Comparison of electrocardiographic analysis for risk of QT interval prolongation using safety pharmacology and toxicological studies

Journal of Pharmacological and Toxicological Methods 60 (2009) 107–116

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Journal of Pharmacological and Toxicological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j p h a r m t ox

Appraisal of state-of-the-art

Comparison of electrocardiographic analysis for risk of QT interval prolongation using safety pharmacology and toxicological studies B.D. Guth a,⁎, A.S. Bass b, R. Briscoe c, S. Chivers d, M. Markert a, P.K.S. Siegl e, J.-P. Valentin f a

Boehringer Ingelheim Pharma GmbH & Co KG, Department of Drug Discovery Support, D-88397 Biberach an der Riss, Germany Schering-Plough Research Institute, Exploratory Drug Safety, K15-2-2880, 2015 Galloping Hill Road, Kenilworth, 07033-0539, New Jersey, USA Merck Research Laboratories, Safety Assessment, West Point, PA 19486, USA d Novartis Pharma AG, Novartis Campus, Basel, CH-4002, Switzerland e Siegl Pharma Consulting LLC, 884 Valley Road, Blue Bell, PA 19422, USA f AstraZeneca R&D Alderley Park, Safety Assessment UK, Macclesfield, SK10 4TG, UK b c

a r t i c l e

i n f o

Article history: Received 12 April 2009 Accepted 4 May 2009 Keywords: hERG QT interval prolongation Electrocardiogram Safety pharmacology Toxicology Integrated risk assessment

a b s t r a c t Testing for possible cardiovascular side effects of new drugs has been an essential part of drug development for years. A more detailed analysis of the electrocardiogram (ECG) to detect effects on ventricular repolarization (effects on the QT interval), as a marker for possible proarrhythmic potential has been added to that evaluation in recent years. State-of-the art evaluation of drug-induced effects on the QT interval have evolved, but due to the complexity of the assessment, the trend in safety pharmacology studies has been to collect large numbers of high quality ECGs to allow for a robust assessment including the influence of heart rate on the QT interval apart from possible drug-induced effects. Since an assessment of the ECG is often included in toxicological studies, one can consider making such an assessment using ECG data from routine toxicological studies. This review summarizes various aspects of both safety pharmacology and toxicology studies with regards to their impact on the quality and quantity of ECG data that one can reasonably derive. We conclude that ECG data from toxicological studies can offer complementary ECG data that can strengthen a risk assessment. However, for the great majority of standard toxicity studies conducted, the ECG data collected do not permit an adequate assessment of drug-induced effects on the QT interval with the sensitivity expected from the ICH S7B guidelines. Furthermore, sponsors should be discouraged from performing any analyses on low quality ECGs to avoid generating misleading data. Substantial improvements in ECG quality and quantity are available, thereby making a QT interval assessment within the context of a standard toxicological study feasible, but these methods may require a larger commitment of resources from the sponsor. From the viewpoint of risk mitigation and limiting the attrition of promising new therapies, a commitment of resources to insure ECG data quality in either toxicology or safety pharmacology studies may be well justified. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Testing for cardiovascular safety of new drugs has been an integral part of drug development for many years. This usually includes an assessment of potential electrocardiographic effects using the electrocardiogram (ECG). The importance of the electrocardiographic assessment has increased in recent years due to the recognition that druginduced effects on ventricular repolarization (prolongation of the QT interval of the ECG) can be a clinically-relevant risk factor for malignant

Abbreviations: ECG, electrocardiogram; GLP, Good Laboratory Practice; hERG, human ether-a-go-go related gene; ICH, International Conference on Harmonizaton; TQT, clinical thorough QT/QTc prolongation study. ⁎ Corresponding author. Tel.: +49 7351 544732; fax: +49 7351 834732. E-mail address: [email protected] (B.D. Guth). 1056-8719/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2009.05.006

tachyarrhythmias such as Torsade de Pointes. As such, electrocardiographic effects of drug candidates in non-clinical models have become an important topic in drug discovery and development (Pugsley, Authier, & Curtis, 2008; Valentin & Hammond, 2008). Indeed, demonstration that a new drug candidate does not affect ventricular repolarization can be critical to a drug's success. In this paper we use QT interval duration as our specific concern for a drug-induced effect on the ECG. It should be kept in mind, however, that all effects on the electrocardiogram that may be of clinical relevance, such as a delay or block of atrio-ventricular conduction, prolonged ventricular conduction, or aberrant firing in the atrium or ventricle should be carefully considered in terms of the potential risk to humans. Thus, all druginduced ECG changes should be included in a comprehensive risk assessment and many of the aspects addressed in this paper are equally valid for collection and analysis of these other ECG-based parameters.

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The overall cardiac safety risk assessment should include all relevant data from both in vitro and in vivo assays. Analysis of electrocardiograms acquired in toxicological studies therefore should contribute to this assessment. However, the question we examine is whether the quality and quantity of the ECG data are adequate for a robust assessment of a drug's potential for affecting ventricular repolarization or other possible electrocardiographic effects. Further, where deficiencies exist in the ECG data, recommendations are offered to mitigate these and enhance the power of analysis of the ECG-derived data collected in the course of toxicological and safety pharmacology investigations. 2. Regulatory background Recommendations for assessing the risk for drug-induced changes in ECG parameters, including QT interval prolongation, can be found in the International Conference on Harmonization (ICH) guidelines; S7A (Anon, 2001), Safety Pharmacology Studies for Human Pharmaceuticals, and, more specifically, ICH S7B (Anon, 2005a), Non-clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals. In this paper we focus on ECG measurements conducted using in vivo models, however, the reader is reminded that the ICH S7B guideline recommends that results from all relevant assays (in vitro and in vivo) should be considered in the integrated risk assessment. ICH S7B also recommends that the sensitivity and specificity of the assays used be well defined and that data with positive and negative reference compounds be included in the risk assessment. This information is particularly important when the overall assessment concludes that there is little or no risk for a drug candidate to affect the ECG in humans. [Although not the focus of this article, we find that these aspects of test system validation are rarely provided, both for safety pharmacology studies as well as for toxicology studies]. In this regard, ECGs collected in a routine toxicology study may not satisfy ICH S7B recommendations. Consequently, we evaluate study design issues and experimental conditions that can contribute to experimental variability and therefore influence the assay sensitivity. Predicting the potential for a drug candidate to prolong the QT interval in humans has two important implications for drug development. A primary issue is, of course, cardiac safety based upon the association of delayed ventricular repolarization with risk of Torsade de Pointes (Bass, Darpo, Valentin, Sager, & Thomas, 2008). A further consideration independent of the prediction of clinical risk per se is that the drug candidate will prolong the QT interval in the clinical thorough QT/QTc prolongation (TQT) assay described in ICH guideline E14 (Anon, 2005b) Clinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential for Non-Antiarrhythmic Drugs. A positive finding in the clinical TQT study has implications for safety, the pace of development of the test agent and ultimate product labeling, any of which may have significant marketing implications. Because the clinical TQT assay is designed to detect small increases in the QT/QTc interval, the sensitivity of the non-clinical assay investigating risk for QT interval prolongation should also be high. Therefore, consideration of how the data will be used for drug development decisions will also impact the level of sensitivity appropriate for the non-clinical assay. The ICH S7A and S7B guidelines provide an option that safety pharmacological endpoints, including the ECG, can be included in toxicological studies. This is an attractive option since toxicological studies are usually done using Good Laboratory Practice (GLP) conditions, thereby qualifying safety pharmacological endpoints to fulfill the requirement for GLP “core battery” studies, as recommended in the ICH S7A guideline. Furthermore, it would evaluate the test agent after repeated dose administrations (a cardiovascular safety pharmacology study is typically a single dose). It can thereby reduce the use of animals and resources (Luft & Bode, 2002). However, one must recognize the inherent limitations of increased variability and decreased sensitivity of the measurements when capturing ECG data from what is primarily a traditional toxicological study.

In recent years we have learned a great deal about how to enhance ECG data collection and analyses in non-clinical studies. Methods are now available that can achieve excellent sensitivity for assessing druginduced effects on the QT interval in non-clinical models. These gains have been obtained through an optimization of the study conditions and improved technology and software for acquiring and analyzing data. These optimal approaches are currently used routinely in dedicated safety pharmacology studies. These aspects will be described in this paper based primarily on experience using the dog as the animal model, but with applicability to other species, including the pig (Stubhan et al., 2008) and non-human primate (Authier et al., 2008). We then address the characteristics of routine toxicological studies and demonstrate why they can be less than ideal for providing QT interval data to the level of ICH S7B for the assessment of risk in humans. Finally, we suggest approaches that could be considered to make the best use of toxicological studies for assessing the ECG, however, necessitating an increased commitment of the sponsor for improving the assay conditions. 3. What has become state-of-the-art for the non-clinical assessment of drug-induced effects on the QT interval in safety pharmacology studies? Much has been learned in recent years in terms of optimizing the detection of drug-induced effects on the ECG. Much of this optimization can be attributed to the intense work done to address the issue of drug-induced effects on ventricular repolarization. Measurement of a drug candidate's potency to directly interact with IKr (also referred to as hERG potassium current) in vitro, combined with an assessment of potential drug candidate effects on the QT interval of the ECG in a well validated animal model form the basis of the non-clinical risk assessment (Anon, 2005b; Hanson et al., 2006). A change in the duration of the QT interval of the ECG is used as the primary nonclinical (and clinical) endpoint to detect prolonged (Pollard, Valentin, & Hammond, 2008) or shortened (Holbrook, Malik, Shah, & Valentin, 2009) ventricular repolarization. A key element for establishing the sensitivity of a test system for detecting drug-induced effects on the QT interval duration is the inherent variability of the parameter measured between individuals and for a given individual over time. Optimization can be achieved through improvement of the quality of the ECG data collected, considering the many environmental influences that might contribute to the animal's excitability, leading to data variability, and the selection of a study design to optimize the statistical power for detecting a given effect and defining the needed sample size. Independent of the experimental approach selected for a given study, there are various aspects of a study that can optimize the quality of the ECG data collected; these include but are not limited to: • • • • •

animals should be calm and acclimatized to the procedure consistent restraint approaches should be utilized, when needed consistent placement of ECG electrodes quiet environment free from sources of electrical interference ECGs should only be acquired on days when other activities are not planned (e.g., do not acquire ECGs on a day when blood sampling is also done) • duration of ECG collection (number of ECG complexes acquired during a session for analysis) should be sufficient to allow for the effects of respiration to be accounted for. When an evaluation of heart rate-induced effects on the QT interval are needed, substantially more ECG complexes will be needed. • when paper recording is used, use a recording speed (e.g.,100 mm/s) to enable sufficient resolution of the beginning and end of the major fiducial points of the electrocardiogram (P wave, QRS complex, T wave). All these aspects are reviewed in detail in the following paragraphs.

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3.1. ECG quality It may seem obvious that a successful study examining the potential drug-induced effects on cardiac electrophysiology should be based on a high quality electrocardiogram. This requires a technically flawless ECG trace (i.e., lack of electrical interference and with well defined wave forms) with accurately-placed and stable electrodes as well as excellent electrical contact to the subject. Whether a cardiovascular safety pharmacology study or toxicology study, this important objective is achievable with reasonable effort and should not be compromised in the course of the investigation. Features of ECGs from safety pharmacology studies that can affect their interpretation have been discussed in the past (Hamlin, 2005). Recommendations for improving ECG quality follow. Inadequate electrical grounding may limit ECG quality if high levels of baseline noise (e.g., 50/60 Hz) are observed. Particularly for determining the end of the T wave, one must assess the return of the ECG signal to the isoelectric line. If the isoelectric line is blurred by electrical, respiratory or motion artifacts, the measurement of the end of the T wave can be impaired. Thorough grounding of the test system can lead to marked improvement of the ECG quality. As seen in Fig. 1, taking the time to inspect the ECG quality can reveal unpleasant surprises. The ECG shown in Fig. 1 was obtained from a contract research organization conducting a single-dose GLPconforming safety pharmacology study in dogs instrumented for the telemetric collection of the ECG and other hemodynamic data. One sees that electrical and/or mechanical artifacts led to a poor quality ECG waveform that was nevertheless evaluated and included in the data analysis. Thus, the first step toward a successful electrocardiographic study is obtaining high quality ECG traces that allow a precise evaluation of the wave form.

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Fig. 2 shows an example of high quality ECGs obtained with an electronic system quite similar to that used in obtaining the ECGs seen in Fig. 1. Thus, the difference in quality is not based on the system used per se, but rather how the electronic system was used and attention to the data quality. 3.2. Sources of ECG variability 3.2.1. Lead placement A minimization of ECG data variability can be aided by recognizing the factors that can lead to inter-individual as well as intra-individual variability. A basic requirement is that the ECG measurements be conducted identically in each individual subject beginning with the same lead placement in each subject and reuse of the exact same electrode placement upon each subsequent measurement, in particular when external limb leads or precordial leads are employed. This approach requires animal restraint causing effects including: a) stress related physiological changes, b) increased blood pressure, heart rate (see Table 1) and body temperature, c) possible modification of drug effects, and d) increased variability of data. Any of these factors can significantly affect quality of the ECG tracings and ultimately data interpretation. The use of implantable telemetry technology has been advantageous with regard to this aspect in that electrodes are fixed in regard to their orientation to the heart and therefore are less sensitive to motion artifacts and do not change between measurements. However, since the use of invasive telemetry procedures in a toxicology study is not practical, at least a consistent placement of the leads and careful positioning of the animal (in the case of restraint procedures) will advance the goal of improving ECG quality. Experience from the use of invasive radiotelemetry has shown how best to position these implanted

Fig. 1. A representative ECG tracing taken as a part of a GLP study in dogs using a full-implant telemetry-based data acquisition system. Markers were set by hand by a supposedly trained individual to identify (left to right) “P start”, “QRS start”, “R Peak”, “QRS end” and “T end”. Thus, despite the use of a “state-of-the-art” telemetry approach, the ECG quality was poor and the positioning of the markers inexplicable.

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Fig. 2. A set of ECGs superimposed at the R-wave from a minipig using a full-implant telemetric data acquisition system (i.e., similar experimental approach to that of Fig.1, but from a different institution) and comparing the influence of increasing doses of moxifloxacin (ECGs take at control and following 10, 30, 100 or 300 mg/kg). In these high quality ECG tracings, the end of the T wave is easily detected such that the prolongation of the QT interval with moxifloxicin can be clearly seen and accurately quantified. The overall trace length is approximately 650 ms.

electrodes in the various species used for such studies (e.g., pig: Stubhan et al., 2008; dog: Markert, Klumpp, Trautmann, & Guth, 2004; cynomolgus monkey: Authier et al., 2008) to produce an ECG tracing that is similar to the limb lead II in terms of its morphology. The electronic systems available for collection of ECGs in toxicology studies allows for 3 (non-invasive telemetry), 6 or 12 lead configurations. 3.2.2. Environmental influences on the ECG A high quality ECG also requires that the subject is in a relaxed and stable physiological state. Muscle tension, tremor or other muscle movement can produce artifacts in the ECG. Arousal due to external influences (noise or activity in the laboratory) can lead to sudden increases in heart rate and blood pressure that do not reflect druginduced effects and these hemodynamic effects can also lead to Table 1 Baseline parameters for heart rate, QT and QTc (van der Water's correction) parameters from three commonly used test systems: non-invasive telemetry, fully implantable telemetry and manual recording in restrained Beagle dogs. Parameters

Manual recording in restrained dogs

Non-invasive telemetry

Fully implantable telemetry

Number of dogs Number of studies Heart rate, beats/min QT, ms QTc, ms

183 7 125 ± 28 188 ± 15 231 ± 11

80 4 74 ± 9 232 ± 11 245 ± 9

24 6 70 ± 15 214 ± 8 223 ± 14

Data derived from these studies was used for power calculation determination to evaluation the sensitivity of the approaches, as seen in Fig. 3. Whereas both types of telemetry-based approaches yielded very similar heart rates and QT intervals, heart rates in restrained animals was markedly higher indicative of the stress involved with the restraint. Furthermore, the variability of the data is much higher.

changes in the ECG. The heart rate dependency of the QT interval duration has been recognized for many years, but more recently it has become apparent that the QT interval is also substantially modulated by autonomic tone (Fossa, 2008). Consequently, attention must be paid to the laboratory setting to insure that the animal subjects are studied under conditions that are comparable from day to day. Simple, visual inspection of the ECG recordings as they are being collected in a study can be highly informative as to the quality of the data generated. A low and steady heart rate, in a range expected for the given species under basal conditions without stress or excitement and that remains constant over the time in which measurements are made is essential. In the dog, the appearance of a sinus arrhythmia and respiratory effects with heart rates below 80 beats/min is a sign that the animal is in an unstressed state. On the other hand, one must collect sufficient numbers of waveforms to smooth out this “physiological variability”. Acclimatization of the animals to the test procedure and the laboratory setting in which measurements are made is indispensable but, of course, requires increased time and resources. While recognizing that these practices may not be fully practical in a toxicology study, their incorporation to the extent possible will offer a basis for improving the quality of the ECG. Control of environmental influences can be further improved with the use of telemetry-based systems. Non-invasive technologies are now available that can be applied to a toxicology study in non-rodents (see Table 1). These systems allow ECG data collection hardware to be located distant to the animals such that they can be held in their home cages with familiar surroundings and without the need to restrain or even interact with the animals during data collection. Data can also be collected continuously over longer periods of time thereby generating a larger set of ECG waveforms for analysis. Times during which there

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are obvious hemodynamic effects or behavioral excitement can thereby be excluded from analysis. Care should be given, however, in reviewing the data to recognize whether these changes affecting the electrocardiogram are random, unrelated events or an indication of test article-related activity. Video monitoring of the animals can be helpful in this regard. Attention to the housing conditions can lead to further optimization of the physiological conditions (Klumpp, Trautmann, Markert, & Guth, 2006). This includes maintaining animals in groups, where possible, or at least with their usual housing partner and with visual contact with other members of the study group. Furthermore, simple approaches including the playing of background music in the vivarium can bring about a more homogeneous auditory environment and block out possible noise from neighboring areas that might arouse the animals. Optimization of such factors can bring about substantial advantages in terms of achieving truly basal hemodynamic conditions; in the well-trained dog, heart rates below 60 beats/min can be achieved (Klumpp et al., 2006) which is substantially lower than the heart rates usually found when ECG data are collected within a traditional toxicological study (Spence, Soper, Hoe, & Coleman, 1998 and Table 1). 3.2.3. Accommodation of animals to the test procedures Although already mentioned above, it deserves repeating that accommodation of animals to the test procedures is essential for an optimized ECG study. As with assuring the quality of the electrodes and consistent electrode placement and a controlled study environment as described above, accommodating animals to the test procedures is readily and easily adaptable to both safety pharmacology and toxicology studies. Training makes the animals familiar with the study routine, the laboratory environment and therefore reduces procedure-related stress. Stress that causes movement or shaking can lead to problems with the lead placement and the increased sympathetic tone associated with the stress can itself have an impact on the QT interval duration (Fossa, 2008). It is highly recommended that animal training begin substantially before the start of a study such that animals are well prepared at the onset of the study. If at all possible, animals that do not appear to be well-suited to the testing procedure should not be included in the study. This is even more critical for inclusion of animals into groups instrumented for telemetric data collection. A considerable time and financial commitment is needed for such animals and a careful selection of the individuals to be included is therefore warranted. 3.2.4. Considering other aspects of study design A primary consideration is the sensitivity of a test system (animal model) for detecting a given change in predefined endpoints; prolongation of QT interval duration in this case. This may be assessed by calculating the statistical power under a specific study design; a minimum of 80% statistical power with a 95% probability is often used as a bench mark for adequate statistical power. Statistical power is dependent upon the variability of the data collected as well as the sample size and the study design. Use of cross-over study designs leads to improved statistical power but is not compatible with toxicological studies which require between-group comparison study designs. The statistical power to detect a predefined change in the QT interval duration in a given study should always be defined, as mentioned in the ICH S7 guidelines. Since the clinical TQT studies are quite sensitive (usually able to detect an upper limit of change of less than 10 ms or ~2.5%), our non-clinical studies should also aim for a similar sensitivity to have the best change of predicting clinical TQT effects. Nevertheless, even a well performed nonclinical study, using numbers of animals acceptable in terms of animal usage considerations, will likely not reach this goal (see power analysis below). This premise is the current view of the authors and will require further discussion and debate in the context of emerging correlative non-

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clinical–clinical data (Trepakova, Koerner, Pettit, Valentin, & on behalf of the HESI Pro-Arrhythmia Committee members, in press). In safety pharmacology studies, cross-over designs may be employed with their inherent advantages in terms of test sensitivity. Each individual receives all treatments allowing for intra-individual comparisons. This further reduces the variability and increases sensitivity of the test system. This is a critical factor for the determination of the required sample size. It has been shown that even modest drug-induced effects on the QT interval duration (e.g., 10%) can be detected with good statistical power with low numbers of animals (e.g., N = 4), under the conditions of an optimized test environment (Stubhan et al., 2008). Interestingly, drug-induced effects on the QT interval duration can be detected with greater sensitivity than other traditional hemodynamic parameters including heart rate, arterial blood pressure and left ventricular pressure in such studies. However, a 10% change, or an absolute change of roughly 25 ms in the QT interval, is substantially larger than the 10 ms change that can usually be detected in a clinical thorough TQT study. 3.2.5. Determination of the statistical power To better illustrate the points above, a power analysis was performed to compare the sensitivities of the three methods most commonly used to collect ECG in toxicology / safety pharmacology studies (see Table 2 for details). The power curves for each method and the parameters heart rate, QT and QTc are presented in Fig. 3. Furthermore, the baseline values of these parameters in the three different models are summarised in Table 1. For this analysis we have used data from actual studies conducted over the last 3-years in the same institution using Beagle dogs. Studies were aimed at assessing effects of proprietary candidate drugs covering a wide range of pharmacological and chemical classes, and therapeutic indications. Ideally, such analyses would require an independent study in which animals are randomised to 1 of the 3 methods, measured for baseline and remeasured after treatment with a compound known to affect the ECG parameters. It should then be clear which method is superior at detecting drug-induced effects. Therefore, we acknowledge that there are some limitations of such a retrospective analysis. Despite these limitations, the following was noted: i) good study-to-study repeatability; ii) tightness of the data (especially heart rate) for the two telemetered methods; iii) very high baseline heart rates for the restrained animals. Accordingly, it should be noted that a 20% effect in heart rate, as an example, means 20% of 74 beats/min for noninvasive telemetry, 20% of 70 beats/min for fully implantable telemetry and 20% of 125 beats/min for restrained animals; i.e., a constant effect in percentage terms, but different magnitudes of effects in absolute terms. Using this data set we conclude that the methods were only capable of detecting large changes in heart rate. Both telemetry systems were equally effective in detecting changes in QT with a 80% chance of detecting a statistically significant (P b 0.05) change in QT of ~9–11% using 4 dogs. In contrast, the restrained animal approach only allows ~20% change in QT to be detected. In general, correcting QT interval for heart rate (i.e., QTc) was associated with an increase in power to detect changes, compared to the sensitivity to detect changes in the uncorrected QT interval. Fig. 3 summarizes the statistical power required to detect changes in heart rate, QT interval and rate corrected QT interval (using the Van de Water correction) based on ECGs recorded by different test systems and technologies. The data demonstrate that heart rate, QT and QTc intervals derived from ECGs recorded using limb leads in restrained animals have a substantially lower statistical power to detect changes compared to the same parameters derived from ECGs recorded using either a non-invasive or a fully implantable telemetry system. 3.2.6. Heart rate dependency of the QT interval duration One of the challenges in assessing drug-induced effects on the QT interval duration is that the QT interval duration is heart rate dependent; as heart rate increases, the QT interval shortens and as heart rate slows,

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Table 2 Technologies available for ECG assessment in large animals (typically dogs and primates) in toxicology studies. Endpoint

Manual recording in restrained dogs

Non-invasive telemetry

Fully implantable telemetry

Recording duration per session

Few seconds to minutes

Days to months

Volume of data Data analysis

Few hours to few days (can be repeated following replacement of the battery) Can be very high Typically automated computer based placement of ECG markers in the data acquisition system.

Low Typically manual paper based placement of ECG interval markers, but can be electronic and automated, depending on the data acquisition system. Yes Yes Yes Yes but reduced

Amenable to GLP Training/acclimation of animals required

Parameters recorded

Parameters measured

ECG Leads I, II, II, aVR, aVL and aVF are possible. Chest leads (V1–V6) are also possible. Typically only Lead II used for analysis. PR, QT and RR intervals and QRS complex duration. HR, QTc Use of correction formulae (e.g., Van de Water's) feasible. Individual correction not feasible due to limited data sample Waveform morphology incl. arrhythmia limited due to snap shot recording

Very high Typically automated computer based placement of ECG markers in the data acquisition system.

Yes Yes, training both for and following instrumentation is possible and recommended. ECG Leads I, II, II, aVR, aVL and aVF are possible. Typically ECG Lead II only. Other leads are Chest leads (V1–V6) are also possible. Typically possible, but limited due to the battery requirements. only Lead II used for analysis.

PR, QT and RR intervals and QRS complex duration. HR, QTc Use of both correction formulae (e.g., Van de Water's) and individual correction feasible

PR, QT and RR intervals and QRS complex duration. Parameters derived HR, QTc Use of both correction formulae (e.g., Van QTc correction de Water's) and individual correction feasible Waveform morphology including Parameters assessed Waveform morphology including arrhythmia — Robust robust analysis feasible if continuous arrhythmia — robust analysis feasible if continuous recording obtained recording obtained Other parameters that can be recorded Blood pressure invasively (ear artery) or Blood pressure invasively (ear artery) or non- Blood pressure, left ventricular pressure, non-invasive (tail cuff) invasive (tail cuff). Respiratory function using core body temperature. Respiratory function using intra-pleural pressure or chest and/or abdominal strain gauges (e.g., diaphragmatic electromyogram (EMG). VivoMetrics life shirt technology). Assessment of onset, duration, Usually not; would require significant Yes Yes dose-dependency and reversibility of effect time points to be recorded and analysed Yes — not considered to adversely interfere Not usually — would require satellite Compatibility with toxicological studies Yes — not considered to adversely groups of animals due to the cost. Although interfere with the primary endpoint of with the primary endpoint of the study toxicology studies have been performed in the study implanted animals. Technical expertise for training and equipping Surgical expertise for implantation and Resource requirements: technical Technical expertise for training and technical/scientific expertise for ECG equipping animals with the equipment animals with the equipment and technical/ acquisition and evaluation. scientific expertise for ECG acquisition and and technical/scientific expertise for evaluation. ECG acquisition and evaluation. Estimated man–hours effort required for a 42 h in-life 14 h in-life 42 h for surgery 1-month toxicological study 35 h for analysis 21 h for analysis 7 h in-life 28 h for analysis Consider including in toxicological studies Yes Yes No, but can be considered on an exceptional case by case basis Amenable to PK/PD assessment Yes in theory but on different days in Yes/no. PK/PD usually conducted on separate Yes/no. PK/PD usually conducted on reality days. separate days. Data collection Paper traces or electronic Electronic Electronic Data storage Archiving of paper or electronic data as Archiving of electronic data Archiving of electronic data raw data Capital investment + (~$20,000) +++ (N $100,000) +++ (N $100,000) The example below refers to the support required for supporting a GLP regulatory 1-month toxicology study. ⁎Assuming a satellite group of 4 dogs on a cross-over dosing regime.

the QT interval lengthens. Linear regression based-formulae have been applied to “correct” the QT interval for changes in heart rate. Some of these were derived from human data (e.g., Bazett's (Bazett, 1920), Fridericia's (Fredericia, 1920)), whereas others come from anesthetized animals (e.g., Van de Water, Verheyen, Xhonneux, & Reneman, 1989)). Unfortunately, the relationship between QT interval and heart rate is not linear over the full range of spontaneous heart rates found in animals and the relationship is further influenced by changes in sympathetic tone which, of course, often accompanies these changes (Fossa, 2008). The use of such formulae proved to be inaccurate for preclinical data from conscious animals (Davis & Middleton, 1999; Matsunaga et al., 1997; Spence et al., 1998) and necessitated novel approaches tailored to the non-clinical data (Meyners & Markert, 2004). Although not ideal, linear regression formulae have been the most commonly employed method for correcting the QT interval duration for heart rate changes by both industry and regulators (Tattersall, Dymond, Hammond, & Valentin, 2006). Other approaches may improve the assessment of drug-induced effects on the QT interval, however.

QT interval data collected in a given animal without treatment over the range of spontaneous heart rates can be used to develop an individual correction formulae. This defines for each individual, the range of QT intervals that one can find at a given heart rate. It is important to note that at a given heart rate, there is a range of QT interval values that may be obtained and not a single value. Furthermore, whereas the QT–HR relationship for a given individual is highly reproducible from day-to-day (assuming otherwise similar study conditions), there can be substantial differences in this relationship between individuals. This approach requires collection and analysis of a substantial amount of data (e.g., over 24 h) and therefore may not be practical in the setting of a toxicological study. On the other hand, ECG data collection via non-invasive, telemetry-based technologies allows continuous collection of ECGs over extended periods of time. In such cases, collection and analysis of the QT–HR relationship in each individual will be possible. In contrast to toxicological studies, safety pharmacology studies may employ a dedicated colony of chronically instrumented animals

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Fig. 3. Statistical power analysis curves for heart rate (top panels A, B and C), QT (middle panels D,E and F) and QTc (Van de Water correction; bottom panels G, H and I) for a population of 4, 6, 8 or 10 dogs (black, red, green and blue lines respectively). Power calculations are based on a population of dogs where ECG was measured in freely moving animals using a non-invasive telemetry system (center panels B, E and H) or a fully implantable telemetry system (right panels C, F and I) or in restrained animals using a manual recording (left panels A, D and G). Baseline data for each parameters in each experimental conditions are presented in Table 1.

from which a subset can be used for a given study. An advantage of using chronically instrumented animals is that one can establish the relationship between HR (or RR interval) and the QT interval duration in each subject and use this predefined relationship in subsequent studies in which the animal is used. One could also consider running a set of such animals as a satellite arm to a toxicological study in those situations where there is a concern regarding the cardiac safety of a compound upon repeated dosing. 3.2.7. Manual versus automated ECG analysis It has long been accepted that the best possible ECG measurements come from manual, visual inspection of the ECGs by a trained cardiologist or safety pharmacologist. Whereas this may be the case for some aspects of ECG analysis (e.g., overall morphological changes of the ECG wave form), the detection of drug-induced effects on the ECG intervals such as the duration of the QT interval, in particular, is greatly enhanced by the use of computer-based analysis of the ECG intervals. This approach enables the use of larger data sets to more precisely evaluate characteristics such as the heart rate dependent effects. New, species-specific ECG analysis algorithms have vastly improved signal detection and interval calculation, but even these require high quality

ECGs. We now come full circle to reemphasize the absolute need for high ECG waveform quality. Even the best algorithms, designed specifically for the ECG morphology found in a given animal model, will fail if ECGs are of poor quality. (Even the trained eye of a cardiologist will not be able to evaluate the ECG if it is affected by artifacts; see Fig. 1). On the other hand, high quality ECG waveforms used together with computer-based algorithms can successfully analyze thousands of individual ECGs within the context of a safety pharmacology or toxicological study. Our experience has shown that well-designed computer algorithms can accurately analyze correctly the QT interval in more than 95% of the ECGs coming from a given study, resulting in up to 30,000 data points over an observation period of 8 h. This gives a solid basis for examining possible drug-induced effects when applying telemetry technology, even if changes in heart rate occur. This does, however, necessitate the use of computer-based data acquisition approaches, with expert review and evaluation by the technical operator, to ensure data quality that eliminates erroneous waveforms. These principles also apply to computer-based analysis of more limited ECG datasets collected by standard methods in toxicology studies. Thus, in summary, experience over that past few years suggests that state-of-the art studies should include accommodation of animals

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to the study conditions, a controlled, stress-free environment and the ability to collect sufficient volume of high quality ECGs to enable the use of appropriate heart rate correction formulae. 4. Use of standard toxicity studies for ICHS7A/B ECG assessments We have considered areas that potentially contribute to the deficiencies in ECGs collected in toxicological and safety pharmacology studies and that can negatively impact the evaluation of ventricular repolarization and other ECG parameters. We now consider in greater depth the methodologies typically used for ECG data collection in toxicological studies and compare them to those employed in optimized studies as presented above. Some of these points have been addressed recently, in regards to integrating safety pharmacology endpoints into toxicology studies (Luft & Bode, 2002). The question ultimately posed is whether the ECG recordings obtained routinely as a part of toxicological studies are adequate to enable a thorough analysis of possible drug-induced effects on the QT interval. From the onset, one must recognize that toxicological studies are performed to address the general safety of a test article (e.g., ante mortem observations, clinical and anatomic pathology) but can, in some cases, also include measures of arterial blood pressure, heart rate and the ECG. Each sponsor should consider whether the ECG data collected from a given toxicological study is of sufficient quality for a detailed waveform analysis or if independent safety pharmacology studies or enhanced ECG data collection in toxicology studies are preferable and will add value for making safety assessments. Indeed, a repeat-dose toxicology study may even be the more relevant option in those situations where QT interval prolongation following subchronic or chronic dosing is of special concern. On the other hand, for compounds that have otherwise been studied rigorously and found to present low risk of QT effects, a less rigorous analysis of the ECG may be all that is warranted. What are the strengths and weaknesses of ECGs collected in toxicological studies and what can be done to mitigate the weaknesses? In large part, much of this discussion has already been mentioned in previous sections and will therefore be only briefly described below.

designed to include an upper dose at which overt toxicity may be observed. The clinical events that the animal demonstrates at overtly toxic doses (e.g., shaking, tremor, hyperactivity, increased resistance, etc.) may also confound interpretation of the data and make it difficult to separate direct from indirect effects on the cardiovascular system. In contrast, safety pharmacology studies usually explore therapeutic drug exposure and modest multiples thereof, up to only moderately adverse effect levels (Anon, 2001, ICH S7A), but usually without producing overt toxic responses that could complicate interpretation of the ECG. Safety pharmacology studies are typically single-dose studies in which a given effect (e.g., QT interval duration) can be measured over time as a consequence of changing plasma exposure of the test substance. Collection of blood for analysis of plasma drug levels may occur in the same study starting after Tmax, during the elimination phase, thereby avoiding the period of Cmax where maximal druginduced effects may be anticipated. Alternatively, a more robust blood sampling could be performed in the same animals used in the cardiovascular study, or in a different group, but on a separate day. Animals accommodated to the study conditions are preferable since they return to baseline levels very quickly after blood sampling. Untrained animals, however, react more markedly to the procedure and need considerably longer to return to a baseline level. Robust toxicokinetic measurements are usually included in toxicological studies, but should not be performed on the same day as ECG measurements due to the influence of stress which may independently affect ECG endpoints. The one major advantage of toxicological studies is that ECG data may be collected sequentially after days or weeks of treatment and can even include washout phases to look at reversibility of any potential effects. Particularly for test substances having a potential for accumulation in cardiac tissue, measuring ECGs following repeated administration may add substantially to the overall risk assessment (for an example, see Fig. 4). One could, of course, design a dedicated cardiovascular safety pharmacology study with multiple administration using a telemetry approach, as described above. 4.3. ECG quality and environmental influences

4.1. Traditional methods of ECG data collection in toxicological studies Traditional methods of data collection in toxicology studies have been described by Dr. David K. Detweiler and others. Based on a careful analysis of comparative cardiac anatomy and physiology across various species and years of personal experience as a veterinary cardiologist consulting with pharmaceutical industry, Detweiler has offered recommendations to optimize the conditions for collection of the ECG in non-rodent species applying surface electrodes to restrained animals (Detweiler, 1983). These recommendations have served as the gold standard for acquiring ECG tracings within toxicological studies. Lead selection for safety pharmacology studies has been addressed recently (Hamlin, 2008). Nevertheless, the demand for a higher level of ECG analysis to detect more subtle effects on the ECG, such as QT interval prolongation, has required a further optimization of the conditions for data collection and evaluation. As described above, this objective can be approached in several different ways.

The topics of ECG quality and environmental conditions have already been presented. It deserves repetition, however, that the need for sufficient numbers of high quality ECG traces and therefore the need for control of study conditions will be governed by the degree of concern for a risk of QT prolongation of a given test agent. However, even if the ECGs obtained in a routine toxicological study have an adequate quality to allow detailed evaluation, we should recognize that they are almost never representative of a true resting condition of the animals due to the need for restraint (see Table 1). Given the fact

4.2. Dose selection, exposure and duration of treatment There can be important differences between toxicological studies and safety pharmacology studies, with the data from each serving a complementary role in understanding the potential cardiac safety risk of a new test substance. These differences include dose selection, duration of administration and the frequency of ECG collection in the course of a study. Furthermore, the collection of blood for analysis of exposure may differ between study types. Toxicological studies are

Fig. 4. Example of drug-induced QTc (van der Water's correction) prolongation over a 28-days toxicology study. The ECG was recorded continuously for 24-hours on day –2 (P-S; pre-study baseline), and then on day 2, 7, 14 21 and 27 of dosing. ECGs were recording in freely moving dogs using a non-invasive telemetry system. Note the overall stability of QTc over the one-month recording period. In this particular example, druginduced QTc prolongation became evident from day 7 onwards.

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that activation of the sympathetic nervous system is associated with effects on the QT interval (Fossa, 2008), this is an important aspect to consider with regards to the assessment of drug-induced effects on ventricular repolarization. Finally, the number of ECGs taken in a standard toxicity study is usually limited to comparatively short strips or “snap shots” that have typically been only a few seconds, and not exceeding a few minutes in the best of cases. It has therefore not been possible to select the optimal time for the ECG measurement, in terms of there being both a clean signal taken at the lowest possible heart rate. A limited number of ECGs for analysis makes determination of the relationship between heart rate and QT interval duration severely limited, again arguing against trying to assess QT interval effects to the level of ICH S7A and S7B expectations in such settings. As shown in the statistical power analysis above, there is substantially more variability in this setting and thereby a markedly lower sensitivity for detecting drug-induced effects. 4.4. Animal restraint and/or training In most routine toxicological studies, there is limited opportunity to acclimatize animals to the laboratory environment and make them accustomed to the procedure for collecting ECG data. The resulting physiological status of restrained dogs reflects a high level of excitement and anxiety as reflected in substantial tachycardia, even at an experienced toxicology unit (Table 1). This can, of course, be improved markedly, if the sponsor is willing to devote the resources to the training of animals prior to a study. Use of sedation (chemical restraint with e.g., ketamine) has some advantages in terms of decreasing motion artifacts but can also introduce other artifacts due to the depth and time course of sedation in an individual and the possible influence of anesthetic on the QT interval (for details see Hammond et al., 2001). On the other hand, dogs can be trained easily to lie quietly on a table for the length of time needed to collect ECG data thereby avoiding any form of chemical restraint. The application of this procedure on the scale of a routine toxicology study may be prohibitive, however. Ultimately, the level of animal training for a given study is dependent upon a cost/benefit assessment that must reflect the overall concern about a cardiac risk assessment and the importance of the particular study in that assessment. 5. Possible new approaches Telemetric techniques have become the ‘gold-standard’ approach to collecting ECG data, but the implanted instrumentation is not a practical option for repeat-dose toxicology studies. Therefore, use of non-invasive technologies (e.g., Holter monitors, non-invasive telemetry) for continuous, ambulatory acquisition of ECG data provides the means to achieve greater volumes of ECG data per subject and this has been used in man and various animal species (Birettoni, Porciello, Rueca, & Fruganti, 2004). As seen in Table 1, ECG tracings acquired in dogs using this approach have lower heart rates than restrained animals, being quite comparable with dogs having fully implanted telemetry units. Given the obvious advantages of this approach, the use of this technology in conjunction with toxicology studies should be encouraged, particularly when a more thorough analysis of the ECG is planned. The sponsor must recognize, however, that running such a study requires a significant upfront capital investment whereas human resource requirement remains unaffected. 6. Risk tolerance versus resources While delayed ventricular replorization may be a risk factor for TdP in humans, it is an imperfect predictor for proarrhythmic risk. Results from the clinical TQT studies will more directly answer the question of risk for QT interval prolongation in humans. Therefore, the issue we are addressing is how much risk is a sponsor willing to take that there will

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be a false-negative signal in the non-clinical QT study prior to entering clinical development? In this case, a positive QT finding in clinical development may force a sponsor to either abandon the compound or significantly revise the clinical development strategy. Resources that had been dedicated to this drug development candidate up to that point might have been redirected to another more promising candidate and thereby represents wasted resources and time, as well as a lost opportunity to have advanced the test agent devoid of QT effects. Alternatively, a false positive QT result from poorly designed non-clinical assays may result in an inappropriate termination of a candidate or even an entire project. A sponsor may want to consider if he/she is more tolerant of false positive or false negative (or neither) outcomes as assays and strategy are chosen. Defining this risk tolerance may help define the level of assay sensitivity with which a sponsor will be comfortable, which may in turn determine whether to devote the resources to collecting ECG QT data in a routine versus enhanced toxicology study. For those institutions that choose to substitute toxicology studies for safety pharmacology studies, this question becomes even more urgent to address. Since there is still a degree of uncertainty even in the most rigorous non-clinical battery of assays, there are never guarantees for the later clinical outcome irrespective of the strategy applied. Therefore, the risk tolerance should be based upon institutional mitigation strategy and spreading risk across the portfolio, with prioritization of some agents for enhanced ECG testing and others for routine testing, but in almost no cases (e.g., possible exception of biologics, Vargas et al., 2008) should routine ECG testing in toxicology studies be a substitute for the core battery safety pharmacology evaluation. 7. Conclusions Based on the study design issues, the application of “state-of-the-art” telemetry and the number and quality of the ECG tracings collected, ECG tracings taken during a dedicated safety pharmacological study are typically superior to those from a traditional toxicological study. Nevertheless, the potential strength of a QT assessment based on ECGs collected within a toxicological study will be dependent upon the care in which these ECGs are acquired, their resultant quality and the ability to define an adequate test sensitivity for detecting a given signal indicative of drug-induced effects. There is no recommendation for the level of assay sensitivity in ICH S7A and S7B, however it is stated that sponsors should document assay sensitivity and demonstrate appropriate responsiveness with positive and negative reference compounds in that test system. Therefore, if protocols for collecting ECG data from a toxicology study meet ICH recommendations, they will be adequate from a regulatory guidance standpoint. Therefore, any of the non-clinical in vivo QT assays (telemetry, jacket or traditional toxicology approach, see Table 2) could be appropriate for program decisions when there is a need for a clear understanding of associated risk. The authors support acquiring ECG data within a toxicological study, but urge strict attention to quality control and ICH S7B recommendations. The strength of the analyses performed will be significantly impacted by the quality and quantity of the ECG tracings obtained. This may, in practice, restrict the analysis of the ECGs collected in routine toxicological studies to those parameters that can be reliably attainable even with less than ideal quality signals, including the heart rate and overt arrhythmias or other large ECG morphological changes. For the great majority of standard toxicity studies conducted, the ECG data collected do not permit an adequate assessment of drug-induced effects on the QT interval with the sensitivity expected from the ICH guidelines. Furthermore, sponsors should be discouraged from performing any analyses on low quality ECG tracings (e.g., Fig. 1) to avoid generating misleading data. Substantial improvements in ECG quality and quantity are available by applying non-invasive telemetry procedures, making a QT interval assessment within the context of a standard toxicological study feasible. It must be recognized that these methods may require a

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larger commitment of resources from the sponsor. From the viewpoint of risk mitigation and limiting the attrition of promising new therapies, a commitment of resources to insure ECG data quality in either toxicology or safety pharmacology studies may be justified. Acknowledgements The authors would like to thank Dr. Jonathan Bright from AstraZeneca for conducting the statistical power analysis, as well as Gregory Friedrichs from Schering-Plough Research Institute for his helpful comments. The authors would also like to acknowledge the role of the Safety Pharmacology Society in fostering the discussion leading to this manuscript and bringing together the co-authors. References Anon (2001). ICHS7A: Safety Pharmacology Studies for Human Pharmaceuticals. Available at: http://www.ich.org/cache/compo/276-254-1.html Anon (2005a). ICHS7B: The Non-clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) By Human Pharmaceuticals. Available at: http://www.ich.org/cache/compo/276-254-1.html Anon (2005b). ICHE14: The Clinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential for Non-Antiarrhythmic Drugs. Available at http://www. ich.org/cache/compo/276-254-1.html Authier, S., Tanguay, J. F., Fournier, S., Gauvin, D., Legaspi, M., Chaurand, F., et al. (2008). Conscious and anesthetized non-human primate safety pharmacology models: hemodynamic sensitivity comparison. Journal of Pharmacological and Toxicological Methods, 58, 94–98. Bass, A. S., Darpo, B., Valentin, J. -P., Sager, P., & Thomas, K. (2008). Frontiers in pharmacology: moving towards better predictors of drug-induced Torsades de Pointes (TdP). British Journal of Pharmacoly, 154, 1550–1553. Bazett, H. C. (1920). An analysis of the time relationships or time-relations of electrocardiograms. Heart, 7, 353–380. Birettoni, F., Porciello, F., Rueca, F., & Fruganti, G. (2004). 24-hour ambulatory electrocardiography in the dog. Veterinary Research Communication, 28(suppl.1), 323–325. Davis, A. S., & Middleton, B. J. (1999). Relatoinship between QT interval and heart rate in Alderley Park Beagles. The Veterinary Record, 145, 248–250. Detweiler, D. K. (1983). Electrocardiographic monitoring in toxicological studies: principles and interpretations. Advances in Experimental Medicine and Biology, 161, 579–607. Fredericia, L. S. (1920). Die systolendauer in elecktrokardiogramm bei normalen menschen and bei herzkranken. Acta Medica Scandinavica, 53, 469–505. Fossa, A. A. (2008). The impact of varying autonomic states on the dynamic beat-to-beat QT–RR and QT–TQ interval relationships. British Journal of Pharmacology, 154(7), 1508–1515. Hamlin, R. L. (2005). Non-drug-related electrocardiographpic features in animal models in safety pharmacology. Journal of Pharmcological and Toxicological Methods, 52, 60–76. Hamlin, R. L. (2008). How many ECG leads are required for in vivo studies in safety pharmacology? Journal of Pharmacological and Toxicological Methods, 57, 161–168.

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