ECG telemetry in conscious guinea pigs

Journal of Pharmacological and Toxicological Methods 81 (2016) 88–98

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Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox

ECG telemetry in conscious guinea pigs Sabine Ruppert a, Thomas Vormberge a, Bernd-Wolfgang Igl b, Michael Hoffmann a,⁎ a b

Safety Pharmacology, Bayer Pharma AG, Wuppertal, Germany Research & Clinical Sciences Statistics, Bayer Pharma AG, Berlin, Germany

a r t i c l e

i n f o

Article history: Received 28 January 2016 Received in revised form 7 April 2016 Accepted 22 April 2016 Available online 24 April 2016 Keywords: ECG Early stage drug development Guinea pigs QT interval correction Social animal housing Telemetry method

a b s t r a c t Introduction: During preclinical drug development, monitoring of the electrocardiogram (ECG) is an important part of cardiac safety assessment. To detect potential pro-arrhythmic liabilities of a drug candidate and for internal decision-making during early stage drug development an in vivo model in small animals with translatability to human cardiac function is required. Methods: Over the last years, modifications/improvements regarding animal housing, ECG electrode placement, and data evaluation have been introduced into an established model for ECG recordings using telemetry in conscious, freely moving guinea pigs. Pharmacological validation using selected reference compounds affecting different mechanisms relevant for cardiac electrophysiology (quinidine, flecainide, atenolol, DL-sotalol, dofetilide, nifedipine, moxifloxacin) was conducted and findings were compared with results obtained in telemetered Beagle dogs. Results and conclusion: Under standardized conditions, reliable ECG data with low variability allowing largely automated evaluation were obtained from the telemetered guinea pig model. The model is sensitive to compounds blocking cardiac sodium channels, hERG K+ channels and calcium channels, and appears to be even more sensitive to β-blockers as observed in dogs at rest. QT interval correction according to Bazett and Sarma appears to be appropriate methods in conscious guinea pigs. Overall, the telemetered guinea pig is a suitable model for the conduct of early stage preclinical ECG assessment. © 2016 Elsevier Inc. All rights reserved.

1. Introduction During the preclinical drug development process, monitoring of the electrocardiogram (ECG) in a relevant animal species is an integral part of cardiac safety assessment. Changes in the ECG intervals PQ (PR), QRS and QT as well as alterations of the ECG waveform and the occurrence of arrhythmias are in the focus of such preclinical investigations in Toxicology and particularly in Safety Pharmacology. For such investigations it is important to select a relevant species which shows high similarity to human cardiac ion channel distribution. Effects on cardiac ion channels translating into changes in ECG intervals are a surrogate for potential pro-arrhythmogenic activity, e.g. inhibition of the cardiac potassium channel (IKr, hERG) resulting in QT interval prolongation and may cause fatal ventricular tachyarrhythmia (Torsades de Pointes). Since adult rodents (rats, mice) are lacking a functional cardiac hERG K+ channel, the ventricular repolarization is not human-like (Kaese & Verheule, 2012; Nerbonne & Kass, 2005; Wang, Feng, Kondo, Sheldon,

Abbreviations: 3Rs, replacement, refinement and reduction; ECG, electrocardiogram; ICH, international conference on harmonization; NCE, new chemical entity; NHP, nonhuman primate; VAP, venous access port. ⁎ Corresponding author at: Safety Pharmacology, Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany. E-mail address: [email protected] (M. Hoffmann).

http://dx.doi.org/10.1016/j.vascn.2016.04.013 1056-8719/© 2016 Elsevier Inc. All rights reserved.

& Duff, 1996). Hence, rats and mice are not appropriate species for cardiac electrophysiological safety assessment (Anonymous, 2005b). According to guidelines (Anonymous, 2009), in toxicological and safety pharmacological studies conducted prior to first use of a new investigational drug in humans (new chemical entity (NCE) or biosimilar) dogs, minipigs or non-human primates (NHP) are employed in most cases, whereas NHP should be used only if no other species is sensitive for the specific drug or target molecule. To detect potential proarrhythmic liabilities of a drug candidate and for internal decision making during early pre-clinical drug development, large animal models have some disadvantages such as high amount of test compound need or expensive animal housing. Cardiovascular telemetry in dogs or NHP is the accepted gold standard in late stage preclinical development (Guth, 2007) but not suitable for cardiovascular safety screening in early stage of drug development. Among the available small animal species, the guinea pig appears to be a suitable model for the assessment of electrophysiological cardiac safety since distribution of cardiac ion channels is human-like (Hamlin, Kijtawornrat, Keene, & Hamlin, 2003). Thus, the predictability of electrophysiology liabilities is considered similar to the non-rodent species dogs, minipigs or NHP. This was reported earlier in anesthetized e.g. (De Clerck et al., 2002; Hauser, Stade, Schmidt, & Hanauer, 2005) and telemetered guinea pig models e.g. (Hess, Rey, Wanner, Steiner, & Clozel, 2007; Shiotani, Harada, Abe, Hamada, & Horii, 2007b). Following the ICH S7 guidance

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(Anonymous, 2000) the use of conscious, freely moving animals in safety pharmacological studies is preferred. In particular when only ECG but no hemodynamic parameters will be assessed, the telemetered guinea pig model has some advantages compared to anesthetized models: (i) no interaction with anesthetics hampers interpretation of results; (ii) long observation periods (theoretically several days of continuous measurement) are possible; (iii) cross-over treatment design including long baseline periods is feasible; (iv) possible circadian rhythm effects can be included. Furthermore, the telemetered guinea pig model, like telemetry models in general, contributes to the 3Rs concept (Russel & Burch, 1959) due to the reuse of animals after an adequate washout period between different studies (reduction) and the use of state-of-theart technique (Kramer & Kinter, 2003) and social housing concepts (refinement). This paper describes the established ECG telemetry model in conscious guinea pigs used in our group over several years (Hoffmann, Ruppert, & Vormberge, 2009) with modifications introduced regarding implementation of modern telemetry technique and housing conditions. Moreover, effects of reference compounds (quinidine, flecainide, atenolol, DL-sotalol, dofetilide, nifedipine, moxifloxacin) affecting different mechanisms relevant for cardiac electrophysiology are shown and compared with results obtained in our in-house telemetered Beagle dog model. 2. Material and methods

89

12 h light/dark cycle. For individual identification all animals were implanted subcutaneously with a transponder ID (Datamars, Bedano, Switzerland) in parallel with transmitter surgery. For ECG recordings, the animals were moved to a separate telemetry room at least one day prior to study start. 2.2. Surgery For monitoring of the ECG, body temperature, and locomotor activity a telemetry device (TA11CTA-F40, Data Science, Inc. (DSI), St. Paul, MN, USA), comprising a transmitter, a temperature sensor, and two wired ECG electrodes, was implanted under sterile conditions. At surgery the guinea pigs were 2–8 months of age and body weight ranged from approx. 600–1000 g. Following ventral midline laparotomy, the body of the transmitter was inserted into the abdominal cavity and fixed with peritoneal suture. The 2 ECG electrodes were placed as described below. Then the abdominal cavity was closed with Ethibond Excel 2-0 and PDS 4-0 suture (Ethicon, Norderstedt, Germany). Prior to surgery the guinea pigs received the antibiotic enrofloxacin (Baytril 2.5%, Bayer, 5 mg/kg subcutaneously), the analgesic carprofen (Rimadyl, Pfizer, 5 mg/kg subcutaneously), and atropine (0.05 mg/kg intramuscularly). An inhalational anesthetic via a face mask with 70% O2 + 30% N2O airflow was induced (4–5%) and maintained with isoflurane (1.5–2%). After surgery the animals were allowed a recovery period of at least 2 weeks.

2.1. Animals 2.3. Placement of ECG electrodes The animal studies reported here were conducted at the Department of Safety Pharmacology of Bayer Pharma AG, Wuppertal, Germany, following the regulatory framework of the domestic animal welfare authorities and current Standard Operating Procedures. Female Dunkin Hartley guinea pigs were obtained from Harlan (Horst, Netherlands or Huntington, UK). After delivery, the animals were acclimatized for at least 2 weeks. During acclimatization and when not employed in a telemetry study the guinea pigs were grouphoused in groups of up to 15 animals on an approx. 40 ft2 floor pen with wood granulate bedding and appropriate environmental enrichment (e.g. shelter, hay). For a period of at least 1 week after surgery and during a telemetry study, the guinea pigs were pair-housed in Noryl cages (Tecniplast, Hohenpeißenberg, Germany, approx. 4.5 ft2 space) with wood granulate bedding (see Fig. 1A). These cages provide a darkened environment with limited direct light exposure. All animals, group-housed or pair-housed, had free access to drinking water and pelleted guinea pig standard food (Ms-H, 4 mm, ssniff, Soest, Germany). The animals were housed under climate-controlled conditions (room temperature: 21–23 °C, relative humidity: 40–60%) with a

The two ECG electrodes were placed corresponding to the direction of standard lead II. 2.3.1. Subcutaneous placement The negative lead was placed subcutaneously on the right side of the thorax, close to the right clavicle. The positive lead was placed subcutaneously left lateral below the last rib. 2.3.2. Subcutaneous/diaphragm placement The negative lead was placed subcutaneously on the right side of the thorax, close to the right clavicle. The positive lead was placed abdominally and fixed at the left lateral side of the diaphragm. 2.3.3. Intravenous solid tip/diaphragm placement The negative lead (solid tip, diameter 1.2 mm, see Fig. 2) was introduced into the superior vena cava via the left jugular vein close to the base of the heart. This was done under ECG waveform control while the solid tip lead was introduced from the jugular vein into the cranial

Fig. 1. Group-housing on open floor space (A), pair-housing with telemetry hardware setup (B).

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Fig. 2. DSI TA11CTA-F40 transmitter with intravenous solid tip lead.

vena cava. The desired position is reached when the P-waveform is between “high P-wave” and “no P-wave”. The positive lead was placed abdominally and fixed at the left lateral side of the diaphragm. 2.4. Data collection and QT interval correction The ECG signals (1000 Hz sampling rate), body temperature and locomotor activity were captured by 4 telemetry receivers (RPC-1, DSI, USA) located underneath each cage (see Fig. 1B). The receivers were connected to a computer with the acquisition software Ponemah P3 Plus (DSI, USA). Collected data were processed by the data acquisition program, averaged over a logging rate of 5 min and stored on hard disk. From ECG signals the PQ (PR) interval, the QRS complex, and the QT interval were evaluated online. Correction of the QT interval for heart rate was done online using Bazett's formula (QTcB [ms] = QT [ms] / (R − R [s])½) (Bazett, 1920). In addition an exponential QT interval correction according to Sarma (QTcS [ms] = QT [ms] + a (exp(b ∗ RR[s]) − exp(b))) (Sarma, Bilitch, & Melinte, 1983) was conducted. The parameters a and b have been estimated from QT:RR plots from previously conducted 310 control experiments in 53 female guinea pigs. ECG data comprising of QT/RR intervals derived from 16,000 single beats (representing approximately 70–90 min of ECG recordings) per animal were used for the QT:RR plots. In these investigations the mean heart rate range was 170–280 beats/min. For QTcS calculations the parameters a and b determined for each individual guinea pig (QTcSi) or the arithmetic mean of all individual parameters a and b calculated from QT:RR plots of the 53 employed guinea pigs were used as “Bayer cohort” parameters (QTcScoh). Since analysis of study data revealed that individually corrected QTcSi data and cohort corrected QTcScoh data differ only marginally by about 3% (data not shown), here the cohort corrected formula QTcS [ms] = QT [ms] + 319.8 (exp(− 7.54 ∗ RR[s]) − exp(−7.54)) was used. 2.5. Study design A cross-over treatment design with 4–6 guinea pigs was conducted. Oral administration of test compounds was done study specific between 10 a.m. and 3 p.m. While the guinea pigs were pair-housed during period of data recordings only one animal was dosed with the test article, the other animal was used as companion for animal welfare reason. Predrug (baseline) telemetry values are the average of read-outs over the period from 120 min before application. After administration, data were calculated every 30 min over a period of 15–24 h. Satellite animals (2–4 animals per dose group) were used for blood sampling and pharmacokinetic investigations. 2.6. Blood sampling and bioanalytics Venous blood sampling for determination of plasma concentrations of administered test compounds was done in satellite animals housed in

groups of 2–3 animals under comparable conditions as described above for telemetry animals. For blood sampling a venous access port (VAP, Plastic SoloPort MIN with a heparin coated 3.5 Fr CBAS catheter, Solomon Scientific, Skokie, IL, USA) was implanted under sterile conditions and inhalational anesthesia as described above. The VAP was placed subcutaneously in the dorsal neck/scapulae region; the attached catheter, filled with sterile heparinized saline, was passed subcutaneously into the left jugular vein. Skin incisions were closed with Ethibond Excel 2-0 suture. After implantation of the VAP a recovery period of at least 4 days followed. Our experience with more than 60 implanted VAPs showed that N 80% of the ports were open longer than 1 week (in average 7 weeks) for blood collection when the catheter was flushed with heparinized saline (30 I.U./mL) regularly every third day. Blood samples were drawn at pre-defined time points into lithium-heparin coated tubes (Microvette 500LH, Sarstedt, Nümbrecht, Germany), stored on ice and then centrifuged. Plasma samples were transferred into labeled tubes and frozen (below −20 °C) until quantitative analysis of test article concentrations. Bioanalytical methods to determine plasma concentrations in dog and guinea pig plasma were developed in-house. The plasma samples were processed by protein precipitation with acetonitrile or acetonitrile/ammonium acetate including an appropriate internal standard. Drug concentrations in plasma of quinidine, flecainide, atenolol, dofetilide, and nifedipine were determined by means of highperformance liquid chromatography (HPLC) and tandem mass spectrometry detection. The lower limit of quantitation was 0.25 μg/L (quinidine, flecainide, dofetilide, atenolol), or 0.05 μg/L (nifedipine), respectively. Determination of moxifloxacin and DL-sotalol was conducted in plasma after addition of an internal standard and protein precipitation with a mixture of acetonitrile and phosphate buffer by HPLC and fluorescence detection. The lower limit of quantitation was 10 μg/L (moxifloxacin) and 12 μg/L (DL-sotalol), respectively. 2.7. Dog telemetry Methods of telemetric ECG measurements and determination of drug plasma concentrations were described previously in detail (Himmel et al., 2012). Briefly, male and female Beagle dogs were equipped with telemetry devices (TL11M2-D70-PCT, DSI, USA) and ECG signals were collected at baseline and for about 15 h after oral administration. Heart rate and ECG intervals PQ, QRS, QT, and QTc according to Van de Water (QTcV) (Van de Water, Verheyen, Xhonneux, & Reneman, 1989) were evaluated. 2.8. Reference compounds and administration The following drugs were used as positive references: Quinidine and atenolol (obtained from Sigma-Aldrich, USA), flecainide (obtained as tablets from Actavis, Germany), DL-sotalol (obtained as tablets from Ratiopharm, Germany), and dofetilide, nifedipine, moxifloxacin (own synthesis, Bayer, Germany). The results from guinea pig telemetry studies were compared with results from dog telemetry studies. Dosage and formulation see Table 1. Dogs were administered orally with gelatin capsules containing 0.25 mL administration formulation per kg body weight or pure powder from grinded tablets. Since the procedure of gavage administration is very stressful, the guinea pigs were administered buccal (into the cheek pouch) with administration volumes of 1.5– 3 mL/kg. 2.9. Data evaluation and statistics The telemetry signals collected with the data acquisition program Ponemah were recorded continuously and average over a pre-defined logging rate of usually 5 min and stored on-line on hard disk followed by a manual off-line check for artifacts based on quality parameters

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3. Results

Table 1 Reference compounds and formulations. Compound Guinea pigs Quinidine Flecainide Atenolol sotalol Moxifloxacin Nifedipine

DL

Dogs Quinidine Flecainide Atenolol sotalol Moxifloxacin Nifedipine

DL

91

Dose range (mg/kg)

Administration vol. (mL/kg)

Vehicle

0–12–40–120 0–15–45–90 0–5–10 0–10–30–100

3.0 3.0 3.0 3.0

0.5% aqueous tylose 0.5% aqueous tylose Water Water

0–30–60–120 0–0.3–1–3

3.0 1.5

0.5% aqueous tylose PEG400/water 90/10

0–5–15–45 0–3–10–30 0–1.5–5–15 0–10–25–50

0.25 Solid powder in capsules 0.25 Solid powder in capsules

EtOH/PEG400 10/90 Water 0.5% aqueous tylose Water

0–15–30–60 0–0.3–1–3

0.25 0.25

0.5% aqueous tylose PEG400

Route of administration: guinea pigs: buccal; dogs: oral with gelatin capsules. Abbreviations: EtOH, ethanol; PEG400, polyethylene glycol 400.

generated by the Ponemah software, e.g. bad waves, good waves, Twave and P-wave counts. After this, the 5 min data were averaged into evenly spaced intervals using Microsoft Excel and transferred to SAS software (Version 9.2 or later) for statistical evaluation and to provide tables und figures as final output. Common intervals are of length 15, 30 or 60 min. On the one hand, a wider interval leads to a stronger smoothing effect with a smaller standard deviation compared to a smaller interval. On the other hand, a small interval may yield larger standard deviations suppressing statistical significance, but also nonrelevant random findings might be supported. We compared the results of our proposed statistical strategy on several different data sets containing different interval lengths and choose a medium length of 30 min per interval since corresponding results seem to be stable and plausible. Each of the telemetry parameters was analyzed separately using a generalized linear model approach for repeated measurements to evaluate the dependency between a response variable on the one hand and several fixed effects on the other. These factors model effects for baseline, treatment, time and their interaction treatment-by-time. The interaction term was included in the model regardless of its level of significance. In general, telemetry studies were performed using a small number of animals. Especially in such scenarios, degrees of freedom for the tests of fixed effects should always be adjusted according to a method developed by Kenward and Roger (1997). A classical Analysis-of-Variance (ANOVA) methodology would concentrate on the (time-) point wise comparison of treatment groups in general and would ignore the correlation between observations stemming from the same animal at different time points. Whether to use this time dependent information is a regularly discussed topic in the statistical community and how to estimate the correlation structure in a small sample setting is a well-known problem (Aylott, Bate, Collins, Jarvis, & Saul, 2011; Chiang, Smith, Main, & Sarazan, 2004). However, in our experiments, measurement time points were equally spaced and correlations were assumed to decline with distance. To this end, we decided to involve a simple correlation structure into our methodology and assume a first-order autoregressive covariance structure to model the dependency structure of repeated measurements over time. Moreover, the baseline value of the primary outcome should be included as a covariate in the primary analysis. In general, if a baseline covariate is involved into the estimation process of a treatment effect; inferential results are identical for both “change from baseline” and the “raw outcome” analysis (Anonymous, 2013). Finally, multiple comparisons between treatment and control data were performed using Dunnett's approach per time point using a significance level of 5% (p b 0.05).

3.1. Baseline and control data In 64 female guinea pigs (age ranging from 2 to 17 months, body weight ranging from 630 to 1440 g) mean baseline values were calculated in total of N 400 experiments conducted under standardized conditions (e.g. ECG measurements in a specific telemetry room with 12 h light/dark cycle, pair-housing during period of ECG recordings, food and drinking water ad libitum). As shown in Table 2, the mean (±SD) heart rate was 213 ± 16 bpm, PQ interval 68 ± 6 ms, QRS complex 30 ± 5 ms, QT interval 157 ± 10 ms, QTcB interval 295 ± 12 ms, and QTcS interval 195 ± 6 ms. A 24 h profile, calculated from 10 experiments with guinea pigs equipped with solid tip/diaphragm electrodes, revealed that baseline values did not change substantially over time and no significant diurnal rhythm was found. Body temperature was constant at about 39 °C; the locomotor activity was slightly changed when lights were switched on or off (see Fig. 3). The locomotor activity is a result of the variability of telemetry signal strength which varies due to orientation and distance of animal movements relative to the receiver. A certain change in signal strength generates activity counts which are summed over the logging rate. The values of mean activity counts over the 24 h period were between 1 and 5, indicating that the animals behaved very quiet. Short periods of high activity can result in counts of about 30. 3.2. Placement of ECG electrodes The 2 ECG electrodes of the F40-transmitter were placed in a standard lead II configuration. A subcutaneous placement of both electrodes results in principle in good and detectable ECG waveforms. However, movements or muscle activity causes some disturbances in ECG recordings. Thus, we tried to minimize such disturbances and placed the positive electrode intraperitonially on the left lateral side of the diaphragm close to the apex of the heart. Later, DSI provided transmitters with a so called “solid tip electrode” (see Fig. 2) which is introduced intravenously into the cranial vena cava via the right jugular vein. These 2 changes in lead configuration markedly improved the ECG recordings. Ponemah's quality parameter “good waves”, meaning the portion of detectable ECG waves relative to all recorded ECG waves after cleaning from artifacts, was improved from about 90% up to nearly 100% (see Table 2). The intravenous solid tip/diaphragm placement is relatively free from movement artifacts and produces clear signals with high wave voltage (R-wave ~ 5 mV) due to the close lead orientation from base to apex of the heart (see Fig. 4A–C). In our group, we have solid tip implanted guinea pigs over more than 1 year without any observed signs of thrombotic events or an increased incidence of cardiac arrhythmia. 3.3. QT interval correction for heart rate In the literature the QT interval correction according to Bazett is often the preferred method in guinea pigs (e.g. Hamlin et al., 2003; Shiotani, Harada, Abe, Hamada, & Horii, 2007a). We investigated some other established formulas in our conscious telemetered guinea pigs. For this purpose the guinea pigs were treated with reference compounds which have effects on heart rate with and without direct effects on QT interval. The compounds investigated were nifedipine (a calcium channel blocker causing vasodilation with reflex tachycardia), atenolol (a β-adrenoceptor blocker causing bradycardia) and DL-sotalol (a hERG K+ channel blocker with β-blocking properties causing heart rate reduction and QT prolongation) at doses known to be pharmacologically active. Although some authors described a good QT correction in guinea pigs with Bazett's formula, for comparison in this investigation the formulas according to Fridericia (1920), Matsunaga (a logarithmic formula developed for the correction of dog QT interval) (Matsunaga et al., 1997) and Sarma (an exponential formula based on individual correction) (Sarma, Sarma, Bilitch, Katz, & Song, 1984) were also tested.

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Table 2 Baseline values at different ECG lead configurations. ECG Lead II configuration

N

HR (bpm)

PQ (ms)

QRS (ms)

QT (ms)

QTcB (ms)

QTcS (ms)

GW %

s.c./s.c. s.c./i.p. s.t./i.p. All

32 24 8 64

214 ± 18 209 ± 12 221 ± 10 213 ± 16

68 ± 7 68 ± 6 69 ± 3 68 ± 6

30 ± 6 32 ± 3 28 ± 2 ±30 ± 5

156 ± 11 162 ± 6 148 ± 7 157 ± 10

293 ± 11 301 ± 10 282 ± 11 295 ± 12

194 ± 5 198 ± 5 188 ± 4 195 ± 6

91 ± 8 98 ± 3 97 ± 3 94 ± 6

Other parameters Lead II configuration

N

BT (°C)

Act (counts)

BW (g)

Age (months)

s.c./s.c. s.c./i.p. s.t./i.p. All

32 24 8 64

39.0 ± 0.2 39.3 ± 0.2 39.2 ± 0.3 39.1 ± 0.3

2.6 ± 1.0 1.4 ± 1.0 3.3 ± 1.3 2.2 ± 1.2

1089 ± 149 832 ± 183 946 ± 85 973 ± 196 (range: 630–1440)

3–17 2–9 4–11 2–17

Values as arithmetic means ± standard deviation (SD); age as range. Lead configuration: s.c./s.c. = (−) and (+) electrodes subcutaneously; s.c./i.p. = (−) subcutaneously, (+) diaphragm; s.t./i.p. = (−) solid tip, (+) diaphragm. Abbreviations: N, number of animals; HR, heart rate; QTcB, QTc according to Bazett; QTcS, QTc according to Sarma; GW, percentage of good ECG waves detected by Ponemah software relative to total ECG waves; BT, body temperature; Act, locomotor activity; BW, body weight.

To verify the adequacy of the different QT correction models the QT/ QTc:RR relationship was plotted using the QT/QTc intervals and the corresponding RR interval, which was measured over 15 min in the 12 control experiments of this study. For each QT/QTc:RR plot the coefficient of determination (R2) and slope of regression was determined. In a regression, R2 is a statistical measure of how well the regression line approximates the real data points. An R2 of 1 indicates that the regression line perfectly fits the data; an R2 of 0 indicates data without dependency.

With regard to QT correction for heart rate, QTc formulas can be classified based on the rule the lower the R2 and the slope, the better the formula. As shown in Table 3, in this study such a classification results in the adequacy order (from best to worst): QTcS ≥ QTcB N QTcF N QTcM. These data shows, that Fridericia's (QTcF) and Matsunaga's (QTcM) formulas does not appropriately correct QT values since the R2 and slope values are not much lower than the uncorrected QT:RR relationship. A much better fit is shown by the formulas according to Bazett

Fig. 3. 24 h profile of pair-housed, telemetered female guinea pigs. ECG lead II configuration: (−) solid tip/(+) diaphragm. Vertical dotted line: administration of a control vehicle (3 mL/kg buccal). Horizontal black line: lights off. Data presented as means ± SD, N = 10. Abbreviations: HR, heart rate; BT, body temperature; QTcB, QTc according to Bazett; QTcS, QTc according to Sarma.

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Fig. 4. Representative ECG traces obtained from different electrodes placement each with 4 animals recorded in parallel. A: subcutaneous/subcutaneous, B: subcutaneous/diaphragm, C: intravenous solid tip/diaphragm. For comparison voltage axis are equally graduated from −1 mV to +6 mV, recording duration: 6 s.

and Sarma with a very similar R2 but a lower slope with Sarma's formula. After administration of the 3 test compounds we found the anticipated effects on heart rate and QT interval: Nifedipine caused a strong

Table 3 R2 and slope of regression from QT/RR plots of control experiments.

R2 Slope

QT

QTcS

QTcB

QTcF

QTcM

0.665 391.7

0.155 108.9

0.151 221.3

0.358 318.7

0.527 297.6

Abbreviations: R2, coefficient of determination; QTcB, QTc according to Bazett; QTcS, QTc according to Sarma; QTcF, QTc according to Fridericia; QTcM, QTc according to Matsunaga.

increase in heart rate and QT shortening, atenolol a decrease in heart rate and slight QT prolongation, DL-sotalol caused a heart rate decrease and pronounced QT prolongation. When the QT interval was corrected for heart rate, the QTc shortening after nifedipine was still pronounced with Bazett but clearly reduced with Sarma's correction. The slight QT prolongation after atenolol was not seen after correction with both, Bazett and Sarma. The QT prolongation caused by DL-sotalol was still present after correction according to Bazett and Sarma (see Fig. 5). Only a small difference on QTcS was found when calculated individually (QTcS i) or on the basis of data from the cohort of 28 guinea pigs (QTcScoh) employed in this investigation. The maximum effects after administration of the 3 test

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Fig. 5. Effects of atenolol, nifedipine and DL-sotalol on heart rate and QT interval in telemetered guinea pigs; Influence of different QT correction formulas. Vertical dotted line: administration of a reference compound. Abbreviations: HR, heart rate; QTcB, QTc according to Bazett; QTcS, QTc according to Sarma based on cohort data.

compounds differs only marginally: DL-sotalol: + 26 ms QTcScoh versus + 23 ms QTcSi , nifedipine: − 14 ms QTcScoh versus − 11 ms QTcSi, atenolol: no detectable difference. The correction according to Matsunaga and Fridericia was less sufficient for heart rate changes after atenolol and nifedipine (data not shown).

3.4. Effects on selected reference compounds A set of orally administered reference compounds was tested for their effects on heart rate and ECG intervals in conscious telemetered guinea pigs (see Table 4) and compared with results from conscious, telemetered Beagle dogs (see Table 5). The dose-ranges tested (see Table 1) were estimated from previous studies in dogs and rats and from the literature. Plasma levels obtained to dogs covered the therapeutic range in humans with the low doses and were above the therapeutic range with the high dose by about 3-fold (quinidine, nifedipine, moxifloxacin), and 10-fold (flecainide, atenolol, dofetilide) up to about 15-fold (DL-sotalol). With the exception of nifedipine (where same dose-range was used), the dose-range adjustment from dogs to guinea pigs was done based on body surface area calculation. Plasma concentrations were determined in satellite animals at each dose level in dogs and at mid and high dose level in guinea pigs. The peak plasma concentrations obtained in guinea pigs were generally about 2-fold lower than in dogs. Due to resources constraints no plasma concentrations were determined in guinea pigs after administration of DL-sotalol, atenolol and nifedipine. In guinea pigs no substantial treatment related effects on locomotor activity and body temperature were found with any of the reference

compounds with the exception of a slight decrease in body temperature by 0.3 °C at the highest tested dose of nifedipine. 3.4.1. Class I anti-arrhythmics quinidine and flecainide Both quinidine and flecainide are inhibitors of the cardiac sodium channel and of the hERG K+ channel (Grace & Camm, 1998) (Aliot, Capucci, Crijns, Goette, & Tamargo, 2011). In the ECG, sodium channel block translates into QRS complex widening, hERG K+ channel block into QTc prolongation. In both guinea pigs and dogs the effects on QRS were slight and not statistically significant after administration of quinidine. In contrast, flecainide caused a clear QRS widening and significant QT prolongation in both guinea pigs and dogs. In guinea pigs effects on QTcS were similar after quinidine and flecainide, whereas in dogs a strong QTcV prolongation was seen after quinidine but only slight effects on QTcV with flecainide. PQ prolongation was found after flecainide in guinea pigs and dogs. The picture was different with regard to heart rate. In guinea pigs no clear effects on heart rate were found with quinidine whereas flecainide caused statistically significant heart rate reduction. In dogs both compounds produced a clear increase in heart rate. 3.4.2. Class II anti-arrhythmic atenolol The class II anti-arrhythmic drug atenolol is a selective blocker of β1adrenoceptors (β-blocker) and lacks any relevant interaction with cardiac ion channels (Himmel et al., 2012). Atenolol caused a delay in atrio-ventricular conduction (prolonged PQ interval) reaching statistically significance in guinea pigs and in dogs. The main effect in humans, i.e. reduction of heart rate, was also seen in guinea pigs as clear, dosedependent and statistically significant effect, whereas in telemetered

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Table 4 ECG effects of reference compounds in telemetered guinea pigs. Compound

Dose mg/kg

HR bpm

Quinidine

BL 12 40 120 BL 15 45 90 BL 5 10 BL 0.03 0.1 0.3 BL 10 30 100 BL 30 60 120 BL 0.3 1 3

224

Flecainide

Atenolol

Dofetilide

DL

a

sotalola

Moxifloxacina

Nifedipine

Δ%

QRS ms

Δ%

28

213

30 −7.3 −17.9⁎ −21.2⁎

27 −6.7 −6.6 −10.3⁎

248

217

+0.5 0.0 189

+4.0 +14.2⁎ +18.9⁎

+3.2 +11.8⁎ +16.3⁎

267 +6.0 +9.1 +9.0⁎

64 −1.1 +5.8 +5.8 33 0.0 +3.0 −3.0

199

284

67

29 −2.2 +3.8 +8.9 204 +15.4⁎ +29.4⁎ +40.2⁎

+2.9 +6.5⁎ +7.6⁎

0.0 +0.7

−3.2 +2.8 −7.0

−3.6 −3.6 0.0

195

303

64

28 −8.1 −15.0⁎ −18.0⁎

+1.5 +4.0⁎ +7.4⁎

+3.7 +7.8⁎ +8.3⁎

+8.2 +15.9⁎

−2.2 +13.3 +34.4⁎

194

295

72

Δ%

QTcS ms

+2.4 +6.6⁎ +9.2⁎

+7.0 +15.1⁎ +23.2⁎

0.0 0.0

Δ%

292

72

32

208

QTcB ms

−3.0 −5.8 −5.9

+3.3 +10.0⁎ +23.3⁎

−18.3⁎ −29.5⁎

Δ%

67 −6.9 −3.6 +7.1

+3.5 +5.0 +5.0

207

PQ ms

181 +8.3 +8.3 +11.2⁎

+5.0 +5.5 +11.3⁎

302 −1.6 +4.6 −4.8 66 −9.2 −10.1 −18.5⁎

200 +3.7 +4.2 +10.6⁎ 303 −3.3 −14.7 −19.8⁎

+2.5 +3.4 +8.0⁎ 199 −2.0 −10.5 −11.1⁎

Cmax μg/L − n.d. 1658 1976 − n.d. 692 1609 − n.d. n.d. − n.d. 2.49 5.77 − n.d. n.d. n.d. − n.d. 2640 3210 − n.d. n.d. n.d.

Data are mean values (n = 4–6 per dose). Pre-drug baseline values (BL) are absolute values in beats per minute (bpm) or ms, whereas all other values are expressed as % change versus BL (Δ%). Maximal drug plasma concentrations (Cmax) were determined in satellite animals (n = 2–4 per dose). Abbreviations: n.d., not determined; HR, heart rate; QTcB, corrected QT interval according to Bazett; QTcS, corrected QT interval according to Sarma. a Data published previously as poster (Hoffmann et al., 2009). ⁎ p b 0.05.

Table 5 ECG effects of reference compounds in telemetered dogs. Compound

Dose mg/kg

HR bpm

Quinidinea

BL 5 15 45 BL 3 10 30 BL 0.3 1.5 5 15 BL 0.01 0.03 0.1 BL 10 25 50 BL 15 30 BL 0.3 1 3

78

Flecainidea

Atenolola

Dofetilideb

DL

sotalola

Moxifloxacinb

a

Nifedipine

Δ%

QRS ms

Δ%

46 +24.9 +39.3 +58.4⁎

76

45

88

117

48

85

51 +3.8 −10.9 +3.6

84

259

107

+4.6⁎ +8.7⁎ +12.4⁎ +11.4⁎ +17.2⁎ +19.1⁎

258 −7.6 −10.4⁎

112 +2.6 +1.7 −6.2

−1.3 +1.6 −2.0 +0.9

+15.9 +18.8⁎ +14.2

−4.0 −6.1 43

−4.0 +36.1 +90.4⁎

254

254

110

48

85

+10.2⁎ +17.1⁎ +18.1⁎ +17.1⁎

+3.9 +4.8⁎ +4.3⁎

−3.8 +4.7 +13.2⁎

+3.2 +2.5 +2.1

+8.0 +13.1

253

108 −4.1 0.0 −4.1

+9.6 −13.8 −11.8

+6.8 +8.0⁎ +15.1⁎

−2.8 +4.0 +15.5⁎

−0.4 +1.3 −1.5 +3.8

Δ%

253

114

49

QTcV ms

−9.9 −10.8 −11.7

−1.4 +8.4 +24.5⁎

−5.9 −11.2 −12.6 −10.1

Δ%

116 +2.8 +6.4 +14.7

+13.0 +20.6 +49.3⁎ 76

PQ ms

+2.3 +5.3⁎ 255

+4.9 −6.5 −16.2⁎

+0.7 −1.3 +0.5

Cmax μg/L – 1631 2538 4229 – 238 693 2700 – 144 594 2388 8069 – 1.55 4.24 16.9 – 5533 12,767 26,302 – 4637 6442 – 16 53 177

Data are mean values (n = 5–6 per dose). Pre-drug baseline values (BL) are absolute values in beats per minute (bpm) or ms, whereas all other values are expressed as % change versus BL (Δ%). Maximal drug plasma concentrations (Cmax) were determined in satellite animals (n = 3–4 per dose). Abbreviations: n.d., not determined; HR, heart rate; QTcV, corrected QT interval according to Van de Water. a Data from Himmel et al. (2012). b In-house data (unpublished). ⁎ p b 0.05.

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dogs only a slight heart rate decrease without statistical significance was found. 3.4.3. Class III anti-arrhythmics DL-sotalol and dofetilide A characteristic of class III anti-arrhythmic drugs is their ability to delay ventricular repolarization as for instance by inhibition of the cardiac ion current IKr (hERG K+ channel) (Numaguchi et al., 2000; Rasmussen, Allen, Blackburn, Butrous, & Dalrymple, 1992). After administration of DL-sotalol or dofetilide a pronounced, dose-dependent and statistically significant prolongation of the QTc intervals was seen in guinea pigs and dogs. In addition, in guinea pigs a QRS widening (with dofetilide only) and slight heart rate reduction was found, whereas in dogs these parameters were generally unaffected. Since dofetilide is a specific blocker of the hERG K+ channel but without inhibition of cardiac sodium channels (Gwilt et al., 1991), the cause of the QRS widening remains unclear. After intravenous administration of dofetilide, Morissette et al. (2013) found no effects on QRS whereas Lu et al. (1995) reported a slight, statistically not significant QRS widening at higher i.v. doses. 3.4.4. Nifedipine The dihydropyridine nifedipine is a blocker of the L-type calcium channel (Himmel et al., 2012). Nifedipine caused dose-dependent, significant heart rate increase and, at high doses, a shortening of the PQ interval in both guinea pigs and dogs. In guinea pigs the QTc intervals were statistically significantly shortened at the highest dose (3 mg/kg) tested. 3.4.5. Moxifloxacin Since the fluoroquinolone antibiotic moxifloxacin is a blocker of the cardiac hERG K+ channel (Alexandrou et al., 2006) without risk for fatal arrhythmias, it is often used as positive reference in thorough QT studies in clinical trials according to ICH E14 guidance (Anonymous, 2005a). After administration of moxifloxacin, a dose-dependent prolongation of QTc intervals was seen in both guinea pigs and dogs. 4. Discussion We established the telemetered guinea pig model in order to have a preclinical in vivo model for early cardiovascular risk and safety assessment in place to be used in conjunction with or as follow-up investigation to in vitro electrophysiology assays. The ECG model was validated with selected reference compounds and modified in a stepwise manner regarding animal housing, ECG electrodes placement and data evaluation. To enhance animal welfare the guinea pigs were housed in large groups when off study and were pair-housed for ECG recordings. In our experimental setting with telemetry recordings in a separate quiet room with darkened environment, baseline values of ECG parameters are of low variability and of good reproducibility. Optimized housing conditions allowing animals to exhibit natural behaviors may reduce possible stress which might have negative influence on study results (Klumpp, Trautmann, Markert, & Guth, 2006; Xing et al., 2015). Housing optimization in particular with regard to telemetry is considered a refinement in accordance with the principle of “3Rs” (refinement, reduction, replacement) (Hawkins, 2014). In contrast to anesthetized animal models, the use of telemetry in conscious, freely moving animals has some advantages, e.g. cross-over treatment design, long-term data recordings or the reuse of animals in further studies which reduces the overall number of animals and thus also contributes to the 3Rs. In anesthetized guinea pig models a PK/PD evaluation can be done on the same animals whereas in the telemetry model satellite animals were used to avoid disturbances during telemetry recordings. Other hemodynamic parameters like systemic blood pressure or cardiac contractility which are often included in anesthetized animal models could in principle also be done in telemetered animals by using specific telemetry devices.

However, our method described here is used to test for ECG changes at an early stage of pre-clinical development; detailed assessment of cardiovascular function in telemetered animals is conducted in species which are used usually in toxicological investigations (e.g. rats or dogs). In the studies with reference compounds as well in our historical data we did not found significant circadian rhythm as seen in rats. The absence of a clear circadian rhythm in telemetered guinea pigs was also described by others (e.g. Hess et al., 2007). We found a small increase in heart rate and locomotor activity at the beginning of the dark period which returned to normal baseline values within 1 h (see Fig. 3). Shiotani et al. (2007a) described clear circadian variations in RR interval in young, 6 weeks old guinea pigs but only small variations in 23 months old animals. Akita, Ishii, Kuwahara, and Tsubone (2001) found circadian rhythm in about 50% of investigated female guinea pigs with a body weight of 500–800 g indicating an age of about 4– 10 weeks. It appears that young guinea pigs with an age below 2 months show a diurnal rhythm with increased heart rate and locomotor activity and decreased body temperature during the dark period whereas such diurnal rhythm is reduced at older age. We found no differences in baseline values at different animal age, except for a lower heart rate (220 versus 205 bpm) in 3–4 months old guinea pigs compared to 12 months old guinea pigs. Continuous long-term ECG recordings (e.g. over 24 h or more) from several animals in parallel produce huge data sets which have to be analyzed and evaluated. To minimize manual workload it is important to ensure the best ECG signal quality possible. This would allow largely automated evaluation of the ECG signals requiring only quality check by random sampling orienting on software generated quality parameters. Telemetry receivers have to be positioned properly, particularly in large cages to capture telemetric signals completely. A second point to consider for improving ECG signal quality is the placement of the ECG electrodes. Different electrode configurations are described in the literature for rats (Sgoifo et al., 1996; Tontodonati, Fasdelli, & Dorigatti, 2011) or guinea pigs (Shiotani et al., 2007b). We have made best experience by placing one electrode abdominally on the diaphragm and the other, a so called solid tip electrode, intravenously into the cranial vena cava close to the base of the heart leading to clear, high-voltage ECG signals enabling an analysis of nearly 100% of all ECG recordings. Such an electrode configuration was also successfully implemented in telemetered Beagle dogs in our group and by others (Walisser et al., 2013). Due to high workload by scanning the large ECG data sets we did not scan each individual ECG recording completely for the occurrence of arrhythmias but only manually by random sampling. However, this harbors the risk to miss arrhythmic events. A solution for this problem could be a software based pre-scan of the ECG recordings with the Notocord ARR30a module (Notocord Systems, France) (Koeppel, Labarre, & Zitoun, 2012) or the recently released Ponameh “Data Insights”-module (DSI, USA). A prerequisite of such semi-automated arrhythmia analyses are high quality ECG waveforms which are obtainable with the solid tip electrode configuration. Our proposed statistical strategy uses evenly spaced time intervals of 30 min length to compare a response variable obtained in different treatment groups with corresponding vehicle data. The generalized linear model approach involves the fixed effects baseline, time, treatment and the interaction treatment-by-time. Additionally, a simple correlation structure of repeated measurements over time is assumed. The model is tested using different settings, such as interval widths, species, etc. and in particular, changes in QT intervals are detected sufficiently and results seem to be stable and plausible. The whole statistical methodology was implemented into a user-friendly automated process involving a SAS-macro producing descriptive and inferential statistical output like tables and figures. The outcome of the pharmacological validation with different positive reference compounds and the comparison with results from telemetered dogs, a model usually used at later stage of preclinical development for safety pharmacology cardiovascular assessment, revealed

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that the sensitivity of guinea pigs is very similar to dogs with regard to compounds which block the hERG K+ channel and prolong the QT interval (dofetilide, DL-sotalol, quinidine, flecainide, moxifloxacin) and was in good accordance with other publications in telemetered (Shiotani et al., 2005) and anesthetized guinea pigs (Hauser et al., 2005; Marks et al., 2012; Morissette et al., 2013). Block of the cardiac sodium channel results in a widening of the QRS complex and slowing of atrio-ventricular conduction (flecainide only) in both guinea pigs and dogs. After flecainide dosing the heart rate was reduced in guinea pigs whereas in dogs an increase in heart rate was seen, most likely secondary to other hemodynamic effects like decreased blood pressure or reduced cardiac output (Mecca, Elam, Nash, & Caldwell, 1980; Roden & Woosley, 1986). Similar to dogs, the effects in guinea pigs on QRS were more pronounced with flecainide than with quinidine. The calcium channel blocker nifedipine caused a heart rate increase which is considered a baroreflex mediated tachycardia as result of vasodilation and drop in blood pressure. A slight QTc shortening found in guinea pigs at high heart rates after administration of nifedipine reflects most likely an inadequate correction by both the Bazett and Sarma formulae. Such an effect was not seen in dogs with van de Water QT correction. Heart rate reduction after administration of β-blockers (e.g. atenolol, DL-sotalol) was hard to detect in our telemetry dogs since the baseline heart rate is low at rest (see Table 5). In guinea pigs a considerable decrease in heart rate was found after administration of DL-sotalol and atenolol. A delay in atrio-ventricular conduction as indicated in a prolonged PQ interval was found in both guinea pigs and dogs. Despite the slight underestimation of pronounced heart rate increase after nifedipine, the QT interval correction according to the square route correction of Bazett or the exponential correction of Sarma appears to correct sufficiently in most cases. Peak plasma concentrations determined from quinidine, flecainide, dofetilide, and moxifloxacin treated guinea pigs were lower than in dogs at equivalent doses. Besides possible different absorption or metabolism, this might be due to the different administration techniques. Plasma concentrations obtained after intravenous administration of flecainide or moxifloxacin published by Morissette et al. (2013) and Marks et al. (2012) were similar to those we obtained in telemetered guinea pigs after buccal administration. However, it cannot ruled out that after buccal administration to guinea pigs potentially not the whole amount of test compound was swallowed in contrast to direct oral capsule administration to dogs. 5. Conclusion During pharmaceutical drug development an in vivo model is required for early go/no go-decision making to substantiate or to disprove evidence for a potential arrhythmia risk based on cardiac ion channel in vitro screening. The telemetered guinea pig is an excellent model for investigating electrophysiological effects. We found that modifications in animal housing (social housing) and ECG electrodes placement (solid tip electrodes) lead to highly reliable and reproducible ECG data with low variability allowing largely automated evaluation. In our experience, QT interval correction according to Bazett and Sarma appears suitable in conscious guinea pigs. The telemetered guinea pig is sensitive to compounds which blocks cardiac sodium channels, hERG K+ channels and calcium channels, and is even more sensitive to β-blockers as dogs. Advantages of the conscious guinea pig model are (i) the low amount of test compound needed, (ii) it is less expensive than dog or NHP models and (iii) it contributes to the 3Rs concept; disadvantages are difficulties with blood sampling and intravenous administration. Overall, the telemetered guinea pig model is appropriate for early stage ECG investigations with similar translatability to human relevance as found in dogs or other large animal models. Findings should be assessed in context with results from in vitro investigations (e.g. cardiac ion channel voltage clamp assays, action potential assays) and, if available, from toxicology studies.

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Statement on conflict of interest and source of funding The authors have no conflicts of interest that could inappropriately influence this work, which was funded by the author's employer Bayer Pharma AG. Acknowledgements We gratefully acknowledge the valuable contributions of Marcus Deitermann (QTc formula calculations) and Volker Harm (SAS-macro programming). Furthermore we thank Michael Kayser (bioanalytical method validations), Dr. Frank-Thorsten Hafner, and Dr. Uwe Thuss for conduction of bioanalytical investigations. We also thank Herbert Himmel for reviewing the manuscript. References Akita, M., Ishii, K., Kuwahara, M., & Tsubone, H. (2001). The daily pattern of heart rate, body temperature, and locomotor activity in guinea pigs. Experimental Animals, 50(5), 409–415. Alexandrou, A. J., Duncan, R. S., Sullivan, A., Hancox, J. C., Leishman, D. J., Witchel, H. J., et al. (2006). Mechanism of hERG K(+) channel blockade by the fluoroquinolone antibiotic moxifloxacin. 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