Validation of the normal, freely moving Göttingen minipig for pharmacological safety testing

Journal of Pharmacological and Toxicological Methods 60 (2009) 79–87

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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

Original article

Validation of the normal, freely moving Göttingen minipig for pharmacological safety testing Michael Markert a,⁎, Miriam Stubhan c, Karin Mayer a, Thomas Trautmann a, Anja Klumpp a, Annette Schuler-Metz b, Kurt Schumacher b, Brian Guth a a b c

Department of Drug Discovery Support, General Pharmacology Group, Boehringer Ingelheim Pharma GmbH & Co KG, Germany Department of Drug Discovery Support, DMPK Group, Boehringer Ingelheim Pharma GmbH & Co KG, Germany Department of Non Clinical Drug Safety, Biological Lab Service Group, Boehringer Ingelheim Pharma GmbH & Co KG, Germany

a r t i c l e

i n f o

Article history: Received 13 November 2008 Accepted 22 December 2008 Keywords: Methods Conscious minipig Contractility Telemetry Hemodynamic QT duration Arterial blood pressure Heart rate

a b s t r a c t Introduction: The objective of this study was to use a newly established cardiovascular model using freely moving minipigs to document the hemodynamic and electrocardiographic effects of known pharmacological agents. The data generated are to serve as the basis of pharmacological drug safety evaluations using this new model. Methods: 6 Göttingen minipigs were equipped with a radiotelemetry system (ITS). Following a recovery period, aortic pressure (AP), left ventricular pressure (LVP), lead II of the ECG and body temperature were continuously recorded throughout an 8 h monitoring period following oral administration of one of the test agents or vehicle. Notocord HEM 4.2 software was used for data acquisition. One known hERG blocker (moxifloxacin (30, 100 or 300 mg/kg)) and one non-selective beta-adrenoreceptor antagonist (propranolol (3,10 or 20 mg/kg)) were tested in the model using a cross-over study design in 6 pigs. Results: We obtained high signal quality and found stable hemodynamic parameters with low intrinsic heart rates in the Göttingen minipig under resting, pre-treatment conditions. After oral dosing of moxifloxacin, a substantial, dose-dependent increase in the QT-interval duration could be shown, as anticipated for this agent. After propranolol administration, a decrease in HR and left ventricular dP/dt was detected as expected for a beta-adrenoceptor blocking agent. Discussion: The present data demonstrate that using this model in conscious, chronically instrumented Göttingen minipigs, a cross-over study with six animals was sensitive enough to detect a dose-dependent QT prolongation when moxifloxacin was administered in oral doses leading to clinically relevant plasma drug concentrations. Additionally, we could demonstrate the expected propranolol-induced effects on heart rate and myocardial contractility, despite the low intrinsic resting heart rates in these minipigs. These data support the use of the Göttingen minipig as a sensitive cardiovascular and electrocardiographic model for the testing of new pharmaceutical agents. © 2009 Elsevier Inc. All rights reserved.

1. Introduction We recently established a telemetric animal model using Göttingen minipigs for safety pharmacology assessment of cardiovascular and electrocardiographic function (Stubhan et al., 2008). A fully implantable telemetric system was used with pressure transducers in the left ventricle and the descending thoracic aorta connected to a transmitter unit and with integrated electrocardiogram (ECG) leads (ITS). This was, to our knowledge, the first application of this system in the minipig. In the present study, we evaluated the effects of 2 well characterized therapeutic compounds on hemodynamic (heart rate, aortic pressure, left ventricular pressure, LVdP/dtmax) and electro-

⁎ Corresponding author. Boehringer Ingelheim Pharma GmbH & Co KG J91 UG, Birkendorferstr. 65, 88397 Biberach, Germany. Tel.: +497351 548727; fax: +497351 838727. E-mail address: [email protected] (M. Markert). 1056-8719/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.vascn.2008.12.004

cardiographic (PR, QRS, QT, RR) parameters, as well as body temperature, in 6 normal, freely moving Göttingen minipigs over 8 h following dosing. LVP measurements are particularly useful in that the derivative of LVP, i.e. LVdP/dt, is a well-established parameter for the assessment of cardiac contractility (Klumpp et al., 2006; Markert et al., 2004; Markert et al., 2007). Drug-induced prolongation of the QT-interval (a marker for a delay in cardiac repolarization) of the electrocardiogram (ECG), has increasingly drawn attention from regulatory agencies and the pharmaceutical industry and detecting these effects has had a great impact on drug discovery and development (Fermini & Fossa, 2004). The delayed repolarization, frequently attributable to blockade of the rapidly activating delayed rectifier potassium channel, IKr, favors the genesis of early after-depolarization (EAD), which can initiate an arrhythmia (Zabel et al., 1997). Additionally, the prolongation of the QT interval by drugs is often associated with increased heterogeneity of cardiac repolarization (Antzelevitch, 2004), a substrate for a reentrant mechanism responsible for a sustained arrhythmia. Preclinical

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testing for tolerability and safety is required in both rodents (usually the rat) and in a second, non-rodent species (Guth et al., 2004; Pugsley & Curtis, 2006). Nevertheless, there are some ‘safe’ drugs that inhibit IKr and cause QT prolongation without inducing TdP (Fermini & Fossa, 2004; Redfern et al., 2003). In any case, it is desirable that models used to test new drugs for potential effects on the QT-interval are shown to have the needed sensitivity to detect subtle changes. In this study, we have tested a known hERG-blocking agent, moxifloxacin, as well as a ß-adrenoceptor blocker, propranolol, in the pig model. Moxifloxacin has been recommended as a positive control for clinical trials assessing QT prolongation potential (ICH S7B, 2005) as it produces a highly reproducible effect on the QT interval duration. Propranolol, is a non-selective ß-adrenocept blocker mainly used in the treatment of hypertension. It is expected to cause a decrease in heart rate and a reduction of myocardial contractility (Lalonde et al., 1987). 2. Methods 2.1. Telemetry system The telemetry system used to measure cardiovascular parameters was manufactured by Konigsberg Instruments, Inc. (Pasadena, CA) and marketed by RMISS (Delaware). The system consists of 5 major components: 1) an implantable transducer unit; 2) a receiver (antenna) located in the animals cage together with an amplifier; 3) an ambient pressure monitor to measure atmospheric pressure; 4) a PC-based “base station” to receive and process the amplified signals; and 5) a PC-based data acquisition system (NOTOCORD Hem 4.2) to process signals. The implantable transducer unit (“T27” total implant) consists of: (1) two high fidelity pressure transducers (4.0 mm diameter), (2) an ECG cable; (3) micro-power battery-operated electronics that process and digitize the information from the pressure transducers and the ECG lead, (4) a radio-frequency transmitter that sends the signals to the telemetry receiver, and (5) a battery. A small cable projecting from the transmitter serves as a switch that allows the device to be turned on and off to prolong battery life. 2.2. Animals Animal handling and treatment followed the German Law on the Protection of Animals and was performed with permission from the Baden-Württemberg Animal Welfare Committee. For this study 6 Göttingen minipigs were purchased from Ellegaard Göttingen minipigs (Aps, Dalmose, Denmark). The Göttingen minipig is a cross between the Vietnamese potbelly pig, Minnesota minipig and the German landrace pig. It is now a genetically outbred minipig available for research. The minipigs used on study were of both sexes (2 males, 4 females), at least one year of age and weighed 22–32 kg. The minipigs were housed individually or in pairs (where possible) in an AAALAC-accredited facility. Room temperature (21 ± 2 °C) and humidity (60 ± 15%) were controlled with a ventilation turnover of 12 cycles/h. The animals had access to water ad libitum and were fed ~ 300 g per animal (~25 kg) of a solid standard minipig diet [SDS SMP (E) from Special Diets Services, Witham, Essex, U.K.; supplier SDS Deutschland Altrip] adapted to body weight once daily. The minipig pens were supplied with wood shavings and sleeping boxes with straw as well as metal chains and balls and rubber nipples for environmental enrichment. Furthermore, the minipigs received daily training from the animal staff and were weighed at least once a week. To monitor the health status of the minipigs, blood was collected every 3 months to monitor haematology and clinical chemistry parameters, including electrolytes and kidney parameters. Before surgery each animal underwent a routine veterinary health inspection

and a brief electrocardiographic assessment (about 15 min using external ECG leads) taken while resting in a sling. The minipigs were previously conditioned to the telemetry lab prior to surgical implantation of the transducer, a process beginning at least 2 months before surgery (e.g. p.o. application, general handling, etc.). In the telemetry lab, room temperature and humidity were controlled (22 ± 2 °C; 60 ± 15% humidity) with a ventilation turnover of 13 cycles/h. The light:dark cycle was maintained as a 12 h light (365 lux) and dark (3 lux, “moonlight”) cycle. Background “noise” was supplied using a radio during the lighted period in an effort to mask any potential noise coming from outside of the pen area. 2.3. One-time surgical implantation The transducers of the T27 implant were calibrated prior to implantation and the unit was sterilized using a low pressure ethylene oxide process. All surgical procedures were conducted under strict aseptic conditions. For perioperative analgesia the minipigs were given meloxicam (Metacam, 0.4 mg/kg, orally). Additionally, a second analgesic component was given: 2–4 Durogesic Smat patches (fentanyl, 25 μg/h) were attached behind the ear the day before surgery after shaving and degreasing the skin. During surgery, a supplemental fentanyl infusion (0.005 mg/kg/h) was given. For prophylactic antibiotic treatment, amoxicillin (Duphamox LA, 15 mg/kg) was given beginning the day before surgery. Animals were sedated with a combination of Ketavet (ketamin hydrochloride, 15 mg/kg i.m.) and midazolam (Dormicum, 0.35 mg/kg i.m.). Propofol (Propofol Lipuro 1%, ~ 2 mg/kg) was then administered i.v. to allow endotracheal intubation. The dose was dependant on effect and administration was performed very slowly to prevent depression of the respiratory system. After preparing the animals skin for surgery under aseptic conditions, anesthesia was maintained throughout the remaining procedure with isoflurane (Forene, 1–3%) inhalation anaesthesia with mechanical ventlilation (66% O2) at a ventilation rate of 14/min. The minipigs were placed in a lateral recumbency with the left side facing the surgeon. An incision was made near the fifth or the sixth rib. The dissection through the connective tissue and the muscles was continued until the rib and the intercostal muscles were reached. As the next step, a small pocket was opened in the abdominal wall for implantation of the transmitter, battery housing and induction switch coil. The cables with both pressure transducers and the ECG leads extending from the transmitter were guided subcutaneously to the lateral incision. The antenna was guided subcutaneously from the transmitter location ventrally towards the linea alba and further parallel to the mamma complexes in a cranial direction. The initial ventral incisions required for battery and transmitter placement were closed. A left thoracotomy was then performed after removing the fifth or sixth rib to provide access to the left ventricle apex and the thoracic aorta. After insertion of the left ventricular Konigsberg transducer and fixing it in place with a purse string suture, the aortic pressure transducer, which also served as one electrode of the ECG, was implanted just below the aortic arch. The other electrode was placed toward the sternum under ECG-signal control to ensure a good (with lead II-like configuration) signal morphology. After the implantation, the lung was inflated, the intercostal muscles were sutured closed and the pneumothorax evacuated. Chest incisions were closed in layers and an intracutaneous suture closed the wound. The gas anaesthesia was then turned off and the minipigs were allowed to wake up. Antibiotics were administered for 10 days postoperatively. To ensure postoperative analgesia, the transdermal fentanyl patches were kept in place for 4 days and meloxicam administration was continued for 10 days. The animals were given at least 21 days for a full recovery prior to the initiation of experiments.

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2.4. Experimental design On the day of the study, the animals were transferred to pens set up for the telemetry measurements and housed in pairs. They were moved in the early morning and left undisturbed for the remainder of the study, except where mentioned below. An initial period of 30– 45 min allowed the minipigs to acclimate to these measurement pens, although earlier training assured that they were well familiar with these pens. After this acclimatization period, the measurements were started. The subsequent hour preceding the administration procedure was taken as a control period for baseline measurements. After this hour, the minipigs were taken out of their pens and test article was administered orally with a special dosing catheter and they were then returned to their pens and left undisturbed for the remainder of the data collection period. Experiments were performed using a crossover design, so that each minipig received each treatment and thereby could serve as its own control. Three doses (3, 10 and 20 mg/kg) of propranolol (dl-propranolol hydrochloride, Sigma) or moxifloxacin (30, 100 and 300 mg/kg, Avalox®, Bayer) were administered orally together with a vehicle treatment (0.5% Natrosol). Blood samples were taken from the jugular vein both prior to treatment and following the 7 h observation period for the measurement of drug plasma concentration (see below). The suspensions were prepared fresh daily. The animals were monitored continuously by video for the duration of the study. They had free access to water and 7 h after application of test article they were fed their standard diet. 2.5. Data acquisition and analysis Digitized telemetry signals were processed by NOTOCORD software (Hem 4.2) on a beat-to-beat basis. The following parameters were continuously measured throughout the experiment: aortic pressure (AP, @250 Hz), left ventricular pressure (LVP, @250 Hz), ECG (@1000 Hz) and temperature (1 Hz). Hemodynamic parameters calculated from these signals during the experiment included: systolic and diastolic aortic pressure, peak systolic and end-diastolic left ventricular pressure, LV dP/ dtmax and min, heart rate and from the ECG wave PQ-, QRS- and QT intervals. EXCEL software (version 2003) was used for data analysis. Data were summarized at predefined time points every 10 min as median values ± SD. For each time point a minimum of 400 sequential beats were used to calculate the median value. The mean of the median values± SD of every parameter are presented graphically over the 7 h study period. Values after placebo administration were compared to the pre-treatment values. For statistical evaluation, a standardized area under the curve was calculated for each parameter over predefined time intervals. Thus, two measurement phases were defined (early phase, 30 min–3 h and late phase, 3 h–7 h), depending on the pharmacokinetic profile of the given compound. Comparisons between these 2 phases were performed by a paired t test in all 6 animals. Statistical significance was accepted when p b 0.05. For evaluation of the QT-house developed computer program (Qtana) was used. Using this program, the data were divided into a training set and a test set. With the data from the training set, the regression algorithm calculated the best parameter estimates (a, b, c, d) for each correction function (nonlinear, shifted logarithmic, exponential, Bazett, Fridericia, Sarma, etc., see Table 1). Different criteria, such as the Pearson`s coefficients (r), Akaike's information criterion (AIC) and PRESS RMSE were considered in order to select the best model (in this particular study the shifted log formula was selected). To illustrate the results, the QT or QTc values are plotted against RR and the respective correlations were determined and graphically displayed as the corresponding regression lines as described elsewhere (Meyners & Markert, 2004). The statistical evaluation was performed using the software package SAS Version 8.2.

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Table 1 Parameter estimates a, b, c and selection criteria Pearson's correlation coefficient r, Akaike's information criterion (AIC) and PRESS RMSE derived from 1040 observations of an individual QT correction. Model

a

r

AIC

RMSE

Bazett QT = a ⁎ RR^(1/2) Fridericia QT = a ⁎ RR^(1/3) Sarma exponential QT = a − b ⁎ exp(c⁎RR) Linear QT = a + b ⁎ RR Hyperbolic QT = a + b / RR Parabolic QT = a ⁎ RR^b Shifted logarithmic QT = log(a + b ⁎ RR) Exponential QT = a + b ⁎ exp(− RR)

341.31

b

c

− 0.78

2684.00

73.67

330.96

− 0.10

2320.60

41.15

0.00

2266.03

37.59

− 2.82

349.36

356.61

206.02

123.59

0.02

2267.05

37.71

391.56

− 63.90

0.00

2264.93

37.58

329.43

0.31

0.04

2260.10

37.29

4.21

1.28

0.73

1814.80

18.27

429.82

− 271.97

− 0.02

2270.48

37.92

2.6. Bioanalytical sample preparation Plasma/Serum sample preparation for analysing moxifloxacin in minipig plasma was carried out via acetonitrile precipitation on a Hamilton Star (Hamilton Bonaduz Switzerland) robot system. Aliquots (10 μl) of the plasma sample (10 µl of blank minipig plasma for calibration standards), 10 µl of calibration solution (20% water and 80% acetonitrile with defined standard solutions for calibration standards) and 30 µl internal standard were mixed and precipitated with 200 µl acetonitrile. The precipitation was kept in the freezer for 15 min and centrifugated at 3000 rpm for 3 min. The supernatant was diluted 1:100 with 25% acetonile 75% of a 0.1% formic acid and injected for analysis by LC–ESI-MS/MS. Standard solutions were prepared as nM dilutions of the original solutions. Calibration range: 100 nM–100,000 nM. 2.6.1. Analysis by LC–ESI-MS/MS The analytical instrumentation consisted of a CTC HTX PAL (CTC Analytics, Switzerland) autosampling system coupled to an Agilent 1100 series liquid chromatography device (Agilent Technologies, Palo Alto, CA, USA) interfaced with an Applied Biosystems/MDS Sciex API 5000 triple quadrupole mass spectrometer (MDS Sciex, Concord, Canada). For the mass transitions of the analyte the following parameters were chosen: Moxifloxacin: monitoring the m/z 402 → 261 MS/MS transition, the dwell times were 100 ms, and both Q1 and Q3 were operated with unit mass resolution. The declustering potential and collision energy were 151 and 29 V, respectively. Mass spectral data were analysed using Analyst 1.4.1 software (Applied Biosystems MDS Sciex, Concord, ON, Canada). 3. Results All animals survived the surgical procedure and recovered quickly. Excellent signal quality was obtained and stable hemodynamic parameters with a low intrinsic heart rate in the Göttingen minipig were noted. After oral dosing, the hemodynamic parameters returned quickly (within 10–15 min) to baseline values indicating that the procedure was well tolerated. 3.1. Moxifloxacin 3.1.1. Hr Baseline heart rate values were 58 ± 5 beats per minute. Moxifloxacin had no effect on heart rate up to 100 mg/kg. With 300 mg/kg there was a significant (p = 0.02) increase in heart rate when compared to the placebo group in the first (30 min–3 h) and a

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Fig. 1. Uncorrected QT values measured over a 7 h monitoring period with a fully implantable telemetric device (ITS) in freely moving Göttingen minipigs. Data of all experiments were summarized as mean ± SD. The dotted line at 0 h indicates oral administration of moxifloxacin whereas the dotted line at 7 h indicates feeding. The arrows indicate when an effect was statistically significant (p b 0.05).

trend (p = 0.07) for an increase in the second time period (3 h–7 h, Figs. 1 and 2). 3.1.2. SAP and DAP Baseline blood pressure values were 122 ± 10 mmHg for the systolic and 85 ± 8 for the diastolic blood pressure. There was no relevant effect on systolic blood pressure up to 300 mg/kg There was no relevant effect on diastolic blood pressure up to 100 mg/kg. With 300 mg/kg there was a trend for an (p = 0.05) increase in the first (30 min–3 h) and a significant (p = 0.002) increase in the second time period (3 h–7 h, Fig. 3). 3.1.3. Lvp Baseline left ventricular pressure was 120 ± 11 mmHg. There was no effect on LVP with moxifloxacin up to 300 mg/kg (data not shown here).

3.1.4. LVdP/dtmax Baseline LVdP/dtmax values were 2200 ± 221 mmHg/s. Moxifloxacin had no effect on myocardial contractility up to 100 mg/kg. With 300 mg/kg there was a significant increase in LVdP/dtmax (p = 0.01) in the first time phase (Fig. 4). 3.1.5. QT interval Baseline QT values were 350 ± 20 ms. There was a dose-dependent significant increase with moxifloxacin at doses of 30 (6%), 100 (17%) and 300 (22%) mg/kg (Figs. 1 and 5). 3.1.6. QTc interval When the QT interval was individually corrected for heart rate (shiftedlogFunction) in the highest dose evaluated (300 mg/kg) there was a 22% increase in the QTc interval compared to placebo values.

Fig. 2. Heart rate values measured over a 7 h monitoring period. Data were summarized as mean ± SD. The dotted line at 0 h indicates oral administration of moxifloxacin whereas the dotted line at 7 h indicates feeding. The arrow indicates when an effect was statistically significant (p b 0.05).

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Fig. 3. Diastolic blood pressure values measured over a 7 h monitoring period. Data were summarized as mean ± SD. The dotted line at 0 h indicates oral administration of moxifloxacin whereas the dotted line at 7 h indicates feeding. The arrow indicates when an effect was statistically significant (p b 0.05).

3.1.7. Body temperature Baseline body temperature was 37.6 ± 0.2 °C. There was no effect on LVP with moxifloxacin up to 300 mg/kg (data not shown here).

20 mg/kg dose led to a maximum decrease to an average heart rate of 34 beats per minute (Fig. 6).

3.1.8. Exposure data The serum concentrations @ 7 h were 4.1, 10.6, and 19.6 μmol (total drug concentration after dosing of 30 mg/kg, 100 mg/kg and 300 mg/kg, respectively).

3.2.2. SAP and DAP Baseline blood pressure values were 121 ± 8 mmHg for the systolic and 83 ± 8 for the diastolic blood pressure. Propranolol at doses up to 20 mg/kg showed no relevant effect on systolic and diastolic blood pressure (data not shown here).

3.2. Propranolol 3.2.1. Hr Baseline heart rate values were 60 ±4 beats per minute. Propranolol caused a significant decrease in heart rate with all doses tested. The

3.2.3. Lvp Baseline left ventricular pressure was 120 ± 11 mmHg. There was no effect on LVP with propranolol up to 300 mg/kg (data not shown here).

Fig. 4. LVdP/dt values measured over a 7 h monitoring period. Data were summarized as mean ± SD. The dotted line at 0 h indicates oral administration of moxifloxacin whereas the dotted line at 7 h indicates feeding. The arrow indicates when an effect was statistically significant (p b 0.05).

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Fig. 5. ECG morphology changes (increase of QT interval) with 30, 100 and 300 mg/kg moxifloxacin treatment (red lines, arrows) compared to pre-dose (blue/black lines). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2.4. LVdP/dtmax Baseline LVdP/dtmax values were 2300 ± 232 mmHg/s. Propranolol caused a significant (p b 0.05) and long-lasting decrease of myocardial contractility in both time phases (30 min–7 h, Fig. 7). 3.2.5. QT interval Baseline QT values were 362 ± 18 ms. There was no significant effect on uncorrected QT interval up to 20 mg/kg with propranolol,

however there was a trend (p = 0.09) for a decrease of the QT interval with the 20 mg/kg dose tested (Fig. 8).

3.2.6. QTc interval When the QT interval was individually corrected for heart rate (shiftedlogFunction), there was a decrease of 9% in QT interval in the highest dose (20 mg/kg) compared to placebo values (Fig. 9).

Fig. 6. Heart rate values measured over a 7 h monitoring period. Data were summarized as mean ± SD. The dotted line at 0 h indicates oral administration of propranolol whereas the dotted line at 7 h indicates feeding. The arrow indicates when an effect was statistically significant (p b 0.05).

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Fig. 7. LVdP/dt values measured over a 7 h monitoring period. Data were summarized as mean ± SD. The dotted line at 0 h indicates oral administration of propranolol whereas the dotted line at 7 h indicates feeding.

3.2.7. Arrhythmia/ECG morphology Propranolol at doses up to 20 mg/kg showed no effect on the ECG waveform morphology nor caused arrhythmias. However, in a pilot study with 30 mg/kg, the minipigs showed toxic side effects (vomiting, general discomfort and restlessness) as well as arrhythmias in the ECG (2nd degree AV-block, Fig. 10). 3.2.8. Body temperature Baseline body temperature was 37.7 ± 0.2 °C. There was no effect on LVP with moxifloxacin up to 300 mg/kg (data not shown here). 4. Discussion Good signal quality was obtained in the conscious minipig instrumented as described for the telemetric collection of hemodynamic

and ECG data. The minipigs had stable hemodynamic parameters over the observation period with a remarkably low intrinsic heart rate. This suggested that the test environment led to little stress for the animals and attempts to exclude disturbing external stimuli were successful. This type of result is very comparable to the data quality from trained Labrador dogs (Klumpp et al., 2006) and the data from a previously published paper describing the minipig model (Stubhan et al., 2008). After oral dosing, the hemodynamic parameters returned quickly (10– 15 min) to pre-dose baseline values, further indicating that the animals were well adapted to the study conditions and were unstressed. Propranolol was selected for testing in this model as a prototypical non-selective beta-adrenoceptor blocker and was expected to block sympathetic input to the heart with anticipated decreases in heart rate and myocardial contractility. Indeed, propranolol at doses of 3, 10 and 20 mg/kg caused a substantial dose-dependent decrease in HR,

Fig. 8. QT values measured over a 7 h monitoring period. Data were summarized as mean ± SD.

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Fig. 9. Individual QTclog values measured over a 7 h monitoring period. Data were summarized as mean ± SD.

myocardial contractility, and a shortening of the log-corrected QT interval. Such effects on heart rate have been well documented in minipig experimental models (Kano et al., 2005) and it has been reported that propranolol shortened the QTc interval in healthy volunteers (Clozel et al., 1984; Sundaram et al., 2008). Results from the pilot study noted that when using higher doses (e.g. 30 mg/kg), the minipigs showed adverse effects (vomiting, general discomfort and restlessness) including arrhythmias (2nd degree AV-block).

Because it causes a clear QT interval prolongation in clinical studies and has a well-known PK profile, moxifloxacin has been recommended as a positive control for clinical trials assessing QT prolongation potential (ICH E14). It has been reported that QT interval at rest (i.e., RR = 1000 ms) significantly increased from 379± 24 ms with placebo to 394 ± 33 ms with 400 mg of moxifloxacin (p b .05) and to 396 ± 28 ms with 800 mg of moxifloxacin (p b .05). These effects corresponded to increases of 4.0%± 5.1% and 4.5%± 3.8%, respectively (Demolis et al.,

Fig. 10. ECG morphology changes (shortening of QT interval and AV-block) with 30 mg/kg propranolol treatment (red line) compared to pre-dose (blue lines). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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2000). In clinical use, a peak level of 4.5 μg/ml (5.6 μM unbound) was reported after 400-mg daily doses for 10 days (Balfour and Wiseman, 1999). In 787 patients in Phase 3 clinical trials, daily repeated doses of 400 mg moxifloxacin increased the QT interval by 6 ± 26 ms (mean± SD) (Prod Info Avelox®, 2003, www.avalox.com). In over 20 specially designed studies in healthy volunteers using standardized approaches and methods, single oral doses of 400 mg moxifloxacin have consistently produced a mean increase in the QTc interval ranging from 5 to 10 ms (Shah, 2005). Our study clearly demonstrated that the concentrationdependent effect of moxifloxacin in the conscious minipig is comparable to clinical outcomes. With moxifloxacin, clinically relevant QTc prolongation is seen at a concentration that produces ~10% inhibition of the hERG current, similar to many IKr/hERG blockers (e.g. dofetilide, E-4031, cisapride, terfenadine, and risperidone). In this telemetry minipig study, the serum concentrations @ 7 h were 4.1, 10.6, and 19.6 μmol (total drug concentration after dosing of 30 mg/kg, 100 mg/kg and 300 mg/kg, respectively). These exposures were associated with mean maximal QT prolongation of 6, 17, and 22% (vs. placebo), respectively. 5. Conclusion Moxifloxacin showed the expected dose-dependent effects on the QT-interval in conscious minipigs. The present data demonstrate that a cross-over study conducted with 6 animals was sensitive enough to detect a statistically significant QT prolongation when moxifloxacin was administered in oral doses leading to clinically relevant plasma drug concentrations. Propranolol showed the expected effects of a prototypical nonselective beta-adrenoceptor blocker and was shown to block sympathetic input to the heart with anticipated effects on heart rate and myocardial contractility. Indeed, propranolol in doses of 3,10 and 20 mg/ kg caused a substantial dose-dependent decrease in HR, myocardial contractility, and a shortening of the log-corrected QT interval. In conclusion, the trained Göttingen minipig appears to be well suited for cardiovascular safety pharmacology studies. References Antzelevitch, C. (2004). Arrhythmogenic mechanisms of QT prolonging drugs: Is QT prolongation really the problem? Journal of Electrocardiology, 37(SUPPL.), 15–24. Balfour, J. A. B., & Wiseman, L. R. (1999). Moxifloxacin. Drugs, 3, 363–374.

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Clozel, J. P., Danchin, N., Genton, P., Thomas, J. L., & Cherrier, F. (1984). Effects of propranolol and of verapamil on heart rate and blood pressure in hyperthyroidism. Clinical Pharmacology and Therapeutics, 36(1), 64–69. Demolis, J. L., Kubitza, D., Tenneze, L., & Funck-Brentano, C. (2000). Effect of a single oral dose of moxifloxacin (400 mg and 800 mg) on ventricular repolarization in healthy subjects. Clinical Pharmacology and Therapeutics, 68, 658–666. Fermini, B., & Fossa, A. A. (2004). Pre-clinical assessment of drug-induced QT interval prolongation. Current issues and impact on drug discovery. Annual Reports in Medicinal Chemistry, 39, 323–334. Guth, B. D., Germeyer, S., Kolb, W., & Markert, M. (2004). Developing a strategy for the nonclinical assessment of proarrhythmic risk of pharmaceuticals due to prolonged ventricular repolarization. Journal of Pharmacological and Toxicological Methods, 49(3), 159–169. ICH (2005). The nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals (S7B). Kano, M., Toyoshi, T., Iwasaki, S., Dato, M., Shimizu, M., & Ota, T. (2005). QT PRODACT: Usability of miniature pigs in safety pharmacology studies: Assessment for druginduced QT interval prolongation. Journal of Pharmacological Sciences, 99(5), 501–511. Klumpp, A., Trautmann, T., Markert, M., & Guth, B. (2006). Optimizing the experimental environment for dog telemetry studies. Journal of Pharmacological and Toxicological Methods, 54(2), 141–149. Lalonde, R. L., Pieper, J. A., Straka, R. J., Bottorff, M. B., & Mirvis, D. M. (1987). Propranolol pharmacokinetics and pharmacodynamics after single dose and at steady state. European Journal of Clinical Pharmacology, 33, 315–318. Markert, M., Klumpp, A., Trautmann, T., & Guth, B. (2004). A novel propellant-free inhalation drug delivery system for cardiovascular drug safety evaluation in conscious dogs. Journal of Pharmacological and Toxicological Methods, 50(2), 109–119. Markert, M., Klumpp, A., Trautmann, T., Mayer, K., Stubhan, M., & Guth, B. (2007). The value added by measuring myocardial contractility ‘in vivo’ in safety pharmacological profiling of drug candidates. Journal of Pharmacological and Toxicological Methods, 56(2), 203–211. Meyners, M., & Markert, M. (2004). Correcting the QT interval for changes in HR in preclinical drug development. Methods of Information in Medicine, 43, 445–450. Pugsley, M. K., & Curtis, M. J. (2006). Safety pharmacology in focus: New methods developed in the light of the ICH S7B guidance document. Journal of Pharmacological and Toxicological Methods, 54(2), 94–98. Redfern, W. S., Carlsson, L., Davis, A. S., Lynch, W. G., MacKenzie, I., Palethorpe, S., Siegl, P. K. S., Strang, I., Sullivan, A. T., & Wallis, R. (2003). Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: Evidence for a provisional safety margin in drug development. Cardiovascular Research, 58(1), 32–45. Shah, R. (2005). Drugs, QT interval prolongation and ICH E14: The need to get it right. Drug Safety, 28(2), 115–125. Stubhan, M., Markert, M., Mayer, K., Trautmann, T., Klumpp, A., Henke, J., & Guth, B. (2008). Evaluation of cardiovascular and ECG parameters in the normal, freely moving Göttingen minipig. Journal of Pharmacological and Toxicological Methods, 57, 2002–2211. Sundaram, S., Carnethon, M., Polito, K., Kadish, H., & Jeffrey, J. (2008). Autonomic effects on QT–RR interval dynamics after exercise. American Journal of Physiology Heart and Circulatory Physiology, 294, H490–H497. Zabel, M., Woosley, R. L., & Franz, M. R. (1997). Is dispersion of ventricular repolarization rate dependent? PACE — Pacing and Clinical Electrophysiology, 20(10 I), 2405–2411.