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articles nature publishing group Covariate Analysis of QTc and T-Wave Morphology: New Possibilities in the Evaluation of Drugs That Affect Cardiac Repolarization C Graff1, JJ Struijk1, J Matz2, JK Kanters3,4,5, MP Andersen1, J Nielsen6 and E Toft7 This study adds the dimension of a T-wave morphology composite score (MCS) to the QTc interval–based evaluation of drugs that affect cardiac repolarization. Electrocardiographic recordings from 62 subjects on placebo and 400 mg moxifloxacin were compared with those from 21 subjects on 160 and 320 mg d,l-sotalol. T-wave morphology changes, as assessed by ΔMCS, are larger after 320 mg d,l-sotalol than after 160 mg d,l-sotalol; and the changes associated with 160 mg d,l-sotalol are, in turn, larger than those associated with moxifloxacin and placebo. Covariate analyses of ΔQTc and ΔMCS showed that changes in T-wave morphology are a significant effect of d,l-sotalol. By contrast, moxifloxacin was found to have no significant effect on T-wave morphology (ΔMCS) at any given change in QTc. This study offers new insights into the repolarization behavior of a drug associated with low cardiac risk vs. one associated with a high risk and describes the added benefits of a T-wave MCS as a covariate to the assessment of the QTc interval. The relationship between the QTc-prolonging property of a drug and the development of torsade de pointes (TdP) ventricular tachycardia is not well defined.1–3 Although QTc prolongation may be associated with an increased risk of arrhythmia, no threshold for QTc prolongation can be used to reliably identify a clinically significant increased risk of TdP. In general, however, the risk of arrhythmia appears to increase with the extent of QTc prolongation. It has been suggested that TdP is unlikely to occur with the use of drugs that prolong the mean QTc (in thorough QT studies) by <5 ms and that the risk of TdP is substantially higher for a prolongation >20 ms.4 In a cohort of patients with inherited long-QT syndrome, there was a 5% exponential increase in the relative risk of a cardiac event for every 10-ms increase in QTc duration beyond 440 ms.5 Whether such data can be generalized to indicate the proarrhythmic potential of drugs remains unclear. It is certainly true that extrapolation can be confounded by the fact that drugs that prolong the QTc interval to the same extent sometimes result in differing incidences of TdP. Moreover, drugs that are associated with a similar incidence of TdP do not necessarily prolong the QTc interval to equivalent extents.6,7 These observations constitute an obvious challenge for characterization of the safety of drugs during development and for the reassessment of the proarrhythmic potential of existing drugs because, although the safety profile of a compound is critically dependent on its QTc-prolonging property, this is not the only relevant factor when evaluating risk of arrhythmia. Evidence suggests that other electrocardiographic indexes, such as the morphology of the T-wave, may also contribute in important ways to the evaluation of cardiac safety. Abnormally shaped T-waves frequently appear even in the absence of overt QTc prolongation,8 and important abnormalities of the repolarization sequence may not be identified by the QT interval, which characterizes only the total duration of depolarization and repolarization. Logistic regression modeling has shown that T-wave morphology can improve the characterization of patients with a history of TdP;9 also, the detection of impaired adaptation of the T-wave amplitude to changes in heart rate has been useful in identifying drug-induced repolarization changes.10 T-wave morphology has been shown to discriminate between KCNQ1 (KvLQT1) and KCNH2 (hERG) mutations in the congenital long-QT syndrome, even with comparable QTc intervals.11,12 A study of acquired bradyarrhythmias demonstrated that an LQT2-like morphology predicts TdP, independent of the QTc interval.13 Our previous work has shown that characteristics of 1Medical Informatics Group (MI), Department of Health Science and Technology, Aalborg University, Aalborg, Denmark; 2H. Lundbeck A/S, Copenhagen, Denmark; 3Department of Cardiology P, Gentofte University Hospital, Gentofte, Denmark; 4Danish National Research Foundation, Centre for Cardiac Arrhythmia (DARC), Laboratory of Experimental Cardiology, University of Copenhagen, Copenhagen, Denmark; 5Department of Cardiology S, Aalborg Hospital, Aarhus University Hospitals, Aalborg, Denmark; 6Unit for Psychiatric Research, Aalborg Psychiatric Hospital, Aarhus University Hospitals, Aalborg, Denmark; 7Center for Sensory Motor Interaction (SMI), Department of Health Science and Technology, Aalborg University, Aalborg, Denmark. Correspondence: C Graff ([email protected]) Received 30 December 2009; accepted 4 March 2010; advance online publication 19 May 2010. doi:10.1038/clpt.2010.51 88 VOLUME 88 NUMBER 1 | July 2010 | www.nature.com/cpt articles T-wave morphology, such as those seen in congenital LQT2, can be used as sensitive descriptors of repolarization abnormality in this syndrome.14 These T-wave characteristics have also been shown to be independent of heart rate15 and to be more sensitive than QTcF (Fridericia’s correction) to repolarization changes induced by various drugs.16–18 In light of such evidence, it is likely that electrocardiographic repolarization indicators other than QTc may contribute importantly to drug safety evaluation. Conceivably, the analysis of QTc prolongation in drug studies could be paralleled by quantitative evaluation of changes from baseline in T-wave morphology, but there are no systematic studies demonstrating the association between quantitative measures of T-wave morphology and QTc prolongation. This study of QTc intervals and T-wave morphology characteristics from placebo, moxifloxacin, and d,l-sotalol data represents a first step in that direction. The antibiotic drug moxifloxacin has a favorable cardio vascular safety profile and is recommended as a positive control in thorough QT studies. Only a handful of moxifloxacin-induced TdP occurrences have been reported in recent literature, and in each case confounding factors were present.19–22 No clinical study has ever demonstrated an increased risk of serious cardiac events related to moxifloxacin. In contrast, the antiarrhythmia drug d,l-sotalol has a less favorable safety profile with a reported incidence of associated TdP between 1.8 and 4.8%.23,24 In the present study, this difference in risk profiles between moxifloxacin and d,l-sotalol is indicated not only by the differences in QTc changes from baseline, but even more so by the dissimilarity between the two drugs with respect to T-wave morphology vs. QTc profiles. Results Effects of drugs on QTcF and T-wave morphology A single oral dose of d,l-sotalol had pronounced effects on cardiac repolarization with considerable prolongation of the QTcF interval and substantial T-wave morphology changes (Figures 1 and 2). Modest repolarization effects followed a single oral dose of 400 mg moxifloxacin. Administration of placebo had no effect on QTcF or T-wave morphology. z-Score equivalents for QTcF and morphology composite score (MCS) indicated that d,l-sotalol had a larger impact on T-wave morphology than on the QTcF interval. The difference between peak z-scores for MCS and QTcF was 2.5 for 320 mg d,l‑sotalol (95% confidence interval (CI), 1.26 to 3.66), Table 1). The peak z-score for MCS after 160 mg d,l-sotalol was similar to the peak z-score for QTcF after a 320 mg dose (95% CI, −1.63 to 0.45, P = 0.25). After the administration of moxifloxacin, there was no indication of a larger repolarization effect on T-wave morpho logy as compared with the QTcF interval, as evidenced by the similarity of peak z-scores (95% CI, −0.54 to 0.19, Table 1). The difference between the two drugs (d,l-sotalol and moxifloxacin) was greater for T-wave morphology than for the QTcF interval. For QTcF, the maximum effect of 160 mg d,l-sotalol was 4 times greater than the effect of 400 mg moxifloxacin and 6 times greater than that of 320 mg d,l-sotalol (Table 1). In contrast, for MCS, the effects of 160 mg d,l-sotalol were seven times greater than those of 400 mg moxifloxacin and 15 times greater than those of 320 mg d,l-sotalol. Covariate effects on QTc and T-wave morphology The slope of the graph representing the relationship between QTcF interval prolongation and change in T-wave morphology was steeper after d,l-sotalol (both doses) than after 400 mg moxifloxacin (Figure 3). Parabolic regression provided better estimates of ΔMCS vs. ΔQTcF in the case of d,l-sotalol than did linear regression lines (comparison of fits: F-ratio = 9.60, P < 0.01), thereby indicating that a linear model would be incorrect. For moxifloxacin, a parabolic fit to data had the smallest error, but the difference between the parabolic and linear models was not significant (F-ratio = 0.58, P = 0.45). The simpler linear model was therefore chosen for moxifloxacin. For placebo data, the quadratic term was not significant, and therefore a linear model was used. The two doses of d,l-sotalol had significantly different morphology–duration profiles (ΔMCS vs. ΔQTcF) that could not be modeled by a common fit for pooled data (individual fits vs. pooled fit: F-ratio = 5.45, P < 0.01). Therefore, across the spectrum of QTcF changes from baseline, a 320 mg dose 400 mg moxifloxacin 1h 2h 3h 4h 160 mg D,L-sotalol 1h 320 mg D,L-sotalol 2h 3h 2h 3h 4h 1h 4h 0.25 mV 100 ms Figure 1 ECG tracings in an individual subject after 400 mg moxifloxacin compared with ECGs from an individual subject following treatment with d,l‑sotalol 160 and 320 mg. T-waves during the first 4 h of treatment are shown. ECG, electrocardiogram. Table 1 Maximum changes from baseline (95% CI) in ΔQTcF and ΔMCS for placebo, moxifloxacin and d,l-sotalol by standard units and their z-scores ΔQTcF Unit Placebo 400 mg moxifloxacin ΔMCS P value ms z-Score MCS score z-Score z-Scorea 1.8 (−0.9, 4.4) 0.11 (−0.05, 0.27) 0.01 (−0.02, 0.05) 0.08 (−0.09, 0.26) 0.82 9.4 (6.5, 12.3) 0.59 (0.41, 0.76) 0.07 (0.02, 0.12) 0.41 (0.12, 0.70) 0.34 160 mg d,l-sotalol 35.1 (26.0, 44.1) 2.18 (1.62, 2.75) 0.53 (0.38, 0.68) 3.01 (2.16, 3.86) 0.05 320 mg d,l-sotalol 57.9 (47.5, 68.2) 3.60 (2.96, 4.25) 1.07 (0.88, 1.26) 6.06 (5.00, 7.12) <0.001 CI, confidence interval; MCS, morphology composite score. aP value comparing z-scores for ΔQTcF with ΔMCS (paired Student’s t-test). Clinical pharmacology & Therapeutics | VOLUME 88 NUMBER 1 | july 2010 89 articles a 320 mg D,L-sotalol 160 mg D,L-sotalol 400 mg moxifloxacin Placebo 1.2 1.0 320 mg D,L-sotalol 160 mg D,L-sotalol 400 mg moxifloxacin Placebo 60 50 40 0.6 30 0.4 20 0.2 10 0 0 0.51 2 3 4 6 8 10 12 14 16 0.5 1 2 3 4 6 Time postdose (h) 8 10 12 14 16 Time postdose (h) d 6 6 4 4 2 2 0 0 0.5 1 2 3 4 6 8 10 12 14 16 0.5 1 2 3 Time postdose (h) 4 6 8 10 12 14 ∆z-score (QTcF) ∆z-score (MCS) c ∆QTcF (ms) ∆MCS 0.8 b 16 Time postdose (h) Figure 2 Time-matched changes from baseline for (a) T-wave morphology and (b) QTcF, and (c,d) their z-score equivalents. Dotted lines in c and d indicate maximum z-scores for MCS after d,l-sotalol and moxifloxacin. All curves show the mean difference from baseline for all study subjects ±1 SE. MCS, morphology composite score. of d,l-sotalol had a greater effect on T-wave morphology than a 160 mg dose did. In contrast, no such difference was found between placebo and the 400 mg dose of moxifloxacin. The common regression line for ΔMCS vs. ΔQTcF with this data indicated that placebo and 400 mg moxifloxacin had equivalent morphology–duration descriptions (individual fits vs. pooled fit: F-ratio = 0.59, P = 0.62). The slopes for moxifloxacin and placebo were similar (Figure 3, t = 1.46, P = 0.14), and the small overall elevation of MCS over the range of QTcF values for moxifloxacin was not significant (t = 0.04, P = 0.97). This observation suggests that comparable QTcF prolongations for placebo and 400 mg moxifloxacin were accompanied by equal degrees of T-wave morphology changes. For moxifloxacin, the morphology–duration slope was steeper in female subjects (0.005; 95% CI, 0.003–0.007) than in male subjects (0.003; 95% CI, 0.002–0.004), and the regression line was elevated in female subjects relative to male subjects in the range of QTcF prolongations above −8 ms. Consequently, for any given prolongation of QTcF, the associated T-wave morphology change in female subjects was generally more pronounced than in male subjects. Bin assessment of modest drug effects Over the range of small QTcF prolongations from baseline, drug-induced changes in T-wave morphology were significantly greater in subjects who received d,l-sotalol, as compared with subjects who received moxifloxacin (Figure 4). For T-wave morphology, a vertical offset was present between moxifloxacin and d,l-sotalol but not between moxifloxacin and placebo. Notably, there was a significant vertical offset in bin 1 for both doses of d,l-sotalol, indicating effects on T-wave morphology 90 but no effect on the QTcF interval. Moreover, the difference between the moxifloxacin group and the d,l-sotalol group with respect to drug-induced T-wave morphology changes increased with rising prolongation of the QTcF interval. Noticeably, the bin 3 (ΔQTcF, >15–25 ms) group receiving 400 mg moxifloxacin had T-wave morphology changes that were smaller than those in the bin 1 (ΔQTcF, −5 to 5 ms) groups receiving 160 and 320 mg d,l-sotalol (ΔMCS400 mg moxifloxacin, Bin 3 = 0.07 vs. ΔMCS160 mg d,l-sotalol,Bin1 = 0.11, P = 0.02, and vs. ΔMCS320 mg d,l-sotalol, Bin 1 = 0.19, P < 0.01). Discussion There is a considerable body of data to support the assertion that drug-induced QTc prolongation is an unreliable surrogate of a drug’s proarrhythmic potential. As a consequence, complementary repolarization indexes that can be used in characterization of drug-induced repolarization changes during the development phases of a new compound and for reassessment of existing compounds are of interest. In this study of moxifloxacin and d,l-sotalol, we showed that a composite marker of LQT2-like T-wave morphology can be used with QTc in a covariate analysis to provide information on repolarization properties of the two drugs that cannot be revealed by analyzing QTc alone. Peak drug effects on QTc and T-wave morphology The d,l-sotalol-induced effects on the morphology of electrocardiographic T-waves were greater than the corresponding changes in QTcF. Moxifloxacin, on the other hand, did not induce prominent complex repolarization patterns. The two drugs were found to be more dissimilar with respect to T-wave VOLUME 88 NUMBER 1 | July 2010 | www.nature.com/cpt articles 2.0 320 mg D,L-sotalol 0.5 1.5 400 mg moxifloxacin 400 mg moxifloxacin Placebo ∆MCS 160 mg D,L-sotalol Placebo 0.4 1.0 0.3 ∆MCS 0.5 0.2 0 0.1 −40 −20 0 20 40 60 80 100 ∆QTcF (ms) 0 2.0 Bin 1 1.5 −5 160 mg D,L-sotalol 5 10 Bin 3 15 20 25 ∆QTcF (ms) Placebo 1.0 ∆MCS 0 Bin 2 Figure 4 ΔMCS vs. ΔQTcF categorized in bins of change in QTcF by −5 to 5 ms, >5–15 ms, and >15–25 ms. Horizontal and vertical error bars indicate 95% confidence intervals for the mean. Dotted lines show regression models. MCS, morphology composite score. 0.5 0 −40 −20 0 20 40 60 80 100 60 80 100 ∆QTcF (ms) 2.0 1.5 320 mg D,L-sotalol Placebo ∆MCS 1.0 0.5 0 −40 −20 0 20 40 ∆QTcF (ms) Figure 3 Covariate assessment of treatment effects for ΔMCS vs. ΔQTcF. Dashed and solid lines indicate regression models for placebo and treatments, respectively. MCS, morphology composite score. associated with greater proarrhythmic potential than homogeneous repolarization is.25 Drug-induced TRIaD (triangulation, instability, and reverse-use dependency) action potential characteristics, which are thought to reflect heterogeneity and increased proarrhythmia risk, can manifest as LQT2-like T-wave morphology on the electrocardiogram (ECG);26 the potential value of TRIaD was largely substantiated in more than 700 trial drugs.27 Temporal repolarization characteristics may also play a role in the evaluation of torsadogenic risk. In the rabbit model, sotalolinduced proarrhythmia can develop before any prolongation of the action potential.28 In dogs, dynamic heterogeneity, as assessed by the beat-to-beat QT–RR interval relationship, was shown to fit the cardiac risk profiles associated with E-4031, nitroprusside, and phenylephrine more accurately than QTc.29 Such experimental data strongly suggest that the proarrhythmic potential of compounds depends on their overall effect on the electrophysiological characteristics of repolarization, not exclusively on the duration of the QTc interval. Covariate analysis of T-wave morphology and QTc morphology changes from baseline than in their effect on QTcF changes. Taken together, these results imply that there are underlying mechanisms of action of drugs, and that in the case of d,l-sotalol these can be better described by T-wave morphology changes than by changes in the QTcF interval. The precise mechanisms that may explain such differential effects on QTc and T-wave morphology for the two drugs may be multifactorial. A prolonged QT interval can result from both homogeneous and heterogeneous prolongation of the predominant myocardial cell types.25 In contrast, T-wave patterns such as those seen in LQT2 are thought to result from a heterogeneous repolarization of the predominant myocardial cell types; this is claimed to be For d,l-sotalol, the slope indicating the relationship between QTc prolongation and T-wave morphology changes was steeper, more nonlinear, and more offset as compared with the same relationship for moxifloxacin. The morphology–duration slope for moxifloxacin was steeper in female subjects than in male subjects, and the mixed-gender composition for moxifloxacin thus essentially reduced the difference between the morphology–duration profiles for moxifloxacin and d,l-sotalol. Therefore, with increasing group homogeneity, a larger difference would be seen between the morphology–duration profiles of the two drugs. In addition, because female subjects were observed to have more pronounced T-wave morphology changes than male subjects despite similar QTcF prolongation, Clinical pharmacology & Therapeutics | VOLUME 88 NUMBER 1 | july 2010 91 articles an assessment of the morphology–duration relationship could present an opportunity to make a gender-specific evaluation of the risk for TdP arrhythmia. The presence of a vertical offset between moxifloxacin and d,l-sotalol for T-wave morphology over the range of QTcF changes from baseline, but not between moxifloxacin and placebo, indicates a drug-specific morphology–duration relationship. These observations show that, for any given QTc prolongation, the T-wave morphology change is greater for 320 mg d,l-sotalol than for 160 mg d,l-sotalol, which in turn is greater than for moxifloxacin and placebo. In the animal model, several drugs that prolong QTc to an equal extent have been associated with markedly different torsadogenic potential.30 Clinically, much smaller QTc prolongations than expected have been reported in patients with drug-induced TdP, whereas the onset of TdP was frequently preceded by marked changes in T-wave shapes.31,32 Beat-to-beat variability has been used to distinguish between patients with a documented history of drug-induced TdP and absence of QTc prolongation from their matched controls.33 The T-type calcium channel antagonist mibefradil, an antihypertensive agent that has been withdrawn from the market, caused distinct T-wave changes, often without QTc prolongation, and produced TdP.34 Similarly, with the antipsychotic drug sertindole, T-wave morphology changes were found to be more pronounced than QTc interval prolongation.18 Collectively, such findings indicate that there may be incompletely understood drug-specific influences on repolarization, in that drugs with the same QTc effect may be associated with different T-wave morphology changes, and that their potential to induce TdP may also vary. Currently, the International Conference on Harmonisation E14 guideline defines a negative thorough QT study as one in which the upper one-sided 95% CI for the maximum timematched mean effect of the drug as compared with placebo is <10 ms.4 However, this threshold was not chosen on the basis of scientific evidence of increased torsadogenic risk associated with such a level of QTc prolongation. This may be problematic because QTc prolongation per se may not represent a risk for arrhythmia. Additionally, a fixed QTc threshold for safety concerns also appears problematic, given the evidence that drugs with similar effects on QTc may have very different safety profiles. Alternatives to the use of the thorough QT study are being investigated, including the possibility of detailed evaluation of the relationship between concentration and QTc effects.35,36 Although commendable, such an approach may have its flaws. The torsadogenic potential of a given compound can depend on the rate of its biological exposure. Almokalant and the experimental drug NS-7 were shown to be highly torsadogenic when they were delivered at a rapid rate of infusion, but not when they were administered at a prolonged infusion rate, despite similar plasma levels and effects on QTc for the two methods of delivery.37,38 d,l-Sotalol is highly proarrhythmic at clinically achievable concentrations,39 whereas moxifloxacin may lack the potential to cause TdP at concentrations encountered 92 clinically.40–42 Moxifloxacin and d,l-sotalol both have linear concentration–QTc relationships,43,44 but there is no clear linear incremental relationship between QTc prolongation and the risk of TdP.3 In congenital long-QT syndrome, for example, TdP risk increases exponentially with prolongation of QTc.5 Therefore, while it may be possible to predict QTc prolongation from concentration data, such linear predictions cannot be used to accurately predict torsadogenic risk. Moreover, in the case of d,l-sotalol, linear concentration–QTc modeling would entirely ignore the nonlinear relationship that was found to exist between QTc and T-wave morphology. Drugs that prolong the QTc interval to clinically relevant magnitudes are likely to be torsadogenic if they also produce relevant changes in the morphology of repolarization waveforms. Therefore, the concurrent analysis of QTc and T-wave morpho logy may serve as a supplementary description of drug effects in thorough QT trials. Certainly, improved characterization of repolarization is needed because of the absence of a clear correlation between QTc and cardiac risk. The assessment of clinical risk with new and existing compounds should be an integrated evaluation of all parameters that are indicative of changes in repolarization and not the QTc interval alone. Integration of quantitative T-wave morphology analysis with QTc could be an important step in the right direction. In conclusion, prolongation of the heart rate-corrected QTc interval does not by itself reveal the full spectrum of druginduced repolarization changes. Rather, it appears that QTc prolongation should be considered together with coexisting T-wave morphology changes. A composite score of asymmetry, flatness, and the appearance of notches, which are the typical ECG patterns in congenital LQT2, provides a measure that, in a concurrent analysis with QTc interval prolongation, may contribute to an improved ECG safety evaluation in drug studies. At this point, it is too early to consider the appropriateness of covariate analysis of QTc and T-wave morphology with drugs that have no effect on the QT interval but may nevertheless affect the duration– morphology profile, because the approach has not been tested in this setting. We propose to further investigate whether concurrent assessment of QTc and T-wave morphology has general validity for drug safety evaluation. Methods Study design. Electrocardiographic recordings from two drug studies were compared. The first study included placebo and moxifloxacin arms from a single-center, randomized, 7-day, parallel-group phase I study (Parexel Clinical Pharmacology Research Unit, Harrow, UK). All study subjects were examined on a baseline day without any treatment and then randomized to either a placebo arm with 7 days of oral placebo treatment or a moxifloxacin arm with 6 days of oral placebo treatment and a single oral dose of 400 mg moxifloxacin on day 7 (Avelox, 400 mg tablets; Bayer Healthcare, Leverkusen, Germany). The moxifloxacin study was approved by the local ethics committee and was conducted in accordance with good clinical practice and the Declaration of Helsinki. The second study, of d,l-sotalol, was a single-center, nonrandomized, 3-day parallel analysis with a fixed treatment sequence. It was conducted VOLUME 88 NUMBER 1 | July 2010 | www.nature.com/cpt articles at the Pfizer Clinical Research Unit (formerly Pharmacia, Kalamazoo, MI). Following a baseline day, when no drug was given, subjects received a single 160 mg dose of d,l-sotalol (Betapace, 80 mg tablets; Berlex Laboratories, Montville, NY) on day 2 of the study and a single 320 mg dose of d,l-sotalol on the last day of the study. The details of the protocol have been published previously.45 An independent institutional review board approved the d,l-sotalol study. All subjects gave written informed consent. Study population. All the subjects in the placebo and moxifloxacin arms were healthy volunteers between the ages of 18 and 45 years. The main inclusion criteria were negative pregnancy test and reliable contraception in women of childbearing potential. The main exclusion criteria were long-QT syndrome or additional risk factors for TdP, concomitant systemic medication, hypersensitivity to fluoroquinolones, or a screening finding precluding the subject from receiving moxifloxacin. In all, 62 subjects (26 women and 36 men) received placebo, and 62 subjects (24 women and 38 men) received a 400 mg dose of moxifloxacin. All the 21 subjects who received d,l-sotalol were healthy male volunteers between the ages of 18 and 45 years. The inclusion/exclusion criteria for subjects in the d,l-sotalol study were comparable to the criteria generally used in ECG phase I investigations, with “healthy” status confirmed by history, physical examination, normal ECG, normal values in laboratory tests, and no use of medication. ECG acquisition. Standard 12-lead ECGs were recorded after administra- tion of placebo and moxifloxacin at time points corresponding to 30 min and 1, 2, 3, 4, 6, 8, 10, 12, 14, 16, and 23.5 h after dosing, which was always at ~8:00 am. Triplicate ECGs were obtained at each nominal time point (not >2 min apart). All ECGs were recorded in 7.5-s segments at a sample rate of 1,000 Hz (H12 Recorder; Mortara Instrument, Milwaukee, WI). The subjects were at rest in a fully supine position for at least 5 min before the recordings were initiated. ECG recordings of the subjects in the d,l-sotalol group were obtained using a 12-lead digital Holter monitor at a sample rate of 180 Hz (H12 Recorder; Mortara Instrument). The subjects received the drug dose at 8:00 am, and ECGs of 10-s durations were derived from a digital Holter at ~30-min intervals thereafter. For each subject, 22.5 h of digital ECG recordings were obtained per day. To ensure stability of repolarization, a representative 10-s segment was extracted from the Holter recording only if it was preceded by 1 min of visually stable heart rate. Care was taken to select the most visually noise-free segments with stable RR intervals, and all the segments were derived from such stable and noise-free 10-s Holter recordings. ECGs were recorded after 5 min of supine rest. The 11 coinciding time points for placebo, moxifloxacin, and d,l-sotalol data were used for analysis (30 min and 1, 2, 3, 4, 6, 8, 10, 12, 14, and 16 h after the dose).The time points and the number of ECG recordings for off-treatment days were similar to those for ontreatment days. ECG processing. Each extracted ECG segment was resampled to 500 Hz and used to form a representative median beat in the recorded leads (I, II, V1–V6) using MUSE/Interval Editor software (GE Healthcare, Milwaukee, WI). T-wave morphology analysis was performed on a principal component beat that was derived from median beats in the recorded leads. A low-pass Kaiser Window FIR filter with a cutoff frequency of 20 Hz was used on the first principal component beat. The filtered beat from the first principal component was subsequently used for analysis of repolarization morphology. Fiducial point detection and QT interval measurements were made automatically using version 21 of the 12SL algorithm (12SL; GE Healthcare). The 12SL algorithm annotates fiducial points on the superimposed representative PQRST complexes from all 12 leads. The algorithm excludes from the analysis any discrete U-waves that occur after the T-wave returns to baseline, whereas complex multiphasic T-waves and Clinical pharmacology & Therapeutics | VOLUME 88 NUMBER 1 | july 2010 T-U complexes are included. QT intervals were corrected for heart rate using Fridericia’s equation: QTcF = QT/RR1/3. T-wave morphology measurements. A morphology combination score (MCS) based on asymmetry, flatness, and notching was used to identify drug-induced shape changes in the T-wave (Equation 1). The MCS was developed to quantify, with equal magnitude, the difference in T-wave morphology between healthy subjects (representing normal repolarization) and patients with LQT2 (as biological models of IKr inhibition and abnormal repolarization). This assignment of equal weight to component variables has been shown to generalize to drug studies for optimal separation between normal and abnormal repolarization16 and to indicate markedly greater effects of drug-induced disturbed repolarization than QTcF.16–18 The linear weighting of component variables is therefore logically appealing, both in terms of generalization of the model and in order to ensure that the composite score shows that these component variables explain different aspects of repolarization MCS = asymmetry + notch + 1.6 × flatness (1) Asymmetry. Asymmetry was defined as the average of the square of the difference between the slopes (first derivatives) of the ascending and descending parts of the T-wave.16–18 Notches. A curvature signal, calculated from the first and second derivatives of T-waves, was used to identify the presence or absence of a notch.16–18 The magnitude of a notch was measured on a unit amplitude T-wave and assigned to one of three categories, as was suggested by Lupoglazoff et al.:46 no deflection = 0, moderate notch (perceptible bulge) = 0.5, and pronounced notch (distinct protuberance above the apex) = 1.0. Flatness. Flatness was calculated as a modified version of the standard kurtosis measure, which is used in statistics to describe the peakedness of a probability distribution.16–18 Statistical analysis. All statistical analyses were performed using Matlab, version 7.4 (Mathworks, Natick, MA). Primary statistical inference was based on drug-induced change from baseline. For triplicate recordings, the median value was used. The baseline value for each postdose assessment was the time-matched assessment for off-treatment. Paired Student’s t-tests for time-matched changes in the mean values for QTcF and MCS were used. Two-sided 95% CIs for the mean change from timematched baseline were determined in all cases, and P < 0.05 was regarded as significant. QTcF and MCS measurements were converted to z-score equivalents by subtraction of the mean (μ) and subsequent division by the SD (σ) of placebo values at baseline (Equation 2). (values − placebo, day-1 ) z-Score = (2) placebo, day-1 z-Score equivalents were used to enable direct comparison of QTcF and MCS on a similar scale. Linear and nonlinear curve fits were derived for ΔMCS vs. ΔQTcF, and the best regression model was determined by minimization of the squared errors. For each treatment, the residuals from the individual curve fitted to ΔMCS vs. ΔQTcF, and the common fits to data were compared using an F-test to examine the null hypothesis that the data from both treatments had the same duration–morphology relationship. Slopes and overall elevation of regression lines for ΔMCS vs. ΔQTcF were compared using the t-test. Categorization of ΔQTcF into bins of change was done in order to investigate modest repolarization effects for ΔMCS vs. ΔQTcF. Acknowledgments The authors thank H. Lundbeck A/S for providing moxifloxacin data and Pfizer Inc. for providing sotalol data. 93 articles Conflict of Interest No external sources of funding were used for this study. C.G., J.J.S., J.K.K., M.P.A., and E.T. are authors of two patents describing the T-wave morphology method. J.N. declared no conflict of interest. J.M. is an employee and shareholder of H. Lundbeck A/S. 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