitled The clinical evaluation of QT/QTc interval prolongation and pro-arrhythmic potential for non-antiarrhythmic drugs was issued by the FDA s CDER and Health Canada s Therapeutic Products Directorate, and was discussed as part of a joint workshop on QT prolongation offered in collaboration with the North American Society of Pacing and Electrophysiology 20, as well as reviewed by an Expert Working Group (EWG) of the International Conference on Harmonization (ICH). The ICH is a joint initiative involving both regulators and research-based industry as equal partners in the scientific and technical discussions of testing procedures that are required to ensure and assess the safety, quality and efficacy of medicines. There are six parties directly involved in the decision making process of the ICH, including representatives NATURE REVIEWS DRUG DISCOVERY VOLUME 2 JUNE

4 a b Ca 2+ Ca 2+ I Ca Ca 2+ EAD Ca 2+ K + I Kr K + Figure 2 Simplified representation of the effects of inhibiting repolarization and the development of early afterdepolarizations. The balance between inward and outward currents determines the morphology and duration of the action potential, and consequently the duration of the QT interval. Drug-induced inhibition of the I Kr current can delay repolarization, and prolong the action potential duration and the QT interval. Lengthening repolarization further delays the inactivation of calcium channels. The resulting late inflow of calcium contributes to the propensity of developing early after-depolarizations (EADs). Prolongation of the action potential also increases the amplitude of the intracellular calcium concentration transient, thereby promoting EADs. When generated in the presence of transmural heterogeneity in ventricular repolarization, EADs are believed to contribute to the generation of extrasystoles that can trigger Torsades de Pointes. HERG Human ether-a-go-go-related gene, the gene that encodes the α-subunit of the I Kr channel, a major determinant of human cardiac repolarization. ION CHANNEL Specialized pores in the membrane of cells that assist in controlling and transferring electrical impulses, called action potentials, in the cell. The function of the ion channel is to regulate the flow of sodium, potassium and calcium ions into and out of the cell. from the European Union, Japan and the United States. For each of the technical topics selected for harmonization, the ICH Steering Committee appoints an EWG to review the differences in requirements between the three regions, and to develop scientific consensus to reconcile those differences. A similar preclinical draft proposed by the ICH, described as S7B and entitled Guideline on safety pharmacology studies for assessing the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals is presently under review, and should be finalized by the end of this year 21. On the basis of the requirements of these guidelines, early identification of the risk of NCEs, or their metabolites, to induce QT prolongation has become an important focus point for the pharmaceutical industry. Preclinical assessment The current version of S7B provides guidelines on suitable levels and methods of preclinical testing in order to further determine the potential for a drug to produce QT prolongation, and they include the following: a review of the activities of other similar agents in the same chemical class or therapeutic group; the assessment from an ionic current assay that measures I Kr or HERG; results from an in vivo assay that measures indices of ventricular repolarization such as QT interval; and the assessment from a ventricular repolarization assay that measures action potential parameters in single or multicellular cardiac preparations. The guidelines suggest that in the event of positive signals in both the ION CHANNEL and in vivo assays, the repolarization assay is optional. However, if either or both assays yield a negative result, the repolarization assay should be performed. In the majority of cases, drugs that prolong the QT interval preferentially inhibit I Kr, the rapid component of the delayed rectifier potassium current, or HERG, the α-subunit of I Kr channels. Cardiac pro-arrhythmic toxicity of drugs resulting solely from the alterations of other ion channels has not yet been reported, but cannot be excluded. Likewise, drugs that cause QT prolongation because of their effects on ion channels other than I Kr might not have the same potential to induce TdP as those that do inhibit I Kr, but this remains to be established. Nonetheless, so far all drugs that cause TdP also inhibit the I Kr channel substantially at therapeutic concentrations. Moreover, wide ranges of chemical structures inhibit the HERG channel, relative to other voltage-dependent potassium channels. Recent studies by Mitcheson and colleagues 22 have revealed that the inner cavity of the HERG channel could be much larger than most voltage-gated potassium channels, which makes it more likely to trap small molecules of different classes. Using homology modelling of the HERG channel, on the basis of the crystallographic structure of the bacterial potassium channel KcsA, they were able to show that each of the four subunits forming the HERG channel also contain two aromatic residues (Tyr652 and Phe656) that face the inner cavity of the pore region, which undoubtedly contributes to many different chemical structures docking and binding to the inner mouth of the channel. With increasing numbers of drugs available for development and a heightened concern for drug safety, 442 JUNE 2003 VOLUME 2

5 Table 1 Marketed drugs that prolong the QT interval and/or might cause Torsades de Pointes Category Drugs Antiarrhythmics Ajmaline, almokalant*, amiodarone*, azimilide, bretylium, clofilium, disopyramide*, dofetilide*, flecainide, ibutilide*, procainamide*, propafenone, quinidine*, sematilide and sotalol* Antihypertensive Ketanserin Calcium channel blockers Bepridil*, isradipine and nicardipine Vasodilators Lidoflazine, prenylamine and papaverine Antidepressants Amitriptyline, citalopram, clomipramine, desipramine, fluoxetine, imipramine, maprotiline, nortriptyline, venlafaxine and zimeldine Antipsychotics Chlorpromazine, fluphenazine, haloperidol*, mesoridazine, pimozide*, prochlorperazine, quetiapine, risperidone, sultopride, sertindole*, thioridazine*, timiperone, trifluoperazine and ziprazidone Antianxiety agents Doxepin and droperidol Antimanic agent Lithium Sedative/hypnotic Chloral hydrate Anticancer agents Arsenic trioxide*, amsacrine, doxorubicin and zorubicin Anti-infectives Amantadine, amphotericin, ciprofloxacin, chloroquine*, clarithromycin*, clindamycin, erythromycin*, fluconazole*, foscarnet, grepafloxacin*, halofantrine*, itraconazole, ketoconazole, Levofloxacin, mefloquine, miconazole, moxifloxacin, pentamidine*, sparfloxacin*, spiramycin, quinine, trimethoprim sulfamethoxazole and troleandomycin Antihistamines Astemizole*, azelastine, clemastine, diphenhydramine, ebastine, hydroxyzine and terfenadine* Anticonvulsants Felbamate and fosphenytoin Diuretics Indapamide*, triamterene* and moexipril/hctz Hormones Octreotide and vasopressin Immunosuppressive Tacrolimus* Migraine serotonin Naratriptan, sumatriptan and zolmitriptan receptor agonists Muscle relaxant Tizanidine Narcotic detoxification Levomethadyl Respiratory Salmeterol sympathomimetic Miscellaneous agents Cisapride*, budipine, furosemide, probucol and terodiline* *Denotes a definitive association with drug-induced Torsades de Pointes. VOLTAGE-CLAMP A procedure used during the study of ion channels that has the effect of keeping the voltage produced on a membrane (due to ion movement) unchanged. It allows the experimenter to measure only current produced by the ion movement through the channel. the need for a high-throughput screening (HTS) assay for the HERG channel is now a necessity in the pharmaceutical industry. Many existing methods are used to study the preclinical effects of lead compounds on the HERG channel, and some of the methods used to address the requirements of the S7B document are briefly described below, and are summarized in BOX 2. Voltage-clamp assay Electrophysiological assays, and more specifically the whole-cell VOLTAGE-CLAMP technique, are extensively used to study the effects of compounds on the HERG channel. No other method can provide such high quality and physiologically relevant data of precise and detailed activity of ion channel function. However, this approach is technically difficult and offers low throughput. Recently, HTS planar patchclamp technology has been developed, and should allow hundreds to thousands of patches per day to be obtained in the whole-cell mode 23. This technology replaces the traditional glass patch electrode with a planar array of recording interfaces miniaturized on the surface of either a silicon, polymer or glass substrate. Nonetheless, it remains to be determined whether this technology can offer the same quality and flexibility as obtained by the standard approach, and whether it is cost effective. Binding assay Radioactive dofetilide binding has been used to study the effects of compounds on the HERG/I Kr channel 24,25. Generally, binding assays are considered non-functional because they detect the ability of a compound to displace a high affinity ligand (in this case 3 H-dofetilide) from the channel, rather than the ability of the compound to alter channel function. This approach also provides no information on agonistic or antagonistic effects of a drug. Furthermore, it is subject to false negatives because simple compound channel binding will not reflect the affinity of a compound for a given state of the channel (that is, open, closed or inactivated). This, in turn, could result in discrepancies between dofetilide-binding studies and results obtained in the standard patch-clamp assay. Finally, the dofetilide-binding assay assumes that compounds that displace dofetilide bind to the same radioligand-binding site, which might not always be the case. Overall, the dofetilide-binding assay provides high throughput, and is useful in the early assessment of NATURE REVIEWS DRUG DISCOVERY VOLUME 2 JUNE

6 QRS T P Pause Long QT Torsades de Pointes Figure 3 Electrocardiogram of Torsades de Pointes. Torsades de Pointes is characterized by an abnormally prolonged QT interval, the presence of morphological changes on the T wave, and the occurrence of rapid polymorphic ventricular tachyarrhythmias with a distinctive twisting morphology of the QRS complex around the isoelectric baseline. The initiation of the arrhythmia is characterized by a short-long-short sequence, followed by a pause and a normal beat with a prolonged QT interval. Adapted with permission from REF. 39 (2002) Annual Reviews. binding potential at the HERG channel. However, the assay does not provide a functional endpoint. Therefore, in terms of generating information regarding potential QT interval effects, this assay is primarily helpful in identifying the candidates that warrant further investigation. Fluorescence assay In general, fluorescence-based methods used to study the effects of drugs on the HERG channel use voltagesensitive dyes to measure membrane potential changes of fluorescence signals. For example, in fluorescence resonance energy transfer (FRET)-based assays, different negatively charged membrane-soluble oxonol dyes are used as voltage-sensing FRET acceptors, and coumarin-tagged phospholipids integrated in the outer leaflet of the membrane are used as donors. Ratiometric changes of FRET responses are observed following membrane hyperpolarization or depolarization 26. Unfortunately, because the changes in fluorescence are relatively slow (measured in seconds), these assays are best suited to the measurement of steady-state changes in membrane potentials. Furthermore, compound fluorescence and compound dye interaction are an important source of artefact, resulting in a high rate of false positives. Finally, fluorescence-based methods are typically less sensitive by one-log value or more than the patch-clamp technique. Nonetheless, these systems adopt at least the 348-well format, are relatively easy to set up, and can achieve HTS status. Assessment of ventricular repolarization The Purkinje fibre APD assay measures action potentials on isolated Purkinje fibres obtained from the myocardium of animal species demonstrating a cardiac ionic profile similar to that of the human heart. The tissues from dog, rabbit and guinea pig have been used for this purpose. This test is recommended by the CPMP and is useful because it allows drug effects on native cardiac channels to be studied. In addition, it can be used to detect effects mediated through ion channels other than the I Kr channel. Therefore, it is likely that candidates with multiple ion channel effects can be characterized in this preparation. In a recent study, Gintant et al. 27 investigated the effects of 12 drugs on APD changes in canine and porcine Purkinje fibres, under physiological conditions. On the basis of concentration-dependent APD prolongation and reverse rate-dependent effects, this model detected six of the seven drugs qualitatively but not quantitatively linked to drug-induced long QT and TdP in humans, and cleared all five drugs not associated with repolarization abnormalities. Nonetheless, this assay is labour intensive, has a very low throughput, and failure to see APD prolongation in this model cannot exclude pro-arrhythmic toxicity or the risk of TdP in humans 28. In vivo QT animal model In this assay, the ECG effects of a drug candidate are monitored in either conscious or anaesthetized guinea pigs, dogs or monkeys. The preferred methodology is the use of unrestrained animals implanted with telemetry systems 29 so that autonomic tone and stress influences are minimized. The heart rate, blood pressure and ECG are continuously recorded over a range of escalating doses in order to detect the occurrence of any arrhythmias or QT interval prolongation 30.Alternatively, the effects of compounds on the ECG and monophasic action potentials can be studied in the anaesthetized state. This model is often used when adverse behaviour or dose-limiting emesis are produced by the drug candidate that interfere with the determination of the pharmacological effect on the QT interval. One important advantage of the anaesthetized model is that ventricular electrical pacing can be used to control the heart rate, eliminating the need to correct the QT interval for heart rate 29,30. However, one of the disadvantages of using anaesthetized animals is that they cannot be used repeatedly. Furthermore, because parenteral routes of administration are necessary, the metabolic profile might not necessarily reflect the clinical profile when normally given orally in the conscious state (that is, no first-pass metabolism through the liver). Another limitation is that most anaesthetics interfere with cardiac conduction and might interact with the drug candidate, which can complicate interpretation of the results. Unless use of another species can be justified, usually on the basis of a metabolic profile closer to humans, the dog cardiovascular ECG analysis is most often considered a requirement before first-in-man testing. Although this assay can provide much information about the cardiac safety of a drug candidate, including the potential effects of any active metabolites, it has a low throughput and is extremely labour intensive. 444 JUNE 2003 VOLUME 2

7 Box 2 Summary of preclinical assays HERG functional assay In vitro assay that measures change in ionic current using the voltage-clamp technique Low throughput, but highly specific assay for the most common mechanism of druginduced QT prolongation High throughput voltage-clamp assays using planar technology have recently become available. This approach eliminates the need for electrodes, microscopes and micromanipulators and is reported to potentially offer the same quality of data as that obtained using standard voltage-clamp techniques. This new technology remains to be fully evaluated 3 H-dofetilide binding assay In vitro assay that assesses the ability of a compound to displace 3 H-dofetilide from HERG channels Will not identify test substances that bind at sites other than the radioligand-binding site High throughput, but does not provide functional data Fluorescence-based assay In vitro assay that measures membrane potential changes of fluorescence signals as a result of changes in ionic fluxes through selected channels Temporal resolution is slow compared with the ion channel properties measured using the voltage-clamp technique due to the redistribution of the dye(s) upon changes in membrane potential High throughput, but less sensitive than standard voltage-clamp results Purkinje fibre action potential duration assay In vitro assay in which action potentials are measured in the Purkinje fibres from animals Low throughout; provides useful data regarding drug effects on action potentials and several native cardiac channels Candidates with multiple ion channel effects might be identified in this assay Less sensitive to HERG-blocking drugs than channel ion assay Differences in the make-up of channels between animal and human might affect its ability to predict clinical outcome Dog cardiovascular electrocardiogram analysis In vivo assay that measures effects of drug candidate on the electrocardiogram of unrestrained, conscious or anaesthetized dogs Integrates effects of drugs at the whole-animal level Usually a requirement before first-in-man testing Human metabolite might be produced in this assay Low throughput, expensive, time consuming and labour intensive PRO-ARRHYTHMIA The more frequent occurrence of pre-existing arrhythmias, or the appearance of new arrhythmias. Pro-arrhythmia is a side effect associated with the administration of some existing anti-arrhythmic drugs, as well as drugs for other indications. BRADYCARDIA Slowing of the heart rate, usually defined as less than 60 beats per minute. Finally, although the use of PRO-ARRHYTHMIA models has been proposed in the draft document from Health Canada 19 (for example, methoxamine-treated rabbits, BRADYCARDIA induced by atrioventricular block or vagal stimulation in dogs, conditions of hypokalemia), the S7B document recognizes that modelling of clinical conditions in which pharmaceuticals elicit arrhythmia in these models is complicated. Although these models might predict arrhythmias in a qualitative manner, they have not been shown to accurately reflect the quantitative clinical incidence of compounds that are known to induce arrhythmias in humans, and are not presently recommended for routine evaluation 21. Summary of preclinical assays Each of the preclinical methods has different advantages and limitations. Most of the in vitro tests give results on a drug s potential to interact with cardiac ion channels rapidly; however, the in vitro approach alone is not sufficient to evaluate a candidate s potential to prolong the QT interval, because some factors that could influence the expression of pharmacological properties are only present under in vivo conditions 30. Compared to the in vitro assays, the in vivo studies are more applicable for assessing a candidate s effect on the QT interval, because they are known to replicate more closely the conditions of the clinical use of the drug 29.However,in vivo studies are generally more expensive and labour intensive than the in vitro tests, and the results can sometimes be more complicated to interpret because of other factors that are present in vivo that might influence the QT interval 30. In determining a plan for a testing sequence, these advantages and limitations should be considered. It should be emphasized that, individually, none of the methods described is superior to any other in its capacity to predict QT prolongation effects in clinical settings. All have the propensity to produce both false-positive and false-negative results. A positive drug effect in any or all of these assays might not necessarily translate to a clinical effect, especially if the clinical concentration is several times lower than the concentration shown to prolong QT in a preclinical model. Finally, it is most probable that a drug that does not produce a signal in any of the preclinical assays, within a 30-fold margin of the clinically relevant concentration 31, is unlikely to produce a QT prolongation effect. Although safety margins should be viewed as a continuum, an extensive review of reported preclinical data in relation to clinical outcome indicates that a 30-fold margin between the effective therapeutic plasma concentration of a drug and its HERG/I Kr IC 50 seems to be a line of demarcation between the majority of drugs associated with TdP, and those that are not 31. Clinical studies Clinical studies for the assessment of the potential for drugs to prolong the QT interval should be initiated as early as possible in the NCE development programme and followed through to the latest stages of product registration. Robust effects on the ECG can often be detected early in small, carefully designed studies in normal healthy volunteers participating in Phase I development programmes, or in specific high-risk patients with a targeted disease in Phase II. In order to improve the chances for early detection of these findings, a number of new clinical protocols for capturing and assessing the data are being explored. However, these are not easily obtained or even agreed upon, because the numerous natural causes of repolarization changes must be carefully separated experimentally from the drug-induced causes that are being assessed concurrently in the NCE study population. Nonetheless, it must be recognized that even the best planned clinical trial will have limitations because the number of patients exposed to the drug in these trials might not be large enough to detect a relatively rare, but potentially fatal, risk. So the design of the clinical protocol to isolate and minimize the variability in the ECG data collection is essential for accurate interpretation of the risk of the NCE alone and in combination with other influencing NATURE REVIEWS DRUG DISCOVERY VOLUME 2 JUNE

8 THERAPEUTIC INDEX Experimental index of the relative safety of a compound, constituting the ratio between the toxic dose (numerator) and the effective dose (denominator) of the compound. factors that the patient might ultimately encounter. Beyond the adequate acquisition of high-quality ECG data lie the equally complicated issues of reading and interpreting the ECG data. With the use of high-speed computers to collect digital ECG data, the question has become whether the computer programs are accurate enough to interpret precise interval reading, or whether all the thousands of ECG data tracings generated still need to be manually over-read by a trained ECG cardiologist. Cardiologists are capable of alerting the NCE sponsor of abnormalities in the ECG morphology more readily than current computer programs. However, the computers can process much more data in a more consistent manner that can ultimately enhance the accuracy of the interpretation of the ECG interval changes. The FDA has also initiated an effort to suggest that NCE sponsors file ECG tracings in a common digital format 32 that can be independently reviewed by the agency to refute or support any interpreted findings. Impact on the cost of developing new drugs Developing a compound with a preclinical QT signal in any one of the studies assessing its cardiac safety is not inconceivable, provided a carefully planned clinical development programme is adopted. However, millions of dollars in additional research and development costs are at stake for such NCEs. To put some of the financial costs in perspective, an internal examination of the Pfizer Clinical Sciences budget indicates that a typical Phase I study for a single compound with a preclinical QT signal approximates nearly US $1 million. Of this, 22% of the total clinical cost is spent on ECG over-reads to determine safety. If the drug is advanced to Phase II for determination of efficacy, the cost for ECG determination of THERAPEUTIC INDICES (a measure of the relationship of the concentration used in the clinical trial versus the onset of significant QT prolongation) increases almost sixfold for a single drug. Pfizer last year initiated close to 50 of these early clinical trials to proceed in parallel. Assuming reported attrition rates of approximately 80 90% for early development candidates, and about 10% for commercial successes 33, the up-front costs required to appropriately address issues related to QT prolongation become very significant. These costs eventually compel sponsors to carefully assess which diseases can be targeted and/or medications can ultimately be developed, despite the need for therapies. Impact on the availability of future new drugs The current regulatory guidelines scrutinize all HERG blockers that delay repolarization to cause QT prolongation. Although, until now, all drugs that have been removed from the marketplace due to TdP have been shown to be HERG blockers, the opposing correlate that all HERG blockers cause TdP has not been well established. As stated previously, a direct link between QT prolongation and arrhythmogenesis is still unclear. Nonetheless, faced with the inability to distinguish between safe versus potential torsadogenic HERG blockers, regulatory agencies have chosen to set guidelines for the development of NCEs with HERG-blocking activity. Pharmaceutical companies must now assess QT prolongation liability using a number of different assays, which inevitably adds to research costs. Once it is established that a drug has the potential to prolong the QTc interval at clinically relevant concentrations, a company must evaluate the approvability, labelling implications and consequent commercial fallout of such an NCE against the benefit of continuing development. Given these high hurdles, it is not inconceivable that several NCEs will not be developed. This, in turn, could lead to a reduction in the number of novel drugs reaching the market in the near future. To challenge this assumption, let us consider how a present marketed compound would perform under the present guidelines. Fluoxetine (Prozac; Eli Lilly) is a drug that revolutionized the treatment of depression when it was first approved for use in It has since become a commercially successful breakthrough (worldwide exposure estimated to be more than 38 million patients, circa 1999), with a low incidence of arrhythmogenic liability. However, a more recent examination of this drug using the new guidelines indicates that fluoxetine is a potent inhibitor of the HERG channel 34 (IC 50 = 1.5 µm), with less than a 30-fold index over its clinical-use concentration. Furthermore, although fluoxetine is considered to be safe, effective and lacking serious cardiovascular side effects, syncope 35, QT prolongation and unexpected deaths 38 were reported in patients taking this drug. Therefore, one can question whether companies would, at present, want to pursue the development of a drug with a similar profile, and/or whether special precautions would be imposed through labelling, potentially affecting its availability to patients. Conclusion The early identification of the risk of NCEs, or their metabolites, to induce QT prolongation has become a principal goal for the pharmaceutical industry, and an integral part of the development process of every new drug. Preclinical evaluation of TdP has progressed significantly over the past few years, and several different assays are available to help identify potential cardiac toxicity of compounds. However, each of these offers advantages and limitations. From the regulatory perspective, the evaluation of QT interval prolongation will continue to be used as assessment for TdP, in the absence of a better clinical surrogate, and any new drug with a known or suspected QT prolongation potential will require extensive preclinical and clinical testing. This, in turn, could impact the number of new and innovative drugs available in the future. Ultimately, the careful planning of clinical studies, and the formation of an integrated assessment of the risk/benefit ratio of a drug that prolongs the QT interval, will weigh heavily in its approvability, especially when the competitive landscape has compounds available with a better cardiac safety profile. There is no doubt that the level of awareness and understanding of the problem of drug-induced QT interval prolongation has progressed tremendously over the past few years, and one can hope that some level of resolution might be achieved in the near future. 446 JUNE 2003 VOLUME 2

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R. & Mendez-Castrillón, J. Fluoxetine-induced QTU interval prolongation, T wave alternans and syncope. Int. J. Cardiol. 65, (1998). 37. Varriale, P. Fluoxetine (Prozac) as a cause of QT prolongation. Arch. Inter. Med. 161, 612 (2001). 38. Spier, S. A. & Frontera, M. A. Unexpected deaths in depressed medical inpatients treated with fluoxetine. J. Clin. Psychiatry 52, (1991). 39. Roden, D. M., Balser, J. R., George, A. L. & Anderson, M. E. Cardiac ion channels. Annu. Rev. Physiol. 64, (2002). Acknowledgements The authors would like to acknowledge the help of Joyce VanWinkle in providing some of the clinical information and Mike Perkins for his thorough review of the manuscript and many insightful suggestions. Online links FURTHER INFORMATION Encyclopedia of Life Sciences Cardiac arrhythmias Access to this interactive links box is free online. NATURE REVIEWS DRUG DISCOVERY VOLUME 2 JUNE